Chemical Constituents and Pharmacology properties of Aristolochia triangularis: a south brazilian highly-consumed botanical with multiple bioactivities

Abstract

Abstract: Aristolochia triangularis Cham., is one of the most frequently used medicinal plant in Southern Brazil. Preparations containing the leaves and/or stems are traditionally used as anti-inflammatory, diuretic, as well as antidote against snakebites. This study screened A. triangularis extracts, fractions and isolated compounds for different bioactivities. A weak antiproliferative activity against human lung cancer cell line (A549) was observed only for chloroform fraction obtained from stems (CF_stems - CC₅₀: 2.93 µg/mL). Also, a moderate antimicrobial activity against Staphylococcus aureus was detected just for chloroform fraction obtained from leaves (CF_leaves -13-16 mm inhibition zone). Additionally, two semi-purified fractions (CF_stems-4 and CF_leaves-4) selectively inhibited HSV-1 replication (IC₅₀ values of 0.40 and 2.61 µg/mL, respectively), while only CF_leaves showed promising results against Leishmania amazonensis. Fractionation of extracts resulted in the isolation of one neolignan (-) cubebin and one lignan (+) galbacin. However, these compounds are not responsible for the in vitro bioactivities herein detected. The presence of aristolochic acid I and aristolochic acid II in the crude ethanol extract of stems (CEEstems) and leaves (CEEleaves) was also investigated. The HPLC analysis of these extracts did not display any peak with retention time or UV spectra comparable to aristolochic acids I and II.

Key words
Aristolochia triangularis ; antiproliferative; antiherpes; antimicrobial; antiprotozoal; aristolochic acids

INTRODUCTION

The Aristolochia genus (Aristolochiaceae) comprises about 500 species of herbaceous perennials, undershrub or shrubs widespread across Europe, tropical Asia, Africa and South America (Neinhuis et al. 2005). Several Aristolochia species have been used worldwide as anti-inflammatory (Muschietti et al. 1996) and to treat rheumatic pains and fevers (Battu et al. 2011) and complications of snakebites (Bhattacharjee and Bhattacharyya 2013).

Several pharmacological activities are attributed to Aristolochia species including antibacterial (de Barros Machado et al. 2005, Kumar et al. 2006, Yu et al. 2007, Alviano et al. 2008), antiproliferative (Chaouki et al. 2010), antispasmodic (Zhang et al. 2008), anti-scorpion venon (Izquierdo et al. 2010), anti-snake venon (Samy et al. 2008), insecticidal (Nascimento et al. 2004, Messiano et al. 2008), anti-inflammatory (Battu et al. 2011), and antimycobacterial (Léon-Diaz et al. 2010).

Concerning the chemical composition of Aristolochia species, flavonoids (Machado and Lopes 2005, Battu et al. 2011), lignans (Zhai et al. 2004, 2005, De Pascoali et al. 2006, León-Diaz et al. 2010), terpenoids (Wu et al. 2005), aristolochic acids and their esters (Cosyns 2003, Chung et al. 2011), and aristolactams (Marti et al. 2013) are described.

Although aristolochic acids are nitrophenantrene derivatives associated to a high risk of nephrotoxicity and upper urinary tract carcinoma disease (Arlt et al. 2002, Debelle et al. 2008) Aristolochia triangularis Cham., known in Brazil as “cipó-mil-homens” or “jarrinha”, is traditionally used for the treatment of inflammatory conditions, diarrhea, asthma, different types of cancer, as well as antidote against snakebites and as diuretic (Simões et al. 1998, Lorenzi and Matos 2002). Regarding to its chemical constituents, Langmann et al. (1979) revealed the presence of tetracyclic diterpenes, kauran derivatives, steroids, lignans and neolignans. In relation to the presence of aristolochic acids in this species, the data reported are conflicting (Ambros and De Siqueira 1971, Langmann 1979, Rücker et al. 1981).

Therefore, as part of our investigations on anti-infective and antitumoral potential of natural products, (De Oliveira et al. 2012, Almeida et al. 2012, Bianco et al. 2013, Guimarães et al. 2013) the in vitro biological profile of extracts, fractions and two isolated compounds obtained from A. triangularis regarding antiproliferative, antimicrobial, antiherpes and antileishmanial activities were investigated. Moreover, a preliminary examination related to the presence of aristolochic acids in stems and leaves of this medicinal plant by HPLC was also conducted.

MATERIALS AND METHODS

PLANT MATERIAL

Stems and leaves of Aristolochia triangularis were collected in Nova Santa Rita (29°52’24”S - 51°15’25”W), Rio Grande do Sul State, Brazil, in March 2013. The plant material was identified by Dr. Sérgio Augusto de Loreto Bordignon, and a voucher specimen was deposited at the Herbarium ICN, voucher number 184663 (Herbarium of the Universidade Federal do Rio Grande do Sul).

EXTRACTION PROCEDURES

Stems and leaves were separated and dried at 40ºC. Thereafter, ethanol maceration (plant: solvent, 1:10, w/v ratio; 2 x 10 days) was carried out. After solvent evaporation, one aliquot of the crude ethanol extract (CEE) was reserved for HPLC analyses while another one was fractionated with chloroform yielding a chloroform fraction (CF) and a residual aqueous fraction (RAF). All these samples were assayed for in vitro biological activities.

