Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the key regulator of low-density lipoprotein cholesterol (LDL-C) plasma levels. We previously observed that treatment of dyslipidemic subjects with nutraceutical combination containing red yeast rice (monacolin K 3.3 mg), Berberis aristata cortex extract (Berberine 531.25 mg) and Morus alba leaves extract (1-deoxynojirimycin 4 mg) (LopiGLIK®) did not alter the plasma PCSK9 levels. Thus, the aim of the present study was to investigate the effect of these three components on PCSK9 expression in HepG2 cell line in relationship to their effects on LDL-C cellular uptake. HepG2 cell line were incubated with Berberis aristata cortex extract (BCE), red yeast rice (RYR) and Morus alba leaves extract (MLE) alone or in combination for 24 h. RYR (50 µg/mL) increased PCSK9 protein expression (WB and ELISA assays), PCSK9 mRNA and its promoter activity. BCE (40 µg/mL) reduced PCSK9 expression, mRNA levels and promoter activity. MLE determined a concentration-dependent inhibition of PCSK9 at mRNA and protein levels, with a maximal reduction at 1 mg/mL; no significant changes in PCSK9 promoter activity were found. MLE also downregulates the expression of fatty acid synthase and HMG-CoA reductase mRNA levels. The combination of RYR, BCE and MLE reduced PCSK9 at mRNA, protein, and promoter activity. Finally, this combination induced the LDL receptor and LDL-C uptake by HepG2 cells. In conclusion, the positive effect of MLE on PCSK9 supports the rational of using this nutraceutical combination to control hyperlipidemic conditions.

Berberis vulgaris L. is currently widely studied for its antioxidant and chemopreventive proper-ties, especially given the beneficial properties of its fruits in this regard. Although the bark and roots have been well known and used in traditional medicine since ancient times, little is known about the other parts of this plant. The aim of the research was to determine the antioxidant and LOX inhibitory activity effects of extracts obtained from leaves, fruits, and stems. Another aim of the work was to carry out the quantitative and qualitative analysis of phenolic acids, flavonoid aglycones, and flavonoid glycosides. Extracts were obtained with the use of ASE (accelerated solvent extraction). The total content of polyphenols was determined and was found to vary de-pending on the organ, with the highest amount of polyphenols found in the leaves extract. The free radical scavenging activity of the extracts was determined spectrophotometrically in relation to the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, with results ranging from 63.88 mgTE/g for leaves to 65.25 mgTE/g for the stem. Antioxidant activity was also assessed using the ABTS test. The lowest value was recorded for the barberry fruit (117.93 mg TE/g) and the highest level was found for the barberry leaves (140.49 mgTE/g). The oxygen radical absorption capacity test (ORAC) showed the lowest value for the stem (167.70 mgTE/g) and the highest level for the leaves (267.81 mgTE/g). The range of percentage inhibition of LOX was determined as well. The percentage inhibition of the enzyme was positively correlated with the sum of flavonoids, TPC, TFC, and the content of selected flavonoids. For the first time, phenolic acids, flavonoid agly-cones, and flavonoid glycosides were determined qualitatively and quantitatively in individual parts of Berberis vulgaris L. The content of phenolic acids, flavonoid aglycones, and flavonoid glycosides was determined with the LC-MS/MS method. For the first time, the following phenol-ic acids were quantitatively and qualitatively identified in individual parts of Berberis vulgaris: 3-caffeoylquinic acid, protocatechuic acid, 5-caffeoylquinic acid, flavonoid glycosides: eleuthero-side E, rutin, isoquercitin, luteoloside, nar-cissoside, naringenin-7-glucoside, isorhamnetin-3-glucoside, afzeline, and quercitrin. Flavonoid aglycones: catechin, luteolin, and eriodictyol were determined qualitatively and quantitatively for the first time as well.

