Biotechnological Approaches on Two High CBD and CBG Cannabis sativa L. (Cannabaceae) Varieties: In Vitro Regeneration and Phytochemical Consistency Evaluation of Micropropagated Plants Using Quantitative 1H-NMR

High cannabidiol (CBD) and cannabigerol (CBG) varieties of Cannabis sativa L., a species with medicinal properties, were regenerated in vitro. Explants of nodal segments including healthy axillary bud, after sterilization, were placed in Murashige-Skoog (MS) culture medium. The shoots formed after 30 days were subcultured in full- or half-strength MS medium supplemented with several concentrations of 6-benzyl-amino-purine (BA) or thidiazuron (TDZ). The highest average number and length of shoots was achieved when both full and half-strength MS media were supplemented with 4.0 μM BA. The presence of 4.0 μM TDZ showed also comparable results. BA and TDZ at concentrations of 4.0, 8.0 μM and 2.0, 4.0 μM respectively, displayed the maximum shooting frequency. The new shoots were transferred on the same media and were either self-rooted or after being enhanced with different concentrations of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Presence of 2.0 or 4.0 μM IBA or 4.0 μM NAA resulted to the optimum rooting rates. The maximum average number and length of roots per shoot was observed when the culture media was supplemented with 4.0 μM IBA or NAA. Approximately 92% of the plantlets were successfully established and acclimatized in field. The consistency of the chemical profile of the acclimatized in vitro propagated clones was assessed using quantitative ¹H-NMR high throughput screening. In each variety, analysis of the micropropagated plant in comparison with the mother plant showed no statistically significant differences (p ≤ 0.05) in CBD+ cannabidiolic acid (CBDA) and CBG+ cannabigerolic acid (CBGA) content respectively, thus indicating stability of their chemical profile.     

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.     

Cannabis-Derived Compounds Cannabichromene and Δ9-Tetrahydrocannabinol Interact and Exhibit Cytotoxic Activity against Urothelial Cell Carcinoma Correlated with Inhibition of Cell Migration and Cytoskeleton Organization

Cannabis sativa contains more than 500 constituents, yet the anticancer properties of the vast majority of cannabis compounds remains unknown. We aimed to identify cannabis compounds and their combinations presenting cytotoxicity against bladder urothelial carcinoma (UC), the most common urinary system cancer. An XTT assay was used to determine cytotoxic activity of C. sativa extracts on T24 and HBT-9 cell lines. Extract chemical content was identified by high-performance liquid chromatography (HPLC). Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and cell cycle, using stained F-actin and nuclei. Scratch and transwell assays were used to determine cell migration and invasion, respectively. Gene expression was determined by quantitative Polymerase chain reaction (PCR). The most active decarboxylated extract fraction (F7) of high-cannabidiol (CBD) C. sativa was found to contain cannabichromene (CBC) and Δ9-tetrahydrocannabinol (THC). Synergistic interaction was demonstrated between CBC + THC whereas cannabinoid receptor (CB) type 1 and type 2 inverse agonists reduced cytotoxic activity. Treatments with CBC + THC or CBD led to cell cycle arrest and cell apoptosis. CBC + THC or CBD treatments inhibited cell migration and affected F-actin integrity. Identification of active plant ingredients (API) from cannabis that induce apoptosis and affect cell migration in UC cell lines forms a basis for pre-clinical trials for UC treatment.     

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.     

Cannabinoids—Characteristics and Potential for Use in Food Production

Scientific demonstrations of the beneficial effects of non-psychoactive cannabinoids on the human body have increased the interest in foods containing hemp components. This review systematizes the latest discoveries relating to the characteristics of cannabinoids from Cannabis sativa L. var. sativa, it also presents a characterization of the mentioned plant. In this review, we present data on the opportunities and limitations of cannabinoids in food production. This article systematizes the data on the legal aspects, mainly the limits of Δ9-THC in food, the most popular analytical techniques (LC-MS and GC-MS) applied to assay cannabinoids in finished products, and the available data on the stability of cannabinoids during heating, storage, and access to light and oxygen. This may constitute a major challenge to their common use in food processing, as well as the potential formation of undesirable degradation products. Hemp-containing foods have great potential to become commercially popular among functional foods, provided that our understanding of cannabinoid stability in different food matrices and cannabinoid interactions with particular food ingredients are expanded. There remains a need for more data on the effects of technological processes and storage on cannabinoid degradation.     

