Development of GC-MS coupled to GC-FID method for the quantification of cannabis terpenes and terpenoids: Application to the analysis of five commercial varieties of medicinal cannabis.

Cannabis terpenes and terpenoids are among the major classes of pharmacologically active secondary metabolites of therapeutic interest. Indeed, these hydrocarbon molecules, responsible for the characteristic aroma of cannabis flowers, are thought to be involved in a synergistic effect known as the "entourage effect", together with cannabinoids. Numerous analytical studies have been carried out to characterize the terpene and terpenoid contents of some cannabis varieties, but they have not proposed any real quantification or have described a limited number of analytical standards or average response factors, which may have led to over- or underestimation of the real content of the cannabis flowers. Real and reliable quantification is necessary to justify the entourage effect. Here, we report a rigorous and precise GC-FID and GC-MS method for the identification and quantification of cannabis terpenes and terpenoids. This method is distinguished by the use of a high number of analytical standards, the determination of retention indices for all compounds studied, an exhaustive comparison of databases and scientific literature, the use of relevant response factors, and internal calibration for reliable results. It was applied to the study of terpenic compounds in five commercial varieties of medicinal cannabis produced by Bedrocan International: a CBD-rich (Bedrolite®), a THC/CBD balanced (Bediol®), and three THC-dominant (Bedrocan®, Bedica® and Bedrobinol®). Two extraction solvents are described (ethanol and hexane) to compare their selectivity towards target molecules, and to describe as exhaustively as possible the terpenic profile of the five pharmaceutical-grade varieties. Twenty-three standards were used for accurate dosages. This work highlights that the choice of solvent and the analysis method reliability are critical for the study of these terpenic compounds, regarding their contribution to the entourage effect.

Aqueous Integrated Process for the Recovery of Oil Bodies or Fatty Acid Emulsions from Sunflower Seeds.

An aqueous integrated process was developed to obtain several valuable products from sunflower seeds. With a high-shear rate crusher, high-pressure homogenization and centrifugation, it is possible to process 600× g of seeds in 1400× g of water to obtain a concentrated cream phase with a dry matter (dm) content of 46%, consisting of 74 (w/w dm) lipids in the form of an oil-body dispersion (droplet size d(0.5): 2.0 µm) rich in proteins (13% w/w dm, with membranous and extraneous proteins). The inclusion of an enzymatic step mediated by a lipase made possible the total hydrolysis of trigylcerides into fatty acids. The resulting cream had a slightly higher lipid concentration, a ratio lipid/water closer to 1, with a dry matter content of 57% consisting of 69% (w/w) lipids, a more complex structure, as observed on Cryo-SEM, with a droplet size slightly greater (d(0.5): 2.5 µm) than that of native oil bodies and a conserved protein concentration (12% w/w dm) but an almost vanished phospholipid content (17.1 ± 4.4 mg/g lipids compared to 144.6 ± 6 mg/g lipids in the oil-body dispersion and 1811.2 ± 122.2 mg/g lipids in the seed). The aqueous phases and pellets were also characterized, and their mineral, lipid and protein contents provide new possibilities for valorization in food or technical applications.

Cannabis sativa Extract Induces Apoptosis in Human Pancreatic 3D Cancer Models: Importance of Major Antioxidant Molecules Present Therein.

In recent years, interest in Cannabis sativa L. has been rising, as legislation is moving in the right direction. This plant has been known and used for thousands of years for its many active ingredients that lead to various therapeutic effects (pain management, anti-inflammatory, antioxidant, etc.). In this report, our objective was to optimize a method for the extraction of cannabinoids from a clone of Cannabis sativa L. #138 resulting from an agronomic test (LaFleur, Angers, FR). Thus, we wished to identify compounds with anticancer activity on human pancreatic tumor cell lines. Three static maceration procedures, with different extraction parameters, were compared based on their median inhibitory concentration (IC50) values and cannabinoid extraction yield. As CBD emerged as the molecule responsible for inducing apoptosis in the human pancreatic cancer cell line, a CBD-rich cannabis strain remains attractive for therapeutic applications. Additionally, while gemcitabine, a gold standard drug in the treatment of pancreatic cancer, only triggers cell cycle arrest in G0/G1, CBD also activates the cell signaling cascade to lead to programmed cell death. Our results emphasize the potential of natural products issued from medicinal hemp for pancreatic cancer therapy, as they lead to an accumulation of intracellular superoxide ions, affect the mitochondrial membrane potential, induce G1 cell cycle arrest, and ultimately drive the pancreatic cancer cell to lethal apoptosis.

