ABSTRACT
Several species of the genus Passiflora are distributed all over South America, and many of these species are used in popular medicine, mainly as sedatives and tranquilizers. This study analyzes the chemical profile of extracts of four Passiflora species used in folk medicine, focusing on the flavonoids, alkaloids and saponins. We employed simple and fast fingerprint analysis methods by high performance liquid chromatography, ultra performance liquid chromatography and capillary electrophoresis techniques. The analysis led to the detection and identification of C-glycosylflavonoids in all the plant extracts, these being the main constituents in P. tripartita var. mollissima and P. bogotensis. Saponins were observed only in P. alata and P. quadrangularis, while harmane alkaloids were not detected in any of the analyzed extracts in concentrations higher than 0.0187 ppm, the detection limit determined for the UPLC method.
Keywords:
Passiflora; C-glycosylflavonoids; Alkaloids; HPLC; UPLC; CE
Introduction
The genus Passiflora is the largest and most important genus of the family Passifloraceae, comprising about 500 species (Lewis and Elvin-Lewis, 1977Lewis, W.H., Elvin-Lewis, M.P.F., 1977. Medical Botany: Plants Affecting Man's Health. Wiley-Interscience, Toronto.). In North America and Europe, the main species, P. incarnata, is popularly known as passion fruit or passionflower, while in South America, several others species of Passiflora are widely distributed and known by distinct names, such as 'maracujá', 'curuba', or 'badea', among others. (Arbelaez, 1996Arbelaez, E.P., 1996. Plantas utiles de Colombia. Jardim Botânico José Celestino Nutis, Bogotá.; Mors et al., 2000Mors, W.B., Rizzini, C.T., Pereira, N.A., 2000. Medicinal Plants of Brazil. Reference Publications, Algonac.) Many of these species (P. edulis var. edulis,P. edulis var. flavicarpa,P. tripartita var. mollissima and others) are cultivated for their edible fruits or for the preparation of juices. Infusions of their leaves are also used in popular medicine in many countries, as a sedative or tranquillizer (Pio Corrêa, 1978Pio Corrêa, M., 1978. Dicionário das plantas úteis do Brasil e das exóticas cultivadas. Impressora Nacional, Rio de Janeiro.; Arbelaez, 1996Arbelaez, E.P., 1996. Plantas utiles de Colombia. Jardim Botânico José Celestino Nutis, Bogotá.).
Different countries in South America have registered pharmaceutical preparations that use Passiflora species as the active component. In Colombia, for example, the leaves of P. tripartita var. mollissima are accepted as sedative and hypnotic component in phytopharmaceutical preparations (Invima, 2006Invima, 2006. Normas Farmacológicas Instituto Nacional de Vigilancia de Medicamentos y Alimentos. Bogotá D.C., http://apps.who.int/medicinedocs/documents/s18383es/s18383es.pdf.
http://apps.who.int/medicinedocs/documen...
). In Brazil, P. alata and P. edulis are included in the most recent version of the Brazilian Pharmacopeia (Farmacopeia Brasileira, 2010Farmacopeia Brasileira, 2010. Agência Nacional de Vigilância Sanitária, Brasília, DF, 5a ed, http://www.anvisa.gov.br/hotsite/cd_farmacopeia/index.htm.
http://www.anvisa.gov.br/hotsite/cd_farm...
).
