Passiflora

Castellanos, Leonardo, Naranjo-Gaybor, Sandra Judith, Forero, Abel M., Morales, Gustavo, Wilson, Erica Georgina, Ramos, Freddy A. & Choi, Young Hae, 2020, Metabolic fingerprinting of banana passion fruits and its correlation with quorum quenching activity, Phytochemistry (112272) 172, pp. 1-13 : 3-7

2.1. NMR metabolic fingerprint of Passiflora species

Freeze-dried samples of Passiflora leaves were extracted with CH 3 OH- d 4 -KH 2 PO 4 buffer in D 2 O (1:1, v/v), in order to obtain a wide range of metabolites that included sugars, amino acids, saponins and flavonoids, as these have been reported to be the major compounds in Passiflora species. The resulting spectra of the Passiflora extracts were analyzed with the Chenomx™ database, along with our in-house database and literature data. Because of the intense overlapping of proton signals, the identity of the proposed compounds was verified by 2DNMR experiments (J-resolved; 1 H– 1 H-correlated spectroscopy-COSY- and heteronuclear multiple bond correlation -HMBC-). The complete NMR data for the identified compounds is presented in the supplementary information (Supp. Table 1 , and Supp. Fig. 6 –16).

The 1 H-NMR spectra of the Passiflora extracts proved to be very similar. As can be observed with the example of the P. tarminiana extract, the presence of amino acids, carbohydrates, and flavonoids was confirmed ( Fig. 1 and in Figure SI-1 supporting information for the other extracts). The main differences between species were observed in the aromatic region, suggesting that the composition of the flavonoids and other phenolics was distinctive between species ( Fig. 2 ).

The analysis of the extracts revealed the presence of nine organic acids, seven amino acids, GABA, sucrose, glucose, myo-inositol and five other unidentified compounds. Their distribution among the studied species is presented as a barcoding of primary metabolites in Fig. 3 , and shows that the species exhibiting most diversity were P. tarminiana , P. cumbalensis , P. mollissima and P. tripartita (Juss.) Poir (syn. P. tripartita var. Tripartita ), while the least complex were those of P. uribei L. K. Escobar and P. lehmannii Mast. The latter was included in this study as an outlier in order to compare the chemical composition of Passiflora spp. In two different subgenera. The content of sugars, polyhydroxyalcohols and other compounds was found to be very similar for the seven extracts, but the content of organic acids and amino acids did not show a distinct distribution pattern.

The amino acid content of two species of the Passiflora genus has been reported. Twenty one amino acids have been identified in P. incarnata ( Gavasheli et al., 1974) and 17 in P. edulis seeds, an ingredient of “Tainung No. 1”, a passion fruit formulation used in China ( Liu et al., 2008). The presence of γ- aminobutyric acid (GABA) was detected in most of the studied species, including P. mollisima , the species that is approved in Colombia as a mild tranquilizer (Ministerio de la Protección Social de Colombia, 2008) and in P. uribei , that appears to be the most abundant source of this compound among the studied samples. It is thought that GABA, that has also been detected in P. incarnata , might be responsible for the anxiolytic and sedative properties of Passion fruit leaf extracts ( Elsas et al., 2010), though the extent of its pharmacological significance is still unclear ( Elsas et al., 2010; Jawna-Zboi ń ska et al., 2016). Trigonelline, which was identified in all samples as a minor compound has been associated to neuroprotective, antimigraine, sedative, memory-boosting and hypoglycemic activities ( Zhou et al., 2012). All these pharmacological properties have been detected in different Passiflora spp. Extracts and support its traditional medicinal use. The presence of 5-carboxymethyl-2,5-dihydrofuran-2- one was unexpected as this compound has only been previously isolated from an unrelated organism, the marine sponge Xestospongia sp. Collected in the island of Viti Levu ( Fiji). This compound has been reported to possess a mild cytotoxic activity against P388 murine leukemia cells ( Quinoa et al., 1986), and has been identified as a key intermediate in the catechol branch of the β- ketoadipate pathway for the degradation of many arenes by a variety of organisms including microorganisms ( Ribbons and Sutherland, 1994). The microbial origin of this compound can explain the variability in its concentration in some of the examined samples, including those of P. caerulea and P. incarnata acquired in The Netherlands (results not shown). Finally, considering that saponins have been reported as major compounds in other species of the subgenus Passiflora of the Passiflora genus, i.e., P. edulis var flavicarpa (Serie Incarnatae) ( Yoshikawa et al., 2000) , P. alata (Serie Quadrangulares) ( Reginatto et al., 2004) , P. quadrangularis (Serie Quadrangulares) and P. ligularis (Serie Tiliaefoliae) , it is noteworthy that no saponins were detected with these methods in the studied species (unpublished results).

