Phytochemistry Letters 9 (2014) 141–148

Oncibauerins A and B, new flavanones from Oncidium baueri(Orchidaceae)

Josiane A. Monteiro a, Ivania T.A. Schuquel a, Thiago L. de Almeida a, Silvana M. de O. Santin a,Cleuza C. da Silva a, Lucas U.R. Chiavelli a, Ana L.T.G. Ruiz b, Joao E. de Carvalho b,Debora B. Vendramini-Costa b, Celso V. Nakamura c, Debora B. Scariot c,Vanessa Kaplum c, Ricardo T. Faria d, Armando M. Pomini a,*a Department of Chemistry, State University of Maringa, Maringa, Parana, Brazilb Division of Pharmacology and Toxicology, State University of Campinas, Campinas, Sao Paulo, Brazilc Department of Basic Health Sciences, State University of Maringa, Maringa, Parana, Brazild Department of Agronomy, State University of Londrina, Londrina, Parana, Brazil

A R T I C L E I N F O

Article history:

Received 5 February 2014

Received in revised form 29 May 2014

Accepted 5 June 2014

Available online 20 June 2014

Keywords:

Orchidaceae

Oncidium baueri

Secondary metabolites

Biological activities

A B S T R A C T

The phytochemical study of Oncidium baueri (Orchidaceae) afforded two new compounds (1 and 2),

along with thirteen known structures (3–15). The chemical structures were established on the basis of

spectral evidence and the new compounds, obtained as a mixture of two glycosylated flavanones were

assigned by comprehensive spectroscopic analysis by 1D and 2D NMR spectroscopy and HRMS.

Moreover, the cytotoxic activity of the crude extract (EBOB), semi-purified fractions (HEOB, CLOB, ACOB,

BUOB and HMOB) and some isolated metabolites against ten human cancer cell lines (U251, UACC-62,

MCF-7, NCI-ADR/RES, 786-0, NCI-H460, PC-3, OVCAR-3, HT29, K562) and the antiprotozoal activity

against Trypanosoma cruzi and Leishmania amazonensis were evaluated. The CLOB fraction showed the

best antitrypanosomal and antileishmanial activities, as well as considerable cytotoxicity against cancer

cell lines UACC-62 (melanoma) and K562 (leukemia) with GI50 values of 6.83 mg/mL and 0.5 mg/mL,

respectively. Furthermore, BUOB and HMOB fractions showed selectivity for UACC-62 (melanoma) with

GI50 < 0.01 mg/mL and the compounds 11, 13 and 14 had inhibited cell growth of almost cancer cell lines

tested with GI50 values close to 30 mg/mL.

� 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

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Phytochemistry Letters

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate /p hyt ol

1. Introduction

Oncidium is one of 850 genera in the Orchidaceae family(Dressler, 1981). Orchid Oncidium baueri Lindl., native to theBrazilian Amazon region, has a high potential for ornamentallandscaping projects and as a cut flower, but little is known aboutthe chemistry and biological activities of this species (Faria et al.,2006). In addition, when one considers the size of the familyrelative to the number of published research articles on its species,the Orchidaceae family is remarkably understudied (Dressler,1990; Fay and Chase, 2009). A study of two species of the Oncidium

genus (O. microchilum and O. isthmi) reported the isolation ofseveral stilbenoids and phenanthrenes. All compounds have beenscreened against NCI-H460 (lung, non-small cell) and M14(melanoma) cell lines and some compounds have inhibitedproliferation of cancer cells (Williams et al., 2012).

* Corresponding author. Tel.: +55 44 3011 3664.

E-mail addresses: ampomini@uem.br, armandopomini@gmail.com (A.M. Pomini).

http://dx.doi.org/10.1016/j.phytol.2014.06.004

1874-3900/� 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rig

Among some interesting species of orchids which have beenphytochemically investigated aiming at the isolation of bioactive

substances, the species Bulbophyllum odoratissimum has afforded

stilbenoid batatasin III (11), and moscatin (14), the latter, a

phenanthrene. These compounds have been evaluated in vitro for

their inhibitory effect on the growth of several cell lines and the

first proved to be particularly active against cell line cancer BEL-

7402 (hepatoma), while the second proved active against cancer

cell line HL-60 (promyelocytic leukemia) (Chen et al., 2008).

Another chemical study performed with orchid Miltonia flavescens

Lindl. has led to the isolation of flavonoid hortensin, which proved

to be active against seven human cancer cell lines and selective for

the NCI/ADR-RES cell line (human resistant ovary sarcoma)

(Pomini et al., 2012).Despite advances in technology, many important drugs are still

derived from natural products or have been obtained based on anatural product as a prototype (Yunes and Cechinel Filho, 2012).Currently, 60% of anticancer drugs and 75% of those used ininfectious diseases are natural or derived from natural products

hts reserved.