Regarding their chemical composition, the compounds more frequently reported for the genus are flavonoids, especially C-glycosylflavonoids, which are usually described as the main components (Ulubelen et al., 1982Ulubelen, A., Oksuz, S., Mabry, T.J., Dellamonica, G., Chopin, J., 1982. C-glycosylflavonoids from Passiflora pittieri, P. alata, P. ambigua and a Adenia manii. J. Nat. Prod. 45, 783.; Li et al., 2011Li, H., Zhou, P., Yang, Q., Shen, Y., Deng, J., Li, L., Zhao, D., 2011. Comparative studies on anxiolytic activities and flavonoid compositions of Passiflora edulis 'edulis' and Passiflora edulis 'flavicarpa'. J. Ethnopharmacol. 133, 1085-1090.; Zucolotto et al., 2012Zucolotto, S.M., Fagundes, C., Reginatto, F.H., Ramos, F.A., Castellanos, L., Duque, C., Schenkel, E.P., 2012. Analysis of C-glycosyl flavonoids from South American Passiflora species by HPLC–DAD and HPLC–MS. Phytochem. Anal. 23, 232-239.). These compounds have recently been associated with several pharmacological effects observed for distinct Passiflora species (Coleta et al., 2006Coleta, M., Batista, M.T., Campos, M.G., Carvalho, R., Cotrim, M.D., Lima, T.C., Cunha, A.P., 2006. Neuropharmacological evaluation of the putative anxiolytic effects of Passiflora edulis Sims, its sub-fractions and flavonoid constituents. Phytother. Res. 20, 1067-1073.; Santos et al., 2006Santos, K.C., Santos, C.A.M., de Oliveira, R.M.W., 2006. Passiflora actinia Hooker extracts and fractions induce catalepsy in mice. J. Ethnopharmacol. 100, 306-309.; Sena et al., 2009Sena, L.M., Zucolotto, S.M., Reginatto, F.H., Schenkel, E.P., De Lima, T.C.M., 2009. Neuropharmacological activity of the pericarp of Passiflora edulis flavicarpa Degener: putative involvement of C-glycosylflavonoids. Exp. Biol. Med. 234, 967-975.; Zucolotto et al., 2009Zucolotto, S.M., Goulart, S., Montanher, A.B., Reginatto, F.H., Schenkel, E.P., Fröde, T.S., 2009. Bioassay-guided isolation of anti-inflammatory C-glycosylflavones from Passiflora edulis. Planta Med. 75, 1-6.; Gazola et al., 2015Gazola, A.C., Costa, G.M., Castellanos, L., Ramos, F.A., Reginatto, F.H., de Lima, T.C.M., Schenkel, E.P., 2015. Involvement of GABAergic pathway in the sedative activity of apigenin, the main flavonoid from Passiflora quadrangularis pericarp. Rev. Bras. Farmacogn. 25, 158-163.). Harmane alkaloids are also frequently associated with Passiflora species, especially P. incarnata (Lutomski and Malek, 1975Lutomski, J., Malek, B., 1975. Pharmakochemische untersuchungen der drogen der gattung Passiflora. IV. Mittlg: Der vergleich des alkaloidgehaltes in verschiedenen harmandrogen. Planta Med. 27, 381-384.; Lutomski et al., 1975Lutomski, J., Malek, B., Rybacka, L., 1975. Pharmacochemical investigation of the raw materials from Passiflora genus. II. The pharmacochemical estimation of juices from the fruits of Passiflora edulis and Passiflora edulis form flavicarpa. Planta Med. 27, 112-121.). Additionally, several saponins have been described for this genus, although their occurrence is restricted to certain species (Orsini et al., 1985Orsini, F., Pelizzoni, F., Verotta, L., 1985. Quadranguloside, a cycloartane triterpene glycoside from Passiflora quadrangularis. Phytochemistry 25, 191-193.; Reginatto et al., 2001Reginatto, F.H., Kauffmann, C., Schripsema, J., Guillaume, D., Gosmann, G., Schenkel, E.P., 2001. Steroidal and triterpenoidal glucosides from Passiflora alata. J. Braz. Chem. Soc. 12, 32-36.; Doyama et al., 2005Doyama, J.T., Rodrigues, H.G., Novelli, E.L.B., Cereda, E., Vilegas, W., 2005. Chemical investigation and effects of the tea of Passiflora alata on biochemical parameters in rats. J. Ethnopharmacol. 96, 371-374.).
As part of our ongoing studies with species of the genus Passiflora, we evaluate, in this study, the variability of metabolite presents in the aqueous extracts of four South American Passiflora species: P. alata,P. quadrangularis,P. bogotensis and P. tripartita var. mollissima, focusing specifically on their C-glycosylflavonoid and alkaloid composition. The presence of saponins in these species was also evaluated. Chemical profiles were obtained by different analytical methods, such as high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC) and capillary electrophoresis (CE).
