Cytotoxic activity of extracts from Tecoma species and isolated lignans

Abstract

A phytochemical study of Tecoma genus (Bignoniaceae) was accomplished by antitumor activity of ethanolic extracts. Species of this genus are composed of small shrubs often used as ornamental plants. The Tecoma stans species is used in folk medicine for different purposes. Recent work shows in vitro anticancer activity against human breast cancer. The ethanolic extracts from leaves and trunks of Tecoma casneifolia, T. garrocha, T. stans var. angustata and T. stans var. stans were tested in vitro. The assays used were against line tumor cells by the MTT method and the most active extracts were further studied. In this way, the ethanolic extract from T. stans var. stans trunks presented the higher cytotoxicity against the tumor cell lines studied (CC₅₀ 0.02 to 0.55 µg/ml) when compared to the other extracts tested (CC₅₀ 0.08 to 200.0 µg/ml). Accordingly, this extract was selected for chromatographic fractionation from which five known lignans were isolated. Further, paulownin, paulownin acetate, sesamin, olivil and cycloolivil were identified using ¹³C and ¹H NMR, IR, UV and spectroscopy and spectrometric MS techniques. These isolated compounds were tested and exhibited CC₅₀ ranging from 13.01 to100.0 µg/ml which is superior to the ethanolic extract of trunk of T. stans.

Keywords:
Tecoma castaneifolia ; Tecoma garrocha ; Tecoma stans ; Lignan

INTRODUCTION

Cancer accounts for about 13% of all causes of death in the world and more than 7 million people die each year from the disease (Who, 2018). The World Health Organization estimated that by the year 2030, 27 million cases of cancer can be expected. The greatest effect of this increase will be on low-and-middle income countries (Who, 2018).

Some prophylactic actions to prevent cancer include health education at all levels of society, early diagnosis prevention and support for research that includes new forms of treatment (drugs, vaccines) (Who, 2018). Hence, research of new bioactive molecules against cancer is an important area of research that can diminish fatality by this disease.

Within this context, the importance of plants as sources of new drugs is universally recognized. Already, many active compounds and their synthetic derivatives are used in the treatment of this illness (Newman, Cragg, 2016). Further studies involving new isolated molecules and/or modified molecules with antineoplastic potential are a promising field (Newman, Cragg, 2016).

Different classes of natural products with cytotoxic activity are currently in clinical use, among alkaloids (vincristine, vinblastine and camptothecin derivatives), diterpenes (taxanes), lignans (podophyllotoxin derivatives) and others (Patrick, 2017). According to Newman and Cragg (2016), from 136 antitumor drugs approved and marketed in United States, between 1981 and 2014, about 83% are of natural origin or synthetic products developed from a natural model.

Bignoniaceae Juss. species are known to be sources of cytotoxic compounds. In this context, naphthoquinones have some active compounds including β-lapachone (Oliveira et al., 1990). Species of this botanical family produce many different compounds, from which they have already isolated terpenoids, flavonoids, xanthones, quinones and lignans (Brandão et al., 2017; Oliveira et al., 1993). Several reported species contain active compounds against major diseases. Cancer (bark of Tabebuia), hepatitis, rabies, diabetes, malaria, leishmaniasis and syphilis (Fischer, Theisen, Lohmann, 2004) are just part of this extended list that can be treated with bignoniaceae obtained compounds.

The genus Tecoma Juss. is composed mostly of shrubs or small trees and rarely subscandent plants. There are about fourteen species occurring in tropical regions of Africa and America, especially in the Andes and Arizona (Gentry, 1992). In Brazil, several species are used for ornamental purpose. Species of this genus have terminal inflorescences racemes or thryrses, yellow, orange-red corolla widely cultivated due to its natural beauty (Gentry, 1992; Anburaj et al., 2016). In addition, some natural occurrence is described in urban areas.

The most widespread species, T. stans, is found in Mexico with at least three varieties. Aerial parts are known to feed animals. Leaves are used in the popular medicine to treat diabetes (Kampati et al., 2018). Description from Mexican Pharmacopoeia of this plant is attributed to digestive properties. Thus, it can be prescribed for gastrointestinal disorders including gastritis from alcoholic derivation. A different traditional use for T. stans is designated by tribal people as anthelmintic (Kampati et al., 2018). Recent extract studies from T. stans demonstrate some anti-cancer activity against human breast cancer (Anburaj et al., 2016).

The present work contain studies from ethanolic extracts of Tecoma sp and their isolated lignans. Both have their cytotoxic activity evaluated against some different tumor cell lines.

MATERIAL AND METHODS

Collection, Taxonomical Determination and Processing of Plant Materials

T. castaneifolia (D. Don) Melchand and T. garrocha Hieron were collected in Santana de Pirapama, MG, Brazil, with the geographic coordinates 19º 00’ 22” S and 44º 02’ 35” W. T. stans var. angustata Rehder and T. stans var. stans (L.) Juss. ex Kunth were collected in Belo Horizonte, MG, Brazil, with the geographic coordinates 19º 55’ 15” S and 43º 56’ 16” W. The plants were taxonomically identified by Dr. JR Stehman, Botany Department of the Institute of Biological Sciences, UFMG, Belo Horizonte, Brazil. Voucher specimens were deposited at the BHCB/UFMG, Belo Horizonte, Minas Gerais, Brazil.