Fig. 1. Structures of the characterized compounds 1 and 2 from Oncidium baueri.

J.A. Monteiro et al. / Phytochemistry Letters 9 (2014) 141–148142

(McChesney et al., 2007). However, only an estimated relativelysmall percentage of native plants, 5–15%, approximately 250,000,have been systematically investigated for the presence of bioactivecompounds (Kinghorn, 2001).

This work is the first phytochemical study conducted withAmazonian orchid Oncidium baueri focused on contributing to abetter knowledge of Brazilian biodiversity applied to the discoveryof new bioactive compounds against cancer, Chagas disease andleishmaniasis. The known compounds 3–15 were readily identi-fied, from their spectral data and by comparison with reportedcorresponding compounds in the literature and the novelcompounds obtained as a mixture, were assigned by comprehen-sive spectroscopic analysis, primarily involving 1D and 2D NMRexperiments and HRMS. Although most of the NMR signals weredouble and indicated a close structural similarity betweenOncibauerins A and B (1 and 2), HMBC, HSQC, COSY, NOESY andTOCSY experiments enabled distinct assignments for the individ-ual components of the mixture.

Herein, the obtaining and structural elucidation of the two newcompounds 1 and 2 and the determination of their antiprotozoalactivity against Trypanosoma cruzi and Leishmania amazonensis andcytotoxicity against ten human cancer cell lines, U251 (glioma,CNS), UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES (ovari-an phenotype of resistance to multiple drugs), 786-0 (kidney), NCI-H460 (lung, non-small cell), PC-3 (prostate), OVCAR-3 (ovariancancer), HT29 (colorectal) and K562 (leukemia), of the crudeextract (EBOB) and its fractions (HEOB, CLOB, ACOB, BUOB andHMOB) and of compounds 9 + 12, 11, 13 and 14 of Oncidium baueri

are reported.

2. Results and discussion

2.1. Structural elucidation

Hydrated plant material (leaves, pseudobulbs, roots andrhizomes) of O. baueri was extracted exhaustively with mixturesof MeOH:CHCl3 (7:3). After organic solvent removal, the resultingextract was submitted the liquid–liquid partition with n-hexane,CHCl3, EtOAc and n-BuOH. The EtOAc fraction was purified bySephadex LH-20 column chromatography afforded two newcompounds, oncibauerins A and B (1 and 2) in admixture(5.4 mg) as a yellow amorphous solid. The mixture of 1 and 2was recognized by analysis of NMR and HRMS. Although most ofthe NMR signals were double, HMBC, HSQC, COSY, NOESY andTOCSY experiments enabled the distinct assignments for 1 and 2.Oncibauerin A (1) was assigned to the molecular formulaC26H30O14 and oncibauerin B (2) to C27H32O14. The relativepercentage of 1 (30%) and 2 (70%) was deduced from 1H NMRintegrals of the signals recorded for H-20/H-60 and H-30/H-50 of thetwo compounds (Fig. 1).

The analysis of the 1H NMR spectrum revealed signals at dH 7.40(2H, d, J = 8.7 Hz, H-20 and H-60) and dH 6.95 (2H, d, J = 8.7 Hz, H-30

and H-50) relating to the B-ring hydrogens of compound 2 and twoless intense signals at dH 7.30 (2H, d, J = 8.7 Hz, H-20 and H-60) anddH 6.81 (2H, d, J = 8.7 Hz, H-30 and H-50) for compound 1. The 1HNMR spectrum showed the characteristic flavanone nucleussignals with resonances at dH 5.36 (1H, dd, J = 3.0 and 12.6 Hz,H-2ax), dH 3.13 (1H, dd, J = 12.6 and 17.1 Hz, H-3ax), and dH 2.73 (1H,dd, J = 3.0 and 17.1 Hz, H-3eq) assigned to 1 and signals at dH 5.39(1H, dd, J = 3.0 and 12.6 Hz, H-2ax), dH 3.13 (1H, dd, J = 12.6 and17.1 Hz, H-3ax), and dH 2.76 (1H, dd, J = 3.0 and 17.1 Hz, H-3eq)attributed to 2, in conjunction with the 13C NMR signals at dC 80.2(C-2) and dC 43.9 (C-3) for compound 1 and at dC 80.3 (C-2) and dC

43.9 (C-3) for compound 2. In addition, characteristic aromaticone-proton signals were observed for ring A at dH 5.96 (1H, s, H-8)and dH 5.97 (1H, s, H-8), for 1 and 2, respectively. These two protons

showed HMBC correlations with five aromatic carbons of the ring A(C-4, C-6, C-7, C-9 and C-10). Furthermore, a typical anomerichydrogen signal observed at 5.28 (1H, d, J = 3.0 Hz, H-10 0 0) wasattributed to both compounds.