Materials and methods
Chemicals
Acetonitrile, methanol and formic acid (HPLC-grade) were provided by Tedia® (Rio de Janeiro, Brazil). Water was purified with a Milli-Q system (Millipore®, Bedford, USA). For the CE analysis, stock solutions were prepared from the electrolytes sodium tetraborate (STB) and ammonium acetate (AcNH4) at 100 mmol/l. Sodium hydroxide solution (NaOH) at 1 and 0.1 mol/l were also prepared. All the salts used were of analytical reagent grade, and were provided by Sigma-Aldrich® (St. Louis, USA). All the solvents and solutions were filtered through a 0.22 µm membrane before use. The reference standards orientin, isoorientin, vitexin, isovitexin, vitexin-2″-O-rhamnoside, harmol, harmane and harmine (all with purity ≥ 96%) were purchased from Sigma-Aldrich®. Swertisin was previously obtained from Wilbrandea ebracteata roots, and identified by NMR spectral data (Santos et al., 1996Santos, R.I., Marlise, A., Schenkel, E.P., 1996. Analysis of the plant drug Wibrandia ebracteata. Int. J. Pharmacogn. 34, 300-330.). The compound 4'-methoxyluteolin-8-C-6″-acetylglucopyranoside was previously isolated from P. tripartita var. mollissima leaves and provided by Prof. Dr. Freddy Ramos (Ramos et al., 2010Ramos, F.A., Castellanos, L., López, C., Palacios, L., Duque, C., Pacheco, R., Guzmán, A., 2010. An orientin derivative isolated from Passiflora tripartita var. mollissima. Lat. Am. J. Pharm. 29, 141-143.). Quadranguloside was previously isolated from Passiflora alata leaves (Costa et al., 2013Costa, G.M., Gazola, A.C., Madóglio, F.A., Zucolotto, S.M., Reginatto, F.H., Castellanos, L., Duque, C., Ramos, F.A., Schenkel, E.P., 2013. Vitexin derivatives as chemical markers in the differentiation of the closely related species Passiflora alata Curtis. and Passiflora quadrangularis Linn. J. Liq. Chromatogr. R. T. 36, 1697-1707.).
Plant material and preparation of extracts and samples
Leaves of adult individuals of species of Passiflora were collected from different regions of Brazil and Colombia (Table 1). Leaves of the different species were air-dried separately at 40 °C, powdered, and extracted by infusion with boiling water (95 °C, plant:solvent 1:10, w/v) for 10 min. The aqueous extract was then filtered, frozen and lyophilized. The samples for HPLC, UPLC and CE analysis were prepared by dissolving the lyophilized crude aqueous extracts or reference standards in methanol:water (1:1, v/v) and filtering through a 0.22 µm membrane before injection. The concentration of the sample extracts was 1000 µg/ml and for the reference standards, the concentration was 100 µg/ml.
Passiflora species, with their respective local common name, place of collection and identification.
HPLC analysis
The HPLC analyses were carried out in a PerkinElmer® Series 200 system, equipped with Diode Array Detection (DAD), quaternary pump, on-line degasser and autosampler. The data were processed using the software Chromera® Workstation. The chromatographic analyses for all samples were performed at room temperature (21 ± 2 °C), with an injection volume of 20 µl. The DAD spectra were acquired at the range of 190–450 nm. The peaks in the samples were characterized by comparing the retention time, UV spectra and co-injection with the reference standards. Vertical® VertSep C18 column (250 mm × 4.6 mm i.d.; 5 µm) was used as stationary phase. In the analysis of flavonoids, a gradient system of acetonitrile [solvent A] and formic acid 0.5% [solvent B] was used as mobile phase, in a single step: 15–35% A (0–20 min). The flow rate was kept constant at 1.2 ml/min and the chromatograms were recorded at 340 nm. For alkaloid analysis, the mobile phase used was composed of an aqueous buffer of sodium phosphate dibasic (50 mmol/l, pH 8.0) [A], methanol [B] and acetonitrile [C] at isocratic conditions of 56% A: 12% B: 32% C (0–20 min). The flow rate was kept at 1 ml/min and the UV detection at 245 nm. The chromatographic conditions for the analysis of saponins were previously described by our group (Costa et al., 2013Costa, G.M., Gazola, A.C., Madóglio, F.A., Zucolotto, S.M., Reginatto, F.H., Castellanos, L., Duque, C., Ramos, F.A., Schenkel, E.P., 2013. Vitexin derivatives as chemical markers in the differentiation of the closely related species Passiflora alata Curtis. and Passiflora quadrangularis Linn. J. Liq. Chromatogr. R. T. 36, 1697-1707.).