Santamarine Shows Anti-Photoaging Properties via Inhibition of MAPK/AP-1 and Stimulation of TGF-β/Smad Signaling in UVA-Irradiated HDFs

Chronic UVA exposure results in elevated reactive oxygen species in skin which leads to photoaging characterized as upregulated matrix metalloproteinase (MMP)-1 and loss of collagen. Therefore, natural antioxidants are hailed as promising agents to be utilized against photoaging. In the current study, reynosin and santamarine, two known sesquiterpene lactones isolated from Artemisia scoparia, were analyzed for their anti-photoaging properties in UVA-irradiated human dermal fibroblasts (HDFs). Results showed that UVA irradiation (8 J/cm²) upregulated the MMP-1 secretion and expression, and suppressed collagen production, which were significantly reverted by santamarine treatment (10 µM). Although both reynosin and santamarine exhibited ROS scavenging abilities, reynosin failed to significantly diminish UVA-stimulated MMP-1 release. UVA-irradiated HDFs showed increased collagen production when treated with santamarine. As a mechanism to suppress MMP-1, santamarine significantly suppressed the UVA-induced phosphorylation of p38 and JNK and nuclear translocation of p-c-Fos and p-c-Jun. Santamarine promoted collagen I production via relieving the UVA-induced suppression on TGF-β and its downstream activator Smad2/3 complex. Antioxidant properties of santamarine were also shown to arise from stimulating Nrf2-dependent expression of antioxidant enzymes SOD-1 and HO-1 in UVA-irradiated HDFs. In conclusion, santamarine was found to be a promising natural antioxidant with anti-photoaging properties against UVA-induced damages in HDFs.     

Phytochemical and In Silico ADME/Tox Analysis of Eruca sativa Extract with Antioxidant,Antibacterial and Anticancer Potential against Caco-2 and HCT-116 Colorectal Carcinoma Cell Lines

Eruca sativa Mill. (E. sativa) leaves recently grabbed the attention of scientific communities around the world due to its potent bioactivity. Therefore, the present study investigates the metabolite profiling of the ethanolic crude extract of E. sativa leaves using high resolution-liquid chromatography-mass spectrometry (HR-LC/MS), including antibacterial, antioxidant and anticancer potential against human colorectal carcinoma cell lines. In addition, computer-aided analysis was performed for determining the pharmacokinetic properties and toxicity prediction of the identified compounds. Our results show that E. sativa contains several bioactive compounds, such as vitamins, fatty acids, alkaloids, flavonoids, terpenoids and phenols. Furthermore, the antibacterial assay of E. sativa extract showed inhibitory effects of the tested pathogenic bacterial strains. Moreover, the antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide (H₂O₂) were found to be IC₅₀ = 66.16 μg/mL and 76.05 μg/mL, respectively. E. sativa also showed promising anticancer activity against both the colorectal cancer cells HCT-116 (IC₅₀ = 64.91 μg/mL) and Caco-2 (IC₅₀ = 83.98 μg/mL) in a dose/time dependent manner. The phytoconstituents identified showed promising pharmacokinetics properties, representing a valuable source for drug or nutraceutical development. These investigations will lead to the further exploration as well as development of E. sativa-based nutraceutical products.     

Calculated and experimental 1H and 13C NMR assignments for cannabicitran

Cannabicitran is an important cannabinoid natural product produced by Cannabis sativa and is often found at surprisingly high levels (up to ~10%) in “purified” commercial cannabidiol (CBD) extract preparations. Despite the prevalence of this molecule in CBD oil and other cannabinoid-related products, and the rapidly expanding interest in cannabinoids for treatment of a wide range of physiological conditions, only unassigned ¹H NMR data and partial unambiguous ¹³C assignments have been published. Herein, we report the complete ¹H and ¹³C NMR assignments of cannabicitran and comparatively evaluate the performance of several density functional theory (DFT) methods with varying levels of theory for the calculation of NMR chemical shifts.     

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.     