A typical methoxyl signal at dH 3.80 (OCH3) indicated thecorrelation of the methoxy group hydrogens to C-40 (dC 161.4),which was observed only for compound 2. The NOESY correlationnoticed between H-30 and H-50 (dH 6.95) with the protons ofmethoxy group (dH 3.80) at C-40 together with the COSY correlationobserved for H-20/H-30 and H-50/H-60, which gives evidence of thelocation of a methoxyl group at C-40. The methoxy proton signal atdH 3.80 showed HMBC correlation with only one carbon at dC 161.4(C-40), which confirmed its position at C-40. The correlation of thesignals dH 3.80 and dC 55.7 was also observed by HSQC. The mainHMBC, COSY and NOESY correlations for compound 2 are shown inFig. 2. Now, for compound 1, the chemical shift of C-40 wasidentified from HMBC correlations of the protons of ring A at dH

7.30 and dH 6.81, with the carbon at dC 159.1.The 13C NMR assignments of compounds 1 and 2 were

confirmed by DEPT, COSY and HSQC experiments. Signals withthe same chemical shift were observed for both compounds at C-5(dC 164.3), C-6 (dC 105.9), C-7 (dC 167.3), C-9 (dC 164.2) and C-10 (dC

103.2). Chemical shifts presented by the glycosidic carbon portionof the atom also coincident in both structures (1 and 2) asdescribed in Tables 1 and 2. Furthermore, the position of thequaternary carbons was established by HMBC correlations and acharacteristic anomeric carbon signal at dC 111.5 (d, 1H, J = 3.0, CH)was observed for both compounds. Two other signals at dC 130.9and dC 132.2 corresponding to C-10 of 1 and 2, respectively, werealso noticed. The assignments of the aglycones of 1 and 2 were alsocompared with literature data and found to be consistent foraglycones naringenin (Andrade et al., 2010) and isosakuranetin(Vasconcelos et al., 1998), respectively.

The chemical shift of C-30 0 (dC 88.3) is very interesting.Literature data for C-glucose linked to other sugars at positions20 0 and 60 0 afforded more shielded chemical shifts for the C-30 0, atabout 80 ppm (Abdel-Kader, 1997; Takayanagi et al., 2003; Wanget al., 2007). The connection of apiose to the glucose C-30 0 can beproven by many correlations: NOESY (H-10 0 0 to H-30 0), COSY (H-10 0 0

to H-20 0 0; H-20 0 to H-10 0 and H-30 0; H-40 0 to H-50 0; H-50 0 to H-40 0 and H-60 0b), which proves the neighborhoods of the hydrogens of theglycosidic portion and HMBC (H-10 0 0 to C-30 0; H-40 0 to C-30 0 and C-60 0;H-10 0 to C-20 0, C-30 0 and C-50 0), and rules out the possibility ofreplacing the apiose in positions 20 0 and 60 0 of glucose.

The relative configuration at C-2 of compounds 1 and 2 wereestablished based on NOESY technique (H-2ax correlates with H-3eq). Moreover, the coupling constant (J2,3 = 12.6 Hz) between theprotons in positions 2 and 3, which indicates axial–axial coupling,revealed that the C-2 hydrogen is axial and that ring B is equatorial.

Fig. 2. Selected HMBC, COSY and NOESY correlations of 2.

Table 1NMR data for oncibauerin A (1), obtained in admixture with compound 2, in CD3OD.

Position dC, type dH, mult. (J in Hz) COSY HMBCd NOESY

2 80.2, CH 5.36, dd (12.6, 3.0) 3a, 3b 10

3ax 43.9, CH2 2.73c, dd (17.1, 3.0) 2, 3b 2, 4 3b

3eq 3.13, dd (17.1, 12.6) 2, 3a 4 3a

4 197.9, C

5 164.3a, C

6 105.9, C

7 167.3, C

8 96.3, CH 5.96, s 4, 6, 7, 9, 10

9 164.2a, C

10 103.2, C

10 132.2, C

20 128.9, CH 7.30, d (8.7) 30 2, 40 , 60 30

30 115.0, CH 6.81, d (8.7) 20 10 , 40 , 50 20

40 161.4, C

50 115.0, CH 6.81, d (8.7) 60 10 , 30 , 40 60

60 128.9, CH 7.30, d (8.7) 50 2, 20 , 40 50

10 0 75.0, CH 4.80, d (9.9) 20 0 5, 6, 7, 20 0 , 30 0 , 50 0

20 0 71.9, CH 4.26, m 10 0 , 30 0 30 0 40 0

30 0 88.3, CH 3.48b, m 20 0 40 0 , 10 ’’ 10 ’’