The direct analysis of the content of phenolic compounds in the NMR spectra of the extracts was hindered by the high complexity of the aromatic region, the shifting of 1 H NMR signals and the low concentration of some of these compounds. Thus, the main phenolics, including some C -glycosyl flavonoids and catechins, had to be isolated from the extracts for their identification. Their chemical shifts in CH 3 OH- d 4 in buffer (90 mM KH 2 PO 4 in D 2 O) solvent are presented in Supp. Table 2 .

The signals for a C -neohesperidoside glycosyl were detected in most of the 1 H-NMR spectra of the Passiflora extracts, except in P. mollissima and P. mixta samples that showed a very low amount if any. The identification of the C -neohesperidoside diglycoside was based on the signals for methyl groups at 0.60 ppm that were assigned to its rhamnose methyl protons. This shift is due to the spatial shielding effect of the A-ring of the flavonoid aglycone when the disaccharide moiety is attached at position C-6 or C-8. The rotational barrier around the C - glycosidic linkage also leads to signal doubling in the NMR spectra, as a result of the presence of two main conformers ( Camargo et al., 2012; Larionova et al., 2010).

The LC-MS analysis of flavonoids using MS and MS/MS data proved to be useful for the structural elucidation of both O- glycosides and Cglycoside flavonoids. This technique has been widely used for flavonoid characterization in Passiflora extracts ( Farag et al., 2016; Simirgiotis et al., 2013; Zucolotto et al., 2012) In all the studied samples, the BuOH fractions were analyzed by reversed-phase UHPLC-DAD/ESI-2 QToF-MS. Peaks were identified by comparison of retention times with those of external standards, mass spectra and UV analysis. The presence of O - or C - glycosylation, hexoses, pentoses, and acetyl groups were assigned by the MS/MS data analysis of well-established fragmentation patterns such as [M-162] +/− (hexoses), [M-132] +/− (pentoses), [M-18] +/− and [M-120/90] +/− cross-ring cleavages [(O–C1 and C2–C3)]. or [(O–C1 and C3–C4)] for C -hexosides, [M-90/60] +/− for C -pentosides, and [M-104/74] for C -deoxyhexosides, among other ions, used for flavonoid characterization ( Figueirinha et al., 2008). This MS-based approach is useful for positional isomer identification. For example, the differentiation between luteolin-6- C -glucoside (isoorientin, 14) and luteolin-8- C -glucoside (orientin, 16) is based on the high abundance of the product ion at m/z 429 [M-18-H] − in 6- C -hexoside, which is less intense in 8- C -glucoside ( Farag et al., 2016).

In total, 34 phenolics were identified. Supp Table 2 includes the NMR data ( CH 3 OH -d 4 in buffer 90 mM KH 2 PO 4 in D 2 O) and retention times, Imax, and experimental m/z and MS/MS data obtained by HRMS-ESI(−). Information on NMR data measured in other solvents such as MeOD and DMSO is recorded in Supp. Table 3.

The LC-MS analysis of the BuOH fraction of Passiflora species ( Fig. 4 ) revealed a wide metabolic diversity in some of the species as shown by the profiles of P. tarminiana , P. mixta , P. tripartita and P. mollissima , as well as some less complex profiles such as those of P. uribei and P. lehmannii extracts, in agreement with their NMR profiles. Interestingly, the profiles of P. tripartita and P. mollissima showed some significant differences while the profile of P. mollisima was similar to that of P. tarminiana ( Fig. 4 ).

