A Rapid Nano-Liquid Chromatographic Method for the Analysis of Cannabinoids in Cannabis sativa L. Extracts

Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H₂O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization–mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia’s protocol on medicinal plants, were carried out, and the results achieved were compared.     

Non-Invasive and Confirmatory Differentiation of Hermaphrodite from Both Male and Female Cannabis Plants Using a Hand-Held Raman Spectrometer

Cannabis (Cannabis sativa L.) is a dioecious plant that produces both male and female inflorescences. In nature, male and female plants can be found with nearly equal frequency, which determines species out-crossing. In cannabis farming, only female plants are preferred due to their high yield of cannabinoids. In addition to unfavorable male plants, commercial production of cannabis faces the appearance of hermaphroditic inflorescences, species displaying both pistillate flowers and anthers. Such plants can out-cross female plants, simultaneously producing undesired seeds. The problem of hermaphroditic cannabis triggered a search for analytical tools that can be used for their rapid detection and identification. In this study, we investigate the potential of Raman spectroscopy (RS), an emerging sensing technique that can be used to probe plant biochemistry. Our results show that the biochemistry of male, female and hermaphroditic cannabis plants is drastically different which allows for their confirmatory identification using a hand-held Raman spectrometer. Furthermore, the coupling of machine learning approaches enables the identification of hermaphrodites with 98.7% accuracy, whereas both male and female plants can be identified with 100% accuracy. Considering the label-free, non-invasive and non-destructive nature of RS, the developed optical sensing approach can transform cannabis farming in the U.S. and overseas.     

The Cannabis Terpenes

Terpenes are the primary constituents of essential oils and are responsible for the aroma characteristics of cannabis. Together with the cannabinoids, terpenes illustrate synergic and/or entourage effect and their interactions have only been speculated in for the last few decades. Hundreds of terpenes are identified that allude to cannabis sensory attributes, contributing largely to the consumer’s experiences and market price. They also enhance many therapeutic benefits, especially as aromatherapy. To shed light on the importance of terpenes in the cannabis industry, the purpose of this review is to morphologically describe sources of cannabis terpenes and to explain the biosynthesis and diversity of terpene profiles in different cannabis chemovars.     

Utilization of matrix-assisted laser desorption/ionization imaging mass spectrometry to search for cannabis in herb mixtures

Herb mixtures including cannabis among the other herbs have recently appeared. When cannabinoids from herb extracts are detected by chemical examinations such as gas chromatography/mass spectrometry, forensic analysts have to determine whether cannabis is actually in the mixture or the cannabinoids are spiked. Morphological examinations are time-consuming, since it is difficult to find several pieces of cannabis among a large number of herb pieces using a microscope. Here, we propose a procedure for efficiently searching for cannabis in herb mixtures using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI/IMS). Pieces of herb mixtures were spread on double-sided adhesive tape attached to a stainless steel plate. The pieces were then covered with a conductive sheet and pressed. After a solution containing a matrix reagent was sprayed, the distribution of cannabinoids in the sample was visualized by MALDI/IMS. Then, just the pieces with cannabinoids could be picked up selectively with tweezers and decolorized. Cystolith hairs and trichomes, which are characteristic of cannabis, were observed in most of these pieces using a biological microscope. This MALDI/IMS procedure enables cannabis to be found in herb mixtures without inefficient random sampling and microscopic morphological examination.     

Mass Spectrometry-Based Metabolomics of Phytocannabinoids from Non-Cannabis Plant Origins

Phytocannabinoids are isoprenylated resorcinyl polyketides produced mostly in glandular trichomes of Cannabis sativa L. These discoveries led to the identification of cannabinoid receptors, which modulate psychotropic and pharmacological reactions and are found primarily in the human central nervous system. As a result of the biogenetic process, aliphatic ketide phytocannabinoids are exclusively found in the cannabis species and have a limited natural distribution, whereas phenethyl-type phytocannabinoids are present in higher plants, liverworts, and fungi. The development of cannabinomics has uncovered evidence of new sources containing various phytocannabinoid derivatives. Phytocannabinoids have been isolated as artifacts from their carboxylated forms (pre-cannabinoids or acidic cannabinoids) from plant sources. In this review, the overview of the phytocannabinoid biosynthesis is presented. Different non-cannabis plant sources are described either from those belonging to the angiosperm species and bryophytes, together with their metabolomic structures. Lastly, we discuss the legal framework for the ingestion of these biological materials which currently receive the attention as a legal high.     

