Influence of Harvesting Stage on Phytochemical Composition, Antioxidant, and Antidiabetic Activity of Immature Ceratonia siliqua L. Pulp from Béni Mellal-Khénifra Region, Morocco: In Silico, In Vitro, and In Vivo Approaches.

Abstract

Ceratonia siliqua L. is a medicinal plant that has long been used in traditional Moroccan medicine to treat many diseases. This study aimed to assess the impact of the stages of the immature phase of carob pulp (M1, M2, M3, M4, and M5) on phytochemical composition, antioxidant activity, and antidiabetic activity of Ceratonia siliqua L. The identification of the phenolic profile by HPLC-UV/MS-MS and the study of the antidiabetic effect by in silico, in vitro, and in vivo studies were carried out for extracts with high contents of phenolic compounds from immature wild carob pulp from the communes of Timoulit (TM), Bin Elouidane (AW), and Ouaouizerth (TG) in the province of Azilal in the Béni Mellal-Khénifra region. The results revealed a gradual increase in total sugar content over the pulp's ripening period, reaching a value of 2134 ± 56.23 mg GE/100 g fresh weight (FW) for TG. The three locations showed peak values for total polyphenol content (TPC), total flavonoid content (TFC), and total condensed tannin (TCT) at the M2 stage. AW had the highest concentrations of TPC (3819 ± 226.4 mg GAE/100 g FM), TFC (1034 ± 57.08 mg QE/100 g FM), and TCT (1472 ± 28.46 mg CE/100 g FM). The DPPH assay (7892 ± 296.1 mg TE/100 g FM) and the FRAP assay (278.2 ± 7.85 mg TE/100 g FM) both demonstrated that the TG zone is a highly potent antioxidant zone. In contrast, the AW site exhibited a markedly elevated value of 725.4 ± 103.6 mg TE/100 g FM in the ABTS assay. HPLC-UV-MS/MS analysis showed that the methanolic extracts of immature carob pulp (MEICP) from the three areas contained several different chemical compounds. The most prevalent were 3-O-p-coumaroyl-5-O-caffeoylquinic acid, quercetin 3-methyl ether, gallic acid, and galloylquinic acid. Immature carob pulp extract (ICPE) from AW showed the strongest in vitro inhibition of pancreatic α-amylase (IC₅₀ = 0.405 µg/mL) and TG extracts were most potent against intestinal α-glucosidase (IC₅₀ = 0.063 µg/mL). In vivo, AW, TG, and TM extracts significantly reduced postprandial glycemia in rats, with AW having the greatest effect. These results highlight the antidiabetic potential of ICPE. The 3-O-p-Coumaroyl-5-O-caffeoylquinic acid showed better affinity for α-amylase compared to acarbose and interacted significantly with several amino acid residues of the enzyme. Similarly, this molecule and 3,4-Dicaffeoylquinic acid demonstrated a strong affinity for α-glucosidase, suggesting their potential as natural inhibitors of enzymes involved in carbohydrate metabolism. Most of the compounds are not substrates of P-glycoprotein and exhibited high intestinal absorption. Furthermore, the majority of these compounds did not act as inhibitors or substrates of CYP450 enzymes, reinforcing their suitability for development as oral medications. These results underscore the potential of immature carob pulp as a promising antidiabetic agent.