UPLC analysis
An UPLC Waters Acquity® H Class system with DAD detector, quaternary pump, on-line degasser and autosampler was used for these analyses. The chromatographic parameters were converted from HPLC to UPLC using the software Empower®. Separations of both flavonoids and alkaloids were carried out in a PerkinElmer® BHE C18 column (100 mm × 2.9 mm i.d.; 1.8 µm). The analyses were also performed at room temperature (21 ± 2 °C), with DAD spectra acquired at the range of 190–450 nm. Flavonoid analysis used a two-steps gradient of acetonitrile [solvent A] and formic acid 0.5% [solvent B]: 15–35% A (0–8 min), followed by 35% A (8–10 min). The flow rate was kept constant at 0.25 ml/min. The injection volume was 3 µl. The chromatogram was recorded at 340 nm. For the alkaloids, the same mobile phase and isocratic system as the HPLC was used, with an analysis time of 7 min. The flow rate was determinate as 0.2 ml/min, with an injection volume of 2 µl. The UV detection was determined at 245 nm.
CE analysis
The analyses were performed on an Agilent 7100 capillary electrophoresis instrument equipped with DAD detector, temperature control device, and autosampler. For all the experiments, a fused-silica capillary (Agilent, model G1600-61232) of 60.5 cm (52 cm effective length), with 50 µm inner diameter and expanded detection window was used. The data were processed using the software Agilent ChemStation®. For the first use, the capillary was pre-treated with a pressure flush with 1 mol/l NaOH solution (30 min). Each day, the capillary was conditioned with NaOH 1 mol/l (5 min), waiting time (1 min), Milli-Q water (5 min) and running buffer (5 min). In between runs, the capillary was flushed with running buffer (2 min). The DAD spectra were acquired at the range of 200–500 nm. A method for the analysis of flavonoids was developed using STB (50 mmol/l; pH 9.5, adjusted with NaOH 1 mol/l), with 20% of MeOH as running buffer. The samples were introduced to the system by hydrodynamic injection (50 mbar/10 s). All separations were performed at a voltage of +25 kV, constant temperature of 30 °C, and direct detection at 390 nm. Apigenin was used as internal standard (I.S.) in order to align the migration time. The alkaloid analysis was performed based on the method previously described by Unger et al. (1997)Unger, M., Stockigt, D., Belder, D., Stockigt, J., 1997. General approach for the analysis of various alkaloid classes using capillary electrophoresis and capillary electrophoresis–mass spectrometry. J. Chromatogr. A 767, 263-276.. Briefly, AcNH4 (100 mmol/l; pH 4.0, adjusted with acetic acid), with 50% ACN was used as running buffer. Hydrodynamic injection (50 mbar/5 s) was used, with separation voltage of +15 kV, constant temperature of 15 °C, and detection at 245 nm.
Experimental determination of the sensitivity of the methods for alkaloid analysis by UPLC and CE
The limits of quantification (LOQ) and detection (LOD) for the analytical methods used in the analysis of alkaloids were experimentally determined using standard solutions from the harmane alkaloid, prepared in different concentrations in the matrix (samples), in the range of 50–0.0187 µg/ml. All solutions were prepared and analyzed in triplicate by both techniques. Linearity was determined using nine-point and five-point regression curves, for UPLC and CE, respectively. LOQ was defined by signal:noise ratio of 10:1 and also by relative standard deviation (RSD > 5%). LOD was defined by a signal:noise ratio of 3:1 (ICH, 2005ICH, 2005. Validation of Analytical Procedures: Text and Methodology – Q2(R1). International Conference on Harmonization. ICH, London.).
Results and discussion
Plant extracts are often complex mixtures whose therapeutic effect cannot always attributed to a single component, and sometimes, the components responsible for a particular effect are not known. As not all the components have reference standards for identification and quantitation, some quality control analyses may be performed by a fingerprint analysis, in which the experimental data from the chemical analysis of different extracts is compared without accurate quantification. This method is accepted by the World Health Organization for the quality control of herbal medicines (WHO, 1991WHO, 1991. Guidelines for the Assessment of Herbal Medicines. World Health Organization, Geneva, Munich.).
