Stilbenoids and cannabinoids from the leaves of Cannabis sativa f. sativa with potential reverse cholesterol transport activity.

Three new stilbenoids (1-3) and 16 known stilbenoids (4-6) and cannabinoids (7-19) were isolated from the leaves of hemp (Cannabis sativa L.). The structures of the three new compounds were identified as α,α'-dihydro-3',4,5'-trihydroxy-4'-methoxy-3-isopentenylstilbene (HM1), α,α'-dihydro-3,4',5-trihydroxy-4-methoxy-2,6-diisopentenylstilbene (HM2), and α,α'-dihydro-3',4,5'-trihydroxy-4'-methoxy-2',3-diisopentenylstilbene (HM3) by 1D and 2D NMR spectroscopy, LC-MS, and HRESIMS. The known α,α'-dihydro-3,4',5-trihydroxy-4,5'-diisopentenylstilbene (5) and combretastatin B-2 (6) were isolated for the first time from C. sativa f. sativa. These isolated compounds exhibited cytotoxic effects on human cancer cells via inhibiting the proliferation of cancer cells and inducing cell death. Among them, compounds 4, 5, 10, 12, 13, 15, and 19 displayed broad-spectrum cytotoxicity, and 1, 7, and 11 displayed selectivity in inhibition efficiency on MCF-7 and A549 cells, which suppressed the proliferation of cancer cells significantly by inducing cell death. The effects of compounds 1-3 on improving reverse cholesterol transport (RCT) were evaluated by isotope-tracing and western blotting. Results showed that the three stilbenoids showed a cytotoxicity above 1.0 mg L-1, especially that of HM3. They could improve [3H]-cholesterol efflux from Raw 264.7 macrophages to high density lipoproteins by enhancing the protein expression of ABCG1 and SR-B1, and HM1 and HM2 showed a significant difference compared with fenofibrate at 1.0 mg L-1. The three stilbenoids could also significantly improve the protein expression of ABCA1. Further study on HepG2 cells indicated that they improve the protein expression of LDLR, SR-B1 and CYP7A1, especially that of HM1 and HM3. However, they showed no significant effect on PCSK9. The above results indicated that these stilbenoids may elevate the transfer of cholesterol to hepatocytes by improving the protein expression of SR-B1 and LDLR, and the synthesis of bile acid by increasing the protein expression of CYP7A1. In conclusion, HM1 showed lower cytotoxicity and higher activity in improving the RCT-related protein expression. Our study suggests that it may be explored as a novel lipid-lowering drug and as a beneficial ingredient in health functional foods and pharmaceuticals.

Bioactive spirans and other constituents from the leaves of Cannabis sativa f. sativa.

In this paper, 17 compounds (1-17) were isolated from the leaves of Hemp (Cannabis sativa f. sativa). Among the isolates, two were determined to be new spirans: cannabispirketal (1), and α-cannabispiranol 4'-O-ß-D-glucopyranose (2) by 1D and 2D NMR spectroscopy, LC-MS, and HRESIMS. The known compounds 7, 8, 10, 13, 15, and 16 were isolated from Hemp (C. sativa f. sativa) for the first time. Furthermore, compounds 8 and 13 were isolated from the nature for the first time. All isolated compounds were evaluated for cytotoxicity on different tissue-derived passage cancer cell lines through cell viability and apoptosis assay. Among these compounds, compounds 5, 9 and 16 exhibited a broad-spectrum antitumor effect via inhibiting cell proliferation and promoting apoptosis. These results obtained have provided valuable clues to the understanding of the cytotoxic profile for these isolated compounds from Hemp (C. sativa f. sativa).

Cannabisin B induces autophagic cell death by inhibiting the AKT/mTOR pathway and S phase cell cycle arrest in HepG2 cells.

This study investigates the anticancer properties of cannabisin B, purified from hempseed hull, in HepG2 human hepatoblastoma cells. The results indicate that cannabisin B significantly inhibited cell proliferation by inducing autophagic cell death rather than typical apoptosis. Cell viability transiently increased upon the addition of a low concentration of cannabisin B but decreased upon the addition of high concentrations. Cannabisin B-induced changes in cell viability were completely inhibited by pre-treatment with 3-methyladenine (3-MA), indicating that the induction of autophagy by cannabisin B caused cell death. Additionally, cannabisin B induced S phase cell cycle arrest in a dose-dependent manner. Moreover, cannabisin B was found to inhibit survival signaling by blocking the activation of AKT and down-stream targets of the mammalian target of rapamycin (mTOR). These findings suggest that cannabisin B possesses considerable antiproliferative activity and that it may be utilised as a promising chemopreventive agent against hepatoblastoma disease.

