Selective Preparation and High Dynamic-Range Analysis of Cannabinoids in "CBD Oil" and Other Cannabis sativa Preparations.

Much confusion exists about the chemical composition of widely sold Cannabis sativa products that utilize the cannabidiol (CBD) acronym and related terms such as "CBD oil", "CBD plus hemp oil", "full spectrum CBD", "broad spectrum CBD", and "cannabinoids". Their rational chemical and subsequent biological assessment requires both knowledge of the chemical complexity and the characterization of significant individual constituents. Applicable to hemp preparations in general, this study demonstrates how the combination of liquid-liquid-based separation techniques, NMR analysis, and quantum mechanical-based NMR interpretation facilitates the process of natural product composition analysis by allowing specific structural characterization and absolute quantitation of cannabinoids present in such products with a large dynamic range. Countercurrent separation of a commercial "CBD oil" yielded high-purity CBD plus a more polar cannabinoid fraction containing cannabigerol and cannabidivarin, as well as a less polar cannabinoid fraction containing cannabichromene, trans-Δ9-tetrahydrocannabinol, cis-Δ9-tetrahydrocannabinol, and cannabinol. Representatives of six cannabinoid classes were identified within a narrow range of polarity, which underscores the relevance of residual complexity in biomedical research on cannabinoids. Characterization of the individual components and their quantitation in mixed fractions were undertaken by TLC, HPLC, 1H (q)NMR spectroscopy, 1H iterative full spin analysis (HiFSA), 13C NMR, and 2D NMR. The developed workflow and resulting analytical data enhance the reproducible evaluation of "CBD et al." products, which inevitably represent complex mixtures of varying molecular populations, structures, abundances, and polarity features.

Effective methane production from the Japanese weed Gyougi-shiba (Cynodon dactylon) is accomplished by colocalization of microbial communities that assimilate water-soluble and -insoluble fractions.

Weed, an abundant biomass, is considered unsuitable as a raw material for methane production. There are few reports on the anaerobic digestion of weeds without the addition of other organic wastes. To solve this problem, a methane-producing microbial community with weed as a sole feedstock was established. This study mainly focused on the degree of contribution between water-soluble and -insoluble fractions of the weed to methane production; thus, methane production from both fractions was tested separately. Methane production after 80-day batch cultures with whole weed, water-soluble and water-insoluble fractions was 184.5, 96.8 and 26.5 NmL g-1 dry matter (DM), respectively. The results of 16S rRNA gene amplicon sequence analysis revealed that Proteiniphilum saccharofermentans and several Methanobacterium species commonly dominated all cultures, whereas the population dynamics of minor species differed in every culture. Moreover, the remixed culture of microbial communities adapted to water-soluble and -insoluble fractions recovered methane production (252.4 NmL g-1 DM). Based on these results, it can be strongly inferred that colocalizing the minor species in water-soluble and -insoluble fractions is important for effective methane production.

Determination of Mogroside V in Luohanguo Extract for Daily Quality Control Operation Using Relative Molar Sensitivity to Single-Reference Caffeine.

Mogroside V is one of the characteristic and effective components of luohanguo extract, a food additive used as a sweetener in Japan as per Japan's Standards and Specifications for Food Additives (JSFA; 9th ed.). JSFA stipulates that the quantitative determination for mogroside V content in luohanguo extract applies HPLC using analytical standard mogroside V. However, no mogroside V reagents with proven purities are commercially available. Therefore the current JSFA determination method is not particularly suited for daily quality control operations involving luohanguo extract. In this study, we applied an alternative quantitative method using a single reference with relative molar sensitivity (RMS). It was possible to calculate the accurate RMS by an offline combination of 1H-quantitative NMR spectroscopy (1H-qNMR) and an HPLC/variable-wavelength detector (VWD). Using the RMS of mogroside V to a commercial certified reference material grade caffeine, the mogroside V contents in luohanguo extracts could be determined using HPLC/VWD without analytical standard mogroside V. There was no significant difference between the mogroside V contents in luohanguo extracts determined using the method employing single-reference caffeine with the RMS and using the JSFA method. The absolute calibration curve for the latter was prepared using an analytical standard mogroside V whose purity was determined by 1H-qNMR. These results demonstrate that our proposed method using a single reference with RMS is suitable for quantitative determination of mogroside V in luohanguo extract and can be used as an alternative method to the current assay method in JSFA.

