Rare Syringyl Acylated Flavonol Glycosides from the Aerial Parts of Leonurus japonicus Houtt

Five new syringyl acylated flavonol glycosides, named leonurusoides A (1), B (2), C (3), D (4), and E (5), together with one known one 6 were obtained from the aerial parts of Leonurus japonicus. Their structures were elucidated by chemical and spectroscopic methods (UV, IR, HRESI-TOF-MS, 1D and 2D NMR). Compounds 1−6 showed triglyceride (TG) accumulation inhibitory effects in free fatty acid-induced HepG2 cells.

Intracellular excess lipid accumulation (especially in liver and muscle) is a mediator of metabolic syndrome, which comprises a cluster of risk factors such as diabetes, hyperlipidemia, and hypertension. Free fatty acid induced TG accumulation HepG2 cells is commonly used for research on lipid metabolism regulation effects. The TG accumulation inhibitory effects of the isolates 1-6 were tested. All compounds showed inhibitory effects on TG accumulation in free fatty acid induced HepG2 cells (Table 4).

Plant Material
The aerial parts of L. japonicus Houtt were purchased from Anguo Medicinal Market, Hebei Province, China and identified by Dr. Li Tianxiang. The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20110909).

TG Accumulation Inhibitory Effects Assay
The hepatic cell line HepG2 (IBMS, CAMS/PUMC, Beijing, China) were maintained in high glucose Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under a humidified atmosphere of 5% CO 2 in air. After growth to 80% confluence, cells were seeded at 4 × 10 4 cells/mL on 48-well dish. After 24 h incubation, the medium was switched to high glucose MEM and supplemented with 10% FBS and 0.2 mM oleic acid sodium salt, together with sample DMSO solution (final concentration of DMSO was less than 0.1%). After 48 h incubation, the amount of intracellular triglycerides was determined with a Triglycerides kit (BioSino Bio-technology and Science Inc., Beijing, China) after cell lysis.

Statistical Analysis
Values are expressed as mean ± S.D. All the grouped data were statistically performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey's Studentized range test was used for post hoc evaluations. p < 0.05 was considered to indicate statistical significance.

Conclusions
In summary, five new syringyl acylated flavonoid glycosides 15, together with the known compound, 2'''-syringylrutin (6) were obtained from the aerial parts of L. japonicas. Although various acylated flavonol glycosides distribute widely in the plant kingdom [13], syringates such as the isolates 16 are quite rare. Compound 6 and quercetin 3-(3'''-syringylrobinobioside), with a syringyl acylated at positions 2 and 3 of rhamnose, respectively, are the first examples of natural flavonol glycosides from the same genus, Leonurus [6,7], and are only found in the genus. All of the syringyl acylated flavonoid glycosides 16 showed TG accumulation inhibitory effects in free fatty acid-induced HepG2 cells.