PHYTOCHEMICAL ANALYSES AND ISOLATION OF THE MAJOR COMPOUNDS FROM CHLOROFORM FRACTIONS

Chloroform fractions (1 g) from stems and leaves (CF_stems and CF_leaves, respectively) were preliminarily fractionated by flash column chromatography on silica gel (70-230 mesh) using n-hexane (200 mL), n-hexane: ethyl acetate (1:1, v/v, 200 mL), ethyl acetate (200 mL) and methanol (200 mL) as mobile phase. The fractions were monitored by TLC, and pooled considering their chemical profiles to yield sub-fractions CF-1 to CF-4. Considering that the TLC profiles of stems and leaves were similar, and the yield of CF_stems sub-fractions were higher than those of CF_leaves, the isolation of major compounds was carried out only with stems sub-fractions. Thus, sub-fraction CF_stems-2 (80 mg) eluted with n-hexane-ethyl acetate (5:5, v/v,) was fractionated by column chromatography on Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) using ethanol as mobile phase. Two pure compounds were isolated and analyzed by NMR. The identification of the neolignan galbacin (compound 1, 23 mg) and the lignan cubebin (compound 2, 16 mg) was carried out by comparing their spectra with spectral data reported in the literature (Lopes and Bolzani 1988, Vieira et al. 1998, Zhang et al. 2008).

BIOLOGICAL ACTIVITY PROFILE

Antiproliferative screening

Human non-small-cell lung cancer A549 cells (ATCC: CCL185) were grown in MEM (Cultilab®, Brazil) supplemented with 5% fetal bovine serum (FBS, Life Technologies®, USA), and maintained at 37°C in a humidified 5% CO₂ atmosphere. Antiproliferative activity was evaluated by the classical colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann 1983). Briefly, 1x10⁴ confluent cells were treated with serial dilutions of samples for 72 h at 37°C. After treatment, medium was replaced by MTT solution (Sigma-Aldrich), and cells were further incubated for 4 h. Optical densities were read at 540 nm (Spectra Max M2 (Molecular Devices, Sunnyvale, CA, USA) immediately after the dissolution of formazan crystals with DMSO (Sigma-Aldrich). The concentration of each sample that reduced cell viability by 50% when compared to untreated controls (CC₅₀) was estimated by non-linear regression of concentration–response curves. Paclitaxel (Sigma-Aldrich) was used as positive control.

Antibacterial and antifungal screening

Microorganisms used were gram-positive bacteria [Clostridium sporogenes (ATCC 11437), Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Streptococcus pneumonie (ATCC 49619), Streptococcus pyogenes (ATCC 19615)]; gram-negative bacteria [Enterobacter cloacae (ATCC 13047), Escherichia coli (ATCC 25922), Klebsiella pneumonie (ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium (ATCC 14028), Shigella flexneri (ATCC 12022)]; and fungus [Candida albicans (ATCC 10231) and Candida tropicalis (ATCC 13803)]. Antimicrobial activity was evaluated by disk diffusion method, as previously described by De Oliveira et al. (2005), with minor modifications. Briefly, filter paper disks (6 mm) were impregnated with 20 μL of sample dilutions (100 mg/mL DMSO) and then placed on Muller-Hinton agar plates (HIMEDIA®), which were inoculated with microorganisms according to the standard protocol described by Clinical Laboratory Standard Institute (2002). Plates were incubated at 35 ± 1°C for 18 h, and the diameters of the inhibition zones were measured. Filter paper disks containing only DMSO were used as negative controls. Standard antibiotic disks were selected according to the sensitivity of each microorganism tested: ampicillin (10 μg), oxacillin (1μg), ceftazidime (30 μg), imipenen (10 μg), chloramphenicol (30 μg), levofloxacin (5 μg), doxacillin (30 μg) and fluconazole (25 μg).

Antiherpes screening

Vero cells (ATCC:CCL-81) were grown in MEM (Cultilab, Brazil) supplemented with 10% FBS (Life Technologies, USA), and maintained at 37°C in a humidified 5% CO₂ atmosphere. Herpes Simplex Virus type 1 (HSV-1 / KOS strain, Faculty of Pharmacy, University of Rennes I, Rennes, France) viral stocks were prepared as previously described, and titrated based on plaque forming units (PFU) (Burleson 1992).

Initially, cytotoxicity was determined by exposing confluent Vero cells to serial dilutions of samples for 72 h at 37°C. Cell viability was determined by MTT assay, as previously described above. Results were expressed as CC₅₀ values, estimated by non-linear regression of concentration-response curves.

Following, antiherpes activity evaluation was carried out by plaque number reduction assay, according to the procedures described by Kratz et al. (2008), with minor modifications. Briefly, 2.5x10⁴ Vero cells were infected with approximately 100 PFU of HSV-1 (KOS strain) for 1h at 37°C. Infected cells were treated with MEM containing 1.5% carboxymethylcellulose in the presence or absence of serial non-toxic dilutions of samples for 72 h at 37°C. After treatment, cells were fixed and stained with naphthol blue-black and viral plaques were counted. The concentration of each sample that reduced plaque number by 50% when compared to untreated controls (IC₅₀) was estimated by non-linear regression of concentration-response curves. The ratio of these indices was used to calculate the selectivity index of each sample (SI = CC₅₀/IC₅₀). Acyclovir (Sigma-Aldrich) was used as positive control.