Preparation of Extracts

Plant material was dried in a circulating air oven at 40°C during 72 h. Aerial parts such as leaves and trunks were ground and extracted by percolation with 96% EtOH at room temperature. The solvent was removed in rotary evaporator under reduced pressure at 50ºC, leaving dark residues which were kept in a vacuum desiccator until constant weight.

Isolation of Chemical Components from trunk Extract

A portion of ethanolic extract of trunk of T. stans (EETTS, 100.0 g) was dissolved in methanol-H₂O (6:4) and this solution was submitted to successive extractions with immiscible solvents using firstly, dichloromethane and then ethyl acetate. There were three isolated parts produced: Organic layer corresponding T. stans dichloromethane trunk extract (TSDT, 63.4 g); T. stans ethyl acetate trunk extract (TSET, 6.2 g) and final T. stans aqueous fraction trunk extract (TSAT, 26.7 g).

The fractioning of TSDT occurred to the formation of a white precipitate (10.7 g) that was separated by decantation. A portion of this precipitate (8.5 g) was chromatographed on a silica gel column employing n-hexane, n-hexane/CH₂Cl₂ (1:1), CH₂Cl₂, CH₂Cl₂/EtOAc (1:1), EtOAc, EtOAc/MeOH (1:1), and MeOH as eluents. The fractions were collected in glass flasks of 20.0 ml obtaining a total of 60 fractions. The fractions were analyzed by a thin layer chromatography (TLC) and the ones that presented similar profiles were combined. In the TLC analyzes, n-hexane/EtOAc (1:1) was used as mobile phase, sulfuric anisaldehyde developer. Subsequently, the soluble part of the dichloromethane fraction (19.0 g) was fractionated using the same conditions utilized previously in the precipitated fractionation. This dichloromethane fraction gave a total of 182 fractions. They were assembled according to their similarity between analogous profiles from TLC analysis using the same conditions employed in the fractionation of the insoluble part. Final fractionations resulted in 19 combined fractions of precipitate and 24 combined fractions from the soluble portion.

Combined fractions 4 (115.5 mg) and 5 (583.5 mg), from insoluble portion of CH₂Cl₂ fraction, were submitted to silica gel chromatography on preparative TLC (silica gel 60 F₂₅₄ -Merck^®; 20x20 cm, layer thickness 1.0 mm) using CH₂Cl₂/EtOAc (8:2) as mobile phase. From the fractionation of Fr 4, a white solid called TST-3 (32.0 mg) was obtained while the fractionation of Fr 5 led to the isolation of a solid called TST-2 (55.0 mg).

The combined fractions 8 (478.5 mg), 9 (358.6 mg) and 10 (679.5 mg) from insoluble portion of CH₂Cl₂ fraction, were subjected to successive recrystallization using absolute ethanol leading to the isolation of a solid called TST-1 (1217.0 mg). Further quantities of TST-1 (292.0 mg) solid were obtained from the recrystallization of the pooled fractions 13 (776.3 mg), 14 (1079.4 mg), 15 (1098.7 mg) and 16 (1045.4 mg) from fractionation of the soluble part of the CH₂Cl₂ fraction.

The EtOAc fraction obtained from the liquid-liquid partition of the ethanolic extract was also fractionated on a silica gel column using the same conditions used in the fractionation of CH₂Cl₂

chromatographic profiles, obtaining 15 combined fractions. The combined fractions 7 (141.6 mg) and 8 (153.5 mg) were rechromatographed on preparative TLC (silica gel 60 F₂₅₄ -Merck^®; 20x20 cm, layer thickness 1.0 mm) using CH₂Cl₂/EtOAc (8:2) as mobile phase, yielding small amounts of called TST-4 (14.0 mg) and TST-5 (11.0 mg).

Structural Determination

Isolated compounds were identified based on spectral analyses and literature comparison. NMR ¹H and ¹³C-NMR including ¹D and ²D spectra such as COSY, HSQC, and HMBC were obtained on a Bruker Avance DRX400 instrument in DMSO-d6 with TMS as internal standard. Spectra of isolated compounds are shown in the supplemental material. Chemical shifts are given as δ (ppm) (Brandão et al., 2013) and coupling constants (J) are given in hertz. Melting points (mps) were measured with a Reichert melting point apparatus and are uncorrected. Infrared spectra were recorded on FT-IR Spectrometer, Varian 640-IR, Varian with system ATR and are reported in wave number (cm^-1). Samples were diluted with methanol- formic acid 0.1 % solution and ESI mass and UV spectra were recorded on a Waters ACQUITY TQD Tandem Quadrupole UPLC-DAD-MS System with direct injection.