High-Throughput Quantitation of Cannabinoids by Liquid Chromatography Triple-Quadrupole Mass Spectrometry

The high-throughput quantitation of cannabinoids is important for the cannabis industry. As medicinal products increase, and research into compounds that have pharmacological benefits increase, and the need to quantitate more than just the main cannabinoids becomes more important. This study aims to provide a rapid, high-throughput method for cannabinoid quantitation using a liquid chromatography triple-quadrupole mass spectrometer (LC-QQQ-MS) with an ultraviolet diode array detector (UV-DAD) for 16 cannabinoids: CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, THC, Δ8-THC, CBL, CBC, THCA-A and CBCA. Linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, recovery and matrix effect were all evaluated. The validated method was used to determine the cannabinoid concentration of four different Cannabis sativa strains and a low THC strain, all of which have different cannabinoid profiles. All cannabinoids eluted within five minutes with a total analysis time of eight minutes, including column re-equilibration. This was twice as fast as published LC-QQQ-MS methods mentioned in the literature, whilst also covering a wide range of cannabinoid compounds.     

Analysis of 30 synthetic cannabinoids in serum by liquid chromatography‐electrospray ionization tandem mass spectrometry after liquid‐liquid extraction

The analysis of synthetic cannabinoids in human matrices is of particular importance in the fields of forensic and clinical toxicology since cannabis users partly shift to the consumption of ‘herbal mixtures’ as a legal alternative to cannabis products in order to circumvent drug testing. However, comprehensive methods covering the majority of synthetic cannabinoids already identified on the drug market are still lacking. In this article, we present a fully validated method for the analysis of 30 synthetic cannabinoids in human serum utilizing liquid‐liquid extraction and liquid chromatography‐electrospray ionization tandem mass spectrometry. The method proved to be suitable for the quantification of 27 substances. The limits of detection ranged from 0.01 to 2.0 ng/mL, whereas the lower limits of quantification were in the range from 0.1 to 2.0 ng/mL. The presented method was successfully applied to 833 authentic serum samples during routine analysis between August 2011 and January 2012. A total of 227 (27%) samples was tested positive for at least one of the following synthetic cannabinoids: JWH‐018, JWH‐019, JWH‐073, JWH‐081, JWH‐122, JWH‐200, JWH‐203, JWH‐210, JWH‐307, AM‐2201 and RCS‐4. The most prevalent compounds in positive samples were JWH‐210 (80%), JWH‐122 (63%) as well as AM‐2201 (29%). Median serum concentrations were all below 1.0 ng/mL. These findings demonstrate a significant shift of the market of synthetic cannabinoids towards substances featuring a higher CB₁ binding affinity and clearly emphasize that the analysis of synthetic cannabinoids in serum or blood samples requires highly sensitive analytical methods covering a wide spectrum of substances. Copyright © 2012 John Wiley & Sons, Ltd.     

Screening of cannabinoids in industrial‐grade hemp using two‐dimensional liquid chromatography coupled with acidic potassium permanganate chemiluminescence detection

Widely known for its recreational use, the cannabis plant also has the potential to act as an antibacterial agent in the medicinal field. The analysis of cannabis plants/products in both pharmacological and forensic studies often requires the separation of compounds of interest and/or accurate identification of the whole cannabinoid profile. In order to provide a complete separation and detection of cannabinoids, a new two‐dimensional liquid chromatography method has been developed using acidic potassium permanganate chemiluminescence detection, which has been shown to be selective for cannabinoids. This was carried out using a Luna 100 Å CN column and a Poroshell 120 EC‐C₁₈ column in the first and second dimensions, respectively. The method has utilized a large amount of the available separation space with a spreading angle of 48.4° and a correlation of 0.66 allowing the determination of more than 120 constituents and mass spectral identification of ten cannabinoids in a single analytical run. The method has the potential to improve research involved in the characterization of sensitive, complex matrices.     

