Chemical Constituents with Inhibitory Activities on Proprotein Convertase Subtilisin/kexin Type 9 Expression from the Aerial Parts of Penthorum chinense

Background: Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, and jaundice. So far there is no report regarding proprotein convertase subtilisin/kexin type 9 inhibitory constituents from this plant. The aim of the present study was to discover new active constituents with PCSK9 expression inhibitory activities from P. chinense Methods: All structures were established by interpreting NMR spectroscopic data and MS data. Further experimental and calculated ECD data were used to determine the absolute conguration of the two new neolignans. To monitor the inhibitory activity on proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression and PCSK9-low density lipoprotein receptor (LDLR) interaction, quantitative real time-PCR, Western blot analysis, and an enzyme-linked immunosorbent assay (ELISA) method by a PCSK9-biotinylated-LDLR binding assay were performed. Results: 39 compounds were isolated and identied including two new oxepine-type neolignans, penthorinols A (1) and B (2) and a naturally occurring chalcone, 6'-hydroxy-2'-methoxychalcone-4'-O-β-D-glucopyranoside (20). Of all tested compounds, penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC 50 values of 15.56 and 11.66 µM, respectively. Furthermore, penthorin A (4) and methyl gallate (25) downregulated PCSK9 protein expression. However, all compounds seemed to be inactive in PCSK9-LDLR interaction. Conclusion: In the present study, two new compounds was discovered from this plant and active constituents with PCSK9 expression inhibitory activities were suggested. P. Penthorum chinense; PCSK9, Proprotein convertase subtilisin-kexin type 9; LDL, low-density lipoprotein; HPLC, high-performance liquid chromatography; MPLC, medium-performance liquid chromatography; MeCN, acetonitrate; ECD, electronic circular dichroism; UV, ultraviolet visible; Ber, Berberine.


Extraction and isolation
The dried aerial parts of P. chinense (1.2 kg) were extracted with 80% MeOH (10L for 2 h, 3times) by sonication. The total extract was condensed in vaccuo to obtain a residue (360 g). This residue was suspended in water and partitioned with n-hexane (4.1 g), n-BuOH (48.22 g) and water. The n-BuOH fraction (48. 22  PC-2 (15.3 g) was chromatographed by a MPLC with a reversed-phase (RP)-C 18  PC-4 (12.53 g) was fractionated by a MPLC with a reversed-phase (RP)-C 18

Calculated ECD prediction
The structures (1 and 2) were generated using ChemBio3D Ultra 13.03 and then summited in Spartan 16 program searched for conformational using with MMFF94 force led minimization. All 13 conformers which had Boltzmann-averaged weight less than 0.95 were selected and optimized using theoretical method of density functional theory (B3LYP) and a basis set of 6-31 + G (d,p) in Gaussian 16 software (Gaussian Inc., Wallingford, CT, USA) in gas phase. Finally, their calculated ECD was carried out using TDDFT with the CAM-B3LYP/6-31 + G (d,p) method and the methanol-selected CPCM in Gaussian 16 software [15,16]. Lastly, the calculated ECD curves of possible structures (1a and 1b) were compared with the experimental ECD curves (Fig. S23 (A) and 23 (B), Supporting Information). The ECD spectra were plotted using SpecDis v. 1.71 software and applying a Gaussian band shape with a sigma/gamma value of 0.20 eV to simulate the experimental curve ( Fig. S23 (C), Supporting Information). The predicted ECD spectrum was obtained using a Boltzmann population-weighted average and was plotted with The plate was subsequently read. The uorescence signal generated with SYBR Green I DNA dye was measured during the annealing steps. The speci city of the ampli cation was con rmed using a melting curve analysis. Data were collected and recorded by CFX Manager Software (Bio-Rad) and expressed as a function of the threshold cycle (C T ). The relative quantity of the gene of interest was then normalized to the relative quantity of GAPDH (ΔΔCT). The mRNA abundance in the sample was calculated using the Eq. 2 -(ΔΔCT) . The following speci c primer sets were used (5′ to 3′): human -GAPDH: GAAGGTGAAGGTCGGAGTCA (forward), AATGAAGGGGTCATTGATGG (reverse); human -PCSK9: GGTACTGACCCCCAACCTG (forward), CCGAGTGTGCTGACCATACA (reverse); Gene-speci c primers were custom-synthesized by Bioneer.