40 0 70.4, CH 3.49b, m 50 0 30 0 , 60 0 20 0 , 50 0

50 0 82.4, CH 3.38, m 40 0 , 60 0b 40 0

60 0 62.9, CH2 a: 3.70, dd (12.0, 5.1) 50 0 50 0

b: 3.86, dd (12.0, 2.1)

10 0 0 111.5, CH 5.28, d (3.0) 20 0 0 30 0 , 40 0 0 3’’

20 0 0 77.8, CH 4.00, d (3.0) 10 0 0 10 0 0

30 0 0 80.5, C

40 0 0 74.9, CH2 a: 3.81, d (9.6) 40 0 0b 10 0 0 , 20 0 0 , 30 0 0 40 0 0b

b: 4.15, d (9.6) 40 0 0a 50 0 0 40 0 0a

50 0 0 65.1, CH2 3.61, br s 20 0 0 , 30 0 0 , 40 0 0

5-OH

7-OH

40-OH

a Signals may be reversed in the same column.b Signals established by HMBC.c The signal of the hydrogen 3a may be reversed with the same hydrogen Table 2.d HMBC correlations from proton(s) stated for the indicated carbon.

J.A. Monteiro et al. / Phytochemistry Letters 9 (2014) 141–148 143

Table 2NMR data for oncibauerin B (2), obtained in admixture with compound 1 in CD3OD.

Position dC, type dH, mult. (J in Hz) COSY HMBCd NOESY

2 80.3, CH 5.39, dd (12.6, 3.0) 3a, 3b 10

3ax 43.9, CH2 2.76c, dd (17.1, 3.0) 2, 3b 2, 4 3b

3eq 3.13, dd (17.1, 12.6) 2, 3a 4 3a

4 197.9, C

5 164.3a, C

6 105.9, C

7 167.3, C

8 96.3, CH 5.97, s 4, 6, 7, 9, 10

9 164.2a, C

10 103.2, C

10 132.2, C

20 128.9, CH 7.40, d (8.7) 30 2, 40 , 60 30

30 115.0, CH 6.95, d (8.7) 20 10 , 40 , 50 20 ,40-OMe

40 161.4, C

50 115.0, CH 6.95, d (8.7) 60 10 , 30 , 40 60 ,40-OMe

60 128.9, CH 7.40, d (8.7) 50 2, 20 , 40 50

10 0 75.0a, CH 4.80, d (9.9) 20 0 5, 6, 7, 20 0 , 30 0 , 50 0

20 0 71.9, CH 4.26, m 10 0 , 30 0 30 0 40 0

30 0 88.3, CH 3.48b, m 20 0 40 0 , 10 0 0 10 0 0

40 0 70.4, CH 3.49b, m 50 0 30 0 , 60 0 20 0 , 50 0

50 0 82.4, CH 3.38, m 40 0 , 60 0b 40 0

60 0 62.9, CH2 a: 3.70, dd (12.0, 5.1) 50 0 50 0

b: 3.86, dd (12.0, 2.1)

10 0 0 111.5, CH 5.28, d (3.0) 20 0 0 3’’, 40 0 0 3’’

20 0 0 77.8, CH 4.00, d (3.0) 10 0 0 10 0 0

30 0 0 80.5, C

40 0 0 74.9a, CH2 a: 3.81, d (9.6) 40 0 0b 10 0 0 , 20 0 0 , 30 0 0 40 0 0b

b: 4.15, d (9.6) 40 0 0a 50 0 0 40 0 0a

50 0 0 65.1, CH2 3.61, br s 20 0 0 , 30 0 0 , 40 0 0

5-OH

7-OH

40-OMe 55.7, CH3 3.80, s 40 30 , 50

a Signals may be reversed in the same column.b Signals established by HMBC.c The signal of the hydrogen 3a may be reversed with the same hydrogen Table 1.d HMBC correlations are from proton(s) stated for the indicated carbon.

J.A. Monteiro et al. / Phytochemistry Letters 9 (2014) 141–148144

Furthermore, the down field aryl proton showing only ortho-coupling (dH 7.30, d, J = 8.7 Hz, H-20 and H-60 for compound 1 anddH 7.40, d, J = 8.7 Hz, H-20 and H-60 for compound 2) also coupledwith C-2 (dC 80.2 for 1 and dC 80.3 for 2) in the HMBC spectrum.Therefore, compounds 1 and 2 differ only in the replacement of C-40, with a hydroxyl group in 1 and a methoxyl group in 2.