The Effects of Food on Cannabidiol Bioaccessibility

Cannabidiol (CBD) is a hydrophobic non-psychoactive compound with therapeutic characteristics. Animal and human studies have shown its poor oral bioavailability in vivo, and the impact of consuming lipid-soluble CBD with and without food on gut bioaccessibility has not been explored. The purpose of this research was to study the bioaccessibility of CBD after a three-phase upper digestion experiment with and without food, and to test lipase activity with different substrate concentrations. Our results showed that lipase enzyme activity and fatty acid absorption increased in the presence of bile salts, which may also contribute to an increase in CBD bioaccessibility. The food matrix used was a mixture of olive oil and baby food. Overall, the fed-state digestion revealed significantly higher micellarization efficiency for CBD (14.15 ± 0.6% for 10 mg and 22.67 ± 2.1% for 100 mg CBD ingested) than the fasted state digestion of CBD (0.65 ± 0.7% for 10 mg and 0.14 ± 0.1% for 100 mg CBD ingested). The increase in bioaccessibility of CBD with food could be explained by the fact that micelle formation from hydrolyzed lipids aid in bioaccessibility of hydrophobic molecules. In conclusion, the bioaccessibility of CBD depends on the food matrix and the presence of lipase and bile salts.     

Yield,Characterization, and Possible Exploitation of Cannabis Sativa L. Roots Grown under Aeroponics Cultivation

Cannabis sativa L. has been used for a long time to obtain food, fiber, and as a medicinal and psychoactive plant. Today, the nutraceutical potential of C. sativa is being increasingly reappraised; however, C. sativa roots remain poorly studied, despite citations in the scientific literature. In this direction, we identified and quantified the presence of valuable bioactives (namely, β-sitosterol, stigmasterol, campesterol, friedelin, and epi-friedelanol) in the root extracts of C. sativa, a finding which might pave the way to the exploitation of the therapeutic potential of all parts of the C. sativa plant. To facilitate root harvesting and processing, aeroponic (AP) and aeroponic-elicited cultures (AEP) were established and compared to soil-cultivated plants (SP). Interestingly, considerably increased plant growth—particularly of the roots—and a significant increase (up to 20-fold in the case of β-sitosterol) in the total content of the aforementioned roots’ bioactive molecules were observed in AP and AEP. In conclusion, aeroponics, an easy, standardized, contaminant-free cultivation technique, facilitates the harvesting/processing of roots along with a greater production of their secondary bioactive metabolites, which could be utilized in the formulation of health-promoting and health-care products.     

Cannabis sativa CBD Extract Shows Promising Antibacterial Activity against Salmonella typhimurium and S. newington

Products derived from Cannabis sativa L. have gained increased interest and popularity. As these products become common amongst the public, the health and potential therapeutic values associated with hemp have become a premier focus of research. While the psychoactive and medicinal properties of Cannabis products have been extensively highlighted in the literature, the antibacterial properties of cannabidiol (CBD) have not been explored in depth. This research serves to examine the antibacterial potential of CBD against Salmonella newington and S. typhimurium. In this study, we observed bacterial response to CBD exposure through biological assays, bacterial kinetics, and fluorescence microscopy. Additionally, comparative studies between CBD and ampicillin were conducted against S. typhimurium and S. newington to determine comparative efficacy. Furthermore, we observed potential resistance development of our Salmonella spp. against CBD treatment.     

Botanically-Derived Δ9-Tetrahydrocannabinol and Cannabidiol,and Their 1:1 Combination,Modulate Toll-like Receptor 3 and 4 Signalling in Immune Cells from People with Multiple Sclerosis

The innate immune response to bacterial and viral molecules involves the coordinated production of cytokines, chemokines, and type I interferons (IFNs), which is orchestrated by toll-like receptors (TLRs). TLRs, and their intracellular signalling intermediates, are closely associated with multiple sclerosis (MS) pathogenesis. Recent data from our laboratory reported that the plant-derived cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD), regulate viral and bacterial inflammatory signalling pathways controlled by TLR3 and TLR4 in macrophages. The aim of this study was to assess the impact of THC and CBD, when delivered in isolation and in combination (1:1), on TLR3- and TLR4-dependent signalling in peripheral blood mononuclear cells (PBMCs) from people with MS (pwMS; n = 21) and healthy controls (HCs; n = 26). We employed the use of poly(I:C) and lipopolysaccharide (LPS) to induce viral TLR3 and bacterial TLR4 signalling, and PBMCs were pre-exposed to plant-derived highly purified THC (10 μM), CBD (10 μM), or a combination of both phytocannabinoids (1:1 ratio, 10:10 μM), prior to LPS/poly(I:C) exposure. TLR3 stimulation promoted the protein expression of the chemokine CXCL10 and the type I IFN-β in PBMCs from both cohorts. THC and CBD (delivered in 1:1 combination at 10 μM) attenuated TLR3-induced CXCL10 and IFN-β protein expression in PBMCs from pwMS and HCs, and this effect was not seen consistently when THC and CBD were delivered alone. In terms of LPS, TLR4 activation promoted TNF-α expression in PBMCs from both cohorts, and, interestingly, CBD when delivered alone at 10 μM, and in combination with THC (in 1:1 combination at 10 μM), exacerbated TLR4-induced TNF-α protein expression in PBMCs from pwMS and HCs. THC and CBD displayed no evidence of toxicity in primary PBMCs. No significant alteration in the relative expression of TLR3 and TLR4 mRNA, or components of the endocannabinoid system, including the cannabinoid receptor CB₁ (encoded by CNR1 gene) and CB₂ (encoded by CNR2 gene), and endocannabinoid metabolising enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGLL), was determined in PBMCs from pwMS versus HCs. Given their role in inflammation, TLRs are clinical targets, and data herein identify CBD and THC as TLR3 and TLR4 modulating drugs in primary immune cells in vitro. This offers insight on the cellular target(s) of phytocannabinoids in targeting inflammation in the context of MS.     

