Metabolomics-based analysis of flavonoid metabolites in Chinese jujube and sour jujube fruits from different harvest periods.

Chinese jujube (Ziziphus jujuba Mill.) and sour jujube (Ziziphus spinosa Hu.) fruits have health benefits because they contain bioactive compounds such as flavonoids. However, differences in the flavonoid metabolites of these two fruits remain unclear. We determined the flavonoids present in Z. jujuba cv. Hupingzao (HPZ) and Z. spinosa cv. Taigusuanzao (TGSZ) from two different harvest periods fruits: HPZ white period (HW) and HPZ red period (HR) as well as TGSZ white period (SW) and TGSZ red period (SR). We identified 123 flavonoid metabolites: 40 flavonols, 37 flavones, 12 anthocyanins, 9 dihydroflavones, 8 flavanols, 7 flavonoid carbonosides, 5 dihydroflavonols, 3 isoflavones, and 2 chalcones. The total flavonoid content of both HPZ and TGSZ decreased with fruit development and was significantly higher in TGSZ than in HPZ fruits. Moreover, we detected 63, 81, 56, and 63 differential flavonoid metabolites (DFMs) between HW and HR (two upregulated and 61 downregulated), SW and SR (four upregulated and 77 downregulated), HW and SW (54 upregulated and two downregulated), and HR and SR (62 upregulated and one downregulated), respectively. KEGG pathway annotation and enrichment analysis showed that 22 DFMs were annotated seven relevant metabolic pathways, among which flavonoid biosynthesis pathway and secondary metabolites biosynthesis pathway were the main pathways, and flavanols were the primary metabolites that influenced the difference in flavonoid accumulation between the fruits. To our knowledge, this is the first study to reveal the differences in flavonoid metabolism between Chinese jujube and sour jujube. Our findings may facilitate the comprehensive use of functional flavonoids. PRACTICAL APPLICATION: Chinese jujube (Ziziphus jujuba Mill.) and sour jujube (Ziziphus spinosa Hu.) fruits have health benefits because they contain bioactive compounds such as flavonoids. However, differences in the flavonoid metabolites of these two fruits remain unclear. We determined the flavonoids present in Z. jujuba cv. Hupingzao (HPZ) and Z. spinosa cv. Taigusuanzao (TGSZ) from two different harvest periods. Our findings may facilitate the comprehensive use and product research of functional flavonoids of jujube.

Emodin-8-O-ß-D-glucopyranoside, a natural hydroxyanthraquinone glycoside from plant, suppresses cancer cell proliferation via p21-CDKs-Rb axis.

Emodin-8-O-ß-D-glucopyranoside (EG), a natural hydroxyanthraquinone glycoside from some traditional medicinal plants, has been demonstrated to have potential antitumor effects in our previous studies. Herein, we confirm that EG remains stable in the cell culture medium. It suppresses cell viability and proliferation and induces G1 cell cycle arrest in human colorectal cancer and neuroblastoma cells in vitro. EG inhibits tumor growth in human colorectal cancer cell HCT 116-bearing xenograft mice with low toxicity in the liver and kidney. The transcriptome analysis shows that the p53 signaling pathway is the most enriched cellular pathway and EG affects the proliferation of HCT 116 cells through modulating cell cycle related genes, such as CDKN1A and Cyclin-dependent kinases (CDKs). We demonstrate that the protein expression level of p21 was up-regulated, and CDK1/CDK2 were reduced significantly in both HCT 116 and SH-SY5Y cells after EG treatment. The switch from hypo- to hyper-phosphorylated Retinoblastoma (Rb), which is believed as a result of activated CDKs, was inhibited when cells were treated with EG. These findings indicate that EG suppresses cancer cell proliferation via p21-CDKs-Rb axis.

Inhibition of CYP3A4 enhances aloe-emodin induced hepatocyte injury.

Aloe-emodin (AE) is a natural hydroxyanthraquinone derivative that was found in many medicinal plants and ethnic medicines. AE showed a wide array of pharmacological activities including anticancer, antifungal, laxative, antiviral, and antibacterial effects. However, increasing number of published studies have shown that AE may have some hepatotoxicity effects but the mechanism is not fully understood. Studies have shown that the liver injury induced by some free hydroxyanthraquinone compounds is associated with the inhibition of some metabolic enzymes. In this study, the CYP3A4 and CYP3A1 were found to be the main metabolic enzymes of AE in human and rat liver microsomes respectively. And AE was metabolized by liver microsomes to produce hydroxyl metabolites and rhein. When CYP3A4 was knocked down in L02 and HepaRG cells, the cytotoxicity of AE was increased significantly. Furthermore, AE increased the rates of apoptosis of L02 and HepaRG cells, accompanied by Ca2+ elevation, mitochondrial membrane potential (MMP) loss and reactive oxygen species (ROS) overproduction. The mRNA expression of heme oxygenase-1 in L02 and HepaRG cells increased significantly in the high-dose of AE (40 µmol/L) group, and the mRNA expression of quinone oxidoreductase-1 was activated by AE in all concentrations. Taken together, the inhibition of CYP3A4 enhances the hepatocyte injury of AE. AE can induce mitochondrial injury and the imbalance of oxidative stress of hepatocytes, which results in hepatocyte apoptosis.