N-Butyrylated Hyaluronic Acid Achieves Anti-Inflammatory Effects In Vitro and in Adjuvant-Induced Immune Activation in Rats

Previously synthesized N-butyrylated hyaluronic acid (BHA) provides anti-inflammatory effects in rat models of acute gouty arthritis and hyperuricemia. However, the mechanism of action remains to be elucidated. Herein, the anti-inflammatory and antioxidative activities of BHA and the targeted signaling pathways were explored with LPS-induced RAW264.7 and an adjuvant-induced inflammation in a rat model. Results indicated that BHA inhibited the generation of pro-inflammatory cytokines TNFα, IL-1β and IL-6, reduced ROS production and down-regulated JAK1-STAT1/3 signaling pathways in LPS-induced RAW264.7. In vivo, BHA alleviated paw and joint swelling, decreased inflammatory cell infiltration in paw tissues, suppressed gene expressions of p38 and p65, down-regulated the NF-κB and MAPK signaling pathways and reduced protein levels of TNFα, IL-1β and IL-6 in joint tissues of arthritis rats. This study demonstrated the pivotal role of BHA in anti-inflammation and anti-oxidation, suggesting the potential clinical value of BHA in the prevention of inflammatory arthritis and is worthy for development as a new pharmacological treatment.     

Chemical Characterization of Flowers and Leaf Extracts Obtained from Turnera subulata and Their Immunomodulatory Effect on LPS-Activated RAW 264.7 Macrophages

The anti-inflammatory properties of Turnera subulata have been evaluated as an alternative drug approach to treating several inflammatory processes. Accordingly, in this study, aqueous and hydroalcoholic extracts of T. subulata flowers and leaves were analyzed regarding their phytocomposition by ultrafast liquid chromatography coupled to mass spectrometry, and their anti-inflammatory properties were assessed by an in vitro inflammation model, using LPS-stimulated RAW-264.7 macrophages. The phytochemical profile indicated vitexin-2-O-rhamnoside as an important constituent in both extracts, while methoxyisoflavones, some bulky amino acids (e.g., tryptophan, tyrosine, phenylalanine), pheophorbides, and octadecatrienoic, stearidonic, and ferulic acids were detected in hydroalcoholic extracts. The extracts displayed the ability to modulate the in vitro inflammatory response by altering the secretion of proinflammatory (TNF-α, IL-1β, and IL-6) and anti-inflammatory (IL-10) cytokines and inhibiting the PGE-2 and NO production. Overall, for the first time, putative compounds from T. subulata flowers and leaves were characterized, which can modulate the inflammatory process. Therefore, the data highlight this plant as an option to obtain extracts for phytotherapic formulations to treat and/or prevent chronic diseases.     

Comprehensive classification of USA cannabis samples based on chemical profiles of major cannabinoids and terpenoids

Abstract

Different USA-origin cannabis samples were analyzed by GC-FID to quantify all possible cannabinoids and terpenoids prior to their clustering. Chromatographic analysis confirmed the presence of seven cannabinoids and sixteen terpenoids with variable levels. Among tested cannabinoids, Δ⁹-Tetrahydrocannabinol Δ⁹-THC and cannabinol CBN were available in excess amounts (1.2–8.0?wt%) and (0.22–1.1?wt%), respectively. Fenchol was the most abundant terpenoid with a range of (0.03–1.0?wt%). The measured chemical profile was used to cluster 23 USA states and to group plant samples using different unsupervised multivariate statistical tools. Clustering of plant samples and states was sensitive to the selected cannabinoids/terpenoids. Principal component analysis (PCA) indicated the importance of Δ⁹-THC, CBN, CBG, CBC, THCV, Δ⁸-THC, CBL, and fenchol for samples clustering. Δ⁹-THC was significant to separate California-origin samples while CBN and fenchol were dominant to separate Oregon-origin samples away from the rest of cannabis samples. A special PCA analysis was performed on cannabinoids after excluding Δ⁹-THC (due to its high variability in the same plant) and CBN (as a degradation byproduct for THC). Results indicated that CBL and Δ⁸-THC were necessary to separate Nevada and Washington samples, while, CBC was necessary to isolate Oregon and Illinois plant samples. PCA based on terpenoids content confirmed the significance of caryophyllene, guaiol, limonene, linalool, and fenchol for clustering target. Fenchol played a major role for clustering plant samples that originated from Washington and Nevada. k-means method was more flexible than PCA and generated three different classes; samples obtained from Oregon and California in comparison to the rest of other samples were obviously separated alone, which attributed to their unique chemical profile. Finally, both PCA and k-means were useful and quick guides for cannabis clustering based on their chemical profile. Thus, less effort, time, and materials will be consumed in addition to decreasing operational conditions for cannabis clustering.     

