Phytochemical Analysis and Biological Evaluation of Carob Leaf (Ceratonia siliqua L.) Crude Extracts Using NMR and Mass Spectroscopic Techniques

Abstract

Carob leaves have gained attention for their bioactive properties and traditional medicinal uses, including as treatment for diabetes, digestive disorders, and microbial infections. The aim of this study was to explore the phytochemical composition of carob leaf acetone extracts using advanced spectroscopic techniques. The combined use of heteronuclear nuclear magnetic resonance (NMR) experiments with 1D selective nuclear Overhauser effect spectroscopy (NOESY) offers detailed structural insights and enables the direct identification and quantification of key bioactive constituents in carob leaf extract. In particular, the NMR and mass spectrometry techniques revealed the presence of myricitrin as a predominant flavonoid, as well as a variety of glycosylated derivatives of myricetin and quercetin, in acetone extract. Furthermore, siliquapyranone and related gallotannins are essential constituents of the extract. The potent inhibitory effects of the carob leaf extract on Staphylococcus aureus (MIC = 50 μg mL^-1) and a-glucosidase enzyme (IC₅₀ = 67.5 ± 2.4 μg mL^-1) were also evaluated. Finally, the antibacterial potency of carob leaf constituents were calculated in silico; digalloyl-parasorboside and gallic acid 4-O-glucoside exert a stronger bactericidal activity than the well-known myricitrin and related flavonoids. In summary, our findings provide valuable insights into the bioactive composition and health-promoting properties of carob leaves and highlight their potential for pharmaceutical and nutraceutical applications.

Keywords: NMR spectroscopy; antimicrobial activity; carbohydrate digestive enzyme inhibition; carob leaf; flavonoids; galloyl derivatives; mass spectrometry; myricetin.

Figure 1

(a) 500 MHz ¹H NMR spectrum of the phenol-OH region of 20.14 mg carob leaf acetone extract in 500 μL DMSO-d₆. (b) The same spectrum as in (a) after titration with trifluoroacetic acid solution in DMSO-d₆. NMR spectra were acquired at 295 K and with 64 scans, 1.9 s acquisition time, and a relaxation delay of 4.0 s.

Figure 2

800 MHz ¹H NMR spectrum of the expanded OH(5) region of flavonoids and their integrals relative to that of the major analyte myricetin-3-O-α- rhamnopyranoside (12.700 ppm).

Figure 3

800 MHz ¹H-¹³C HMBC spectrum of the selected region of 20.14 mg carob leaf acetone extract in 500 μL DMSO-d₆. Selected region of 800 MHz ¹H-¹³C HMBC spectrum of 20.14 mg carob leaf acetone extract in 500 μL DMSO-d₆. The double arrows denote the connectivities of -OH(5) and -OH(7) with characteristic carbon atoms of myricetin-3-O-α-l-rhamnopyranoside. The spectrum was acquired at 298 K and with 56 scans and 1024 increments. The total experimental time was 14 h and 30 min.

Figure 4

800 MHz HSQC-TOCSY spectrum of selected region of 20.14 mg carob leaf acetone extract in 500 μL DMSO-d₆. Characteristic connectivities between the methyl carbon and all protons of the α-rhamnopyranoside ring are shown. The spectrum was acquired at 298 K and with 64 scans, 1024 increments, and a mixing time of 100 ms. The total experimental time was 1 day and 7 h.

Figure 5

800 MHz 1D selective NOESY NMR spectrum of 20.14 mg carob leaf acetone extract in 500 μL DMSO-d₆. The irradiated proton of myricetin-3-O-α-l-rhamnopyranoside is denoted with thunder and the protons showing connectivities are circled. The spectrum was acquired at 298 K and with 256 scans and a mixing time of 600 ms. The total experimental time was 30 min.