C-glycosylflavonoid analysis
For the development of the chromatographic methods, seven authentic samples of C-glycosylflavonoids were used as reference compounds. After evaluating several parameters, including the distinct chromatographic systems described in literature, the initial conditions used were determined as those that allowed good resolution between all reference compounds, and enabled the differentiation of the extracts analyzed, according to the flavonoid profile. The analytical parameters were then optimized, based on visualization of the maximum number of peaks, their resolution index, the time required for the analysis, and the simplicity of the method. The flavonoid fingerprints obtained for each species in the final selected method are presented in Fig. 1, and the peak assignments for each extract are described in Table 2.
HPLC chromatograms of flavonoids standards (upper chromatogram; normalized view) and aqueous extracts of the leaves of Passiflora species. 1: isoorientin, 2: orientin, 3: vitexin-2″-O-rhamnoside, 4: vitexin-2″-O-xyloside, 5: vitexin, 6: isovitexin, 7: swertisin, and 8: 4'-methoxyluteolin-8-C-6″-acetylglucopyranoside. *A: orientin-2″-O-glucoside, *B: orientin-2″-O-xyloside, *C: vitexin-2″-O-glucoside (identified by Gazola, 2014Gazola, A.C., (Ph.D. thesis) 2014. Avaliação química e neurofarmacológica de espécies de Passiflora da América do Sul. Florianópolis. Universidade Federal de Santa Catarina, Brazil, pp. 252.), *D: isoorientin-2″-O-rhamnoside, *E: isovitexin-2″-O-rhamnoside, *F: luteolin-6-C-α-L-rhamnopyranosyl-(1→2)-(6″-O-acetyl)-β-D-glucopiranoside, and *G: apigenin-6-C-α-L-rhamnopyranosyl-(1→2)-(6″-O-acetyl)-β-D-glucopiranoside (identified by Costa et al., 2015Costa, G.M., Cárdenas, P.A., Gazola, A.C., Aragón, D.M., Castellanos, L., Reginatto, F.H., Ramos, F.A., Schenkel, E.P., 2015. Isolation of C-glycosylflavonoids with α-glucosidase inhibitory activity from Passiflora bogotensis Benth by gradient high-speed counter-current chromatography. J. Chromatogr. B 990, 104-110.). For details of the chromatographic method, see the 'Materials and methods' section.
Among the four analyzed species, P. alata and P. quadrangularis presented the least complex flavonoid profile. It is notable that the major flavonoids of both species have almost the same retention times. This observation has previously been described by our research group, the two distinct major compounds being identified as vitexin-2″-O-rhamnoside (3) (P. alata) and vitexin-2″-O-xyloside (4) (P. quadrangularis), proposed as chemical markers for these species (Costa et al., 2013Costa, G.M., Gazola, A.C., Madóglio, F.A., Zucolotto, S.M., Reginatto, F.H., Castellanos, L., Duque, C., Ramos, F.A., Schenkel, E.P., 2013. Vitexin derivatives as chemical markers in the differentiation of the closely related species Passiflora alata Curtis. and Passiflora quadrangularis Linn. J. Liq. Chromatogr. R. T. 36, 1697-1707.). Additionally, orientin (2) and isovitexin (6) were identified in P. alata, while vitexin (5) was observed only in P. quadrangularis. Other C-glycosylflavonoids from P. quadrangularis, which have been isolated and identified (Gazola, 2014Gazola, A.C., (Ph.D. thesis) 2014. Avaliação química e neurofarmacológica de espécies de Passiflora da América do Sul. Florianópolis. Universidade Federal de Santa Catarina, Brazil, pp. 252.), were also assigned (Fig. 1, peaks *A–*C). Some of these compounds (orientin-2″-O-xyloside, vitexin-2″-O-glucoside, vitexin-2″-O-xyloside) have also been described by Sakalem and co-workers (2012)Sakalem, M.E., Negri, G., Tabach, R., 2012. Chemical composition of hydroethanolic extracts from five species of the Passiflora genus. Rev. Bras. Farmacogn. 22, 1219-1232..