In Situ Decarboxylation-Pressurized Hot Water Extraction for Selective Extraction of Cannabinoids from Cannabis sativa. Chemometric Approach

Isolation of the therapeutic cannabinoid compounds from Cannabis Sativa L. (C. Sativa) is important for the development of cannabis-based pharmaceuticals for cancer treatment, among other ailments. The main pharmacological cannabinoids are THC and CBD. However, THC also induces undesirable psychoactive effects. The decarboxylation process converts the naturally occurring acidic forms of cannabinoids, such as cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA), to their more active neutral forms, known as cannabidiol (CBD) and tetrahydrocannabinol (THC). The purpose of this study was to selectively extract cannabinoids using a novel in situ decarboxylation pressurized hot water extraction (PHWE) system. The decarboxylation step was evaluated at different temperature (80–150 °C) and time (5–60 min) settings to obtain the optimal conditions for the decarboxylation-PHWE system using response surface methodology (RSM). The system was optimized to produce cannabis extracts with high CBD content, while suppressing the THC and CBN content. The identification and quantification of cannabinoid compounds were determined using UHPLC-MS/MS with external calibration. As a result, the RSM has shown good predictive capability with a p-value < 0.05, and the chosen parameters revealed to have a significant effect on the CBD, CBN and THC content. The optimal decarboxylation conditions for an extract richer in CBD than THC were set at 149.9 °C and 42 min as decarboxylation temperature and decarboxylation time, respectively. The extraction recoveries ranged between 96.56 and 103.42%, 95.22 and 99.95%, 99.62 and 99.81% for CBD, CBN and THC, respectively.     

Synthesis and antioxidative/anti-inflammatory activity of novel fullerene–polyamine conjugates

(E)-4-(Fullerenopyrrolidin-1-yl)-3-methylbut-2-enoic acid and its corresponding succinimidyl ester, readily obtained through Prato-type modification of C₆₀, were used for the selective N-acylation of polyamines. The thus obtained conjugates were evaluated for their antioxidative and anti-inflammatory activity and their cytotoxicity was determined. Members of this family of compounds showed interesting anti-lipid peroxidation, anti-lipoxygenase and anti-inflammatory activity and comparable cytocompatibility to spermidine.     

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.     

Metal phenolic network-stabilized nanocrystals of andrographolide to alleviate macrophage-mediated inflammation in-vitro

Macrophages play a crucial role in initiating, maintaining, and resolving inflammation through the phenotypic shift, inducing or inhibiting the production of inflammatory cytokines. Therefore, macrophages are potential targets for treating inflammatory diseases. Andrographolide (AND) is a potent anti-inflammatory drug that can reduce pro-inflammatory cytokines and suppress NF-κB /MAPK pathway in activated macrophages. Although AND has many medicinal properties, its lower water solubility and first-pass effect in the liver have hindered its clinical application. In this context, by using a metal phenolic network as a stabilizer, we designed and prepared highly stabilized AND nanocrystals (AND-MPN Ns) with high drug loading capacity to facilitate the clinical application of AND. Our findings showed that AND-MPN Ns could be used to enhance the anti-inflammation in-vitro via macrophage polarization, reducing pro-inflammatory cytokines IL-6 and TNF-α, and suppressing the NF-κB signaling pathway activation. The results demonstrated the potential of AND-MPN Ns to combat inflammatory diseases effectively.     

Polyphenols’ Cardioprotective Potential: Review of Rat Fibroblasts as Well as Rat and Human Cardiomyocyte Cell Lines Research

According to the World Health Organization, cardiovascular diseases are responsible for 31% of global deaths. A reduction in mortality can be achieved by promoting a healthy lifestyle, developing prevention strategies, and developing new therapies. Polyphenols are present in food and drinks such as tea, cocoa, fruits, berries, and vegetables. These compounds have strong antioxidative properties, which might have a cardioprotective effect. The aim of this paper is to examine the potential of polyphenols in cardioprotective use based on in vitro human and rat cardiomyocytes as well as fibroblast research. Based on the papers discussed in this review, polyphenols have the potential for cardioprotective use due to their multilevel points of action which include, among others, anti-inflammatory, antioxidant, antithrombotic, and vasodilatory. Polyphenols may have potential use in new and effective preventions or therapies for cardiovascular diseases, yet more clinical studies are needed.     

