Article
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25-143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ(9)-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ(9)-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... It represents the substrate of three enzymes: tetrahydrocannabinolic acid synthase convertingcannabigerolic acid to ∆9-THCA [16], cannabidiolic acid synthase yielding cannabidiolicacid [20], and cannabinochromenic acid synthase producing cannabinochromenic acid [21]. Recent research could identify in planta both the acidic forms of the cannabinoids [22] as well as the decarboxylated forms (THC, CBD, cannabichromene, cannabigerol, cannabinol)-albeit in much lower amounts [23]. Non-enzymatic decarboxylation of these compounds is promoted by heating (during smoking or baking), sunlight, and storage. ...
... It is believed that the high number of diverse cannabinoids identified so far from Cannabis occur due to non-enzymatic modifications [24]. Studies performed on laser-microdissected capitate-stalked trichomes could suggest that cannabinoids are also present in the multicellular stipes of capitate-stalked hairs in addition to the secretory portion of the trichomes [23]. The exact contribution of the stalk cells to the biosynthesis of cannabinoids and the source of these compounds in the stems remains to be elucidated. ...
... cannabinoids identified so far from Cannabis occur due to non-enzymatic modifications [24]. Studies performed on laser-microdissected capitate-stalked trichomes could suggest that cannabinoids are also present in the multicellular stipes of capitate-stalked hairs in addition to the secretory portion of the trichomes [23]. The exact contribution of the stalk cells to the biosynthesis of cannabinoids and the source of these compounds in the stems remains to be elucidated. ...
Article
Full-text available
Cannabis sativa L. is a plant long used for its textile fibers, seed oil, and oleoresin with medicinal and psychoactive properties. It is the main source of phytocannabinoids, with over 100 compounds detected so far. In recent years, a lot of attention has been given to the main phytochemicals present in Cannabis sativa L., namely, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). Compared to THC, CBD has non-psychoactive effects, an advantage for clinical applications of anti-tumor benefits. The review is designed to provide an update regarding the multi-target effects of CBD in different types of cancer. The main focus is on the latest in vitro and in vivo studies that present data regarding the anti-proliferative, pro-apoptotic, cytotoxic, anti-invasive, anti-antiangiogenic, anti-inflammatory, and immunomodulatory properties of CBD together with their mechanisms of action. The latest clinical evidence of the anticancer effects of CBD is also outlined. Moreover, the main aspects of the pharmacological and toxicological profiles are given.
... Specifically, hemp is endowed with secretory structures, namely trichomes, representing the main site to produce secondary metabolites. Among them, cannabinoids and terpenes are contained in a sort of resin released by capitate-stalked glandular hairs localized on flower bracts and, to a minor extent, by capitate sessile and bulbous trichomes occurring also on other vegetative organs [6]. Notably, volatile terpenes of hemp glandular trichomes are recovered as a yellowish and odorous EO [7,8]. ...
... In previous literature contributions, glandular trichomes of various morphotypes have been defined under diverse, controversial terms over time [6,9,[59][60][61]. Therefore, an update of trichome terminology would be highly desirable to redefine the gland morphotypes. ...
... The bulbous hairs occasionally exhibited faintly positive responses to terpenes and polyphenols. Cannabinoid production, however, takes place mainly in the capitate trichomes, especially the stalked ones, as was largely confirmed by gas-liquid chromatographic evidence, by the identification of the candidate biosynthetic genes [6], and by CARS microscopy [10]. Hooked hair-like lithocysts were observed on the bract, bracteole, and inflorescence axis surfaces. ...
Article
Full-text available
New hemp (Cannabis sativa L.) strains developed by crossbreeding selected varieties represent a novel research topic worthy of attention and investigation. This study focused on the phytochemical characterization of nine hemp commercial cultivars. Hydrodistillation was performed in order to collect the essential oils (EO), and also the residual water and deterpenated biomass. The volatile fraction was analyzed by GC-FID, GC-MS, and SPME-GC-MS, revealing three main chemotypes. The polyphenolic profile was studied in the residual water and deterpenated biomass by spectrophotometric assays, and HPLC-DAD-MSn and 1H-NMR analyses. The latter were employed for quali–quantitative determination of cannabinoids in the deterpenated material in comparison with the one not subjected to hydrodistillation. In addition, the glandular and non-glandular indumentum of the nine commercial varieties was studied by means of light microscopy and scanning electron microscopy in the attempt to find a possible correlation with the phytochemical and morphological traits. The EO and residual water were found to be rich in monoterpene and sesquiterpene hydrocarbons, and flavonol glycosides, respectively, while the deterpenated material was found to be a source of neutral cannabinoids. The micromorphological survey allowed us to partly associate the phytochemistry of these varieties with the hair morphotypes. This research sheds light on the valorization of different products from the hydrodistillation of hemp varieties, namely, essential oil, residual water, and deterpenated biomass, which proved to be worthy of exploitation in industrial and health applications.