Spectroscopic Data for Isolated Lignans

Paulownin (TST-1): White solid (MeOH); m.p. 107.0-108.5 °C; Lit. 105-106 °C (Ragasa et al., 2015); [α]_D = +30.8; UV (MeOH) l_max 234, 285 nm; IR n_max 3487, 2935, 2875, 1608, 1592, 1493, 1457, 1405, 1358, 1254, 1231, 1081, 1023, 1007, 959, 938, 910, 881, 805, 761, 715 cm^-1; ¹H NMR (CDCl₃, 400 MHz): δ 6.93 (2H, dd, J = 1.2, sl Hz, H-2, H-2’), 6.78-6.88 (4H, m, H-5, H-3’, H-6, H-6’), 4.83 (1H, d, J = 5.2 Hz, H-7), 3.04 (1H, m, H-8), 3.83 (1H, dd, J = 6.0, 2.8 Hz, H-9a), 4.50 (1H, dd, J = 8.4, 8.8 Hz, H-9b), 4.81 (1H, s, H-7’), 3.91 (1H, d, J = 9.6 Hz, H-9’a), 4.04 (1H, d, J = 9.2 Hz, H-9’b), 5.97 (2H, s, -OCH₂O-), 5.95 (2H, s, -OCH₂O-); ¹³C NMR (CDCl₃ , 100 MHz): δ 129.24 (C-1), 119.81 (C-2), 108.23 (C-3), 147.32 (C-4), 148.05 (C-5), 106.91 (C-6), 85.83 (C-7), 60.47 (C-8), 71.63 (C-9), 134.66 (C-1’), 120.13 (C-2’), 108.60 (C-3’), 147.95 (C-4’), 148.19 (C-5’), 107.43 (C-6’), 87.49 (C-7’), 91.69 (C-8’), 74.87 (C-9’), 101.27 and 101.13 (2x -OCH₂O-); ESI-MS m/z 371.27 [M+H]⁺, m/z 393.22 [M+Na] (calcd for C₂₀H₁₉O₇, 371.1130).

Paulownin acetate (TST-2): white solid (MeOH); m.p. 144.5-145.5 °C; Lit. 143-144 °C (Takahashi, Hayashi, Takani, 1970); UV (MeOH) l_max 237, 286 nm; IR n_max 3017, 2987, 2875, 1745, 1602, 1504, 1481, 1455, 1389, 1250, 1200, 1171, 1054, 932, 805, 780, 745 cm^-1; ¹H NMR (CDCl₃, 400 MHz): δ 6.92 (2H, dd, J = sl, sl Hz, H-2, H-2’), 6.75-6.88 (4H, m, H-5, H-6, H-3’, H-6’), 4.73 (1H, d, J = 4.0 Hz, H-7), 3.27 (1H, m, H-8), 4.24 (1H, m, H-9a), 4.40 (1H, m, H-9b), 5.03 (1H, s, H-7’), 3.76 (1H, d, J =4.0 Hz, H-9’a), 3.78 (1H, d, J = 4.0 Hz, H-9’b), 5.97 (2H s, -OCH₂O-), 5.94 (2H, s, -OCH₂O-), 1.74 (3H, s, -CH₃); ¹³C NMR (CDCl₃, 100 MHz): δ 130.26 (C-1), 119.84 (C- 2), 108.22 (C-3), 147.41 (C-4), 147.58 (C-5), 106.81 (C-6), 85.78 (C-7), 59.01 (C-8), 69.93 (C-9), 134.08 (C-1’), 122.35 (C-2’), 108.87 (C-3’), 147.43 (C-4’), 148.10 (C-5’), 107.99 (C-6’), 86.84 (C-7’), 97.20 (C-8’), 75.18 (C-9’), 101.17 and 101.10 (2x -OCH₂O-), 169.45 (-C=O), 20.93 (-CH₃); ESIMS m/z 413.48 [M+H]+ (calcd for C₂₂H₂₁O₈, 413.1158).

Sesamin (TST-3): white powder (MeOH); m.p. 123.0-125.5 °C; Lit. 123-124 °C (Ragasa et al., 2015); UV (MeOH) l_max 235, 286 nm; IR n_max 3065, 2998, 2875, 1508, 1452, 1385, 1254, 1200, 1100, 1079, 1045, 986, 931, 815, 765, 708 cm^-1; ¹H NMR (CDCl₃, 400 MHz): 1H NMR (400 MHz, CDCl₃): δ 6.87 (2H, d, J = SL, H-2, H-2’), 6.79-6.82 (4H, m, H-5, H-5’, H-6, H-6’), 4.75 (2H, d, J = 4.0 Hz, H-7, H-7’), 3.07 (2H, m, H-8, H-8’), 3.90 (2H, dd, J = 3.6, 5.6 Hz, H-9, H-9’), 4.26 (2H, dd, J = 6.8, 2.0 Hz, H-9, H-9’), 5.97 (2x -OCH₂O-); ¹³C NMR (100 MHz, CDCl₃): δ 135.12 (C-1 e C-1’), 106.50 (C-2 e C-2’), 147.99 (C-3 e C-3’), 147.13 (C-4 e C-4’), 108.18 (C-5 e C-5’), 119.34 (C-6 e C-6’), 85.80 (C-7 e C-7’), 54.36 (C-8 e C-8’), 71.73 (C-9 e C-9’), 101.27 and 101.06 (2× -OCH₂O-); ESI-MS m/z 355.26 [M+H]⁺ (calcd for C₂₀H₁₉O₆, 355.1181).