Antileishmanial screening

Macrophage-like THP-1 (ATCC:TIB 202) cells were grown in RPMI-1640 without phenol red (Sigma), supplemented with 10% FBS (Life Technologies, USA), 12.5 mM HEPES, penicillin (100 U/mL), streptomycin (100 μg/mL) and Glutamax (2 mM), at 37°C in a humidified 5% CO₂ atmosphere. Leishmania amazonensis (MHOM/BR/77/LTB0016) promastigotes, expressing beta-galactosidase, were grown at 26°C in Schneider’s insect medium (Sigma-Aldrich) supplemented with 5% FBS and 2% of human urine.

For the antileishmanial screening against intracellular L. amazonensis amastigotes, THP-1 cells (3.0 x 10⁴ per well) were cultivated in 96 well plates with RPMI-1640 medium supplemented as described above, and treated with 100 µg/mL of phorbol 12-myristate 13-acetate (PMA) for 72 h at 37°C to allow THP-1 cells differentiation into non-dividing macrophages (Schwende et al. 1996).

Four day culture promastigotes (4.0 x 10⁶ parasites/mL) were washed with phosphate buffered saline, pH 7.4 (PBS) an incubated in RPMI-1640 supplemented with 10% human AB+ serum for 1 h at 34°C for parasite opsonization. THP-1 cells were then incubated with a parasite/cell ratio of 10:1 for 3 h at 34°C and 5% CO₂. After this period, non-adherent parasites were removed by one wash with PBS, and infected cells were incubated with 180 µL of full supplemented RPMI-1640 medium for more 24 h to allow the transformation of promastigotes into intracellular amastigotes.

Infected cell monolayers were treated by addition of 20 μL of each sample in different concentration (50 µg/mL to 1.56 µg/mL, in triplicate, followed by incubation for 48 h, at 34°C in 5% CO₂. After treatment, cells were carefully washed with PBS and incubated for additional 16 h at 37°C with 250 µL of chlorophenolred-ß-D-galactopyranoside (CPRG) (Sigma-Aldrich) at 100 µM and nonidet P-40 0.1% (NP-40) (Amresco Inc, USA). Optical density was read at 570/630 nm in an Infinite M-200 multiplate reader (TECAN, Austria). The concentration of each sample that reduced parasite viability by 50% when compared to untreated controls (IC₅₀) was estimated by non-linear regression of concentration-response curves. Amphotericin B (Sigma) was used as positive control.

For cell toxicity evaluation, THP-1 cells were seeded in 96 well plates and incubated for 48 h at 37°C with serial dilutions of samples. The assays were carried out in triplicate, and cell viability was determined by MTT assay as described above.

Statistical analyses

Data analysis was performed with PRISM 5 software (GraphPad Software, San Diego, CA, USA.). CC₅₀ and IC₅₀ values were calculated by fitting variable slope normalized sigmoidal concentration-response curves. Data are presented as means ± standard deviations with 95% confidence interval (n = 3).

HPLC ANALYSES

HPLC analyses of CEE obtained from stems and leaves was conducted to detect the presence of aristolochic acids AA-I and AA-II, and were performed in a LC PerkinElmer Series 200, composed by a Diode Array Detector (DAD), quaternary pump, autosampler, and online degasser. The separation was achieved on a Perkin Elmer Brownlee Choice C₁₈ column (250 mm x 4.6 mm i.d. x 5 µm), and the mobile phase was a gradient of solvent A (acetonitrile-acetic acid 0.2%) and solvent B (acetic acid 0.2%) as follows: 20-40% A (0-32 min), isocratic 40% A (32-67 min), and gradient 40-90% A (67-95 min). The flow rate was kept constant at 1.0 mL/min. The chromatograms were recorded at 254 nm, and the UV spectra were monitored over a range of 200-500 nm. Standards of AA-I and a mixture of AA-I and AA-II were purchased from Sigma-Aldrich® (St. Louis, MO, USA). Peaks were characterized by comparing retention times of the samples and their UV spectra with reference standards and by co-injection of authentic samples. All analyses were performed in triplicate.

RESULTS AND DISCUSSION

In South Brazil, according to a study conducted by EPAGRI (Santa Catarina Agricultural Agency) (Silva-Junior and Salerno 2012), A. triangularis was cited as the most frequently used medicinal plant in Santa Catarina State. In this way, a multiple in vitro biological screening of extracts, fractions and two isolated compounds from this plant was performed. Considering the controversial literature data about the presence of aristolochic acids in A. triangularis, the existence of these substances in the crude ethanol extract (CEE) of this plant was also investigated.