In the present study, were characterized 13 previously knowncompounds (3–15) from O. baueri. Comparison of NMR data withthose in previous reports indicated that these compounds weresitosterol (3) (Goulart et al., 1993), stigmasterol (4) (Goulart et al.,1993), campesterol (5) (Goulart et al., 1993), daucosterol (6)(Manetti et al., 2010), phytol (7) (Song et al., 2008), squalene (8)(Khursid et al., 2002; Miranda et al., 2012), acacetin-7-O-rutino-side (9) (Piao et al., 2003), 1-glyceryl monostearate (10) (Galiciaet al., 2007), batatasin III (11) (Majumder and Ghosal, 1993),pectolinarin (12) (Lim et al., 2008), phloretic acid (13) (Owen et al.,2003), moscatin (14) (Honda and Yamaki, 2000) and 30,4-O-dimethylcedrusin 9-O-b-glucopyranoside (15) (Pieters et al.,1993; Calis et al., 2005). Compounds 3–5, 7, 8, 9 and 12 wereobtained in admixture. However, pure compound 9 was isolatedfrom CLOB fraction (Fig. 3).

2.2. Biological activities

The antiprotozoal activity against Trypanosoma cruzi andLeishmania amazonensis (Table 3) and also antiproliferative effectagainst various human cancer cell lines (Table 4) of crude extract(EBOB) and its hexane (HEOB), chloroform (CLOB), ethyl acetate(ACOB), butanol (BUOB), hydromethanol (HMOB) fractions wereassessed.

The chloroform fraction (CLOB) showed the best results inantitrypanosomal and antileishmanial activity tests with IC50

(inhibitory concentration of 50% of parasite growth) values of10.0 mg/mL against the axenic amastigote form of Leishmania

amazonensis (Table 3) and 105.0 mg/mL for the epimastigote formof Trypanosoma cruzi. Preliminary biological evaluation of the otherfractions and of compound 9 indicated no significant antiprotozoalactivity against T. cruzi and L. amazonensis. Some compounds couldnot be tested due to the low amounts available.

Antiproliferative assays were also conducted with severalhuman cancer cell lines using doxorubicin as a positive control.According to Table 4, the chloroform fraction (CLOB) was the mostactive fraction with significant activity against human cancer celllines, with GI50 (concentration that inhibits 50% of cell growth)values lower than 41 mg/mL for all the cancer cell lines tested. Inaddition, hexane fraction also showed good results againstmelanoma (UACC-62) and leukemia (K562). Furthermore, BUOBand HMOB fractions showed selectivity for UACC-62 (melanoma)with GI50 < 0.01 mg/mL. Testing of compounds 9 and 12 inadmixture showed no antiproliferative activity against any ofthe cancer cell lines evaluated in this study, while compounds 11,13 and 14 had inhibited cell growth of almost cell lines tested withGI50 values close to 30 mg/mL (Table 4).

Batatasin III and moscatin, two compounds isolated from CLOBfraction, which had the most relevant results, were active in vitro

evaluation of their inhibitory effect against the growth of variouscell lines. The first compound proved to be particularly activeagainst the cell line cancer BEL-7402 (hepatoma) with IC50 value of7.48 mg/mL, while the second inhibited cell growth of HL-60(promyelocytic leukemia) with IC50 value of 4.60 mg/mL (Chen

Fig. 3. Structures of natural products 9, 11, 12, 13 and 14 biologically evaluated.

J.A. Monteiro et al. / Phytochemistry Letters 9 (2014) 141–148 145

et al., 2008). These relates may suggest that the observedantiproliferative activity for chloroform fraction of Oncidium baueri

could be partially attributed to the presence of batatasin III andmoscatin.

In summary, although the high values presented by manyfractions in all tests may seem inappropriate, they can beconsidered appropriate, since they are not pure compounds.

Table 3In vitro antiprotozoal activities of crude extract, fractions and isolated compound from

Crude extract/fractions/compund Trypanosoma cruzi

Epimastigote

IC50 (mg/mL)a

Try

EC

EBOB 218.3 � 25.46 >2

HEOB 210.4 � 14.14 >2

CLOB 105.0 � 7.00 13

ACOB >1000 >2

BUOB 750.1 � 70.71 >2

HMOB >1000 >2

(9) >100 >2

Benznidazolec 6.5 � 0.70e 34

Amphotericin Bd

a The IC50 and EC50 values are expressed as mean of three determinations.b NT = not tested.c Standard antitrypanosomal agent.d Standard antileishmanial agent.e Values expressed as mM.

