Inhibitory Effects of Constituents from the Aerial Parts of Rosmarinus officinalis L. on Triglyceride Accumulation

Sixteen flavonoids (1–16) including two new ones, named officinoflavonosides A (1) and B (2) were obtained from the aerial parts of Rosmarinus officinalis. Among the known ones, 6, 10, and 13 were isolated from the rosmarinus genus for the first time. Their structures were elucidated by chemical and spectroscopic methods. Moreover, the effects on sodium oleate-induced triglyceride accumulation (TG) in HepG2 cells of the above-mentioned compounds and 16 other isolates (17–32) reported previously to have been obtained in the plant were analyzed. Results show that eight kinds of flavonoids (compounds 1, 2, 3, 6–9 and 11) and seven kinds of other known isolates (compounds 17–20, 23, 26 and 31) possessed significant inhibitory effects on intracellular TG content in HepG2 cells. Among them, the activities of compounds 1 and 20 were comparable to that of orlistat, which suggested that these compounds in this plant might be involved in lipid metabolism.


Introduction
Rosmarinus officinalis L., is a perennial edible herb, belonging to Labiatae, commonly known as rosemary. R. officinalis extract has been reported to have antioxidant, anti-inflammatory, antidiabetic and anticancer properties [1]. In the course of our characterization studies on bioactive constituents from the aerial parts of R. officinalis, the isolation and structure elucidation of 16 terpenoids including normonoterpenoid, diterpenoid and triterpenoid glycosides [2], had been reported by us. Their structures were elucidated by chemical and spectroscopic methods. As a continuing study on the plant, we obtained 16 flavonoids including two new ones, named officinoflavonosides A (1) and B (2). In this paper, we describe the isolation and structure elucidation of these new flavanoids, along with the triglyceride (TG) accumulation inhibitory effects of the above-mentioned 45 compounds in HepG2 cells.
Although a limited number of compounds were tested for inhibitory effects on TG accumulation in HepG2 cells, we can summarize the structure-activities relationship as follows: in flavonoids, 5,7-dihydroxyl substitution showed strong activities (compounds 1, 6-9), and the activities would be reduced when7-hydroxyl group was glucosylated (Compound 9 > 10). Among the diterpenes, compound 20 showed strongest inhibitory effect on TG accumulation. Methylation or configuration changing at position 20 would quench the activity, which indicated that 20α-hydroxy is important for inhibitory activity. The intracellular TG content in each well was examined using a TG assay kit. Each value represents the mean ± S.E.M., n = 5, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. model group (Mod.).
Studies of dose dependency of six selected compounds (compounds 1, 2, 9, 20, 26 and 31), which comparatively showed the best TG-lowering activities, were then conducted. In this system, orlistat, a lipase inhibitor showed significant TG accumulation effects at concentration of 5 µmol/L. As shown in Figure 6, the results revealed that all the above isolates could inhibit the SO-induced intracellular TG accumulation in a dose dependent manner. Among them, compound 20 exhibited the strongest activity that it had demonstrated a clearance ratio of more than 5.6% from 1 µmol/L. Then, Oil red O staining further visually confirmed the results that a large amount of lipid droplets were seen in SO-treated HepG2 cells, indicating lipid accumulation, but the accumulation was inhibited by compound 20 in a dose dependent manner (shown in Figure 7). Although the colorimetric assay results showed that the concentration of 1 µmol/L exhibited no significant difference compared to the model group, the possible reasons for this might be the lower precision of this detection method or the activity-instability at this low concentration.
Although a limited number of compounds were tested for inhibitory effects on TG accumulation in HepG2 cells, we can summarize the structure-activities relationship as follows: in flavonoids, 5,7-dihydroxyl substitution showed strong activities (compounds 1, 6-9), and the activities would be reduced when 7-hydroxyl group was glucosylated (Compound 9 > 10). Among the diterpenes, compound 20 showed strongest inhibitory effect on TG accumulation. Methylation or configuration changing at position 20 would quench the activity, which indicated that 20α-hydroxy is important for inhibitory activity.

Plant Material
The dried aerial parts of R. officinalis were collected from Butarie, Rwanda, and identified by Dr. Li Tianxiang (Experiment Teaching Department, Tianjin University of Traditional Chinese Medicine). The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20110910).

Extraction and Isolation
The dried aerial parts of R. officinalis (2.5 kg) were dealt by using the same method as reported before [2,3]. As results, CHCl 3 partition layer (269 g), H 2 O (47 g) and 95% EtOH (45 g) eluted fractions were obtained.

Statistical Analysis
Statistical analysis was performed using one-way ANOVA with the Tukey's test for multiple comparisons. All data were expressed as mean ± S.E.M. and values of p < 0.05 were considered significant.

Conclusions
Taken together, 16 flavonoids (1-16) including two new ones, officinoflavonosides A (1) and B (2), were obtained from the aerial parts of R. officinalis. Among the known ones, 6, 10, and 13 were isolated from the rosmarinus genus for the first time, and the NMR data of 13 and 14 in DMSO-d 6 were reported for the first time. Their structures were elucidated by chemical and spectroscopic methods and their effects on TG accumulation were analyzed in HepG2 cells. The results showed that six kinds of flavonoids (compounds 3, 6, 7, 8, 9 and 11) together with the two novel ones (compounds 1 and 2) and seven kinds of other known isolates (compounds 17, 18, 19, 20, 23, 26 and 31) possessed significant inhibitory activities on TG accumulation in vitro. Among them, the activities of compounds 1 and 20 were comparable to that of orlistat, which suggested that these compounds contained in the R. officinalis might be involved in the lipid metabolism.