Supporting Information

Unraveling the active hypoglycemic agent trigonelline in Balanites aegyptiaca date fruit using metabolite fingerprinting by NMR

Mohamed A. Farag12*, Andrea Porzel2, Ludger Wessjohann2

1Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, Egypt, Kasr el Aini st. P.B. 11562

2Leibniz Institute of Plant Biochemistry, Dept. Bioorganic Chemistry, Weinberg 3, 06120Halle (Saale), Germany.

*Corresponding author:

NMR quantification

In order to ensure full relaxation of all 1H NMR resonances (including the reference compound), between consecutive scans, a prerequisite for NMR based quantification, a rather large sum of relaxation delay and acquisition time of 23 s was used, as the longest relaxation time was measured to be 4.5 s for the HMDS (reference compound) protons). For the quantification of trigonelline using NMR spectroscopy, the peak area of selected proton signals belonging to the target compound (H-2), and the peak area of the IS (HMDS) were integrated manually for all the samples. The following equation was applied for the calculations.
mT = mass of the target compound in the solution used for 1H NMR measurement [mg]

MT molecular weight of target compound [g/mol]

IT relative integral value of 1H NMR signal of the target compound

ISt relative integral value of 1H NMR signal of the standard compound

xSt number of protons belonging to the 1H NMR signal of the standard compound

xT number of protons belonging to the 1H NMR signalof the target compound

cSt concentration of standard compound in the solution used for 1H NMR measurement [mmol/L]

vSt volume of solution used for 1H NMR measurement [mL]

Fig. 1S

Assignment of trigonelline in the methanol extract of B. aegyptiaca fruit using the 1H-13C correlations observed in the HSQC spectrum. HSQC correlations were traced for the aromatic signals of H-2, H-4, H-5 and H-6 spanning from13C (128-167 ppm) and 1H (8.1-9.2 ppm).

Fig. 2S

Assignment of trigonelline in the methanol extract of B. aegyptiaca fruit using the 1H-13C correlations observed in the HMBC spectrum. HMBC correlations were traced starting for the methyl proton observed at d 1H=4.43 ppm assigned for H-7 in trigonelline.

Fig. 3S

Assignment of trigonelline in the methanol extract of B. aegyptiaca fruit using the 1H-13C correlations observed in the HMBC spectrum. HMBC correlations were traced starting for the methyl proton observed at d 1H=8.062 ppm assigned for H-5 in trigonelline.

Fig. 4S

Comparison of the 1H-NMR spectrum of cultivated Balanites aegyptiaca “date” fruit and Pheonix dactylifera date fruit (Deglet Nour cv.) demonstrating both signal richness and dynamic range from three regions: (0.0- 2.5 ppm) for saponins, (3-5 ppm) for sugars and (7-9 ppm) for aromatics. Note the absence for trigonelline signals in the NMR spectrum of Pheonix dactylifera date fruit (7-9 ppm).