Quantitative analysis of trigonelline in some Annona species by proton NMR spectroscopy

A quantitative 1H NMR method (qHNMR) was used to measure the trigonelline content in the leaves of six species of the Annona genus. The methodology employed compared the intensities of the signals at δ 9.14 (H-2) and δ 0.00, the internal standard TSP-d4. This measuring method was able to establish the concentration of trigonelline in the range from 0.67 to 10.04 mg·g−1 depending on the investigated extract.


Introduction
Betaines are derivatives amino acids with quaternary nitrogen atom which are commonly found in a variety of living organisms, such as animals, plant, fungi, bacteria and algae 1,2 . Physiologically, these substances have osmotic regulatory properties and can act as a methyl group donor 3 . Among these mesoionic-type substances, trigonelline ( Figure 1) is somewhat unique as it displays hypoglycemic, hypocholesterolemic, antitumoral and anti-septic properties 4,5 . Moreover, it is well known that trigonelline plays an important role in the resistance process of plants against several pathogens 6 .
Trigonelline is widely distributed in mainly herbaceous plants of saline dried habitats 7 , and in particular is isolated from leaves of Trigonella foenum graecum 8 . In the studies of Moringa oleifera performed by Mantur and Kamal 9 using HPLC, different contents (3.55, 2.60, 2.15, 1.90 and 1.60 mg·g -1 ) were found in the pods, leaves, roots, stems and flowers, respectively. Many analytical techniques were employed for the characterization and identification of trigonelline 3,9 . Machado et al. 10 observed by NMR spectroscopy a content of 1.20 mg·g -1 of trigonelline in Coffea Arabica roots. NMR in particular, is a non-targeted analytical technique where there is no need for previous separations, derivates and even purification with processes being quick and efficient and capable to simultaneously detect a large amount of metabolites in only one analysis 11 .
The genus Annona has a great commercial importance because it comprises from a large variety of edible fruits. It is noteworthy to mention that the utilization of several species of this genus in popular medicine is due to its diverse pharmacological properties such as its anti-parasitic, anti-ulcer, antidiarrheal and anti-spasmodic amongst other therapeutic actions 12 . This work herein describes the identification and the quantification of trigonelline in the extracts of various Annona species by proton NMR spectroscopy in one and two dimensional experiments.

Results and Discussion
The proton signals of trigonelline were promptly assigned in all spectra mainly due to their special chemical shifts in the 8.00 to 10.00 ppm region. Figure 2 illustrates one of the various proton NMR spectra obtained for the extracts with the appropriate assignments that were supported by COSY experiments that exhibited the correlations between H-5 to H-4 and H-6.
Generally, the methods described in literature for quantitative analysis by NMR were not performed to determine the longitudinal relaxation time of the trigonelline and TSP-d 4 hydrogens. These measurements were crucial to the establishment of the recuperation delay (d1) which were  13 . This delay was set to 5 * longest T1 for the return to the Boltzman equilibrium where all liquid magnetizations were reestablished. Therefore, the usual inversion-recovery experiment was applied to determine the T1 values for the hydrogens involved in the quantification (H-2 of trigonelline and methyl group of TSP-d 4 ) (Figure 3). The T1 values of H-2 and methyl hydrogens were 3.06 and 4.05 s, respectively. Thus the value of the recuperation delay (d1) was set to 21s to ensure complete relaxation. Table 1 contains the results of this quantification and it can be seen that Annona laurifolia has one with the highest amount of trigonelline (i.e. 10.04 mg·g -1 ). This amount of trigonelline can be compared to one of the main natural sources which is Trigonella foenum graecum L., where this metabolite is found at the concentration of 13.3 mg·g -114 .This species may be an alternative source of this compound.
In conclusion, the present investigation involved the identification and quantification of trigonelline in leaves of A. muricata, A. laurifolia, A. dolabripetala, A. sylvatica, A. cherimolia and A. coriacea. The efficacy of NMR spectroscopy as an analytical tool has been, once more demonstrated, with its peculiarities, i.e., non-targeted, non-expensive and non-time consuming methodology.

Experimental Section
Vegetable Material. Six species of Annona were collected and immediately frozen in liquid nitrogen. Botanical identification was performed by João Renato Stehmann of the Departamento de Botânica do Instituto de Ciências Biológicas of Universidade Federal de Minas Gerais.
Sample Generic Preparation. Extracts were prepared according to methodology reported by Kim et al. 15 In the triplicate extractions, 50 mg of leaves were submitted to the extraction by a mixture of methanol-d 4 (0.75 mL) and a buffer solution (0.75 mL) of KH 2 PO 4 in D 2 O (90 mM, pH = 6.0) containing TSP-d 4 0.01%. The extraction was conducted by 1 minute vortex stirring followed by 20 minutes sonication and finally, centrifugation. Next, 0.8 mL of the supernatant was transferred to 5 mm NMR tubes. NMR Experiments. NMR experiments were recorded in a Bruker Avance DRX400 (9.4 Tesla) equipped with an inverse multinuclear 5 mm probehead at 303 K. Firstly, the proton longitudinal relaxation times (T1) were determined for the methyl hydrogens of TSP-d 4 and H-2 of trigonelline by the inversion-recovery method for optimization of the recuperation delay, d1 13 . The 64k data points proton NMR spectra were acquired twice for each sample using 16 ppm sweep width. The pulse program 'zgcppr' was the best choice for the water suppression and the 0.3 Hz line broadening were applied for the Fourier transform. The phase and baseline corrections were performed and the TSP-d 4 signal calibrated at 0.00 ppm. To ensure the H-2 chemical shift assignment 1 H NMR spectra besides homonuclear correlation spectroscopy (COSY) were recorded after addition of standard trigonelline to the selected extracts.