[Determination of the Plant Origin of Licorice Oil Extract, a Natural Food Additive, by Principal Component Analysis Based on Chemical Components].

"Licorice oil extract" (LOE) (antioxidant agent) is described in the notice of Japanese food additive regulations as a material obtained from the roots and/or rhizomes of Glycyrrhiza uralensis, G. inflata or G. glabra. In this study, we aimed to identify the original Glycyrrhiza species of eight food additive products using LC/MS. Glabridin, a characteristic compound in G. glabra, was specifically detected in seven products, and licochalcone A, a characteristic compound in G. inflata, was detected in one product. In addition, Principal Component Analysis (PCA) (a kind of multivariate analysis) using the data of LC/MS or (1)H-NMR analysis was performed. The data of thirty-one samples, including LOE products used as food additives, ethanol extracts of various Glycyrrhiza species and commercially available Glycyrrhiza species-derived products were assessed. Based on the PCA results, the majority of LOE products was confirmed to be derived from G. glabra. This study suggests that PCA using (1)H-NMR analysis data is a simple and useful method to identify the plant species of origin of natural food additive products.

Cycloartane triterpenes and ingol diterpenes isolated from Euphorbia neriifolia in a screening program for death-receptor expression-enhancing activity.

In our screening program for natural products that increase death-receptor 5 expression, seven new cycloartane triterpenes, euphonerins A-G (1-7), and 3-O-acetyl-8-O-tigloylingol (8), a new ingol diterpene, were isolated from the MeOH extract of Euphorbia neriifolia leaves, together with 3,12-di-O-acetyl-8-O-tigloylingol (9), (24R)-cycloartane-3ß,24,25-triol (10), and three known flavonols (11-13). The structures of 1-8 were elucidated by spectroscopic analysis. Among these compounds, 1-11 showed death-receptor 5 expression enhancing activity.

Cryptolepine, isolated from Sida acuta, sensitizes human gastric adenocarcinoma cells to TRAIL-induced apoptosis.

Bioassay guided separation of Sida acuta whole plants led to the isolation of an alkaloid, cryptolepine (1), along with two kaempferol glycosides (2-3). Compound 1 showed strong activity in overcoming TRAIL-resistance in human gastric adenocarcinoma (AGS) cells at 1.25, 2.5 and 5 µm. Combined treatment of 1 and TRAIL sensitized AGS cells to TRAIL-induced apoptosis at the aforementioned concentrations.

Constituents of Amoora cucullata with TRAIL resistance-overcoming activity.

In search of bioactive natural products for overcoming TRAIL resistance from natural resources, we previously reported a number of active compounds. Bioassay-guided fractionation of mangrove, Amoora cucullata, collected from Sundarbans Mangrove Forest, Bangladesh, led to the isolation of four new compounds (1-4), along with seven known compounds (5-11). Of the isolates, compounds 1, 5, 8, and 9 showed TRAIL resistance-overcoming activity, among which 8 showed the most potent activity and enhanced TRAIL-induced apoptosis in TRAIL-resistant human gastric adenocarcinoma (AGS) cells through the activation of caspase-3/7, enhancing the expression of DR4 and DR5 mRNA in AGS cells. Cell death caused by the combined treatment of 8 and TRAIL was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, indicating that 8 sensitizes TRAIL-resistant AGS cells to TRAIL through the induction of DR4 and DR5.

Activity of mangosteen xanthones and teleocidin a-2 in death receptor expression enhancement and tumor necrosis factor related apoptosis-inducing ligand assays.

