Cannabinoids as New Drug Candidates for the Treatment of Glaucoma.

Glaucoma is a blinding eye disease that affects about 70 million patients globally today. The cannabinoid receptors and the endocannabinoid system have found attention for new drug concepts. This review will analyze the potential of cannabinoids, primarily tetrahydrocannabinol, THCVS, and cannabinol, as drug candidates and the role of CB1/CB2 receptors with regard to the pathophysiology of glaucoma. The mode of action of cannabinoids as innovative drug candidates and recent formulations for topical delivery will be discussed. Cannabinoid receptors with associated TRPV channels will be evaluated for their potential as drug targets. Especially the role of the endocannabinoid system (fatty acid amide hydrolase, monoacylglycerol lipase) impacting the prostaglandin network (cyclooxygenase, PGE, PGF) and neuroprotection by inhibition of nitric oxide radical formation is in the focus of this review. Delivery systems, including recent clinical trials, will be analyzed to evaluate the potential for innovative future ophthalmological drugs.

Activity of THC, CBD, and CBN on Human ACE2 and SARS-CoV1/2 Main Protease to Understand Antiviral Defense Mechanism.

THC, CBD, and CBN were reported as promising candidates against SARS-CoV2 infection, but the mechanism of action of these three cannabinoids is not understood. This study aims to determine the mechanism of action of THC, CBD, and CBN by selecting two essential targets that directly affect the coronavirus infections as viral main proteases and human angiotensin-converting enzyme2. Tested THC and CBD presented a dual-action action against both selected targets. Only CBD acted as a potent viral main protease inhibitor at the IC50 value of 1.86 ± 0.04 µM and exhibited only moderate activity against human angiotensin-converting enzyme2 at the IC50 value of 14.65 ± 0.47 µM. THC acted as a moderate inhibitor against both viral main protease and human angiotensin-converting enzymes2 at the IC50 value of 16.23 ± 1.71 µM and 11.47 ± 3.60 µM, respectively. Here, we discuss cannabinoid-associated antiviral activity mechanisms based on in silico docking studies and in vitro receptor binding studies.

Cannabis sativa research trends, challenges, and new-age perspectives.

Cannabis sativa L. has been one of the oldest medicinal plants cultivated for 10,000 years for several agricultural and industrial applications. However, the plant became controversial owing to some psychoactive components that have adverse effects on human health. In this review, we analyzed the trends in cannabis research for the past two centuries. We discussed the historical transitions of cannabis from the category of herbal medicine to an illicit drug and back to a medicinal product post-legalization. In addition, we address the new-age application of immuno-suppressive and anti-inflammatory extracts for the treatment of COVID-19 inflammation. We further address the influence of the legal aspects of cannabis cultivation for medicinal, pharmaceutical, and biotechnological research. We reviewed the up-to-date cannabis genomic resources and advanced technologies for their potential application in genomic-based cannabis improvement. Overall, this review discusses the diverse aspects of cannabis research developments ranging from traditional use as herbal medicine to the latest potential in COVID-19, legal practices with updated patent status, and current state of art genetic and genomic tools reshaping cannabis biotechnology in modern age agriculture and pharmaceutical industry.

In vitro metabolism of tebuconazole, flurtamone, fenhexamid, metalaxyl-M and spirodiclofen in Cannabis sativa L. (hemp) callus cultures.

BACKGROUND: Cannabis sativa L. (hemp) is a medicinal plant producing various cannabinoids. Its consumption is legalized for medical use due to the alleged positive health effects of these cannabinoids. To satisfy the demand, C. sativa plants are propagated in contained growth chambers. During indoor propagation, pesticides usually are used to ensure efficient production. However, pesticide registration and safe application in C. sativa has not been investigated in detail. RESULTS: With this study the metabolic degradation of pesticides in recently established C. sativa callus cultures was examined. Tebuconazole, metalaxyl-M fenhexamid, flurtamone and spirodiclofen were applied at 10 µm for 21 days. Results were compared with metabolism data obtained from Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Tritium aestivum L. callus cultures as well as in metabolism guideline studies. The successfully established C. sativa callus cultures were able to degrade pesticides by oxidation, demethylation, and cleavage of ester bonds in phase I, as well as glycosylation and conjugation with malonic acid in phase II and III. Initial metabolites were detected after Day (D)7 and were traced at D21. CONCLUSION: The resulting pathways demonstrate the same main degradation strategies as crop plants. Because metabolites could be the main residue, the exposure of consumers to these residues will be of high importance. We present here an in vitro assay for a first estimation of pesticide metabolism in C. sativa. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Challenges at the Time of COVID-19: Opportunities and Innovations in Antivirals from Nature.