Targeting Inflammation by Anthocyanins as the Novel Therapeutic Potential for Chronic Diseases: An Update

Low-grade chronic inflammation (LGCI) and oxidative stress act as cooperative and synergistic partners in the pathogenesis of a wide variety of diseases. Polyphenols, including anthocyanins, are involved in regulating the inflammatory state and activating the endogenous antioxidant defenses. Anthocyanins’ effects on inflammatory markers are promising and may have the potential to exert an anti-inflammatory effect in vitro and in vivo. Therefore, translating these research findings into clinical practice would effectively contribute to the prevention and treatment of chronic disease. The present narrative review summarizes the results of clinical studies from the last 5 years in the context of the anti-inflammatory and anti-oxidative role of anthocyanins in both health and disease. There is evidence to indicate that anthocyanins supplementation in the regulation of pro-inflammatory markers among the healthy and chronic disease population. Although the inconsistencies between the result of randomized control trials (RCTs) and meta-analyses were also observed. Regarding anthocyanins’ effects on inflammatory markers, there is a need for long-term clinical trials allowing for the quantifiable progression of inflammation. The present review can help clinicians and other health care professionals understand the importance of anthocyanins use in patients with chronic diseases.     

Applications of Cannabis Sativa L. in Food and Its Therapeutic Potential: From a Prohibited Drug to a Nutritional Supplement

Hemp (Cannabis sativa L.) is a herbaceous anemophilous plant that belongs to the Cannabinaceae family. The cannabis seed (hemp) has long been utilized as a food source and is commercially important as an edible oil source. In this review, the positive and negative health effects of cannabis, the relationship between cannabis and various diseases, and the use of cannabis in various food products have been discussed. In addition, the scientific literature on the potential use of cannabis and its derivatives as a dietary supplement for the prevention and treatment of inflammatory and chronic degenerative diseases in animals and humans has been reviewed. Cannabis is being developed as a key ingredient in a variety of food items, including bakery, confectionery, beverages, dairy, fruits, vegetables, and meat. Hemp seeds are high in readily digestible proteins, lipids, polyunsaturated fatty acids (PUFA), insoluble fiber, carbs, and favorable omega-6 PUFA acid to omega-3 PUFA ratio and have high nutritional value. The antioxidants of cannabis, such as polyphenols, help with anxiety, oxidative stress, and the risk of chronic illnesses, including cancer, neurological disorders, digestive problems, and skin diseases. Cannabis has been shown to have negative health impacts on the respiratory system, driving, and psychomotor functions, and the reproductive system. Overall, the purpose of this research is to stimulate more in-depth research on cannabis’s adaptation in various foods and for the treatment of chronic illnesses.     

Anti-Inflammatory Effects of Phytochemical Components of Clinacanthus nutans

Recent studies on the ethnomedicinal use of Clinacanthus nutans suggest promising anti-inflammatory, anti-tumorigenic, and antiviral properties for this plant. Extraction of the leaves with polar and nonpolar solvents has yielded many C-glycosyl flavones, including schaftoside, isoorientin, orientin, isovitexin, and vitexin. Aside from studies with different extracts, there is increasing interest to understand the properties of these components, especially regarding their ability to exert anti-inflammatory effects on cells and tissues. A major focus for this review is to obtain information on the effects of C. nutans extracts and its phytochemical components on inflammatory signaling pathways in the peripheral and central nervous system. Particular emphasis is placed on their role to target the Toll-like receptor 4 (TLR4)-NF-kB pathway and pro-inflammatory cytokines, the antioxidant defense pathway involving nuclear factor erythroid-2-related factor 2 (NRF2) and heme oxygenase 1 (HO-1); and the phospholipase A₂ (PLA₂) pathway linking to cyclooxygenase-2 (COX-2) and production of eicosanoids. The ability to provide a better understanding of the molecular targets and mechanism of action of C. nutans extracts and their phytochemical components should encourage future studies to develop new therapeutic strategies for better use of this herb to combat inflammatory diseases.