The flavonoids identified in the studied samples (Supp. Table 2 ) included luteolin-derivatives (10, 14–16, 20, 22–24, 28, 30, 34) apigenin-derivatives (1, 2, 9, 11–13, 17–19, 21, 31) and chrysin (25, 27, 33) aglycones, along with some catechins (3–5, 8) and procyanidins (6, 7). Luteolin derivatives were found to be dominant in P. mollissima , but less abundant in P. uribei and P. mixta . The compound 4′-methoxyluteolin-8- C -6″acetylglucopyranoside (34) described previously by us ( Ramos et al., 2010) has been proposed as a chemical marker for P. mollissima ( Simirgiotis et al., 2013) . However, it was found also in P. mixta , P. tarminiana and P. uribei . But not in P. tripartita .

Apigenin-related flavonoids have been selected as chemical markers for P. alata by the Brazilian Pharmacopoeia ( Farmacopéia, 2010). However, in the studied Passiflora samples, these were detected in all species, except in P. cumbalensis extracts, in which chrysin C -glycosides were found instead as highly abundant compounds. These chrysin derivatives were also found in P. tripartita , and P. mixta extracts, but in small quantities. Chrysin had been previously isolated from P. caerulea and proposed as an anxiolytic compound ( Wolfman et al., 1994). Catechin derivatives were detected in large amounts in P. tarminiana and in the two varieties of P. tripartita . Interestingly, catechins have also been reported to induce anxiolytic activity ( Vignes et al., 2006). However, the biological activity of these particular catechins still has to be determined.

Quorum quenching active butanolic extracts of P. lehmannii and P. uribei yielded two previously unreported flavonoids, 1 and 2 respectively, as the major compounds. The (–)-HRESIMS spectra of flavonoid 1 of the P. lehmannii extract showed an ion at m/z 635.1624 [M-H] - suggesting a molecular formula of C 29 H 32 O 16. The MS/MS spectrum of the parent ion at m/z 635 yielded ions at m/z 473 [M-hexose] - and 413 [M-H-hexose-CH 3 COO] -. The 1 H-NMR spectrum (400 MHz, Methanol-d 4) (Supp Fig 6 ) of this compound showed characteristic signals of apigenin with a monohydroxilated aromatic B ring (δH 8.03, 2H, d, J = 8.4 Hz; δH 7.26, 2H, d, J = 8.4 Hz), a penta-substituted A ring (δH 6.28, 1H, bs) and the characteristic H-3 proton of the C ring (δH 6.68, 1H, bs), along with two β - anomeric protons (δH 5.04, 1H, d, J = 7.4 Hz and 4.99, 1H, d, J = 10.2 Hz). The analysis of the coupling constants showed that both sugar moieties correspond to β- glucose residues. Assignment of the glucose residues was supported by the HMBC correlation from the glucose protons H-1″ (δH 4.96) and H-2″ (δH 4.12), with the aromatic C-8 carbon at δC 104.6, suggesting a C -glycosidic bond in the A ring (Supp Fig 8). A similar analysis showed the HMBC correlation from the anomeric proton H-1‴ (δH 5.03), with the aromatic carbon C-4′ at äC 157.4, suggesting an O -glycosidic bond to the B ring of the flavonoid moiety. The correlation from both H-6″ protons at δH 4.47 and 4.28, to the carbon assigned to the acetate carboxyl at δC 173.1 suggested the presence of an acetyl group on 6″ of the C -glucopyranoside residue ( Fig. 5A ). Thus, compound 1 was identified as the previously undescribed flavonoid apigenin-4′ -O- β -glucopyranosyl,8- C - β -(6″acetyl)-glucopyranoside. The NMR data are summarized in Table 2 .