A screening study using a luciferase assay to identify natural products that enhance death receptor 5 (DR5) expression was carried out, and bioassay-guided fractionation of two organisms, the pericarp of Garcinia mangostana (mangosteen) and actinomycete CKK609 strain, led to the isolation of eight xanthone derivatives (1-8) and teleocidin A-2 (9). Among them, compounds 1, 2, and 5, isolated from G. mangostana, and 9, from the actinomycete, showed potent DR5 promoter activity. Furthermore, we revealed that combined treatment with gartanin (5) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) showed a potentiation effect in sensitizing TRAIL-resistant human gastric adenocarcinoma (AGS) cells. Thus, the present results suggested that 5 has the ability to overcome TRAIL resistance via the up-regulation of DR5 and may be an effective sensitizer of TRAIL-resistant cells.

Death receptor 5 promoter-enhancing compounds isolated from Catimbium speciosum and their enhancement effect on TRAIL-induced apoptosis.

The TRAIL/death-receptor signaling pathway has been considered a promising target for selective cancer therapy, although some malignant tumors exhibit TRAIL resistance. We previously found that isoflavonoid enhanced TRAIL-induced apoptosis in TRAIL-resistant cells, which is achieved through up-regulation of death receptor 5 (DR5). In our screening program targeting DR5 promoter enhancement activity, activity-guided fractionations of the extract of Catimbium speciosum led to the isolation of six compounds. Of the isolates, cardamomin (6), the most potent compound, enhanced the expressions of DR5 and DR4 and decreased the Bcl-xL level in TRAIL-resistant DLD1 cells. The combination of 6 and TRAIL synergistically enhanced TRAIL-induced apoptosis against TRAIL-resistant cells upon the activation of caspase-8, 9, and 3. In addition, enhancement of apoptosis by 6 was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, TRAIL-neutralizing fusion proteins, indicating that 6 sensitize TRAIL-resistant cells to TRAIL through the induction of DR5 and DR4. Also, up-regulation of DR5 by 6 paralleled that of CCAAT/enhancer-binding protein-homologous protein (CHOP).

Cardenolide glycosides of Thevetia peruviana and triterpenoid saponins of Sapindus emarginatus as TRAIL resistance-overcoming compounds.

A screening study for TRAIL resistance-overcoming activity was carried out, and activity-guided fractionations of Thevetia peruviana and Sapindus emarginatus led to the isolation of four cardenolide glycosides (1-4) and four triterpenoid saponins (5-8), respectively. In particular, cardenolide glycosides (1 and 2) from T. peruviana were shown to have a significant reversal effect on TRAIL resistance in human gastric adenocarcinoma cells, and real-time PCR showed that thevefolin (2) enhanced mRNA expression of death receptor 4 (DR4) and DR5. In addition, 1H and 13C NMR characterizations are shown for thevefolin (2) for the first time.

Structure of new monoterpene glycoside from Sibiraea angustata RCHD. and its anti-obestic effect.

A new monoterpene glycoside (1) isolated from the aerial part of Sibirae angustata RCHD. (Rosaceae) was found to be 1-O-beta-D-glucopyranosyl-geraniol-5,10-olide and named as sibiskoside. Acute toxicity study revealed that oral administration of 1 (2.5 g/kg body weight) to mice resulted in no death and no evidence of abnormalities in internal organs. Its oral administration to the mice reared with high-fat diet resulted in weight-loss, which was also reflected in serum triglyceride and sugar level, and the weight of abdominal fat. Sibiskoside could be considered to be an active ingredient of Liucha for exerting weight-loss effect in a drink of S. angustata.

New Wnt/beta-catenin signaling inhibitors isolated from Eleutherine palmifolia.

Aberrant Wnt/beta-catenin signaling has recently been implicated in tumorigenesis. On the basis of our screening program targeting inhibition of TCF/beta-catenin transcriptional activity, a plant extract of Eleutherine palmifolia was selected as a hit sample. Activity-guided fractionations led to the isolation of 15 naphthalene derivatives (1-15), including 4 new glucosides, eleutherinosides B-E (1-4), and 10 of the 15 compounds showed strong activities with high viability among 293T cells. Our data showed that 2 and 9 inhibited the transcription of TCF/beta-catenin in SW480 colon cancer cells in a dose-dependent manner. These two compounds also showed selective cytotoxicity against three colorectal cancer cell lines. In addition, treatment with 9 led to a significant decrease in the level of nuclear beta-catenin protein, suggesting this reduction to have resulted in the inhibitory effect of 9 on the transcription of TCF/beta-catenin.