... They refer interchangeably to these botanical parts, containing the most active phytochemicals, as pistillate 'tops', 'flowers', 'inflorescences', or 'fruits'. More accurately, the active ingredients (mostly phytocannabinoids, terpenoids, phenols) are biosynthesised inside the multicellular, glandular heads of epidermal multiseriate stalk trichomes (Chandra et al., 2017;Clarke, 1981;ECDD, 2018;Evans, 2009: 525-527, 553-557;Flemming et al., 2007: 8;Frank, 2018;Happyana et al., 2013;Heinrich et al., 2017: 18, 149-150;Turner et al., 1981). While these epidermal glandular trichomes (EGT) are also present in a much lesser extent on leaves, stem, and other parts of the plants (explaining the results of Kimura and Okamoto, 1970; see also Turner et al., 1981), their dense concentration around the reproductive parts of pistillate plants (the so-called 'buds' or 'heads') is a major factor in the choice of the parts to harvest and transform for psychoactive purposes. ...
... All of these compounds correspond to those described as characteristics of plant resins in literature. They are obtained by the secretion from a plant's glands and are all known for their ether soluble properties (Clarke, 1981;Evans, 2009: 525-527, 557;Happyana et al., 2013). In other words, all acceptations of 'resin' (glandular origin, composition, solubility in alcohol) match with the material contained in C. sativa trichomes' glandular heads. ...
... Variations dramatically affecting the pharmacological properties of a product can be induced by basic thermomechanical stimuli (Agarwal et al., 2018;Naz et al., 2017), for instance, heating at 200 C for seven minutes (Verhoeckx et al., 2006) or ageing (Fairbairn, 1976: 15;Mechoulam and Hanu s, 2000;Zamengo et al., 2019). This is the case when phytocannabinoids, obtained in acidic form when separated from the plant (Happyana et al., 2013;Kimura and Okamoto, 1970;Perrotin-Brunel et al., 2010, Pertwee, 2006 decarboxylate into compounds with enhanced psychopharmacological effects (Reekie et al., 2018;Verhoeckx et al., 2006). From many perspectives, the most compelling evidence suggests the 'method of obtention' is a common, and overlooked criterion in classification systems that fail to overarch C. sativa products. ...
Article
Full-text available
Objective: Identify a coherent nomenclature of products containing cannabinoids (whether derived from Cannabis sativa L. or not). Design: Research undertaken in parallel to the three-year assessment of Cannabis derivatives by the World Health Organisation. The scope is limited to Cannabis products intended for human incorporation (internal and topical con- sumption). Primarily embedded in pharmacognosy, the study incorporates a wide range of scholarly and grey literature, folk knowledge, archives, pharmacopœias, international law, field pharmacy, clinical and herbal medicine data, under a philosophical scrutiny. Generic and Cannabis-specific nomenclatural frames are compared to determine the extent to which they coincide or conflict. Results: All lexica reviewed use weak, ambiguous, or inconsistent terms. There is insufficient scientific basis for terms and concepts related to Cannabis at all levels. No sound classification exists: current models conflict by adopting idiosyncratic, partial, outdated, or utilitarian schemes to arrange the extraordinarily numerous and diverse derivatives of the C. sativa plant. In law and policy, no clear or unequivocal boundary between herbal and non-herbal drugs, nor natural and synthetic cannabinoids was found; current nomenclatures used need updates. In science, the botanical Cannabis lexicon overlooks parthenocarpy, and wide disagreement remains as to the taxonomy and systematics of the plant; chemical research should address differences in kinds between synthetic cannabinoids; pharmacopœias include little information related to Cannabis, and disagree on broader classes of herbal medicines, virtually failing to embrace many known Cannabis medicines. Since existing products and compounds fail to be categorised in an evidence-based manner, confusions will likely increase as novel cannabinoid compounds, genetic and biotechnological modifications surge. Conclusions: The lack of clarity is comprehensive: for patients, physicians, and regulators. The study proposes an update of terms at several levels. It points at gaps in morphological descriptions in botany and pharmacognosy and a need for a metaphysical address of cannabinoids. Methods of obtention are identified as a common criterion to distinguish products; the way forward suggests a mutually exclusive nomenclatural pattern based on the smallest common denominator of obtention methods. In the context of a swelling number of Cannabis products being consumed (be it via medical prescription, adult-use, ‘hemp’ foodstuff and cosmetics, or other purposes), this study can assist research, contribute to transparent labelling of products, consumer safety and awareness, pharmacovigilance, medical standards of care, and an update of prevention and harm reduction approaches. It can also better inform regulatory policies surrounding C. sativa, its derivatives, and other cannabinoid-containing products. Original article available at: https://journals.sagepub.com/doi/full/10.1177/2050324520945797
... For the purpose of phytocannabinoid profiling, NMR spectroscopy is used as (semi)quantitative method alone [20,209] or as an orthogonal technique to LC [216][217][218] or GC [219] for the purpose of qualitative peak assignment of major phytocannabinoids [20], chemical and morphological examination [220], chemotaxonomic classification [20,219,220], metabolomics-based chemovar distinction [51] or quantitative analysis of cannabis plant material without the need of pre-purification step [221], chromatographic separation or use of certified reference standards [219]. Cryogenic NMR spectroscopy combines improved sensitivity and noise reduction with a cryogenic cooling system for the receiver coil and preamplifiers. ...