The (–)-HRESIMS spectra of compound 2 yielded an ion at m/z 739.2091 [M-H] -, corresponding to a possible molecular formula of C 33 H 40 O 19, that together with the ion at m/z 413 [M-hexose-deoxyhexose-H] - obtained with the MS/MS data of the parent ion, suggested the presence of a flavonoid bearing two hexoses and one deoxyhexose residue. The 1 H-NMR data (400 MHz, Methanol- d 4) (Supp Fig 11) for this compound, revealed signals that are characteristic of apigenin showing two main conformers with paired signals at δH 7.97 (d, J = 8.7 Hz) [7.81 (d, J = 8.5 Hz)]; 6.93 (d, J = 8.7 Hz) [6.94 (d, J = 8.5 Hz)]; 6.59 (s) [6.62]; 6.27 (s) [6.25 (s)], together with three anomeric protons (δH 5.15 (d, J = 1.3 Hz) [5.31 (d, J = 1.7 Hz)]; 5.03 (d, J = 9.9 Hz) [5.15 (d, J = 9.8 Hz)]; 4.39 (d, J = 7.7 Hz) [4.27(d, J = 7.9 Hz)] and a highly overlapping region for the carbinolic protons of the three sugar moieties. The presence of a neohesperidoside moiety was determined by the HMBC (Sup Fig. 11) correlation from the α-rhamnopyranosyl anomeric proton at δH 5.15 (d, J = 1.8 Hz) [5.31 (d, J = 1.7) Hz]/ δC 102.0 [101.3] to the C-2” (δC 77.6 [76.7]) of glucopyranoside. The bonding of the neohesperidoside moiety to the carbon C-8 was revealed by the HMBC correlation of the β- anomeric glucoside proton at δH 5.03 (d, J = 9.9 Hz) [5.15 (d, J = 9.8 Hz)]/ δC 73.7 [75.3], and H-2″ glucopyranose proton at δH 4.26 (dd, J = 9.9; 8.5) [4.07 (bt, J = 9,3)] to the C-8 carbon at δC 104.7. The presence of a shielded methyl group at δH 0.73 (d, J = 6.2) [0.88 (d, J = 6.2)] of the rhamnopyranosyl CH 3 -6‴in a C-8 linked neohesperidoside moiety (α-rhamnopyranosyl-(1 → 2)-β- glucopyranoside), due to the strong diamagnetical shift caused by the anisotropic effects of one of the aromatic rings of the apigenin moiety in the preferred conformation of the compound ( Larionova et al., 2010). Finally, the position of glycosylation was determined to be C-4′ by the HMBC correlation between the anomeric proton at δH 4.39 (d, J = 7.44) [4.27 (d, J = 7.9] and C-4′ at δC 162.7 as is shown in Fig. 5B . The complete assignment of NMR signals was done using COSY, HSQC and J -resolved spectra and are summarized in Table 2 (see supporting information). Compound 2 was thus identified as apigenin-4- O -β -glucopyranosyl-8- C- β -neohesperidoside. The presence of this compound in P. coactilis had been proposed by Escobar et al., using enzymatic hydrolysis, TLC co-chromatography, 100 MHz NMR and UV analysis for its identification ( Escobar et al., 1983). We have now completed this identification with complete NMR and MS data.

Figures

Fig. 6. PCA plot: The score plot of the principal component analysis (PCA) of 8 different species of banana passion fruits species shows a separation into four main groups.

Fig. 1. 1H NMR spectrum of Passiflora leave extract showing aliphatic, sugar and aromatic region and some assignments: ascorbic acid (I), proline (II), threonine (III), ethanol (IV), leucine (V) pipecolic acid (VI) and acetic acid (VIII) (A and B). Sugar region ascorbic acid (I) and glucose (X) (B and C). Phenolic region glucose (X), tyrosine (IX), 5-carboxymethyl-2,5-dihydrofuran-2-one (XI) shikimic acid (XII) (D). The whole NMR signals assignation can be consulted at Table 1 supporting information.

Fig. 2. The main differences for Passiflora samples can be observed in the aromatic region, suggesting a different composition of flavonoids and other polyphenolic compounds for each species.

Fig. 3. Barcoding of primary metabolites for banana passion fruit species.

Fig. 4. UHPLC chromatograms (340 nm) of the butanolic extract of Passiflora species. Bold numbers correspond to identified compounds, and numbers in italics to the m/z of unidentified compounds.

Fig. 5. Unreported flavonoids identified as major components from Passiflora lehmannii Apigenin-4′-O-β-glucopyranosyl, 8-C-β-(6″acetyl)-glucopyranoside (1) (A) and Passiflora uribei Apigenin-4-O-β-glucopyranosyl-8-C-β-neohesperidoside (2) (B). Arrows represent key HMBC correlations.