Constituents from Hoya parasitica and their cell growth inhibitory activity.

An androstanoid, hoyasterone (1), a sesquiterpene, 15-bulnesolic acid (2), and a phenolic compound, 1-(4-hydroxy-3-methoxyphenyl)-1-methoxypropan-2-ol (3), together with a known triterpene, dihydrocanaric acid, were isolated from Hoya parasitica. Structures were elucidated by 1 D and 2 D NMR and mass spectroscopic analysis. Among the isolated compounds, only dihydrocanaric acid exhibited growth inhibitory activity against both HeLa and SW480 cells.

Acylated triterpenoid saponins from Schima noronhae and their cell growth inhibitory activity.

Two new acylated triterpenoid saponins were isolated from the branches of Schima noronhae by bioassay-guided purification. Their chemical structures were established on the basis of spectroscopic analysis and chemical means as 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl 22-O-angeloyl-A1-barrigenol (1) and 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl 22-O-angeloylerythrodiol (2). Compounds 1 and 2 showed cell growth inhibitory activity against both HeLa and DLD1 cells at a concentration of less than 10 microM.

Quinic acid esters from Pluchea indica with collagenase, MMP-2 and MMP-9 inhibitory activities.

Investigation of collagenase inhibitory natural components afforded two quinic acid esters (1 and 2) and quercetin (3) from the leaves of Pluchea indica (Compositae). Of these, compounds 1 and 2 exhibited collagenase inhibitory activity (IC(50)) at a concentration of less than 10 microm, and 1 showed matrix metalloproteinase (MMP)-2 and -9 inhibitory activity (IC(50)) at 2.5 and 6.4 microm, respectively.

First isolation of sesquiterpenes and flavonoids from Zingiber spectabile and identification of zerumbone as the major cell growth inhibitory component.

Nine sesquiterpenes and eight flavonoids were isolated from Zingiber spectabile for the first time. Structures were determined by spectroscopic methods. The major compound zerumbone (1) was found to be the most active (IC(50) 13 microg mL(-1)) in cell growth inhibitory assay against colon carcinoma SW480 cells.

Mangiferin identified in a screening study guided by neuraminidase inhibitory activity.

A screening study on neuraminidase inhibitory constituents was carried out, and activity-guided fractionations of three plants, Gouania obtusifolia, Zizyphus cambodiana, and Mangifera odorata, led to the isolation of eleven compounds (1-11). Mangiferin was identified as a significant neuraminidase inhibitor.

Secoiridoid components from Jasminum grandiflorum.

Secoiridoid glucosides, 2''-epifraxamoside and demethyl-2''-epifraxamoside, and the secoiridoid, jasminanhydride were isolated from Jasminum grandiflorum together with four previously known phenolics and a triterpene. Structures were elucidated by detailed spectroscopic analysis. Stereochemistry of the compounds was determined by differential NOE experiment.

Difference of growth-inhibitory effect of Scutellaria baicalensis-producing flavonoid wogonin among human cancer cells and normal diploid cell.

Methanol extract from cultured Scutellaria baicalensis cells inhibited the proliferation of human monocytic leukemia cell line THP-1 and human osteogenic sarcoma cell line HOS. The inhibitory effects of baicalin, baicalein and wogonin, the three major flavonoids contained in the extract, were studied. It should be noted that wogonin did not show the inhibitory effect on human fetal lung normal diploid cell line TIG-1, as compared to the inhibition observed in cancer cells. Physiological analyses in THP-1 cells showed that wogonin induced cell cycle arrest at G(2)/M phase and apoptosis. This is the first report discovering a cancer-specific apoptosis-inducing activity of wogonin.

Flavonoids from Sonneratia caseolaris.

According to the traditional medicinal usage of Sonneratia caseolaris, we tested the extract of S. caseolaris for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging effect on thin-layer chromatography. Following activity-oriented separation, two flavonoids, luteolin (1) and luteolin 7-O-ß-glucoside (2), were isolated. Both of the compounds were found to possess antioxidant activity.