PHYTOCHEMICAL ANALYSES AND ISOLATION OF MAJOR COMPOUNDS

Chloroform fractions (CF_stems and CF_leaves) were submitted, separately, to flash column chromatography procedures yielding four fractions (CF-1, CF-2, CF-3 and CF-4), which were analyzed by TLC. It was observed the presence of two major compounds 1 and 2 in CF_stems-4, which could be isolated by chromatographic fractionation over silica gel and Sephadex LH-20. These compounds were characterized comparing their NMR spectra with spectral data previously reported (Lopes and Bolzani 1988, Vieira et al. 1998, Zhang et al. 2008). Compounds 1 (23 mg) and 2 (16 mg) were identified as the neolignan (+) galbacin (5-[(2R,3R,4R,5R)-5-(1,3-benzodioxol-5-yl)-3,4-dimethyloxolan-2-yl]-1,3-benzodioxole (1): ¹H-NMR (CDCl₃, 125 MHz) δ 5.94 (2H, s, -O-CH₂-O-), δ 4.61(2H, d, J-9.5 Hz, H-2 and H-5), δ 1.75 (2H, m, H-3 and H-4), δ 1.01 (6H, s, two methyl groups)), and the lignan (-) cubebin [(2R,3R)-2,3-di-(3,4-methylenedioxybenzyl)-butyrolactol](2): ¹H-NMR (CDCl₃, 125 MHz) δ 6.47 - 6.50 (6H, m, Aryl-H), δ 5.90 (4H, s, (2x) -O-CH2-O-), δ 5.23 (1H, brs, H-9’), δ 3.82-3.55 (1H, m, H-9’), δ 2.64-2.40 (4H, m, (2x) H-7 and (2x) H-6), δ 2,10 (2H, m, H-8 and H-8’), respectively (Figure 1). Lignans and neolignans with great structural diversity were described for Aristolochia species (Zhai et al. 2005, De Pascoali et al. 2006, León-Diaz et al. 2010). In this way, our results corroborate these reported data as well as the results obtained by Langmann et al. (1979) with A. triangularis extracts, who also isolated cubebin and galbacin.

Figure 1
Structure of the neolignan and lignan isolated from Aristolochia triangularis, galbacin (1) and cubebin (2).

ANTIPROLIFERATIVE SCREENING

The antiproliferative activity against the human lung cancer cell line (A549) is summarized in Table I.

TABLE I
Antiproliferative activity against A549 cells of Aristolochia triangularis extracts, fractions and compounds 1 and 2.

Samples	Stems		Leaves
	CC50 (μg/mL)	CI 95%	CC50 (μg/mL)	CI 95%
CEE	9.21	7.97-10.65	> 50	-
CF	2.93	2.25-3.81	12.96	11.54-14.55
CF-1	22.94	14.45-36.42	43.97	31.95-61.29
CF-2	22.04	15.46-31.42	24.53	14.66-41.04
CF-3	16.74	6.27-44.72	13.80	4.79-39.74
CF-4	7.24	0.43-12.31	7.22	4.17-12.51
1	13.42		7.51-23.99
2	23.51		7.19-60.51

• CI 95%: confidence interval at 95%; Paclitaxel - CC₅₀ = 0.72; CI = 0.42-1.24; CEE: crude ethanol extract; CF: chloroform fraction; CF-1: n-hexane; CF-2: n-hexane-ethyl acetate (5:5); CF-3: Ethyl acetate; CF-4: methanol; 1: galbacin; 2: cubebin.

Results showed that CF_stems was the most cytotoxic sample. CF fractions, from stems and leaves, showed similar antiproliferative effects, but were less cytotoxic than the respective crude extracts. In addition, compounds 1 and 2 were less cytotoxic when compared to CF fractions.

The antitumor potential of Aristolochia species and related compounds has been previously described (Chaouki et al. 2010, Hedge et al. 2010, Marti et al. 2013, Zhou et al. 2013). For A. triangularis, antiproliferative effects were described for dichloromethane extract against human epidermoid carcinoma (KB cells) (Mongelli et al. 2000). Moreover, several neolignans have been isolated from Aristolochia species, and showed a diverse pattern of cytotoxicity against various cancer cell lines (Zhou et al. 2013). As far as we are aware, this is the first report of antiproliferative activity for A. triangularis against A549 cells. Concerning the antiproliferative activity of compounds 1 and 2, the results indicated a weak action suggesting that these compounds, individually, are not responsible for the detected activity. Indeed, few studies demonstrated that galbacin and cubebin, alone, showed merely moderate antiproliferative activity against other cancer cell lines (Lee et al. 2004, Nascimento et al. 2004).

ANTIBACTERIAL AND ANTIFUNGAL SCREENING

CEE_stems and CF_stems-4 showed weak antimicrobial activity against S. aureus (9-12 mm inhibition zone). The most interesting results were obtained with CF_leaves (13-16 mm inhibition zone). Compounds 1 and 2 were inactive against all bacterial and fungal species evaluated.

Few studies reporting antimicrobial activity of plants from Aristolochia genus have been published (Camporese et al. 2003, Machado and Lopes 2005). From A. triangularis, there is only one report concerning the antibacterial activity of its roots against S. aureus (Mesa-Alicia et al. 1950).

ANTIHERPES SCREENING

In this study, the antiviral activity against Herpes Simplex Virus type 1 (HSV-1, KOS strain) was also evaluated. Cytotoxic effects on Vero cells, which are permissive to herpesvirus replication, were preliminarily investigated by MTT assay. Only non-cytotoxic concentrations were used for the antiviral screening. The results indicated that only semi-purified fractions CF_stems-4 and CF_leaves-4 could inhibit viral replication (99.0 and 97.9%, respectively). These results agree with the findings of Garcia et al. (1990), who showed that a crude ethanolic extract from A. triangularis did not present antiherpes activity. Furthermore, the antiherpes activity of both semi-purified fractions CF_stems-4 and CF_leaves-4 inhibited viral replication in a concentration-dependent manner with IC₅₀ values of 0.40 and 2.61 µg/mL, respectively (Figure 2), and selectivity indices of 5.0 and 4.1, respectively. Compounds 1 and 2 did not show antiviral activity (inhibition < 10%).