A higher accumulation of flavonoids was observed in the extract of P. bogotensis, although only two flavonoids could be identified by spiking experiments with commercial standards (isoorientin (1) and isovitexin (6)). Recently, work on the flavonoid composition of P. bogotensis leaves has reported the presence of these compounds, together with other C-glycosylflavonoids, indicated in Fig. 1 (peaks *D–*G) (Costa et al., 2015Costa, G.M., Cárdenas, P.A., Gazola, A.C., Aragón, D.M., Castellanos, L., Reginatto, F.H., Ramos, F.A., Schenkel, E.P., 2015. Isolation of C-glycosylflavonoids with α-glucosidase inhibitory activity from Passiflora bogotensis Benth by gradient high-speed counter-current chromatography. J. Chromatogr. B 990, 104-110.).
Among the extracts analyzed, P. tripartita var mollissima displayed the most complex flavonoid profile. Isoorientin, orientin, vitexin, swertisin and 4'-methoxyluteolin-8-C-6″-acetylglucopyranoside could be identified by co-injection and UV spectra. In a previous work, the same authors have evaluated the C-glycosylflavonoid profile of P. tripartita var. mollissima leaves and pericarp by a distinct HPLC method (Zucolotto et al., 2012Zucolotto, S.M., Fagundes, C., Reginatto, F.H., Ramos, F.A., Castellanos, L., Duque, C., Schenkel, E.P., 2012. Analysis of C-glycosyl flavonoids from South American Passiflora species by HPLC–DAD and HPLC–MS. Phytochem. Anal. 23, 232-239.). Although both are suitable for their purposes, the method described herein allows the great diversity of flavonoids to be better observed, with an analysis time of only 20 min. Simirgiotis and co-workers (2013)Simirgiotis, M.J., Schmeda-Hirschmann, G., Bórquez, J., Kennelly, E.J., 2013. The Passiflora tripartita (banana passion) fruit: a source of bioactive flavonoid C-glycosides isolated by HSCCC and characterized by HPLC–DAD–ESI/MS/MS. Molecules 18, 1672-1692. have studied the peel and fruit juice of this species, which although different parts of the plant, presented several flavonoids that were structurally related to those observed in our work.
The parameters developed for the HPLC analysis of the reference compounds (authentic samples of C-glycosylflavonoids) and the extracts (Fig. 1), were subsequently used in the UPLC analysis, yielding similar results (Fig. 2). Although the HPLC analysis allowed rapid analysis with an adequate resolution between peaks, comparatively, the analysis time by UPLC was reduced by 50% (10 min), with virtually unchanged fingerprints. As for the HPLC analysis, the UPLC analysis also enabled us to differentiate the four extracts, especially in the difficult distinction of the mayor flavonoids from P. alata and P. quadrangularis.
UPLC chromatogram of flavonoids from aqueous extracts of the leaves of Passiflora species. For details of the chromatographic method, see the 'Experimental' section.
In addition to the analysis by chromatographic methods, the flavonoid composition was also analyzed by capillary electrophoretic (CE). The literature mostly reports the use of borate buffers as running electrolytes in the analysis of flavonoids by CE (Molnár-Perl and Füzfai, 2005Molnár-Perl, I., Füzfai, Z.S., 2005. Chromatographic, capillary eletrophoretic and capillary eletrochromatographic techniques in the analysis of flavonoids. J. Chromatogr. A 1073, 201-227.; Marchart et al., 2003Marchart, E., Krenn, L., Kopp, B., 2003. Quantification of the flavonoid glycosides in Passiflora incarnata by capillary electrophoresis. Planta Med. 69, 452-456.; Rijke et al., 2006Rijke, E., Out, P., Niessen, W.M.A., Ariese, F., Gooijer, C., Brinkman, U.A.T., 2006. Analytical separation and detection methods for flavonoids. J. Chromatogr. A 1112, 31-63.). Considering that the previously chromatographic analyses have revealed a higher complexity of flavonoids in P. tripartita var. mollissima, the CE method was initially developed for this extract. Some parameters were evaluated for the change in electrosmotic flow, such as the electrolyte concentration, voltage, capillary temperature and injection volume. The conditions that provide the best separation for P. tripartita var. mollissima (see 'Materials and methods' section) were applied to the other extracts (Fig. 3), enabling to distinguish the flavonoid fingerprints for each species.