Berberis vulgaris L. (Berberidaceae) is a shrub that has been widely used in European folk medicine as an anti-inflammatory and antimicrobial agent. The purpose of our study was to elucidate the mechanisms of antiproliferative action of the plant’s methanolic root extract (BVR) against colon cancer cells. Cytotoxicity and pro-apoptotic activity were determined in human colon adenocarcinoma cell lines (LS180 and HT-29) and control colon epithelial CCD841 CoN cells. According to the MTT assay, after 48 h of cell exposure, the IC50 values were as follows: 4.3; 46.1 and 50.2 µg/mL for LS180, HT-29, and CCD841 CoN cells, respectively, showing the greater sensitivity of cancer cells to BVR. The Cell Death Detection ELISAPLUS kit demonstrated that BVR induced programmed cell death only against HT-29 cells. RT-qPCR with relative quantification method showed significant changes in the expression of genes related to apoptosis in both colon cancer cell lines. The genes BCL2L1 (126.84-366.66%), BCL2L2 (277.08-330.12%), CASP3 (180.36-261.36%) and CASP9 (158.08-213.46%) had significantly elevated expression, while BCL2 (23.08-52.06%) had reduced expression. Furthermore, in a panel of antioxidant tests, BVR showed positive effects (63.93±0.01, 122.92±0.01, and 220.29±0.02 mg Trolox equivalents (TE)/g in the DPPH•, ABTS•+ and ORAC assays, respectively). In the lipoxygenase (LOX) inhibition test, BVR revealed 62,60±0.87% of enzyme inhibition. The chemical composition of BVR was determined using UHPLC-UV-CAD-MS/MS analysis and confirmed the presence of several known alkaloids, including berberine, as well as other alkaloids and two derivatives of hydroxycinnamic acid (ferulic and sinapic acid hexosides). This is the first such extensive assessment of the biological activity of BVR. The results are very promising and encourage for the use of BVR as a comprehensive chemopreventive agent (anti-inflammatory, antioxidant, and pro-apoptotic) in colorectal cancer.

Berberis vulgaris (L.) has remarkable ethnopharmacological properties and is widely used in traditional medicine. The present study investigates B. vulgaris stem bark (Berberidis cortex) by extraction with 50% ethanol. The main secondary metabolites were quantified, resulting in a polyphenols content of 17.6780 ± 3.9320 mg Eq Tannic acid / 100 g extract, phenolic acids amount of 3.3886 ± 0.3481 mg Eq Chlorogenic acid / 100 g extract and 78.95 µg/g berberine. The dried hydro-ethanolic extract (BVE) was thoroughly analyzed using Ultra-High Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry (UHPLC–HRMS/MS) and HPLC and 40 bioactive phenolic constituents were identified. Then, the antioxidant potential of BVE was evaluated using three methods; our results could explain the protective effects of Berberidis cortex EC50FRAP = 0.1398 mg/mL, IC50ABTS = 0.0442 mg/mL, IC50DPPH = 0.2610 mg/mL compared to ascorbic acid (IC50 = 0.0165 mg/mL). Next, the acute toxicity and teratogenicity of BVE and Berberine - Berberine sulfate hydrate (BS) - investigated on Daphnia sp reported significant BS toxicity after 24 h; BVE revealed considerable toxicity after 48 hours and induced embryonic developmental delays. Finally, the anticancer effects of BVE and BS were evaluated on different tumor cell lines after 24 and 48 hours of treatments. The MTS assay evidenced a dose- and time-dependent antiproliferative activity, higher on BS than BVE. The strongest diminution of tumor cell viability was recorded on breast (MDA-MB-231), colon (LoVo) cancer, and OSCC (PE/CA-PJ49) cell lines after 48 h of exposure (IC50 < 100 µg/mL). However, no cytotoxicity was reported on normal epithelial cells (HUVEC) and hepatocellular carcinoma (HT-29) cell lines. Extensive data analysis supports our results, showing a significant correlation between the BVE's concentration, phenolic compounds content, antioxidant activity, exposure time, and the viability rate of various normal cells and cancer cell lines.