Figure 2
Antiherpes activity (HSV-1, KOS strain) of the semi-purified fractions CF_stems-4 (a) and CF_leaves-4 (b) from Aristolochia triangularis.

These findings could be regarded as promising for semi-purified fractions or extracts of medicinal plants (Cos et al. 2006). In addition, since compounds 1 and 2 were inactive, the antiherpes activity detected is possibly related to the synergistic effects of multiple compounds present in the extracts or fractions (Williamson 2001, Wagner 2010), although the antiherpes activity of other neolignans and lignans has been described (Sawasdee et al. 2013).

ANTILEISHMANIAL SCREENING

CF_stems showed promising results against L. amazonensis (85.3% growth inhibition at 50 µg/mL) (Table II.). This fraction was also tested against the intracellular forms of L. amazonensis showing a selectivity index (SI) of 31.80, which could be also regarded as promising (Cos et al. 2006). Additionally, CF_stems-1 to CF_stems-4 were tested, but they were less active. In relation to the isolated compounds, only 1 was active showing a moderated growth inhibition at 50 µg/mL, and an IC₅₀ of 16.69 µg/mL.

TABLE II
Antileishmanial screening (50 µg/mL) against Leishmania amazonensis.

Samples	Growth inhibition (%)	IC50 (µg/mL)	CI 95% (µg/mL)	CC50 (µg/mL)	CI 95% (µg/mL)	SI
CEEstems	NI	-	-	-	-	-
CFstems	85.3 ± 0.5	2.28	0.86 - 6.07	72.5	45.21 - 116.2	31.80
CFstems-1	23.8 ± 7.1	-	-	-	-	-
CFstems-2	59.7 ± 9.2	56.13	54.50 - 57.80	154.6	150.8 - 158.5	2.75
CFstems-3	13.1 ± 5.3	-	-	-	-	-
CFstems-4	NI	-	-	-	-	-
CEEleaves	NI	-	-	-	-	-
CFleaves	NI	-	-	-	-	-
CFleaves-1	59.8 ± 5.5	12.82	9.70 - 16.95	268.9	214.0 - 337.8	20.98
CFleaves-2	NI	-	-	-	-	-
CFleaves-3	65.7 ± 2.2	12.06	7.38 - 19.69	49.53	6.05 - 405.6	4.11
CFleaves-4	47.8 ± 5.2	-	-	-	-	-
1	59.47 ± 4.7	16.69	13.61 - 20.45	> 50	-	> 3.00
2	9.69 ± 4.0	-	-	-	-	-

• NI: no inhibitory activity; CI 95%: confidence interval at 95%; Positive control (Amphotericin B, 2 µM); IC₅₀: concentration that show parasite growth inhibition in 50%; CC₅₀: concentration that reduces cell viability by 50%; SI: selectivity index (CC₅₀/IC₅₀);92.8 ± 1.7%. CEE_stems: crude ethanolic extract; CF_stems: chloroform fraction; CF_stems-1: n-hexane; CF_stems-2: n-hexane-ethyl acetate (5:5); CF_stems-3: ethyl acetate; CF_stems-4: methanol; compound 1: galbacin; compound 2: cubebin; CEE_leaves: crude ethanolic extract; CF_leaves: chloroform fraction; CF_leaves-1: n-hexane; CF_leaves-2: n-hexane-ethyl acetate (5:5); CF_leaves-3: ethyl acetate; CF_leaves-4: methanol.

Tempone et al. (2008) reported leishmanicidal and trypanocidal activities for Aristolochia cymbifera, also popularly known in Brazil as “cipó-mil-homens”. Another study conducted by Sartorelli et al. (2010) strengthened these results, and correlated the detected trypanocidal activity to the presence of diterpenes and lignans in this species. Thus, our results are, at least in part, in agreement with the literature.

HPLC ANALYSES OF CRUDE ETHANOL EXTRACT (CEE)

Considering the reported contradictory results concerning the presence of aristolochic acids (AA) in A.triangularis (Ambros and De Siqueira 1971, Langmann 1979, Rücker et al. 1981), its popular utilization in South Brazil for different purposes, and the possible toxic effects associated with these acids (Arlt et al. 2002, Debelle et al. 2008), we performed HPLC analyses the crude ethanol extract of stems and leaves.

Under the chromatographic conditions employed, AA-I and AA-II standards showed retention times of 62.7 min and 53.5 min, respectively (Figure 3). Chromatograms from CEE_stems (Figure 3a) and CEE_leaves (Figure 3b) did not display any peak with retention time comparable to that of AA-II (limits of detection and quantitation of 0.99 and 3.03 µg/mL for AA-I, and 0.13 and 0.39 µg/mL for AA-II). On the other hand, the chromatograms revealed the presence of a compound with a similar retention time of AA-I at 62 min, especially in CEE_stems. However, this putative compound showed important differences in its UV spectra (λ 275 and 225 nm) when compared to those of AA-I (λ 320 and 270 nm). Therefore, according to our data, A. triangularis did not show the presence of AA-I and AA-II in CEE_stems and CEE_leaves. Thus, these findings agree with those published by Langmann (1979) and Rücker et al. (1981), who evaluated two different samples of A. triangularis by distinct chromatographic techniques, TLC and GC/MS, respectively, and also did not detected the presence of AAs.

