Prenylated Flavonoid Glycosides with PCSK9 mRNA Expression Inhibitory Activity from the Aerial Parts of Epimedium koreanum

Phytochemical investigation on the n-BuOH-soluble fraction of the aerial parts of Epimedium koreanum using the PCSK9 mRNA monitoring assay led to the identification of four previously undescribed acylated flavonoid glycosides and 18 known compounds. The structures of new compounds were elucidated by NMR, MS, and other chemical methods. All isolated compounds were tested for their inhibitory activity against PCSK9 mRNA expression in HepG2 cells. Of the isolates, compounds 6, 7, 10, 15, and 17–22 were found to significantly inhibit PCSK9 mRNA expression. In particular, compound 7 was shown to increase LDLR mRNA expression. Thus, compound 7 may potentially increase LDL uptake and lower cholesterol levels in the blood.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in degrading LDLR via clathrin-dependent endocytosis and preventing LDLR recycling, resultantly decreasing the capacity of LDL uptake into cells [14]. Thus, high expression of PCSK9 is often associated with the incidence of hypercholesterolemia, and inhibition of PCSK9 expression or activity has been suggested as a tool to treat patients with familial hypercholesterolemia [15]. Currently, two antibody drugs are prescribed clinically since 2015 [16].
As part of our ongoing project to discover PCSK9 expression inhibitory compounds from medicinal plants [17][18][19][20], the n-BuOH-soluble fraction of the aerial parts of E. koreanum was selected for further investigation due to its initial PCSK9 mRNA expression inhibitory activity (Supplementary Material Figure S1-1). However, there are no reports regarding PCSK9 inhibitory substances from this plant. Thus, herein, we describe the isolation and identification of four new acylated flavonoid glycosides and 18 known compounds, and their effects on PCSK9 and LDLR mRNA expression in the HepG2 cells.

Bioactivity Evaluation
All isolates  were tested for their PCSK9 and LDLR mRNA expression in the HepG2 cells. As shown in the Figure 3, compounds 6, 7, 10, 15, and 17-22 were found to inhibit PCSK9 mRNA expression significantly while other flavonoid glycosides seemed to be inactive. Of the active compounds, compound 7 (ikarisoside A) also significantly increased LDLR mRNA expression. Thus, it seems that compound 7 may have potential to increase LDL uptake and lower cholesterol levels in the blood.

Discussion and Conclusions
The uptake of LDL-cholesterol into the hepatocytes may control cholesterol levels in the blood; this LDL-cholesterol uptake is mediated by LDLR. Hence, adequate LDLR expression in the cells may clear cholesterol in the blood. PCSK9 facilitates the degradation of LDLR after endocytosis of the PCSK-LDLR complex. Upon endocytosis of LDLR-LDL in the absence of PCSK9, LDLR usually dissociates with LDL in the endosomes and then moves back to the cell surface; meanwhile, in the presence of PCSK9, LDLR is degraded in the lysosomes and, resultantly, less LDLR in the cell surface appear, leading to a decrease in the uptake of LDL into cells [35]. Recently, two antibody drugs which interfere the binding of PCSK9 and LDLR were approved for cholesterol-lowering drugs. However, due to some adverse effects of these antibody drugs, small molecules from synthetic molecules or natural molecules were pursued as PCSK9 inhibitory substances [36]. In particular, small molecules from natural sources were found to participate in inhibiting PCSK9 transcriptional or translational expression, PCSK9 secretion, and interaction of PCSK9 and LDLR [37,38]. In this study, PCSK9 transcriptional expressions by the compounds 6, 7, 10, 15, and 17-22 isolated from E. koreanum were significantly downregulated. Concomitantly, LDLR transcriptional expression was upregulated by ikarisoside A (7). Previously, prenylated flavonoids [20] were able to downregulate PCSK9 expression, but their upregulation of LDLR expression was not documented. As natural compounds with downregulation of PCSK9 expression and upregulation of LDLR expression, α-mangostin [37] and sauchinone [38] were reported and demonstrated an increase in LDL uptake, implying the potential in lowering blood cholesterol. Likewise, ikarisoside A (7) may have the positive potential for a cholesterol-lowering effect. Thus, ikarisoside A (7) may have strong merits for further investigation in vitro and in vivo.

Plant Material
The aerial parts of E. koreanum were purchased from Daerim Pharmaceutical Wholesale Company (Cheongju, Korea) and identified by one of the authors (J. Kim). A voucher specimen (CYWSNUKP-00019) was deposited at the medicinal plant garden in the College of Pharmacy, Seoul National University.

Cell Culture, Drugs and Chemicals
HepG2 (human hepatocellular liver cell line) was obtained from the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea) and grown in Eagle's minimum essential medium (EMEM), supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin sulfate. Cells were incubated in a humidified incubator at 37 • C in a 5% CO 2 atmosphere. EMEM, penicillin, and streptomycin were purchased from HyClone Laboratories (Logan, UT, USA). Oligonucleotide primers for LDLR, PCSK9, and GAPDH were purchased from Bioneer Corp. (Daejeon, Korea). Berberine·HCl was purchased from Chengdu Biopurify Phytochemicals Ltd. (Sichuan, China).

Quantitative Real-Time RT-PCR
Total cellular RNA was isolated using a Trizol RNA extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Total RNA (1 µg) was then converted to cDNA using 200 units of iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) at 25 • C for 5 min and at 46 • C for 20 min. The reaction was stopped by incubating the solution at 95 • C for 1 min, after which 1 µL of cDNA mixture was used for enzymatic amplification. PCR reactions were performed using 4 µL of the cDNA and 6 µL master mix containing iQ SYBR Green Supermix (Bio-Rad), 5 pmol of forward primer, and 5 pmol of reverse primer, in a CFX96 real-time PCR detection system (Bio-Rad). Reaction conditions were 3 min at 95 • C, followed by 40 cycles of 10 s at 95 • C and 30 s at 55 • C. The plate was read subsequently. The fluorescence signal generated with SYBR Green I DNA dye was measured during the annealing step. The specificity of the amplification was confirmed using a melting curve analysis. Data were collected and recorded with CFX Manager Software (Bio-Rad) and expressed as a function of the threshold cycle (CT). The relative quantity of the gene of interest was then normalized to the relative quantity of GAPDH (∆∆CT). The mRNA abundance in the sample was calculated using the 2−(∆∆CT) method. The following specific primer sets were used (5 to 3 ): human GAPDH: GAAG-GTGAAGGTCGGAGTCA (forward), AATGAAGGGGTCATTGATGG (reverse); human LDLR: GTGCTCCTCGTCTTCCTTTG (forward), TAGCTGTAGCCGTCCTGGTT (reverse); human PCSK9: GGTACTGACCCCCAACCTG (forward), CCGAGTGTGCTGACCATACA (reverse). Gene-specific primers were custom-synthesized by Bioneer (Daejeon, Korea).

Statistical Analysis
For multiple comparisons, one-way analysis of variance (ANOVA) was performed followed by Dunnett's t test. Data from experiments are presented as means ± standard error of the mean. The number of independent experiments analyzed is given in the figure captions. P-values of less than 0.05 were regarded as statistically significant.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.