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded ¹H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The ¹H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same ¹H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The ¹H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in ¹H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by ¹H qNMR was not feasible.     

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.     

Oxidative Stress and Multi-Organel Damage Induced by Two Novel Phytocannabinoids,CBDB and CBDP,in Breast Cancer Cells

Over the last few years, much attention has been paid to phytocannabinoids derived from Cannabis for their therapeutic potential. Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) are the most abundant compounds of the Cannabis sativa L. plant. Recently, novel phytocannabinoids, such as cannabidibutol (CBDB) and cannabidiphorol (CBDP), have been discovered. These new molecules exhibit the same terpenophenolic core of CBD and differ only for the length of the alkyl side chain. Roles of CBD homologs in physiological and pathological processes are emerging but the exact molecular mechanisms remain to be fully elucidated. Here, we investigated the biological effects of the newly discovered CBDB or CBDP, compared to the well-known natural and synthetic CBD (nat CBD and syn CBD) in human breast carcinoma cells that express CB receptors. In detail, our data demonstrated that the treatment of cells with the novel phytocannabinoids affects cell viability, increases the production of reactive oxygen species (ROS) and activates cellular pathways related to ROS signaling, as already demonstrated for natural CBD. Moreover, we observed that the biological activity is significantly increased upon combining CBD homologs with drugs that inhibit the activity of enzymes involved in the metabolism of endocannabinoids, such as the monoacylglycerol lipase (MAGL) inhibitor, or with drugs that induces the activation of cellular stress pathways, such as the phorbol ester 12-myristate 13-acetate (PMA).     

The Medicinal Natural Products of Cannabis sativa Linn.: A Review

Cannabis sativa is known among many cultures for its medicinal potential. Its complexity contributes to the historical application of various parts of the plant in ethno-medicines and pharmacotherapy. C. sativa has been used for the treatment of rheumatism, epilepsy, asthma, skin burns, pain, the management of sexually transmitted diseases, difficulties during child labor, postpartum hemorrhage, and gastrointestinal activity. However, the use of C. sativa is still limited, and it is illegal in most countries. Thus, this review aims to highlight the biological potential of the plant parts, as well as the techniques for the extraction, isolation, and characterization of C. sativa compounds. The plant produces a unique class of terpenophenolic compounds, called cannabinoids, as well as non-cannabinoid compounds. The exhaustive profiling of bioactive compounds and the chemical characterization and analysis of C. sativa compounds, which modern research has not yet fully achieved, is needed for the consistency, standardization, and the justified application of Cannabis sativa products for therapeutic purposes. Studies on the clinical relevance and applications of cannabinoids and non-cannabinoid phenols in the prevention and treatment of life-threatening diseases is indeed significant. Furthermore, psychoactive cannabinoids, when chemically standardized and administered under medical supervision, can be the legal answer to the use of C. sativa.     

Oat (Avena sativa) Extract against Oxidative Stress-Induced Apoptosis in Human Keratinocytes