Figure 3
HPLC chromatograms, at 254 nm, of crude ethanolic extract (CEE) from stems (a) and leaves (b) of Aristolochia triangularis with reference standards: aristolochic acid I (AA-I, 62.7 min) and aristolochic acid II (AA-II, 53.5 min).

CONCLUSIONS

The biological screening performed with extracts, semi-purified fractions and isolated compounds showed multiple in vitro activities. In summary, we found that chloroform fractions from stems and leaves of A. triangularis showed in all tested assays results that could be regarded as promising for semi-purified fractions or extracts of medicinal plants. However, the neolignan/lignan isolated (galbacin and cubebin) did not show anti-HSV-1 activity and displayed only a weak antiproliferative activity and the neolignan galbacin was active against L. amazonensis, showing a moderated growth inhibition at 50 µg/mL, and an IC₅₀ of 16.69 µg/mL. Further pharmacological evaluation of A. triangularis remains highly desirable, especially in the light of its high usage among the population and the paucity of scientific data.

Finally, in view of the abundant studies reporting the toxicity of aristolochic acid derivatives and that they are frequently reported for Aristolochia species, the presence of aristolochic acid I and aristolochic acid II was also investigated in the crude ethanol extract of stems and leaves, and these compounds could not be detected by the HPLC analysis. Thus, these findings agree with those published by Langmann (1979) and Rücker et al. (1981), who evaluated two different samples of A. triangularis, and also did not detect the presence of aristolochic acid derivatives.

ACKNOWLEGMENTS

S.Q.O. and J.M.K. thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Ciência, Tecnologia, Inovações e Comunicações (MCTIC),Ministério da Ciência, Tecnologia, Inovações e Comunicações, Pós-Doutorado Júnior - grant number 385066/2013-9 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), (Ministério da Educação, Programa Nacional de Pós-Doutorado grant number 2207/2009), respectively, for their postdoctoral fellowships. M.S., F.H.R., C.M.O.S., and E.P.S are also grateful to CNPq for their research fellowships. T.R.G. is also grateful to CAPES for her PhD fellowship. A.V. and S.D. were supported by a Marie Curie International Research Staff Exchange Scheme Fellowship within the Seventh European Community Framework Programme (hERGscreen, 295174).