Composition and content of phenolic acids and flavonoids among the different varieties, development stages, and tissues of Chinese Jujube (Ziziphus jujuba Mill.).

The composition and content of phenolic acids and flavonoids among the different varieties, development stages, and tissues of Chinese jujube (Ziziphus jujuba Mill.) were systematically examined using ultra-high-performance liquid chromatography to provide a reference for the evaluation and selection of high-value resources. Five key results were identified: (1) Overall, 13 different phenolic acids and flavonoids were detected from among the 20 excellent jujube varieties tested, of which 12 were from the fruits, 11 from the leaves, and 10 from the stems. Seven phenolic acids and flavonoids, including (+)-catechin, rutin, quercetin, luteolin, spinosin, gallic acid, and chlorogenic acid, were detected in all tissues. (2) The total and individual phenolic acids and flavonoids contents significantly decreased during fruit development in Ziziphus jujuba cv.Hupingzao. (3) The total phenolic acids and flavonoids content was the highest in the leaves of Ziziphus jujuba cv.Hupingzao, followed by the stems and fruits with significant differences among the content of these tissues. The main composition of the tissues also differed, with quercetin and rutin present in the leaves; (+)-catechin and rutin in the stems; and (+)-catechin, epicatechin, and rutin in the fruits. (4) The total content of phenolic acid and flavonoid ranged from 359.38 to 1041.33 µg/g FW across all examined varieties, with Ziziphus jujuba cv.Jishanbanzao having the highest content, and (+)-catechin as the main composition in all 20 varieties, followed by epicatechin, rutin, and quercetin. (5) Principal component analysis showed that (+)-catechin, epicatechin, gallic acid, and rutin contributed to the first two principal components for each variety. Together, these findings will assist with varietal selection when developing phenolic acids and f lavonoids functional products.

Exploration of components and mechanisms of Polygoni Multiflori Radix-induced hepatotoxicity using siRNA -mediated CYP3A4 or UGT1A1 knockdown liver cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygoni Multiflori Radix, the dried root of Polygonum multiflorum Thunb., and its processed products have been used as restoratives for centuries in China. However, the reports of Polygoni Multiflori Radix-induced liver injury (PMR-ILI) have received wide attention in recent years, and the components and mechanism of PMR-ILI are not completely clear yet. Our previous studies found that the PMR-ILI was related to the down-regulation of some drug metabolism enzymes (DME). AIM OF THE STUDY: To explore the effect of the inhibition of CYP3A4 or UGT1A1 on PMR-ILI, screen the relevant hepatotoxic components and unveil its mechanism. METHODS: RT-qPCR was used to detect the effects of water extract of Polygoni Multiflori Radix (PMR) and its main components on the mRNA expression of CYP3A4 and UGT1A1 in human hepatic parenchyma cell line L02. High-performance liquid chromatography (HPLC) was employed to detect the content of major components in the PMR. And then, the stable CYP3A4 or UGT1A1 knockdown cells were generated using short hairpin RNAs (shRNA) in L02 and HepaRG cells. Hepatotoxic components were identified by cell viability assay when PMR and its four representative components, 2,3,5,4'-tetrahydroxy stilbene glycoside (TSG), emodin (EM), emodin-8-O-ß-D-glucoside (EG), and gallic acid (GA), acted on CYP3A4 or UGT1A1 knockdown cell lines. The PMR-ILI mechanism of oxidative stress injury and apoptosis in L02 and HepaRG cells were detected by flow cytometry. Finally, the network toxicology prediction analysis was employed to excavate the targets of its possible toxic components and the influence on the metabolic pathway. RESULTS: PMR and EM significantly inhibited the mRNA expression of CYP3A4 and UGT1A1 in L02 cells, while TSG and GA activated the mRNA expression of CYP3A4 and UGT1A1, and EG activated CYP3A4 expression while inhibited UGT1A1 expression. The contents of TSG, EG, EM and GA were 34.93 mg/g, 1.39 mg/g, 0.43 mg/g and 0.44 mg/g, respectively. The CYP3A4 or UGT1A1 knockdown cells were successfully constructed in both L02 and HepaRG cells. Low expression of CYP3A4 or UGT1A1 increased PMR cytotoxicity remarkably. Same as PMR, the toxicity of EM and GA increased in shCYP3A4 and shUGT1A1 cells, which suggested EM and GA may be the main components of hepatotoxicity in PMR. Besides, EM not only inhibited the expression of metabolic enzymes but also reduced the cytotoxicity threshold. EM and GA affected the level of ROS, mitochondrial membrane potential, Ca2+ concentration, and dose-dependent induced hepatocyte apoptosis in L02 and HepaRG cells. The network toxicology analysis showed that PMR-ILI was related to drug metabolism-cytochrome P450, glutathione metabolism, and steroid hormone biosynthesis. CONCLUSION: The inhibition of mRNA expression of CYP3A4 or UGT1A1 enhanced hepatotoxicity of PMR. EM and GA, especially EM, may be the main hepatotoxic components in PMR. The mechanism of PMR, EM and GA induced hepatotoxicity was proved to be related to elevated levels of ROS, mitochondrial membrane potential, Ca2+ concentration, and induction of apoptosis in liver cells.