As viral infections are an increasing threat to human societies, the need for new therapeutic strategies is becoming even more obvious. As no vaccine is available for COVID-19, the development of directly acting antiviral agents and preventive strategies have to be considered. Nature provides a huge reservoir of anti-infectious compounds, from which we can deduce innovative ideas, therapies, and products. Anti-adhesive natural products interact with the receptor-mediated recognition and early interaction of viruses with the host cells, leading to a reduced internalisation of the virus and reduced infections (e.g., procyanidin-B-2-di-O-gallate against influenza and herpes virus). Lignans like podophyllotoxin and bicyclol show strong antiviral activities against different viruses, and essential oils can directly interact with viral membranes and reduce the host's inflammatory responses (e.g., 1,8-cineol). Echinacea extracts stimulate the immune system, and bioavailable alkamides are key players by interacting with immunomodulating cannabinoid receptors. COVID-19 and SARS-CoV-2 infections have, in part, successfully been treated in China by preparations from traditional Chinese medicine and, while it is too early to draw conclusions, some promising data are available. There is huge potential, but intensified research is needed to develop evidence-based medicines with a clearly defined chemical profile. Intensified research and development, and therefore funding, are needed for exploiting nature's reservoir against viral infections. Combined action for basic research, chemistry, pharmacognosy, virology, and clinical studies, but also supply chain, sustainable sourcing, and economic aspects have to be considered. This review calls for intensified innovative science on natural products for the patients and for a healthier world!

Best practice in research - Overcoming common challenges in phytopharmacological research.

BACKGROUND: The pharmacology, toxicology and pharmacokinetics of bioactive preparations derived from natural sources has become a flourishing field of research. However, researching complex extracts and natural products faces numerous challenges. More broadly in recent years the critique of pharmacological research, and specifically its design, the methods used and reporting has intensified. AIMS: This consensus document provides a perspective on what constitutes best practice in pharmacological research on bioactive preparations derived from natural sources, providing a perspective of what the leading specialist journals in the field consider as the core characteristics of good research. APPROACH ('METHODS'): The editors-in-chief of seven journals developed this best practice statement in an iterative process. A first draft of the guidelines (prepared by MH) was then discussed and amended by the other editors. OUTCOMES: Core to this contribution is a table which provides detailed advice including simple points like a use of appropriate controls and the full taxonomic validity of the material under investigation (see also below), to the relevance of the model for the question being researched (e.g., can specific in silico or in vitro models really address the species anti-inflammatory activity?). Therefore, obviously, researchers must pay detailed attention to reporting and discussing such studies. This information must be discussed critically (as much as it is possible based on the published papers) in terms of their scientific quality and validity. While these points are obvious, as editors, we are aware that they are often not properly implemented. CONCLUSION: We call for an approach which incorporates a careful design, meticulous execution and a detailed reporting of studies focusing on the pharmacology/bioactivity of bioactive preparations. Clearly testable research questions must be developed and investigated experimentally. As the founder of pharmacology Claude Bernard put it already in 1865: '…. either the experimenter's hypothesis will be disproved or it will be proved by experiment. When experiment disproves its preconceived ideas, the experimenter must discard or modify it.'

Tropane Alkaloids: Chemistry, Pharmacology, Biosynthesis and Production.

Tropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis in planta and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.

Traditional use of ethnomedicinal native plants in the Kingdom of Saudi Arabia.

The Arabian Peninsula is recognized as an arid area dominated by deserts and poor biodiversity. However, the Kingdom of Saudi Arabia (henceforth abbreviated into KSA) has a wide range of flora, consisting of different species of trees, herbs, and shrubs and containing numerous edible and medicinal plants. The KSA is characterized by its vast area of diverse geographical landscapes and climates. Consequently, there is enormous variation in the distribution of plants across the Kingdom. The traditional use of ethnomedical plants in the KSA represents a strong interconnection among familiar remedies, health, diet, and traditional healing practices characterized by specific cultures. The present paper reviews a collection of medicinal plants in KSA used in ethnomedicine. This review might be useful in developing strategies for the sustainable use of medicinal plants which are among the threatened important natural resources in folk medicine in the KSA. The present study reports 309 genera which cover 471 species from a total of 2253 known species belonging to 89 families. The most dominating families are Asteraceae, Fabaceae, Lamiaceae, Euphorbiaceae, Solanaceae, Apiaceae, Brassicaceae, Chenopodiaceae, Poaceae, Amaranthaceae, Boraginaceae, Apocynaceae, Convolvoulaceae, Asclepiadaceae, Capparaceae, Polygonaceae, and Zygophyllaceae.