CE electropherogram of flavonoids from aqueous extracts of the leaves of Passiflora species. Internal standard (I.S.): apigenin. For details of the capillary electrophoretic method, see the 'Materials and methods' section.
Compared with the chromatographic techniques, the profiles obtained by CE showed also a short time analysis, good peak resolution and symmetry. Qualitatively, another substantial difference between the chromatographic and electrophoretic methods is the detection wavelength. In HPLC, flavonoids are usually detected in the range of 330–350 nm, which is the band of maximum absorption for these compounds, providing selectivity to the method. However, the detection in CE was performed at 390 nm, due to the borate-flavonoid complex at pH 9.5, which leads to a bathochromic effect for this band.
Alkaloids analysis
Some previous studies report the presence of alkaloids in P. incarnata and P. edulis (Poethke et al., 1970Poethke, V.W., Schwarz, C., Gerlach, H., 1970. Substances of Passiflora incarnata 1. (Constituents of Passiflora bryonioides). Alkaloids. Planta Med. 18, 303-314.; Lutomski et al., 1975Lutomski, J., Malek, B., Rybacka, L., 1975. Pharmacochemical investigation of the raw materials from Passiflora genus. II. The pharmacochemical estimation of juices from the fruits of Passiflora edulis and Passiflora edulis form flavicarpa. Planta Med. 27, 112-121.). Nevertheless, more recent works, with more sensitive methods, have questioned the presence or levels of these compounds (Speroni and Minghetti, 1988Speroni, E., Minghetti, A., 1988. Neuropharmacological activity of extracts from Passiflora incarnata. Planta Med. 54, 488-491.; Rehwald et al., 1995Rehwald, A., Sticher, O., Meier, B., 1995. Trace analysis of harman alkaloids in Passiflora incarnata by reversed-phase high performance liquid chromatography. Phytochem. Anal. 6, 96-100.; Grice et al., 2001Grice, I.D., Ferreira, L.A., Griffiths, L.R., 2001. Identification and simultaneous analysis of hamane, harmina, harmol, isovitexin, and vitexin in Passiflora incarnata extracts with a novel HPLC method. J. Liq. Chromatogr. R. T. 24, 2513-2523.).
In the alkaloid analysis presented in this study, chromatographic and electrophoretic methods were developed using the standards harmol, harmane and harmine, compounds that have previously been described for P. incarnata and P. edulis (Poethke et al., 1970Poethke, V.W., Schwarz, C., Gerlach, H., 1970. Substances of Passiflora incarnata 1. (Constituents of Passiflora bryonioides). Alkaloids. Planta Med. 18, 303-314.; Lutomski et al., 1975Lutomski, J., Malek, B., Rybacka, L., 1975. Pharmacochemical investigation of the raw materials from Passiflora genus. II. The pharmacochemical estimation of juices from the fruits of Passiflora edulis and Passiflora edulis form flavicarpa. Planta Med. 27, 112-121.). Qualitatively, the presence of these alkaloids was not observed in the crude aqueous extract from the leaves of these species by HPLC, UPLC or CE (Fig. 4). For this reason, a calibration curve for harmane was built in UPLC and CE (the techniques with the fastest methods) to determine the linearity and sensitivity of these methods. The results are presented in Table 3.
Chromatograms (I: HPLC; II: UPLC) and electropherogram (III) of alkaloids standards (A: harmol; B: harmane; C: harmine) and of aqueous extracts of the leaves of Passiflora species. For details of the methods, see the 'Experimental' section.
The quantitative data showed an excellent linear relationship between peak area and concentration (r2 > 0.999) for both techniques. About the sensitivity of these methodologies, it was observed that UPLC presented a sensitivity 15-fold higher then CE in these conditions, which can be mainly explained by the difference of the interparticules spaces in the UPLC column and the internal diameter of the capillary column in CE.