Biochemical Composition of Cumin Seeds, and Biorefining Study.

A new biorefinery approach has been developed in the present study, and applied on cumin (Cuminum cyminum) seeds as a potential source of phytochemicals of interest. Cumin is a popular spice used widely for its distinctive aroma. It is a rich reserve of both vegetable and essential oils. The biorefinery approach here focused on the evaluation of the influence of four different geographical origins (i.e., Lebanon, France, Algeria and Syria) on oil yield and quality in cumin seed, and on the valorization of remaining by-products by investigating their nutritional content and biological activity for the first time. Vegetable and essential oils were extracted, and their compositions were determined. Nutritional traits were also assessed. The delipidated and hydrodistillated cakes just as aromatic water were characterized for their fiber, sugar, protein, phenol and flavonoid contents. Antibacterial and antioxidant activities were also determined. Cumin seeds showed high contents in both vegetable and essential oils, proteins and sugars regardless their origin. Moreover, this Apiaceae species exhibited high levels of petroselinic fatty acid (an isomer of oleic acid) and sterols. Cakes and aromatic water also presented high levels of proteins, fibers, sugars and phenols. These residues revealed interesting antioxidant and antibacterial activities. These results emphasized the potential use of cumin in a biorefinery concept, with a multi-purpose industrial process. In addition, large differences were observed between the four geographical origins for phytochemical contents and compositions. These findings highlight the perspectives for developing selection programs for nutritional traits and industrial interests. All obtained results validate the health promoting effect of cumin composition as well as its industrial importance along with the residues.

Fatty acid composition and oil content during coriander fruit development.

Coriander contains petroselinic acid, an isomer fatty acid of oleic acid. Coriander seed oil has been proposed as novel food ingredient in the European Union. Field experiments were performed at Auch (France) during two seasons (2010 and 2011). From flowering to maturity, fruits were harvested weekly and oil content and fatty acid (FA) compositions were determined. Fruits presented 2% more oil in 2010 than in 2011. Petroselinic acid (PA) contents was higher in 2011 than in 2010. Oil accumulation began earlier after flowering (2 DAF) in 2011. A first step in accumulation was identified between two and 21 DAF characterized by high SFA and PUFA, which decreased 21 DAF. Subsequently, PA increased to its highest concentration (30-55 DAF) and SFA and PUFA reached their lowest. These results suggest that higher concentrations of PA can be achieved by collecting fruits before full maturity.

Recovery of slaughterhouse Animal Fatty Wastewater Sludge by conversion into Fatty Acid Butyl Esters by acid-catalyzed esterification.

Two types of Animal Fatty Wastewater Sludges (AFWS 1 and 2) were analyzed and fully characterized to determine their suitability for conversion into biofuel. AFWS 1 was determined to be unsuitable as it contains 68.8wt.% water and only 32.3wt.% dry material, of which only around 80% is lipids to be converted. AFWS 2 has only 15.7wt.% water and 84.3wt.% dry material of which is assumed to 100% lipids as the protein and ash contents were determined to be negligible. The 4-dodecylbenzenesulfonic acid (DBSA) catalyzed esterification of AFWS with 1-butanol was performed in a novel batch reactor fitted with a drying chimney for the "in situ" removal of water and optimized using a non-conventional Doehlert surface response methodology. The optimized condition was found to be 1.66mol equivalent of 1-butanol (with respect to total fatty acid chains), 10wt.% of DBSA catalyst (with respect to AFWS) at 105°C for 3h. Fatty Acid Butyl Esters (FABEs) were isolated in good yields (95%+) as well as a blend of FABEs with 1-butanol (16%). The two potential biofuels were analyzed in comparison with current and analogous biofuels (FAME based biodiesel, and FABE products made from vegetable oils) and were found to exhibit high cetane numbers and flash point values.

Characterization of French Coriander Oil as Source of Petroselinic Acid.