Olivil (TST-4): white powder (MeOH); m.p. decomposes at 269.0-279.0 °C; Lit. 135 °C (Ghogomu-Tih et al., 1985), UV (MeOH) l_max 222, 257, 275 (sh), 320 (sh), 366 nm; IR n_max 3583, 2920, 1598, 1512, 1444, 1361, 1260, 1171, 1066, 1047, 1022, 883, 833 cm^-1; ¹H NMR (DMSO-d6, 400 MHz): δ 6.91 (2H, dd, J = 1.8, 16.8 Hz), 6.77-6.86 (m, 4H), 5.97 (s, 2H, -OCH₂O- ), 5.94 (s, 2H, -OCH₂O-), 4.80 (1H, s, H-1), 4.03 (1H, d, J = 9.0 Hz, H-3), 3.92 (1H,d, J = 9.6 Hz, H-3), 4.82 (1H, d, J = 4.8 Hz, H-4), 3.03 (1H, m H-5), 3.82 (1H, dd, J = 6.0, 9.0 Hz, H-6), 4.50 (1H, dd, J = 8.4, 9.0 Hz, H-6); ¹³C NMR (DMSO-d6, 100 MHz δ 129.63 (C-1), 115.17 (C-2), 147.75 (C-3), 146.08 (C-4), 115.34 (C-5), 122.96 (C-6), 83.76 (C-7), 61.06 (C- 8), 59.41 (C-9), 134.91 (C-1’), 111.53 (C-2’), 147.34 (C-3’), 145.17 (C-4’), 115.26 (C-5’), 119.65 (C-6’), 39.63 (C-7’), 80.99 (C-8’), 76.67 (C-9’), 56.05 and 56.04 (2× -OCH₃-); ESI-MS m/z 375.53 [M-H]- (calcd for C₂₀H₂₃O₇, 375.1122).

Ciclolivil (TST-5): white solid (MeOH); m.p. 285.6-287.9 °C; Lit. 289-291 °C (Ghogomu-Tih et al., 1985); UV (MeOH) l_max 267, 313 (sh) nm; IR n_max 3452, 2980, 1608, 1516, 1447, 1352, 1259, 1168, 1049, 1032, 1023, 873, 821 cm^-1; ¹H NMR (DMSO-d6, 400 MHz): δ 7.24 (1H, d, J = 2.0 Hz, H-2), 6.62 (1H, d, J = SL, H-5), 6.62 (1H, dd, J = 2.0 e 6.4 Hz, H-6), 4.50 (1H, d, J = 12.0 Hz, H-7), 2.50 (1H, m, H-8), 4.21 (1H, m, H-9a), 4.01 (1H, m, H-9b), 7.06 (1H, s, H-2’), 7.22 (1H, s, H-5’), 3.67 (1H, d, J = 16,40 Hz, H-7’a), 3.02 (1H, d, J = 14.0 Hz, H-7’b), 4.29 (1H, m, H-9’a), 4.01 (1H, m, H-9’b), 4.24 and 4.23 (6H, s, 2× -OCH₃); ¹³C NMR (DMSO-d6, 100 MHz): δ 143.21 (C-1), 123.56 (C-2), 158.00 (C-3), 155.65 (C-4), 126.46 (C-5), 132.87 (C-6), 54.27 (C-7), 57.17 (C-8), 79.26 (C-9), 147.96 (C-1’), 122.35 (C-2’), 156.35 (C-3’), 154.84 (C-4’), 125.29 (C-5’), 136.18 (C-6’), 49.89 (C-7’), 83.69 (C-8’), 70.40 (C-9’), 65.92 and 66.03 (2× OCH3); ESI-MS m/z 375.51 [M-H]- (calcd for C₂₀H₂₃O₇, 375.1122).

Cell lines

A panel of human cancer cell lines were used for the cytotoxicity studies. ATCC^® cell lines including hepatocellular carcinoma Hep G2 (ATCC^® HB-8065^™), ovarian cell carcinoma TOV-21G (ATCC^® CRL-11730^™), urinary bladder transitional cell carcinoma T24 (ATCC^® HTB-4^™), cervix cell carcinoma HeLa (ATCC^® CCL- 2^™) and breast cell carcinoma MDA-MB-231 (ATCC^® HTB-26^™), as well as normal human lung fibroblast cell MRC-5 (ATCC^® CCL-117^™) were used in the assays. The cells were cultivated in complete cell medium consisting of Dulbecco’s modified Eagle medium (DMEM, Cultilab, Campinas, SP, Brazil), supplemented with 5% fetal bovine serum, 50 μg/mL gentamicin, 100 U/mL penicillin and 5 μg/mL amphotericin B (Brandão et al., 2013). The cells were grown at 37 °C in a humidified atmosphere containing 5% CO₂ and harvested in log-phase for experimental use.