REFERENCES

• ALMEIDA MTR et al. 2012. Anti-infective pregnane steroid from the octocoral Carijoa riisei collected in South Brazil. Lat Am J Pharm 31: 1489-1495.
• ALVIANO WS, ALVIANO DS, DINIZ CG, ANTONIOLLI AR, ALVIANO CS, FARIAS LM, CARVALHO MA, SOUZA MM and BOLOGNESE AM. 2008. In vitro antioxidant potential of medicinal plant extracts and their activities against oral bacteria based on Brazilian folk medicine. Arch Oral Biol 53: 545-552.
• AMBROS ML and DE SIQUEIRA NS. 1971. Aristolochic acid in Aristolochia triangularis. Rev Brasil Farm 52: 61-63.
• ARLT VM, STIBOROVA M and SCHMEISER HH. 2002. Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 17: 265-277.
• BATTU GR, PARIMI R and SHEKAR KBC. 2011. In vivo and in vitro pharmacological activity of Aristolochia tagala (syn: Aristolochia acuminata) root extracts. Pharma Biol 49: 1210-1214.
• BHATTACHARJEE P and BHATTACHARYYA D. 2013. Characterization of the aqueous extract of the root of Aristolochia indica: Evaluation of its traditional use as an antidote for snakebites. J Ethnopharmacol 145: 220-226.
• BIANCO E et al. 2013. Anti-Infective Potential of Marine Invertebrates and Seaweeds from the Brazilian Coast. Molecules (Basel. Online) 18: 5761-5778.
• BURLENSON FG, CHAMBERTS TM and WIEDBRAUK DL. 1992.Virology: a Laboratory Manual; Academic Press: San Diego, CA, USA, p. 250.
• CAMPORESE A, BALICK MJ, ARVIGO R, ESPOSITO RG, MORSELLINO N, DE SIMONE F and TUBARO A. 2003. Screening of anti-bacterial activity of medicinal plants from Belize (Central America). J Ethnopharmacol 87: 103-107.
• CHAOUKI W, LEGER DY, ELJASTIMI J, BENEYTOUT JL and HMAMOUCHI M. 2010. Antiproliferative effect of extracts from Aristolochia baetica and Origanum compactum on human breast cancer cell line MCF-7. Pharm Biol 48: 269-274.
• CHUNG YM et al. 2011. A novel alkaloid, aristopyridinone A and anti-inflammatory phenanthrenes isolated from Aristolochia manshuriensis. Bioorg Med Chem Lett 21: 1792-1794.
• CLINICAL and LABORATORY STANDARDS INSTITUTE. 2002. Performance standards for antimicrobial disk susceptibility tests: approved standard M2-A8. In: Performance Standards for Antimicrobial Susceptibility Testing, Clinical and Laboratory Standards Institute, 8th ed., CLSI document M2-A8; CLSI: Wayne, PA, USA, p. 1-58.
• COS P, VLIETINCK AJ, BERGHE DV and MAES L. 2006. Anti-infective potential of natural products: how to develop a stronger in vitro ‘proof-of-concept’. J Ethnopharmacol 106: 290-302.
• COSYNS JP. 2003. Aristolochic acid and ‘Chinese herbs nephropathy’: a review of the evidence to date. Drug Safety 26: 33-48.
• DE BARROS MACHADO T, LEAL IC, KUSTER RM, AMARAL AC, KOKIS V, DE SILVA MG and DOS SANTOS KR. 2005. Brazilian phytopharmaceuticals - evaluation against hospital bacteria. Phytoter Res 19: 519-525.
• DEBELLE FD, VANHERWEGHEM JL and NORTIER JL. 2008. Aristolochic acid nephropathy: A worldwide problem. Kidney Int 74: 158-169.
• DE OLIVEIRA SQ, ALMEIDA MTR, MARASLIS FT, SILVA IT, SINCERO TCM, PALERMO JA, CABRERA GM, CARO MSB, SIMÕES CMO and SCHENKEL EP. 2012. Isolation of three new ent-labdane diterpenes from Dodonaea viscosa Jacquin (Sapindaceae): Preliminary evaluation of antiherpes activity. Phytochem Lett 5: 500-505.
• DE OLIVEIRA SQ, TRENTIN VH, KAPPEL VD, BARELLI C, GOSMANN G and REGINATTO FH. 2005. Screening of antibacterial activity of south Brazilian Baccharis species. Pharm Biol 43: 434-438.
• DE PASCOLI I, NASCIMENTO IR and LOPES LMX. 2006. Configurational analysis of cubebins and bicubebin from Aristolochia lagesiana and Aristolochia pubescens. Phytochemistry 67: 735-742.
• GARCIA GH, CAMPOS R, DE TORRES RA, BROUSSALIS A, FERRARO G, MARTINO V and COUSSIO J. 1990. Antiherpetic activity of some Argentine medicinal plants. Fitoterapia 61: 542-546.
• GUIMARÃES T et al. 2013. Anti HSV-1 Activity of Halistanol Sulfate and Halistanol Sulfate C Isolated from Brazilian Marine Sponge Petromica citrina (Demospongiae). Mar Drugs 11: 4176-4192.
• HEGDE VR, BORGES S, PATEL M, DAS PR, WU B, GULLO VP and CHAN TM. 2010. New potential antitumor compounds from the plant Aristolochia manshuriensis as inhibitors of the CDK2 enzyme. Bioorg Med Chem Lett 20: 1344-1346.
• IZQUIERDO AM, ZAPATA EV, JIMÉNEZ-FERRER JE, MUÑOZ CB, APARICIO AJ, TORRES KB and TORRES LO. 2010. Scorpion antivenom effect of micropropagated Aristolochia elegans. Pharm Biol 48: 891-896.
• KRATZ JM et al. 2008. Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate. Mem Inst Oswaldo Cruz 103: 437-442.
• KUMAR VP, CHAUHAN NS, PADH H and RAJANI M. 2006. Search for antibacterial and antifungal agents from selected Indian medicinal plants. J Ethnopharmacol 107: 182-188.
• LANGMANN B. 1979. Über die inhaltsstoffe von Aristolochia triangularis chamisso. (Aristolchiaceae). Doctoral thesis, Münster University, 206 p.
• LEE JS, KIM J, YU YU and KIM YC. 2004. Inhibition of Phospholipase Cy1 and Cancer Cell Proliferation by Lignans and Flavans from Machilus thunbergfi. Arch Pharm Res 27: 1043-1047.