3. Experimental

3.1. General experimental procedures

1H and 13C NMR and 2D spectra were obtained on a VarianSpectrometer (Mercury Plus) operating at 300.06 and 75.45 MHzfor 1H and 13C NMR, respectively. Chemical shifts are given in ppm

Oncidium baueri.

Leishmania amazonensis

pomastigote

50(mg/mL)a

Promastigote

IC50 (mg/mL)

Axenic amastigote

IC50 (mg/mL)

00 45.7 � 4.57 NTb

00 22.1 � 1.44 NTb

7.5 � 6.37 41.2 � 5.13 10.0 � 0.93

00 150.0 � 3.33 NTb

00 156.4 � 5.29 NTb

00 188.0 � 1.74 NTb

00 >100 NTb

.5 � 7.60e

0.76 � 0.13e 0.49 � 0.19e

Table 4In vitro antiproliferative activity of Oncidium baueri crude extract, fractions and compounds against ten cancer cell lines (GI50 values).

Crude extract/fractions/compounds Cancer cell linesa,e

A B C D E F G H I J K

EBOB 144.76 3.97 40.09 29.54 214.32 50.66 68.21 165.88 155.68 3.49 41.28

HEOB 30.04 5.81 27.71 67.55 30.57 51.08 49.47 72.68 73.64 4.15 32.17

CLOB 29.90 6.83 29.11 24.62 40.04 26.34 26.89 26.76 32.06 0.48 26.58

ACOB >250 >250 >250 2.88 >250 >250 >250 >250 >250 1.70 >250

BUOB >250 <0.01 >250 >250 >250 >250 >250 >250 >250 27.70 >250

HMOB >250 <0.01 155.75 >250 >250 >250 >250 >250 >250 0.52 14.39

(9 + 12)f >250 >250 >250 >250 >250 >250 >250 >250 >250 >250 >250

Doxorubicinb 0.02 0.02 0.03 0.11 0.03 0.01 0.15 0.58 0.23 0.02 0.02

(11) 25.1 NTg 30.4 26.9 27.7 52.4 NTg NTg 72.4 NTg 30.9

(13) 29.7 NTg 29.0 26.1 27.5 30.7 NTg NTg 31.3 NTg 29.9

Doxorubicinc 0.02 NTg 0.02 0.21 0.03 0.01 NTg NTg 0.09 NTg 0.04

(14) 26.1 NTg 112.3 25.7 25.5 27.3 25.7 NTg 29.5 NTg 26.5

Doxorubicind 0.03 NTg 0.08 0.28 0.03 0.01 0.10 NTg 0.20 NTg 0.03

a A = U251 (glioma, CNS); B = UACC-62 (melanoma); C = MCF-7 (breast); D = NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance); E = 786-0 (renal);

F = NCI-H460 (lung, non-small cell); G = PC-3 (prostate); H = OVCAR-3 (ovarian); I = HT29 (colon); J = K562 (leukemia); K = HaCat (human keratinocytes, immortalized non

tumoral cell).b Used as a positive control for extract, fractions and compounds 9 and 12.c Used as a positive control for the compound 14.d Used as a positive control for the compounds 11 and 13.e Results are expressed as GI50 values (mg/mL).f Compounds 9 and 12 in admixture.g NT = not tested.

J.A. Monteiro et al. / Phytochemistry Letters 9 (2014) 141–148146

with tetramethylsilane (TMS) as an internal standard. Thedeuterated solvents CDCl3, CD3OD, D2O e DMSO-d6 from Aldrichor CIL were used. The chromatograms and low-resolution massspectra were obtained on a Focus GC (Thermo-Finnigan) apparatuscoupled to a DSQ II (Thermo-Finnigan) mass spectrometer. Datawere processed with Xcalibur and NIST MS Search spectral libraryversion 2.0. The carrier gas utilized was helium (He), purity99.999% (Praxair) and the equipment had a DB-5 capillary column(5% phenyl, 95% methylpolysiloxane). The ionization source andinjector temperature were maintained at 250 8C. Injections weredone in splitless mode, with an oven temperature ramp from 50 8Cto 290 8C at a rate of 10 8C min�1, maintained for 10 min. Positive-ion high-resolution mass spectra were acquired on an ThermoScientific ESI-FTMS system (orbtrap) using MeOH as solvent(5.0 � 10�3 mg mL�1), spray conditions ionization voltage of 3 kV,capillary temperature 300 8C, capillary voltage of 0 V and 190 V fortube lens.