Cytotoxicity Assay

Cell lines were exposed to different concentrations of extracts/fractions/compounds for 72 h ⁶. After incubation, cell viability was assessed by the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck) assay at a concentration of 2 mg/mL in PBS (Brandão et al., 2013; Twentyman, Luscombe, 1987). Each sample was assayed in three replicates for concentrations ranging from 200.0 to 3.125 μg/mL. The cytotoxicity of each sample was expressed as CC50, i.e. the concentration of the samplethat inhibited cell growth by 50% (Brandão et al., 2013).

RESULTS AND DISCUSSION

Identification of compounds isolated from trunk ethanolic extract of T. stans var. stans

The ethanolic extract from the trunk of T. stans var. stans showed the most cytotoxic results for the five tumor cell lines. Therefore, it was selected for a chromatographic fractionation. This fractionation resulted in isolation of five known lignans (Figure 1). The lignans were identified using a ¹H and ¹³C NMR spectroscopy. Later, structural elucidation was confirmed by comparison of bibliography data. Spectra of ¹D and ²D were used such as COSY, HSQC and HMBC (Brandão et al., 2013). Literature data of ¹D and ²D was also used during the identification (Takahashi, Hayashi, Takani, 1970; Ghogomu-Tih et al., 1985; Ragasa et al., 2015), Spectra were attached in supplementary file. The presence of lignans in the species of Bignoniaceae is widely reported (Cipriani et al., 2008).

FIGURE 1
Chemical structures of paulownin (A), paulownin acetate (B), sesamin (C), olivil (D) and cycloolivil (E).

Paulownin A (TST-1) was isolated in large quantities from the dichloromethane fraction after chromatographic separation. About 1.5 g of this amorphous white solid was obtained. The ¹H NMR spectrum of TST-1 showed six signals of aromatic hydrogen at δ 6-94 to 6.78 ppm corresponding to two trisubstituted aromatic rings. The two singlets at δ 5.97 and δ 5.95 ppm, referring to two hydrogens, characterizing two groups of methylenedioxy coupled to aromatic ring in its structure. Furthermore, two oxymethine hydrogens were observed as a duplet at δ 4.84 ppm referring to the H-7 and as a singlet at δ 4.81 ppm corresponding to the H-7’, the couplings of this signals were confirmed at the COSY contour map. The ¹³C NMR spectrum presents the signals at δ 101.27 ppm and δ 101.13 ppm, the confirmed groups of methylenedioxy switched on to the aromatic system. The signals observed at δ 129.24 ppm and δ 134.66 ppm, referring to the carbons C-1 and C-1’, with these values they were characteristic of the furofuran basic structure with an aril group type 3,4-methylenedioxyphelinic at equatorial position. Moreover, the difference at 5.5 ppm between C-1 and C-1’ observed indicates the presence of a hydroxyl group at the carbon C-8’ (Agrawal, Thakur, 1985). The MS of TST-1 showed an [M + Na]⁺ ion at m/z 393.22 and [M + H ] ⁺ ion at m/z 371 peaks. The molecular formula of TST-1 was determined as C₂₀H₂₀O₇ (371.1130). The data allowed identification of TST-1 as a natural paulownin product (1,4-bis(3,4-methylenedioxyphenil)- tetrahydro-1H,3H-furo[3,4-c]furan-3-ol).

The ¹H and ¹³C NMR spectra of paulownin acetate B (TST-2) (55.0 mg) showed similar signals observed by TST-1. Likewise, this different molecule is also a furofuran lignan containing an aril group type 3,4-methylenedioxyphelinic at equatorial position. Furthermore, the ¹³C NMR spectrum presents the signals at δ 169.45 ppm referring to the carbonile coupled to the C-8’ and the methyl group at δ 20.93 ppm. The MS spectrum of TST-2 showed an [M]⁺ ion at m/z 413.4777 as a base peak. The molecular formula of TST-2 was determined as C22H21O8 (413.12). Obtained data allowed for the identification of TST-2 as paulownin acetate (1,4-bis(3,4-methylenedioxyphenil)-tetrahydro-1H,3H-furo[3,4-c] furan-3-il).

A different furofuran lignan isolated from dichloromethane fraction was sesamin C (TST-3) (32.0 mg), where the ¹H and ¹³C NMR spectra showed six signals of aromatic hydrogen at δ 6-87 to 6.79 ppm corresponding to two trisubstituted aromatic rings. Signals characteristic of furofuran lignans were observed as double-doublet at δ 4.26 and δ 3.90 ppm, attributed to the oxymethylenic hydrogens H-9 and H-9’ at equatorial and axial position, respectively. The ¹³C NMR spectrum showed ten signals that suggest a symmetric molecule, like the methylenedioxy signal at δ 101.06 ppm coupled to the aromatic systems. The MS of TST-3 showed an [M]⁺ ion at m/z 355.2642 as a base peak. The molecular formula of TST-3 was determined as C₂₀H₁₉O₆ (355.1181). These data allowed the identification of TST-3 as sesamin (1,4-bis(methylenedioxy)-tetrahydro- 1H,3H-furo[3,4-c] furan-3-o).