Virus-induced gene silencing (VIGS) in Cannabis sativa L.

BACKGROUND: The raised demand of cannabis as a medicinal plant in recent years led to an increased interest in understanding the biosynthetic routes of cannabis metabolites. Since there is no established protocol to generate stable gene knockouts in cannabis, the use of a virus-induced gene silencing (VIGS) method, resulting in a gene knockdown, to study gene functions is desirable. RESULTS: For this, a computational approach was employed to analyze the Cannabis sativa L. transcriptomic and genomic resources. Reporter genes expected to give rise to easily scorable phenotypes upon silencing, i.e. the phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI), were identified in C. sativa. Subsequently, the targets of specific small interfering RNAs (siRNAs) and silencing fragments were predicted and tested in a post-transcriptional gene silencing (PTGS) approach. Here we show for the first time a gene knockdown in C. sativa using the Cotton leaf crumple virus (CLCrV) in a silencing vector system. Plants transiently transformed with the Agrobacterium tumefaciens strain AGL1, carrying the VIGS-vectors, showed the desired phenotypes, spotted bleaching of the leaves. The successful knockdown of the genes was additionally validated by quantitative PCR resulting in reduced expression of transcripts from 70 to 73% for ChlI and PDS, respectively. This is accompanied with the reduction of the chlorophyll a and carotenoid content, respectively. In summary, the data clearly demonstrate the potential for functional gene studies in cannabis using the CLCrV-based vector system. CONCLUSIONS: The applied VIGS-method can be used for reverse genetic studies in C. sativa to identify unknown gene functions. This will gain deeper inside into unknown biosynthetic routes and will help to close the gap between available genomic data and biochemical information of this important medicinal plant.

Chemical fingerprinting of single glandular trichomes of Cannabis sativa by Coherent anti-Stokes Raman scattering (CARS) microscopy.

BACKGROUND: Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type. RESULTS: Hyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ9-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures. CONCLUSION: Here we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.

The Phytochemical and Biological Investigation of Jatropha pelargoniifolia Root Native to the Kingdom of Saudi Arabia.

Extensive phytochemical analysis of different root fractions of Jatropha pelargoniifolia Courb. (Euphorbiaceae) has resulted in the isolation and identification of 22 secondary metabolites. 6-hydroxy-8-methoxycoumarin-7-O-ß-d-glycopyranoside (15) and 2-hydroxymethyl N-methyltryptamine (18) were isolated and identified as new compounds along with the known diterpenoid (1, 3, 4, and 7), triterpenoid (2 and 6), flavonoid (5, 11, 13, 14, and 16), coumarinolignan (8⁻10), coumarin (15), pyrimidine (12), indole (17, 18), and tyramine-derived molecules (19⁻22). The anti-inflammatory, analgesic, and antipyretic activities were evaluated for fifteen of the adequately available isolated compounds (1⁻6, 8⁻11, 13, 14, 16, 21, and 22). Seven (4, 6, 10, 5, 13, 16, and 22) of the tested compounds showed a significant analgesic effect ranging from 40% to 80% at 10 mg/kg in two in vivo models. Compound 1 could also prove its analgesic property (67.21%) when it was evaluated on a third in vivo model at the same dose. The in vitro anti-inflammatory activity was also recorded where all compounds showed the ability to scavenge nitric oxide (NO) radical in a dose-dependent manner. However, eight compounds (1, 4, 5, 6, 10, 13, 16, and 22) out of the fifteen tested compounds exhibited considerable in vivo anti-inflammatory activity which reached 64.91% for compound 10 at a dose of 10 mg/kg. Moreover, the tested compounds exhibited an antipyretic effect in a yeast-induced hyperthermia in mice. The activity was found to be highly pronounced with compounds 1, 5, 6, 10, 13, and 16 which decreased the rectal temperature to about 37 °C after 2 h of the induced hyperthermia (~39 °C) at a dose of 10 mg/kg. This study could provide scientific evidence for the traditional use of J. pelargoniifolia as an anti-inflammatory, analgesic, and antipyretic.

Ethnobotany and Medicinal Plant Biotechnology: From Tradition to Modern Aspects of Drug Development.