Antiprotozoal assays were performed with a BIO-TEK PowerWave XS Spectrophotometer. Column chromatographic separa-tions were carried out using silica gel 60G (from Merck or Fluka),and Sephadex LH-20 as packing materials. The column dimensionsvaried according to the amount of material adsorbed to the columnand the monitoring was performed using thin layer chromatogra-phy. All solvents were purchased from commercial suppliers withanalytical purity or were previously distilled.

3.2. Plant material

The hydrated plant material (leaves, pseudobulbs, roots andrhizomes) of O. baueri was acquired during the flowering periodfrom the Orchidarium of the State University of Londrina, Brazil inMarch 2012, where a voucher specimen was deposited andidentified by Professor Ricardo Tadeu Faria. The access number isnot currently available.

3.3. Extraction and characterization

The aerial parts of Oncidium baueri (4.0 kg) except the flowers,were ground in a blender and the plant material was extracted

with MeOH:CHCl3 (7:3). A crude extract (48.0 g) was obtained afterremoval of the organic solvent. A portion of the crude extract(36.15 g) was dissolved in 500.0 mL of MeOH:H2O (1:1) andpartitioned with increasing polarity solvents: n-hexane (10�200 mL), chloroform (4� 200 mL), ethyl acetate (3� 200 mL) andn-butanol (4� 100 mL). Evaporation of the solvents gave hexane(HEOB, 9.70 g), chloroform (CLOB, 3.30 g), ethyl acetate (ACOB,3.25 g) and butanol (BUOB, 10.0 g) fractions. The remainingfraction was labeled hydromethanol (HMOB, 9.90 g).

The study of the HEOB fraction resulted in the characterizationof six compounds: daucosterol, a mixture of steroids sitosterol,stigmasterol and campesterol and also a mixture of phytol andsqualene. All structures were in agreement with literature data andconfirmed by GC–MS analysis. The study of the CLOB fractionresulted in the characterization of five compounds: 1-glycerylmonostearate, acacetin-7-O-rutinoside (flavanone), the neolignanglucoside 30,4-O-dimethylcedrusin 9-O-b-glucopyranoside andalso a stilbenoid and a phenanthrene characterized as batatasinIII and moscatin, respectively. All structures were in agreementwith literature data. Compounds 1-glyceryl monostearate, bata-tasin III and moscatin also were confirmed by GC–MS analysis.

Pre-fractioning of the ACOB fraction with hexane, chloroformand methanol provided three new subfractions, labeled ACOB-H,ACOB-C and ACOB-M, respectively. The study of the ACOB-Msubfraction resulted in the characterization of two novel com-pounds, Oncibauerins A and B (5.4 mg). These compounds wereobtained after purification using LH-20 column chromatographyand methanol as an eluent. This fraction also afforded pectolinarin(characterized in admixture with compound acacetin-7-O-rutino-side) and phloretic acid. A mixture of compounds acacetin-7-O-rutinoside and pectolinarin was also obtained from fractions BUOBand HMOB, together with sucrose.

3.3.1. Oncibauerins A and B (1 and 2)

Oncibauerins A and B (1 and 2) were obtained in admixture(5.4 mg) as a yellow amorphous solid (MeOH); 1D and 2D NMRdata given in Tables 1 and 2 and Supplementary data; HREIMS m/z

589.1527 [M+Na]+ (calcd. for C26H30O14Na, 589.1527) for 1 and603.1684 [M+Na]+ (calcd. for C27H32O14Na, 603.1684) for 2.

J.A. Monteiro et al. / Phytochemistry Letters 9 (2014) 141–148 147

3.4. Bioassay proceduce

3.4.1. Antitrypanosomal activity assay

The LLCMK2 cell line (epithelial cells from the kidney of Macaca

mulatta monkey) was grown according to the standard proceduresfrom the American Type Culture Collection (ATCC1). The in vitro

antiproliferative assay against the epimastigote forms of Trypa-

nosoma cruzi and the in vitro viability assay of the trypomastigoteforms of the same parasite were performed according to theliterature (Brener, 1962; Mosmann, 1983).

Log phase epimastigote were adjusted to a final inoculum of1.0 � 106 parasites mL�1 in LIT medium plus 10% fetal bovineserum (FBS). Then, the inoculum was added to 24-well platescontaining different concentrations of crude extract and fractions.The plates were incubated at 28 8C for 96 h, when the parasiteswere counted in a Neubauer’ chamber observed under an opticalmicroscope. Antiproliferative activity is expressed as the percent-age of growth inhibition compared with control parasites grown inLIT medium. The 50% inhibitory concentration (IC50) was deter-mined by linear regression.