Coriander vegetable oil was extracted from fruits of French origin in a 23% yield. The oil was of good quality, with a low amount of free fatty acids (1.8%) and a concurrently high amount of triacylglycerols (98%). It is a rich source of petroselinic acid (C18:1n-12), an important renewable building block, making up 73% of all fatty acids, with also significant amounts of linoleic acid (14%), oleic acid (6%), and palmitic acid (3%). The oil was characterized by a high unsaponifiable fraction, comprising a substantial amount of phytosterols (6.70 g/kg). The main sterol markers were ß-sitosterol (35% of total sterols), stigmasterol (24%), and Δ7-stigmastenol (18%). Squalene was detected at an amount of 0.2 g/kg. A considerable amount of tocols were identified (500 mg/kg) and consisted mainly of tocotrienols, with γ-tocotrienol as the major compound. The phospholipid content was low at 0.3%, of which the main phospholipid classes were phosphatidic acid (33%), phosphatidylcholine (25%), phosphatidylinositol (17%), and phosphatidylethanolamine (17%). About 50% of all phospholipids were non-hydratable. The ß-carotene content was low at 10 mg/kg, while a significant amount of chlorophyll was detected at about 11 mg/kg. An iron content of 1.4 mg/kg was determined through element analysis of the vegetable oil. The influence of fruit origin on the vegetable oil composition was shown to be very important, particularly in terms of the phospholipids, sterols, and tocols composition.

Aframomum stipulatum (Gagnep) K. Schum and Aframomum giganteum (Oliv. & Hanb) K. Schum as Aroma Tincto Oleo Crops resources: essential oil, fatty acids, sterols, tocopherols, and tocotrienols composition of different fruit parts of Congo varieties.

BACKGROUND: Today, few known plant species provide both an essential oil (EO) and a vegetable oil (VO). Seed and husk of two Aframomum species were investigated and compared in terms of EO, fatty acids, tocopherols, and tocotrienols. RESULTS: EO yield reaches 15.3 g kg(-1) in the seeds and 3.2 g kg(-1) in the husks, while VO yield is 180.0 g kg(-1) in the seeds and 25.0 g kg(-1) in the husks. ß-Pinene, 1,8-cineol, α-selinene, terpine-4-ol, linalool, myrtenal and ß-caryophyllene are the major compounds of seed and husk EO. Fatty acid analysis of two Aframomum species shows that oleic, linoleic, and palmitic acids were the major compounds of VO. Total sterol contents reached 4.3 g kg(-1) in seed VO and 8.5 g kg(-1) in husk VO. An appreciable amount of tocopherols (0.52 g kg(-1) ) was found in seed VO. CONCLUSION: The seed and husk oil of A. stipulatum and A. giganteum fruits are rich sources of many bioactive constituents such as fatty acids, sterols, tocopherols and tocotrienols. These tropical wild fruits can be considered as new Aroma Tincto Oleo Crops (ATOC) resources that contain both EOs and VOs.

Genetic analysis of phytosterol content in sunflower seeds.

Interest in phytosterol contents due to their potential benefits for human health has been largely documented in several crop species. Studies were focused mainly on total sterol content and their concentration or distribution in seed. This study aimed at providing new insight into the genetic control of total and individual sterol contents in sunflower seed through QTL analyses in a RIL population characterized over 2 years showing contrasted rainfall during seed filling. Results indicated that 13 regions on 9 linkage groups were involved in different phytosterol traits. Most of the QTL mapped were stable across years in spite of contrasted growing conditions. Some of them explained up to 30 % of phenotypic variation. Two QTL, located on LG10, near b1, and on LG14, were found to co-localize with QTL for oil content, indicating that likely, a part of the genetic variation for sterol content is only the result of genetic variation for oil content. However, three other QTL, stable over the 2 years, were found on LG1, LG4 and LG7 each associated with a particular class of sterols, suggesting that some enzymes known to be involved in the sterol metabolic pathway may determine the specificity of sterol profiles in sunflower seeds. These results suggest that it may be possible to introduce these traits as criteria in breeding programmes for quality in sunflower. The molecular markers linked to genetic factors controlling phytosterol contents could help selection during breeding programs.

Antifungal Properties of Chenopodium ambrosioides Essential Oil Against Candida Species.

The essential oil of the aerial part (leaves, flowers and stem) of Chenopodium ambrosioides was obtained by hydrodistillation and its chemical composition analyzed by GC and GC/MS, which permitted the identification of 14 components, representing 98.8% of the total oil. Major components were α-terpinene (51.3%), p-cymene (23.4%) and p-mentha-1,8-diène (15.3%). The antifungal properties of this essential oil were investigated in vitro by the well diffusion and broth microdilution methods. The in vitro antifungal activity was concentration dependent and minimum inhibitory concentration values varied from 0.25 to 2 mg/mL. The in vivo antifungal activity was evaluated on an induced vaginal candidiasis rat model. The in vivo activity of the oil on mice vaginal candidiasis was not dose-dependent. Indeed, all the three tested doses; 0.1%, 1% and 10% led to the recovery of mice from the induced infection after 12 days of treatment. The effect of the essential oil on C. albicans ATCC 1663 fatty acid profile was studied. This oil has a relatively important dose-dependent effect on the fatty acids profile.