Genomic analyses of diverse wild and cultivated accessions provide insights into the evolutionary history of jujube.

The Chinese jujube (Ziziphus jujuba Mill.), a member of the Rhamnaceae family, is an important perennial fruit tree crop of substantial economic, ecological and nutritional value, and is also used as a traditional herbal medicine. Here, we report the resequencing of 493 jujube accessions, including 202 wild and 291 cultivated accessions at >16× depth. Our population genomic analyses revealed that the Shanxi-Shaanxi area of China was jujube's primary domestication centre and that jujube was then disseminated into East China before finally extending into South China. Divergence events analysis indicated that Ziziphus acidojujuba and Ziziphus jujuba diverged around 2.7 Mya, suggesting the interesting possibility that a long pre-domestication period may have occurred prior to human intervention. Using the large genetic polymorphism data set, we identified a 15-bp tandem insertion in the promoter of the jujube ortholog of the POLLEN DEFECTIVE IN GUIDANCE 1 (POD1) gene, which was strongly associated with seed-setting rate. Integrating genome-wide association study (GWAS), transcriptome data, expression analysis and transgenic validation in tomato, we identified a DA3/UBIQUITIN-SPECIFIC PROTEASE 14 (UBP14) ortholog, which negatively regulate fruit weight in jujube. We also identified candidate genes, which have likely influenced the selection of fruit sweetness and crispness texture traits among fresh and dry jujubes. Our study not only illuminates the genetic basis of jujube evolution and domestication and provides a deep and rich genomic resource to facilitate both crop improvement and hypothesis-driven basic research, but also identifies multiple agriculturally important genes for this unique perennial tree fruit species.

Hepatotoxicity in Rats Induced by Aqueous Extract of Polygoni Multiflori Radix, Root of Polygonum multiflorum Related to the Activity Inhibition of CYP1A2 or CYP2E1.

The objective of this study is to investigate the relationship between the hepatotoxicity induced by Polygoni Multiflori Radix (PMR, root of Polygonum multiflorum Thunb., He Shou Wu) and the activity of CYP1A2 or CYP2E1 in the rat liver. Levels of rat serum transaminases ALT and AST were not altered but the activity of CYP1A2 or CYP2E1 in the rat liver was significantly inhibited after oral administration of aqueous extract of PMR under the experimental dosage. However, levels of ALT and AST were significantly increased and the activity of CYP1A2 or CYP2E1 was significantly decreased after injection of specific inhibitor for CYP1A2 or CYP2E1 combined with oral administration of aqueous extract of PMR, especially under the repeated treatment over interval times. Liver histopathological observation showed that a moderate liver injury occurred in rats receiving PMR treatment with the activity of CYP1A2 or CYP2E1 inhibited, but there was no significant liver damage in rats receiving PMR treatment or CYP inhibitor alone. These suggested that low level activity of CYP1A2 or CYP2E1 from genetic polymorphism among people might be one of the important reasons for the hepatotoxicity induced by PMR in clinical practice.

[Study on inhibitory effect of water extract of Polygonum multiflorum on CYP1A2 and CYP2E1 enzymatic activities and mRNA expressions in rat liver].

Rats were continuously given different doses of water extract of Polygonum multiflorum (1, 10 g x kg(-1)) for 7 days to prepare liver microsomes. Cocktail in vitro incubation approach and Real-time quantitative PCR technology were used to observe the effect of water extract of P. multiflorum on CYP450 enzymatic activities and mRNA expressions in rat liver. Compared with the blank control group, both 1, 10 g x kg(-1) water extract of P. multiflorum treated groups showed significant inhibitions in CYP2E1 enzymatic activities and mRNA expressions (enzymatic activities of CYP2E1, P < 0.01; mRNA expression of CYP2E1, P < 0.05 in 1 g x kg(-1) group, P < 0.01 in 10 g x kg(-1) group). They revealed a significant increase in the enzymatic activity of CYP3A1 (P < 0.01), but without significant change in mRNA expressions. The 10 g x kg(-1) group showed a significant inhibition in CYP1A2 enzymatic activities and mRNA expressions in rat livers (P < 0.01).