The fractionation of the ethyl acetate portion isolated small amounts of olivil D (TST-4) (14.0 mg). NMR spectrum of ¹H showed aromatic hydrogens due to the signals at δ 7.03 ppm, δ 6.65 ppm and δ 6.78 ppm and the multiplet from δ 6.85 to 678 ppm. Analysis of ¹³C NMR data and HSQC contour map suggest the presence of two aromatic rings. These spectra showed the presence of carbon signals at δ 115.17 ppm (C-2), δ 111.53 ppm (C-2’), δ 115.34 ppm (C-5), δ 115.26 ppm (C-5’), δ 122.96 ppm (C-6) and δ 119.65 (C-6’), which correspond to the aromatic region. These followed correlations of spectra allowed for the conclusion that aromatic rings are 1,3,4-trisubstituted, identified as a tetrahydrofuran lignan skeleton. The MS of TST-4 showed an [M]^- ion at m/z 375.3220 as a base peak. The molecular formula of TST-4 was determined as C₂₀H₂₃O₇ (375.1152). These data allowed the identification of TST-4 as a natural olivil product (3-(4-hidroxy-3-methoxybenzil)-5-(4-hidroxy-3-methoxyphenyl)-4-(hidroxymethyl) tetrahydrofuran-3-ol).

NMR ¹H spectrum of cycloolivil E (TST-5) (11.0 mg), isolated from the ethyl acetate fraction, showed five aromatic hydrogens. The coupling constants from these signals corresponded to two different rings. The signals at δ 7.12 ppm (1H, J = 2.0 and 6.40 Hz) couple with the signals of doublet-doublet at δ 7.24 ppm (1H, d, J = 2.0 Hz) and δ 6.62 ppm (1H, J = 6.40), and were attributed to the hydrogens of the 1,3,4-trisubstituted ring. The second set of signals at 7.06 (1H, s) and 7.22 (1H, s) suggests the presence of a 1,3,4,6-tetrasubstituted ring. These described signals characterize a lignan skeleton tetrahydronaphthalene type. The MS of TST- 5 showed an [M]^- ion at m/z 375.3220 as a base peak. The molecular formula of TST-5 was determined as C₂₀H₂₃O₇ (375.1152). These data allowed for the identification of TST-5 as cycloolivil (4-(4-hidroxy-3- methoxyphenyl)-2,3-bis(hidroxymethyl)-7-methoxy- 1,2,3,4-tetrahydronaphthalene-2,6-diol).

Cytotoxic essay of Tecoma species extract and constituents

Ethanolic extracts of species from genus Tecoma, T. stans var. stans, T. stans var. angustata, T. castaneifolia and T. garrocha were evaluated for in vitro cytotoxic activity. Biological essay was evaluated against five tumor cell lines including: Hep G2, T24, TOV-21G, HeLa and MDA- MB-231 and normal cell line, MRC-5, in concentrations ranging from 200.0 to 3.125 μg/mL. The T. stans var. stans trunk extract presented CC₅₀ between 0.0156 and 0.5533 μg/mL against tumor cell lines while leaf extract presented CC₅₀ from 39.89 to 200.0 μg/mL. The T. stans var angustata trunk extract also were very cytotoxic presenting CC₅₀ from 0.084 to 56.03 μg/mL whereas leaves extract presented a moderate CC₅₀ cytotoxicity between 24.22 and 200.0 μg/ mL. The extract of T. castaneifolia stems presented CC₅₀ ranging from 15.90 to 110.80 μg/mL and the extract of leaves from this species had values between 18.31 to 200.0 μg/mL. Cytotoxicity of T. garrocha extracts also showed similar results previously ranging from 12.96 to 200.0 μg/ mL for the trunk extract and from 27.93 to 200.0 μg/mL for the leaves extract.

Trunks extracts from all species of the genus Tecoma were more cytotoxic against tumor cell lines when compared to leaves extracts. Thus, the most active T. stans var. stans ethanolic extract was selected for fractionation by liquid-liquid partition. Five lignans were isolated: paulownin, paulownin acetate, sesamin, olivil and cycloolivil. These compounds were also evaluated for cytotoxic activity against the five tumor cell lines at concentrations ranging from 100 to 1.5625 μg/mL.

Paulownin presented mean cytotoxicity values between 29.35 and 100.0 μg/mL, whereas paulownin acetate showed CC₅₀ from 28.15 to 100.0 μg/mL, the CC₅₀ values of sesamin ranged from 13.01 to 100.0 μg/ mL, while the olivil was not cytotoxic at the highest concentration tested (100.0 μg/mL) to any of the cell lines tested. Finally, the cycloolivil presented CC₅₀ between 45.98 and 100.0 μg/mL. Extracts and isolated compounds showed CC₅₀ values for normal cell line, while MRC-5 was higher than those found for tumor cell lines. The results of mean cytotoxic concentrations (CC₅₀) for each extract and isolated compound are described in Table I.

