Anthocyanin biosynthesis is one of the best studied secondary metabolisms. However, related pathways were generally concluded based on anthocyanin components; most studies focused on the backbone forming of anthocyanidins (cyanidin, delphinidin, and pelargonidin) of model or commercial plants, while anthocyanin modification was less discussed, and non-model plants with abundant colorations were less researched either. Ranunculus asiaticus L. has great diversity in flower colorations, not only indicating its value in researching anthocyanin biosynthesis but also implying it is unique in this regard. Based on transcriptome sequencing and gene annotation of three varieties (10 samples) of Ranunculus asiaticus L., 176 unigenes from 151,136 unigenes were identified as involved in anthocyanin biosynthesis, among which, 74 unigenes were related to anthocyanin modification; 61 unigenes were responsible for glycosylation at C3 and C5 with 3-monosaccharides of glucose, 3-biosides of rutinose, sophorose, or sambubiose to form 3Gly-, 3Gly5Gly-, 3Gly3′Gly-, 3Gly2′′Gly-, 3Gly2′′Xly-, 3Gly2′′Rly-glycosylated anthocyanins, etc.; 2 unigenes transferred –CH₃; 11 unigenes of BAHD family catalyzd the aromatic or malonyl acylation at 6′′ / 6′′′′position of 3/5-O-glucoside. Based on gene composition, a putative pathway was established. The pathway was validated by flower colorations, and gene expression patterns where F3H, F3′H, 3GT, 5GT, and FMT2 were highly expressed in varieties colored as lateritious and carmine, while variety with purple flowers had high expression of F3′5′H and 3MAT. In view of anthocyanin biosynthesis pathway of Ranunculus asiaticus L., great diversity in its flower colorations was illustrated via the complete branches (F3H, F3′H and F3′5′H) as well as complete modifications (glycosylation, methylation, and acylation), and besides, via the higher percentage of C3 glycosylation than C5 glycosylation.

The current study was conducted to evaluate the ameliorative and protective potentials of Moringea oleifera leaves ethanolic extract (MOLE) against thioacetamide (TAA) toxicity. A total of 58 male albino rats were randomly assigned into six experimental groups. G1, rats received distilled water. G2, rats were injected with a single dose of TAA (200 mg/kg BW) i.p. G3, rats were given MOLE (300 mg/kg BW) orally for 26 days. G4, rats were injected TAA as in G2 and treated with MOLE as G3. G5, rats were kept for 26 days without treatment then on day 27 injected with TAA as in G2. G6, rats were given MOLE for 26 days then on day 27 injected with TAA. Phytochemical analysis of MOLE indicated the presence of kaempferol, kaempferol malonylglucoside, kaempferol hexoside, kaempferol -3-O-glucoside, kaempferol-3-O-acetyl-glucoside, cyanidin -3-O-hexoside, ellagic acid, quercetin, quercetin-3-O-glucoside, and apigenin glucoside. Intoxication of rats with TAA significantly elevated activities of serum AST, ALT, and ALP; concentrations of malondialdehyde, nitric oxide, and hepatic tissue protein expression of caspase 3 and COX2 with alteration of the histological structures of hepatic tissues, while it decreased serum levels of total protein, albumin, and hepatic tissue contents of reduced glutathione. Also, TAA intoxication resulted in 62.5% mortality in rats of G5. Treatment of TAA intoxicated rats (G4) with MOLE ameliorated the toxic effects of TAA on hepatic tissue structure and function. It decreased serum activities of AST, ALT, and ALP; enhanced hepatic GSH concentration; reduced pathological alterations and lipid peroxidation; and downregulated caspase 3 and COX2 proteins expression in hepatic tissue. In addition, MOLE protected rats of G6 from TAA-induced hepatic tissues injury and dysfunction, and increased survival rate of rats. In conclusion, MOLE had both ameliorating and protecting potentials against TAA-induced rats liver damage through regulation of antioxidant, anti-apoptotic, and inflammatory biomarkers. Graphical abstract