Trypomastigote forms were obtained from the supernatant of amonolayer of infected LLCMK2 cells in DMEM supplemented with10% FBS at 37 8C in a humidified 5% CO2 atmosphere. To evaluatethe in vitro viability, firstly the cells LLMCK2 culture (sixth day ofinfection) supernatant was centrifuged to obtain T. cruzi trypo-mastigotes, which were then resuspended in DMEM up to aconcentration of 1.0 � 107 parasites mL�1. Each compartment of96-well plates received aliquots of protozoan suspension andcrude extract and fractions in different concentrations. The sameprocedure was performed for the positive (benznidazole) andnegative (only DMEM) controls. The plates were incubated at 37 8Cfor 4 h in 5% CO2. The parasites were counted according to thePizzi–Brener method. From each well, 5.0 mL were transferred to aglass slide, which was covered with a coverslip for counting withthe aid of a microscope. Motility was interpreted as parasiteviability. All assays were performed in triplicate and benznidazolewas used as a control.

3.4.2. Antileishmanial activity assay

Antileishmanial activity was determined by direct counting offree-living parasites after exposure to extract, fractions andcompounds. The concentration that inhibited of 50% parasitegrowth compared to the control (IC50) was evaluated graphicallyby plotting the concentration vs. percentage growth inhibitioncurve. The experimental procedures were performed accordingwith the literature (Mosmann, 1983; Monks et al., 1991; Ueda-Nakamura et al., 2001; Tiuman et al., 2005; Volpato et al., 2013).

Leishmania amazonensis promastigotes were cultured in War-ren’s medium supplemented with 10% FBS and incubated at 25 8C.Axenic amastigotes were obtained from in vitro transformations ofpromastigotes and cultured in Schneider’s insect medium supple-mented with 20% FBS and incubated at 32 8C. Nest, promastigotes(1 � 106 cells mL�1) and axenic amastigote forms (1 � 106 para-parasites mL�1) were grown until logarithm phase in 24-well andin 12-well plates, respectively. Both protozoan forms wereincubated for 72 h in the presence of different concentrations ofthe crude extract, fractions and isolated compound (Table 3),previously dissolved in DMSO. Cell density was determined in ahemocytometer, followed by IC50 determination. All assays wereperformed in triplicate and amphotericin B was used as control.

3.4.3. Antiproliferative activity assay

Human tumor cell lines U251 (glioma), UACC-62 (melanoma),MCF-7 (breast), NCI-ADR/RES (ovarian expressing phenotypemultiple drugs resistance), 786-0 (renal), NCI-H460 (lung, non-small cells), PC-3 (prostate), OVCAR-03 (ovarian), HT-29 (colon)

and K562 (leukemia) were kindly provided by Frederick CancerResearch & Development Center – National Cancer Institute –Frederick, MA, USA. Human keratinocyte (HaCaT) cell linewas donated by Dr. Ricardo Della Coletta (Piracicaba DentalSchool, University of Campinas). Stock cultures were grownin medium containing 5 mL RPMI 1640 (GIBCOR BRL) supple-mented with 5% fetal bovine serum. Penicillin:streptomycin(1000 mg mL�1:1000 UI mL–1, 1 mL L�1) was added to experimen-tal cultures. Cells in 96 well plates (100.0 mL cells well�1) wereexposed to sample concentrations in DMSO/RPMI (0.25, 2.5, 25 and250 mg mL�1) (DMSO, dimethyl sulfoxide) at 37 8C, 5% of CO2 in airfor 48 h. Final DMSO concentration did not affect cell viability.Afterwards, cells were fixed with 50% trichloroacetic acid and cellproliferation determined by spectrophotometric quantification(540 nm) of cellular protein content using sulforhodamine B assay,by measuring absorbance at the beginning of incubation (timezero, T0) and 48 h post-incubation for compound-free (T1)and tested (T) cells Cell proliferation was determined accordingto the equation 100 � [(T � T0)/T1 � T0], for T0 < T � T1, and100 � [(T � T0)/T0], for T � T0. Using the concentration–responsecurve for each cell line, TGI (concentration that causes total growthinhibition) was determined through non-linear regression analysis(Table 4) using software ORIGIN 8.0 (OriginLab Corporation)(Monks et al., 1991).

Acknowledgments

The authors acknowledge financial support for this workgenerously provided by CAPES (Coordination of Improvement ofHigher Education Personnel). JEC is fellow researchers of CNPq(National Council for Scientific and Technological Development).We are also indebted to Prof. Anita J. Marsaioli and Celio F. F.Angolini for HRMS analyses.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.phytol.2014.06.004.

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