TABLE I
Cytotoxic activity (CC₅₀) of Tecoma species extract and lignans

Extract/ Compound MRC-5		CC50 µg mL-1 (µM)
		HeLa	IS	Hep G2	IS	T24	IS	TOV-21G	IS	MDA-MB-231	IS
Tecoma castaneifolia	Trunk	131.30 ± 1.39	110.80 ± 1.12	1.19	25.56 ± 1.37	5.14	15.90 ± 1.61	8.26	17.51 ± 1.66	7.50	23.24 ± 1.49	5.65
	Leaves	171.60 ± 1.18	58.86 ± 1.16	2.92	48.03 ± 1.40	3.57	18.31 ± 1.32	9.37	83.40 ±1.42	2.06	> 200.00	< 0.86
Tecoma garrocha	Trunk	> 200.00	> 200.00	> 1.00	92.53 ± 1.32	> 2.16	12.96 ± 1.98	> 15.43	16.76 ± 1.47	> 11.93	53.07 ± 1.34	> 3.77
	Leaves	> 200.00	> 200.00	1.00	119.10 ± 1.27	> 1.68	27.93 ± 1.31	> 7.16	88.94 ± 1.45	> 2.25	> 200.00	1.00
Tecoma stans var. angustata	Trunk	80.25 ± 1.18	56.03 ± 1.17	1.43	0.1963 ± 1.40	408.81	0.0841 ± 1.51	954.22	0.1697 ± 1.69	472.89	19.59 ± 1.49	4.10
	Leaves	144.10 ± 1.33	185.80 ± 1.50	0.78	64.41 ± 1.68	2.24	24.22 ± 5.95	4.49	140.30 ± 1.47	1.03	> 200.00	< 0.72
Tecoma stans var. stans	Trunk	0.4258 ± 1.30	0.5533 ± 5,69	0.77	0.1198 ± 1.96	3.55	0.0156 ± 1.96	27.30	0.1043 ± 1.56	4.08	0.2680 ± 1.15	1.59
	Leaves	> 200.00	> 200.00	1.00	62.48 ± 1.68	> 3.20	39.89 ± 1.12	> 5.01	69.28 ± 1.23	> 2.89	> 200.00	1.00
Paulownin (TST-1)		> 100.00	> 100.00	1.00	31.88 ± 1.71 (86.08 ± 1.71)	> 3.14	29.35± 1.21 (79.25 ± 1.21)	> 3.40	29.51± 1.49 (79.68 ± 1.49)	> 3.39	> 100.00	1.00
Paulownin acetate (TST-2)		> 100.00	> 100.00	1.00	34.13 ± 2.07 (82.76 ± 2.07)	> 2.93	31.31 ± 1.35 (75.92 ± 1.35)	> 3.19	28.15 ± 1.55 (68.26 ± 1.55)	> 3.55	> 100.00	1.00
Sesamin (TST-3)		> 100.00	> 100.00	1.00	73.34 ± 1.76 (206.97 ± 1.76)	> 1.36	13.01 ± 1.44 (36.72 ± 1.44)	> 7.69	94.73 ± 1.39 (267.33 ± 1.39)	> 1.06	> 100.00	1.00
Olivil (TST-4)		> 100.00	> 100.00	1.00	> 100.00	> 1.00	> 100.00	> 1.00	> 100.00	> 1.00	> 100.00	1.00
Cycloolivil (TST-5)		> 100.00	> 100.00	1.00	78.57 ± 1.65 (208.74 ± 1.65)	> 1.27	45.98 ± 118 (122.16 ± 1.18)	> 2.17	65.62 ± 2.14 (174.34 ± 2.14)	> 1.52	> 100.00	1.00
Podofilotoxin		0.1339 ± 1,49 (0.3231 ± 1.49)	0.1293 ± 1.48 (0.3120 ± 1.48)	1.04	0.0043 ± 1.47 (0.0104 ± 1.47)	31.14	0.0025 ± 1.43 (0.0062 ± 1.43)	53.56	0.0048 ± 1.75 (0.0117 ± 1.75)	27.90	0.0986 ± 1.85 (0.2379 ± 1.85)	1.36

• CC₅₀: Mean cytotoxic concentration; MRC-5: normal human lung fibroblast cell; HeLa: cervix cell carcinoma; Hep G2: hepatocellular carcinoma, T24: urinary bladder cell carcinoma; TOV-21G: ovarian cell carcinoma; MDA-MB-231: breast cell carcinoma; SI (Selectivity Index): CC50 MRC-5/ CC50 Tumor cell line

The cytotoxic activity of some lignans are widely known, including podophyllotoxin, which interferes with the mitotic spindle interacting with tubulin (Gordaliza et al., 2004). Semisynthetic derivatives from podophyllotoxin etoposide and teniposide are potent anticancer agents with different mechanisms of action. These in relation to modified lignans stabilize the covalent intermediate formed between DNA and topoisomerase II, and are also thought to produce strand breakage by free radical production (Gordaliza et al., 2004; Nobili et al., 2009).