Secondary natural products from plants are important drug leads for the development of new drug candidates for rational clinical therapy and exhibit a variety of biological activities in experimental pharmacology and serve as structural template in medicinal chemistry. The exploration of plants and discovery of natural compounds based on ethnopharmacology in combination with high sophisticated analytics is still today an important drug discovery to characterize and validate potential leads. Due to structural complexity, low abundance in biological material, and high costs in chemical synthesis, alternative ways in production like plant cell cultures, heterologous biosynthesis, and synthetic biotechnology are applied. The basis for any biotechnological process is deep knowledge in genetic regulation of pathways and protein expression with regard to todays "omics" technologies. The high number genetic techniques allowed the implementation of combinatorial biosynthesis and wide genome sequencing. Consequently, genetics allowed functional expression of biosynthetic cascades from plants and to reconstitute low-performing pathways in more productive heterologous microorganisms. Thus, de novo biosynthesis in heterologous hosts requires fundamental understanding of pathway reconstruction and multitude of genes in a foreign organism. Here, actual concepts and strategies are discussed for pathway reconstruction and genome sequencing techniques cloning tools to bridge the gap between ethnopharmaceutical drug discovery to industrial biotechnology.

Current Perspectives on Biotechnological Cannabinoid Production in Plants.

The plant Cannabis sativa contains a number of psychoactive chemical compounds, the cannabinoids, which possess a significant pharmaceutical potential. Recently, the usage of Cannabis for medicinal purposes was legalized in many countries. Thus, the study on the influence of different cannabinoids in combination with other Cannabis-derived compounds with respect to the treatment of various diseases becomes increasingly important. Besides the production of distinct cannabinoids in a heterologous host, like tobacco or yeast, transgenic Cannabis plants would be a suitable alternative to modify and therefore optimize the cannabinoid profile. This perspective highlights the current efforts on Cannabis cell culture systems, in vitro propagation, and transformation of the plant and reveals the resulting opportunities concerning biotechnological production of cannabinoids. Furthermore, alternative platform organisms for the heterologous production of cannabinoids, like tobacco, are considered and evaluated.

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6ß-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

Influence of Light, Temperature, and Macronutrients on Growth and Scopolamine Biosynthesis in Duboisia species.

Scopolamine is used in the pharmaceutical industry as a precursor in the organic synthesis of different classes of important active substances and is extracted in large scale from field grown Duboisia plants. Previous research revealed that plant growth as well as production of scopolamine and its derivatives varies strongly depending on abiotic factors. However, only a small amount of systematic research has been done on the influence of environmental conditions on scopolamine and biomass production, so far. In order to extend knowledge in this field, plants of three different genotypes (wild type Duboisia myoporoides and hybrids of D. myoporoides and Duboisia leichhardtii) were grown in climate chambers under controlled conditions in order to systematically analyse the influence of temperature (20, 24, 28 °C), light (50-300 µmol/m2 × s, 12, 18, 24 h per day) and macronutrients (nitrogen, calcium, potassium) on growth and scopolamine biosynthesis. The data indicate that light intensity and daily exposure to light have a major impact on scopolamine production and plant development, whereas temperature only shows a minor influence. Nitrogen (N) positively affects biomass production with increasing levels up to 4 mM, but is negatively correlated with scopolamine content. Calcium (Ca) shows a negative influence on scopolamine biosynthesis at increased levels above 1 mM as well. Potassium (K) neither affects biomass nor scopolamine production within the tested concentration range (0.05-4 mM). All in all, it can be concluded that light intensity and nitrogen supply are especially important regulating variables that can be applied in a targeted manner for influencing scopolamine and biomass production.

Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using 1H NMR-Based Metabolomics and Real-Time PCR.

Cannabis sativa trichomes are glandular structures predominantly responsible for the biosynthesis of cannabinoids, the biologically active compounds unique to this plant. To the best of our knowledge, most metabolomic works on C. sativa that have been reported previously focused their investigations on the flowers and leaves of this plant. In this study, (1)H NMR-based metabolomics and real-time PCR analysis were applied for monitoring the metabolite profiles of C. sativa trichomes, variety Bediol, during the last 4 weeks of the flowering period. Partial least squares discriminant analysis models successfully classified metabolites of the trichomes based on the harvest time. Δ (9)-Tetrahydrocannabinolic acid (1) and cannabidiolic acid (2) constituted the vital differential components of the organic preparations, while asparagine, glutamine, fructose, and glucose proved to be their water-extracted counterparts. According to RT-PCR analysis, gene expression levels of olivetol synthase and olivetolic acid cyclase influenced the accumulation of cannabinoids in the Cannabis trichomes during the monitoring time. Moreover, quantitative (1)H NMR and RT-PCR analysis of the Cannabis trichomes suggested that the gene regulation of cannabinoid biosynthesis in the C. sativa variety Bediol is unique when compared with other C. sativa varieties.