• LÉON-DIAZ R, MECKES M, SAID-FERNÁNDEZ S, MOLINA-SALINAS GM, VARGAS-VILLARREAL J, TORRES J, LUNA-HERRERA J and JIMÉNEZ-ARELLANES A. 2010. Antimycobacterial neolignans isolated from Aristolochia taliscana. Mem Inst Oswaldo Cruz 105: 45-51.
• LOPES LMX and BOLZANI VS. 1988. Lignans and diterpenes of three Aristolochia species. Phytochemistry 27: 2265-2268.
• LORENZI H and MATOS FJA. 2002. Plantas medicinais no Brasil nativas e exóticas. São Paulo: Instituto Plantarum de Estudos da Flora Ltda, p. 77-79.
• MACHADO MB and LOPES LMX. 2005. Chalcone-flavone tetramer and biflavones from Aristolochia ridicula. Phytochemistry 66: 669-674.
• MARTI G et al. 2013. Natural aristolactams and aporphine alkaloids as inhibitors of CDK1/Cyclin B and DYRK1A. Molecules 18: 3018-3027.
• MESA-ALICIA M, GUSMAN JESUS CR and CALVO DE LA TORI J. 1950. Antibiotic properties of Aristolochia. Ciencia e Invest 6: 471-476.
• MESSIANO GB, VIEIRA L, MACHADO MB, LOPES LM, DE BORTOLI SA and ZUKERMAN-SCHPECTOR J. 2008. Evaluation of insecticidal activity of diterpenes and lignans from Aristolochia malmeana against Anticarsia gemmatalis. J Agric Food Chem 56: 2655-2659.
• MONGELLI E, PAMPURO S, COUSSIO J, SALOMON H and CICCIA G. 2000. Cytotoxic and DNA interaction activities of extracts from medicinal plants used in Argentina. J Ethnopharmacol 71: 145-151.
• MOSMANN T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.
• MUSCHIETTI L, MARTINO V, FERRARO G, COUSSIO J, SEGURA L, CARTAIIA C, CANIGUERAL C and ADZET T. 1996. The anti-inflammatory effect of some species from South America. Phytother Res 10: 84-86.
• NASCIMENTO MSJ, PEDRO M, CERQUEIRA F, BASTOS M, VIEIRA LM, KIJJOA A and PINTO MMM. 2004. Effect of natural 2,5-Diaryl-3,4-dimethyltetrahydrofuran lignans on complement activation, lymphocyte proliferation, and growth of tumor cell lines. Pharm Biol 42: 449-453.
• NEINHUIS C, WANKE S, HILU KW, MÜLLER K and BORSCH T. 2005. Phylogeny of Aristolochiaceae based on parsimony, likelihood, and Bayesian analyses of trnL-trnF sequences. Plant Syst Evol 250: 7-26.
• RÜCKER G, LANGMANN B and DE SIQUEIRA NS. 1981. Constituents of Aristolochia triangularis. Planta Med 41: 143-149.
• SAMY RP, THWIN MM, GOPALAKRISHNAKONE P and IGNACIMUTHU S. 2008. Ethnobotanical survey of folk plants for the treatment of snakebites in Southern part of Tamilnadu, India. J Ethnopharmacol 115: 302-312.
• SARTORELLI P, CARVALHO CS, REIMÃO JQ, LORENZI H and TEMPONE AG. 2010. Antitrypanosomal activity of a diterpene and Lignans isolated from Aristolochia cymbifera. Planta Med 76: 1454-1456.
• SAWASDEE K, CHAOWASKU T, LIPIPUN V, DUFAT T, MICHEL S and LIKHITWITAYAWUID K. 2013. Neolignans from leaves of Miliusa mollis. Fitoterapia 85: 49-56.
• SCHWENDE H, FITZKE E, AMBS P and DIETER P. 1996. Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. J. Leukoc. Biol. 59: 555-561.
• SILVA-JÚNIOR AA and SALERNO AR. 2012. O inventário florestal e as plantas bioativas de Santa Catarina. Agropecu Catarin 25: 35-38.
• SIMÕES CMO, MENTZ LA, SCHENKEL EP, IRGANG BE and STEHMANN JR. 1998. Plantas da Medicina Popular no Rio Grande do Sul, 5ª ed., Porto Alegre: Ed. Da Universidade/ UFRGS, p. 32, 36, 50, 85, 94, 110-111.
• TEMPONE AG, SARTORELLI P, TEIXEIRA D, PRADO FD, CALIXTO I, LORENZI H and MELHEM MSC. 2008. Brazilian flora extracts as source of novel antileishmanial and antifungal compounds. Mem Inst Oswaldo Cruz 103: 443-449.
• VIEIRA LM, KIJJOA A, SILVA AMS, MONDRANONDRA IO and HERZ W. 1998. 2,5-diaryl-3,4-dimethyltetrahydrofuran lignans from Talauma hodgsonii. Phytochemistry 37: 1079-1081.
• WAGNER H. 2010. Synergy research: approaching a new generation of phytopharmaceuticals. Fitoterapia 82: 34-47.
• WILLIAMSON EM. 2001. Synergy and other interactions in phytomedicines. Phytomedicine 8: 401-409.
• WU TS, DAMU AG, SU C and KUO PC. 2005. Chemical constituents and pharmacology of Aristolochia Species. In: Attaur-Rahman, editor. Studies in natural products chemistry (bioactive natural products) Amsterdam: Elsevier 32: 855-1018.
• YU JQ, LIAO ZX, CAI XQ, LEI JC and ZOU GL. 2007. Composition, antimicrobial activity and cytotoxicity of essential oils from Aristolochia mollissima. Environ Toxicol Pharmacol 23: 162-167.
• ZHAI H, INOUE T, MORIYAMA M, ESUMI T, MITSUMOTO Y and FUKUYAMA Y. 2005. Neuroprotective effects of 2,5-diaryl-3,4-dimethyltetrahydrofuran neolignans. Biol Pharm Bull 28: 289-293.
• ZHAI H, NAKATSUKASA M, MITSUMOTO Y and FUKUYAMA Y. 2004. Neurotrophic effects of talaumidin, a neolignan from Aristolochia arcuata, in primary cultured rat cortical neurons. Planta Med 70: 598-602.
• ZHANG G et al. 2008. Chemical constituents of Aristolochia constricta: antispasmodic effects of its constituents in guinea-pig ileum and isolation of a diterpeno-lignan hybrid. J Nat Prod 71: 1167-1172.
• ZHOU Z, LUO J, PAN K, SHAN S, ZHANG W and KONG L. 2013. Bioactive Benzofuran Neolignans from Aristolochia fordiana. Planta Med 79: 1730-1735.

Publication Dates

• Publication in this collection
  12 Aug 2019
• Date of issue
  2019

History

• Received
  13 July 2018
• Accepted
  6 Sept 2018