The presence of paulownin in extracts of Bignoniaceae species was previously reported at Kigelia africana (Sidjui et al., 2015) and Markhamia lutea (Ali et al., 2015). Paulownin (TST-1) had cytotoxic concentrations ranging from 79.25 to 86.08 μM. A study by Huang et al. (2013) demonstrated paulownin with antitumor activity against human chronic myelogenous leukemia (K-562) cell lines with CC₅₀ of 70.6 μM and lung cancer cells (A549) with CC₅₀ of 22.6 μM.

The evaluation of the paulownin acetate cytotoxicity resulted in cytotoxic concentrations between 68.29 and 82.76 μM, demonstrating that paulownin acetate has antitumor activity similar to paulownin. This lignan was found in Gmelia arborea (Verbenaceae) as one of the major chemical constituents (Acharya et al., 2015). However, in the Bignoniaceae family, the only report in scientific literature about the isolation of paulownin acetate was found in the work performed by Caetano (1983), who also isolated it from the trunk part of T. stans species. It has been the first report of cytotoxic activity of this compound in cell culture.

In the Bignoniaceae family, sesamin was isolated from the species Kigelia africana (Sidjui et al., 2015) and Markhamia lutea (Ali et al., 2015). In the present study, this compound had a cytotoxic concentration ranging from 36.72 to 267.33 μM. Akl et al. (2013) evaluated antitumor activity from sesamin against breast cancer cell lines, MCF-7 and MDA-MB-231, obtaining CC₅₀ of 98.0 μM and CC₅₀ of 43.9 μM, respectively. In another study, Hirano et al. (1994) considered sesamin to have low potency against leukemia tumor cells (HL-60 and MOLT-4), since it had CC₅₀> 0.0028 μM. Using this same parameter, sesamin can be considered with low potency evaluated against cell lines that was used in this work.

The olivil is a lignan with wide distribution between botanic families. Occurrence reports in Bignoniaceae family involving this compound can be found in Stereospermum species such as S. cylindricum (Kanchanapoom et al., 2006) and S. kunthianum (Ghogomu-Tih et al., 1985). Olivil biological data did not demonstrate any cytotoxic effect at the concentrations tested (CC₅₀> 100.0 μg/mL) against any tested cell lines in this present study. Previous research has also failed to detect the cytotoxic effect of these compounds against breast cancer cell line (MCF-7), lung cancer cells (A- 549) and normal lung fibroblast cell line (WI-38) at concentrations below 100.0 μg/mL (Wangteeraprasert et al., 2012).

The fifth lignan isolated from T. stans var. stans trunk extract was identified as cycloolivil. The cycloolivil is a lignan commonly found in the family Bignoniaceae, as in species of the genus Tabebuia, such as T. heptaphylla (Schmeda-Hirschmanna, Papastergioub, 2003), and species of the genus Stereospermum: S. kunthianum (Ghogomu-Tih et al., 1985) and S. cylindricum (Kanchanapoom et al., 2006). Wangteeraprasert et al. (2012) evaluated the cytotoxic effect of cycloolivil against cell lines of breast cancer (MCF-7), lung cancer (A-549) and normal cell line of lung fibroblasts (WI- 38) and an antiproliferative activity at the highest tested concentration of 100 μg/mL was not observed. In the present study, the cycloolivil showed CC₅₀ of 78.57, 45.98 and 65.62 μg/mL against Hep G2, T24 and TOV-21G cell lines, respectively. Finally, cycloolivil did not demonstrate any cytotoxic effect against HeLa and MDA-MB-231 even at the highest tested concentration (100 μg/mL).

Our results reveal that the trunk of the T. stans var stans is rich in lignans and may be a source for obtaining paulownin. We can also conclude by the cytotoxicity tests results using tumor cell lines evaluating ethanolic extracts of T. stans var. stans and T. stans var angustata as potential sources of cytotoxic compounds. However, some lignans such as podophyllotoxin and its derivatives etoposide and teniposide show marked cytotoxic activity (Patrick, 2017). The lignans isolated from the ethanolic extract of T. stans trunks (paulownin, paulownin acetate, sesamin, olivil and cycloolivil) have only moderate cytotoxic activity when compared to the activity of ethanolic extract of origin. These data may suggest the presence of other bioactive compounds, not yet isolated from the extracts and with more cytotoxic activity. Another hypothesis can be the synergic effect promoted by a set of substances.

ACKNOWLEDGMENTS

This work was supported by funds from FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil, (process numbers CDS- APQ-00270-13 and CDS - APQ-01529-15) and CAPES - Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (Brazil). PROPP-UFOP - Pró-Reitoria de Pesquisa e Pós- Graduação da UFOP (process number 23109.003267/2017- 01). Thanks to Dr. J.R. Stehman, Botany Department, Institute of Biological Sciences, UFMG, Belo Horizonte, Brazil, for collection and taxonomical determination of Tecoma species.

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Publication Dates

• Publication in this collection
  09 Jan 2023
• Date of issue
  2022

History

• Received
  21 Dec 2019
• Accepted
  24 Feb 2022