PCSK9-LDLR binding assay
All the isolated compounds were evaluated using an enzyme-linked immunosorbent assay (ELISA) method by a PCSK9-biotinylated-LDLR binding assay kit (BPS Bioscience, Inc., San Diego, USA; cat no. #72002) according to the manufacturer's protocol. A positive control, alirocumab (TAB-719) was purchased from Creative Biolabs (Shirley, NY, USA).

Statistical Analysis
For multiple comparisons, one-way analysis of variance (ANOVA) was performed followed by Dunnett's ttest. Data from experiments are presented as means ± standard error of the mean (SEM). The number of independent experiments analyzed is given in the gure captions.

PCSK9 inhibitory activities of isolated compounds
All the compounds 1-39 were tested in PCSK9 mRNA expression using HepG2 cell (Fig. 4). Of the isolates, penthorin A (4) and methyl gallate (25) were found to signi cantly inhibit PCSK9 mRNA expression with IC 50 values of 15.56 and 11.66 µM (Fig. 5A), respectively, in assay system (IC 50 value of positive control, berberine, 9.84 µM) while other compounds deemed inactive. In further Western blotting analysis, penthorin A (4) and methyl gallate (25) demonstrated to inhibit PCSK9 expression in HepG2 cells when compared with a positive control (berberine). In addition, all isolated compounds (1-39) were evaluated for their inhibitory activities against PCSK9-LDLR interaction. As shown in Fig. S31, all compounds were not signi cantly to inhibit PCSK9-LDLR interaction at a concentration of 50 µM.

Discussion
Phytochemical investigations on P. chinense have been widely documented and reviewed elsewhere [4,5,7,8,35]. The oxepine-type lignans including two new compounds 1 and 2 were isolated from this plants.
Of the known structures, compound 20, 6'-hydroxy-2'-methoxychalcone-4'-O-β-D-glucopyranoside, previously this compound was produced by microbial glycosylation [30] and as a naturally occurring molecule, the current study was the rst report to isolate and identify this compound from natural sources. The structure of compound 20 was con rmed by interpreting 1D and 2D NMR spectroscopic data, and analysis of acidic hydrolysates, which was compared with the published values [30]. In the present study, three new naturally occurring compounds were identi ed from this plant for the rst time.
PCSK9 is a gene that is involved in lipid metabolism and atherosclerosis [40]. In particular, PCSK9 protein binds to LDLR and then facilitates the degradtion of LDLR, leading to blocking the recycle of LDLR and consequently inhibiting LDL uptake from blood into hepatocyte. For this function of PCSK9, PCSK9 inhibitors are emerging as an additional lipid-lowering therapy for patients with artery disease [41].
Interestingly, a few statin drugs including atorvastatin are known to induce transcriptional expression of PCSK9, which may explain of the limitation of statin treatment in some patients [42,43]. So far, as PCSK9 inhibitor drugs, antibody drugs which inhibit PCSK9-LDLR binding have been launched while natural product-derived small molecules or synthetic small molecules are underway [44]. In the present study, penthorin A (4) and methyl gallate (25) were found to downregulate PCSK9 mRNA and protein expression. However, penthorin A (4) seemed to inhibit PCSK9 expression without affecting LDLR while methyl gallate (25) slightly increased LDLR protein expression (Fig. 5B), comparable to the positive control, berberine. Methyl gallate is widely distrubuted in the plant kingdom and the present nding may give more applications of speci c plants with high quantity of methyl gallate in the future. Even though in the limited structures with gallic acid moiety (Fig. S1), it could be highlighted that methyl gallate (25) was important structure in modulating PCSK9 and LDLR expression.

Conclusion
In the present study, three new naturally occurring compounds (1, 2, and 20) were isolated from P. chinense and two compounds (4 and 25) were found to downregulate PCSK9 expression. Considering most PCSK9 are expressed in liver and the medicinal applications of this plant are related to hepatic disorders, these ndings may help add values of this plant in the future.