Considering the determined limits for these methods, it can be stated that the aqueous extracts analyzed in this work do not have harmane type alkaloids at levels exceeding 0.0187 µg/ml (=0.0187 ppm). This result is in accordance with some previous quantitative works by HPLC, which also did not detect alkaloids in the Passiflora species investigated, at concentrations higher than 0.1 ppm (Rehwald et al., 1995Rehwald, A., Sticher, O., Meier, B., 1995. Trace analysis of harman alkaloids in Passiflora incarnata by reversed-phase high performance liquid chromatography. Phytochem. Anal. 6, 96-100.). Grice and co-workers (2001)Grice, I.D., Ferreira, L.A., Griffiths, L.R., 2001. Identification and simultaneous analysis of hamane, harmina, harmol, isovitexin, and vitexin in Passiflora incarnata extracts with a novel HPLC method. J. Liq. Chromatogr. R. T. 24, 2513-2523. detected alkaloids in commercial samples of P. incarnata at levels lowers than 0.018 ppm, but using a fluorescence detector instead of a diode array detector.
Nevertheless, the non-detection of alkaloids, despite appearing as a negative result, has great relevance. The aqueous extracts evaluated were prepared according to the use of Passiflora leaves in folk medicine. Thus, it was demonstrated that these compounds are absent in the traditional preparations. However, these data do not rule out the presence of harmane alkaloids in these species, which may be present in other organs of the plant, or even in the leaves, in which a specific alkaloid extractive method could be used for this purpose.
Saponin analysis
Even though many saponins have been described for the genus Passiflora, the occurrence of these metabolites is restricted to just a few species. The presence of saponins in P. alata and P. quadrangularis has already been reported in previous works of our group (Reginatto et al., 2004Reginatto, F.H., Gosmann, G., Shripsema, J., Schenkel, E.P., 2004. Assay of quadranguloside, the major saponins of leaves of Passiflora alata, by HPLC. Phytochem. Anal. 15, 195-197.; Birk et al., 2005Birk, C.D., Provensi, G., Gosmann, G., Reginatto, F.H., Schenkel, E.P., 2005. TLC fingerprint of flavonoids and saponins from Passiflora species. J. Liq. Chromatogr. R. T. 28, 2285-2291.), as has a comparative analysis by HPLC of these two species (Costa et al., 2013Costa, G.M., Gazola, A.C., Madóglio, F.A., Zucolotto, S.M., Reginatto, F.H., Castellanos, L., Duque, C., Ramos, F.A., Schenkel, E.P., 2013. Vitexin derivatives as chemical markers in the differentiation of the closely related species Passiflora alata Curtis. and Passiflora quadrangularis Linn. J. Liq. Chromatogr. R. T. 36, 1697-1707.). The additional results presented herein, using the same chromatographic conditions, indicate that the extracts of P. bogotensis and P. tripartita var. mollissima leaves showed no evidence of these compounds (see supplementary material Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2016.02.005. ).
In conclusion, fast and simple analytical methods for the fingerprinting of flavonoids and alkaloids from P. alata,P. quadrangularis,P. bogotensis and P. tripartita var. mollissima extracts were successfully established by three different techniques, showing good resolution and sensitivity. A wide diversity of flavonoids was observed for these four species, while saponins were accumulated only in P. alata and P. quadrangularis extracts. Alkaloids, whose presence is controversial in previous papers, were not detected by any of the methods used. The analytical methods and techniques reported herein are suitable for quality control analysis of these metabolites in plant samples, and would be of great help in future works with other Passiflora species.
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Ethical disclosuresProtection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).Confidentiality of data. The authors declare that they have followed the protocols of their work center on the publication of patient data.Right to privacy and informed consent. The authors have obtained the written informed consent of the patients or subjects mentioned in the article. The corresponding author is in possession of this document.
Appendix A Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2016.02.005.
Acknowledgements
The authors greatly appreciate the financial support provided by Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación, Francisco José de Caldas, contract No. 0459 – 2013, Red Nacional para la Bioprospección de Frutas Tropicales-RIFRUTBIO. The authors E.P. Schenkel and F.H. Reginatto thank the CNPq-National Council of Scientific and Technology Development (Brazil) for providing the research fellowships.
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Publication Dates
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Publication in this collection
Jul-Aug 2016
History
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Received
17 Nov 2015 -
Accepted
23 Feb 2016