Correlation of screening and confirmatory results in tiered immunogenicity testing by solution-phase bridging assays.

Biotherapeutic proteins induce undesired immune responses that can affect drug efficacy and safety. For this reason, immunogenicity assessment is an integral part of drug development and is mandated by the regulatory authorities. Immunogenicity is typically evaluated by a tiered approach consisting of a screening assay followed by a competitive inhibition with unlabeled drug serving as confirmatory assay and additional characterization of the immune response. The confirmatory assay is intended to reduce the number of false positive responses generated in the screening tier and ensure that all samples are correctly classified as positive or negative. The positive-negative sample decisions are based on screening and confirmatory assay cut points that are statistically derived through evaluation of drug-naive samples. In this paper, we describe the analysis of cut point data for the presence of statistical correlation between the screening and confirmatory results. Data were obtained from validations of solution-phase bridging assays for detection of anti-drug antibodies against monoclonal antibody therapeutics. All data sets showed moderate to strong positive correlation, indicating that the screening and confirmatory assays were not independent and were likely to generate similar information. We present theoretical evidence that correlated results may be a general feature of the tiered approach when the same test platform is used for both screening and confirmatory assays. The competitive inhibition test, therefore, may be of limited value beyond reduction of the overall false positive rate. Our results indicate that similar sample results could be obtained by using just the screening assay with the false positive rate set to 1%.

The isolation and identification of two compounds with predominant radical scavenging activity in hempseed (seed of Cannabis sativa L.).

Forty samples were extracted from defatted kernels and hulls of two varieties of hempseed (Bama and Yunma No. 1) using 10 different polar solvent systems. The radical scavenging capacity of the extracts was evaluated using 2,2-diphenyl-1-pikrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays and the total phenolic content was determined by Folin-Ciocalteu's phenol reagent. The correlation analysis indicated that the antioxidants in hempseed belonged to phenolic and DPPH() assay was suitable for evaluating the radical scavenging activity. Two compounds, with predominant antiradical activity, were isolated in 60% ethanol extract of hempseed hull using macroporous resin absorption, LH-20 gel chromatography, and high performance liquid chromatography methods, which were identified as N-trans-caffeoyltyramine and cannabisin B by high-resolution mass spectra, nuclear magnetic resonance spectra, and ultraviolet data. The two compounds exhibited significant high DPPH() scavenging activity and protective effect against in vitro oxidation of human low-density lipoprotein compared with extracts from flaxseed, grape seed, and soybean. This suggests that hempseed hull extract is a potential source of natural antioxidants, which could be added to dietary supplements to help prevent oxidative stress.

One-pot process combining transesterification and selective hydrogenation for biodiesel production from starting material of high degree of unsaturation.

A one-pot process combining transesterification and selective hydrogenation was established to produce biodiesel from hemp (Cannabis sativa L.) seed oil which is eliminated as a potential feedstock by a specification of iodine value (IV; 120 g I(2)/100g maximum) contained in EN 14214. A series of alkaline earth metal oxides and alkaline earth metal supported copper oxide were prepared and tested as catalysts. SrO supported 10 wt.% CuO showed the superior catalytic activity for transesterification with a biodiesel yield of 96% and hydrogenation with a reduced iodine value of 113 and also exhibited a promising selectivity for eliminating methyl linolenate and increasing methyl oleate without rising methyl stearate in the selective hydrogenation. The fuel properties of the selective hydrogenated methyl esters are within biodiesel specifications. Furthermore, cetane numbers and iodine values were well correlated with the compositions of the hydrogenated methyl esters according to degrees of unsaturation.

Analytical characterization of Hempseed (seed of Cannabis sativa L.) oil from eight regions in China.

In this study, eight cultivars of hempseed were collected from different regions of China for analysis of physiochemical properties and chemical composition, as well as for seed indexes and proximate composition of seed kernel. The results indicated that Yunma No. 1 and Bama Huoma, with more than 50% oil and 30% protein in dehulled seed, could be considered as oil extraction material and protein source with respect to kernel yield. Iodine values ranging from 153.6 to 169.1 g/100 g reflected the high degree of unsaturation. The concentration of unsaturated fatty acids exceeded 90%, higher than most conventional vegetable oils. Moreover, polyunsaturated fatty acids ranged from 76.26% to 82.75% and were mainly composed of linoleic acid and α-linolenic acid with a ratio close to 3:1. γ-Tocopherol was found at an average concentration of 28.23 mg/100 g of hempseed oil. The results indicated that hempseed oil is a potentially valuable vegetable oil.