Oat (Avena sativa) is well known for its various health benefits. The protective effect of oat extract against oxidative stress-induced apoptosis in human keratinocytes HaCaT was determined. First, extracts of two varieties of oat, Daeyang and Choyang, were analyzed for fat-soluble antioxidants such as α-tocotrienol, γ-oryzanols, lutein and zeaxanthin using an UPLC system and for antioxidant activity using a DPPH assay. Specifically, an 80% ethanol extract of Daeyang oat (Avena sativa cv. Daeyang), which had high amounts of antioxidants and potent radical scavenging activity, was further evaluated for protective effect against oxidative stress-induced cell death, intracellular reactive oxygen species levels, the phosphorylation of DNA damage mediating genes such as H2AX, checkpoint kinase 1 and 2, and p53 and the activation of apoptotic genes such as cleaved caspase-3 and 7 and poly (ADP-ribose) polymerase in HaCaT cells. The Daeyang and Choyang oat 80% ethanol extracts had 26.9 and 24.1 mg/100 g γ-oryzanols, 7.69 and 8.38 mg/100 g α-tocotrienol, 1.25 and 0.34 mg/100 g of lutein and 1.20 and 0.17 mg/100 g of zeaxanthin, respectively. The oat 80% ethanol extract treatment (Avena sativa cv. Daeyang) had a protective effect on oxidative stress-induced cell death in HaCaT cells. In addition, the oat 80% ethanol extracts led to a significant decrease in the intracellular ROS level at a concentration of 50–200 μg/mL, the attenuation of DNA damage mediating genes and the inhibition of apoptotic caspase activities in a dose dependent manner (50–200 μg/mL). Thus, the current study indicates that an oat (Avena sativa cv. Daeyang) extract rich in antioxidants, such as polyphenols, avenanthramides, γ-oryzanols, tocotrienols and carotenoids, has a protective role against oxidative stress-induced keratinocyte injuries and that oat may a useful source for oxidative stress-associated skin damage.     

A New Butyrate Releaser Exerts a Protective Action against SARS-CoV-2 Infection in Human Intestine

Butyrate is a major gut microbiome metabolite that regulates several defense mechanisms against infectious diseases. Alterations in the gut microbiome, leading to reduced butyrate production, have been reported in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A new butyrate releaser, useful for all the known applications of butyrate, presenting physiochemical characteristics suitable for easy oral administration, (N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), has been recently developed. We investigated the protective action of FBA against SARS-CoV-2 infection in the human small intestine and enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), neuropilin-1 (NRP1), pro-inflammatory cytokines expression, genes involved in the antiviral response and the activation of Nf-kB nuclear factor (erythroid-derived 2-like) 2 (Nfr2) pathways. We found that FBA positively modulates the crucial aspects of the infection in small intestinal biopsies and human enterocytes, reducing the expression of ACE2, TMPRSS2 and NRP1, pro-inflammatory cytokines interleukin (IL)-15, monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and regulating several genes involved in antiviral pathways. FBA was also able to reduce the number of SARS-CoV-2-infected cells, and ACE2, TMPRSS2 and NRP1 expression. Lastly, through the inhibition of Nf-kB and the up-regulation of Nfr2, it was also able to reduce the expression of pro-inflammatory cytokines IL-15, MCP-1 and TNF-α in human enterocytes. The new butyrate releaser, FBA, exerts a preventive action against SARS-CoV-2 infection. It could be considered as an innovative strategy to limit COVID-19.     

Impact of enzymatic and alkaline hydrolysis on CBD concentration in urine

A sensitive and specific analytical method for cannabidiol (CBD) in urine was needed to define urinary CBD pharmacokinetics after controlled CBD administration, and to confirm compliance with CBD medications including Sativex—a cannabis plant extract containing 1:1 ?⁹-tetrahydrocannabinol (THC) and CBD. Non-psychoactive CBD has a wide range of therapeutic applications and may also influence psychotropic smoked cannabis effects. Few methods exist for the quantification of CBD excretion in urine, and no data are available for phase II metabolism of CBD to CBD-glucuronide or CBD-sulfate. We optimized the hydrolysis of CBD-glucuronide and/or -sulfate, and developed and validated a GC-MS method for urinary CBD quantification. Solid-phase extraction isolated and concentrated analytes prior to GC-MS. Method validation included overnight hydrolysis (16 h) at 37 °C with 2,500 units β-glucuronidase from Red Abalone. Calibration curves were fit by linear least squares regression with 1/x ² weighting with linear ranges (r ²?>?0.990) of 2.5–100 ng/mL for non-hydrolyzed CBD and 2.5–500 ng/mL for enzyme-hydrolyzed CBD. Bias was 88.7–105.3 %, imprecision 1.4–6.4 % CV and extraction efficiency 82.5–92.7 % (no hydrolysis) and 34.3–47.0 % (enzyme hydrolysis). Enzyme-hydrolyzed urine specimens exhibited more than a 250-fold CBD concentration increase compared to alkaline and non-hydrolyzed specimens. This method can be applied for urinary CBD quantification and further pharmacokinetics characterization following controlled CBD administration.