... For the purpose of phytocannabinoid profiling, NMR spectroscopy is used as (semi)quantitative method alone [20,209] or as an orthogonal technique to LC [216][217][218] or GC [219] for the purpose of qualitative peak assignment of major phytocannabinoids [20], chemical and morphological examination [220], chemotaxonomic classification [20,219,220], metabolomics-based chemovar distinction [51] or quantitative analysis of cannabis plant material without the need of pre-purification step [221], chromatographic separation or use of certified reference standards [219]. Cryogenic NMR spectroscopy combines improved sensitivity and noise reduction with a cryogenic cooling system for the receiver coil and preamplifiers. ...
... Cryogenic NMR spectroscopy combines improved sensitivity and noise reduction with a cryogenic cooling system for the receiver coil and preamplifiers. Its improved spectral quality is employed in compound identification from mass limited samples and as orthogonal analytical technique to HPLC in phytocannabinoid profiling in laser-micro dissected samples of capitate-stalked and capitate-sessile trichomes [220]. ...
Article
Full-text available
Cannabis is gaining increasing attention due to the high pharmacological potential and updated legislation authorizing multiple uses. The development of time- and cost-efficient analytical methods is of crucial importance for phytocannabinoid profiling. This review aims to capture the versatility of analytical methods for phytocannabinoid profiling of cannabis and cannabis-based products in the past four decades (1980–2021). The thorough overview of more than 220 scientific papers reporting different analytical techniques for phytocannabinoid profiling points out their respective advantages and drawbacks in terms of their complexity, duration, selectivity, sensitivity and robustness for their specific application, along with the most widely used sample preparation strategies. In particular, chromatographic and spectroscopic methods, are presented and discussed. Acquired knowledge of phytocannabinoid profile became extremely relevant and further enhanced chemotaxonomic classification, cultivation set-ups examination, association of medical and adverse health effects with potency and/or interplay of certain phytocannabinoids and other active constituents, quality control (QC), and stability studies, as well as development and harmonization of global quality standards. Further improvement in phytocannabinoid profiling should be focused on untargeted analysis using orthogonal analytical methods, which, joined with cheminformatics approaches for compound identification and MSLs, would lead to the identification of a multitude of new phytocannabinoids.
... Ãåíåðàòèâí³ òà âåãåòàòèâí³ îðãàíè ðîñëèí C. sativa âêðèò³ çàëîçèñòèìè âîëîñêàìè, òîìó äëÿ äîñë³äaeåííÿ ñòðóêòóðíî¿ îðãàí³çà-ö³¿ çàëîç òà ¿õ ìîðôîëîã³÷íèõ îçíàê ïðîàíà-ë³çîâàíî òèïîâ³ ðîñëèíè ñó÷àñíèõ ñîðò³â â³ò-÷èçíÿíî¿ òà çàðóá³aeíî¿ ñåëåêö³¿. Ïåðåäóñ³ì ñîðòè êëàñèô³êîâàíî çà åêîëîãî-ãåîãðàô³÷íèìè ãðóïàìè òà âì³ñòîì êàíàá³íî¿äíèõ ñïîëóê [15,16]. ...
... Chemical analysis for the content of cannabinoid substances was carried out in the laboratory of the breeding and seed production department of the Institute of Bast Crops of the NAAS of Ukraine (Glukhiv, 2014-2019). The content of neutral substances [cannabinol (CBN), cannabidiol (CBD), tetrahydrocannabinol (THC)] and acids (CBDA, THCA) was 16 ...
Article
Full-text available
Purpose. Identifying the features of the seasonal growth rhythm, development of the plants, morphological and biological differences in monoecious introduced and domestic varieties of Cannabis sativa L. in the North-east region of Ukraine.Methods. The research was conducted from 2014 till 2019. The following research methods were employed: field (phenological observations, sampling for laboratory analysis), microscopic (study of the morphological structure of glandular and cystolithic hairs on vegetative and generative organs, determination of the size and density of hair formation), laboratory and analytical (determination of the content of cannabinoid substances by thin layer and gas-liquid chromatography), biomorphological me­thods (analysis of morphological characteristics of plants). Chemical analysis for the content of cannabinoid substan­ces was carried out in the laboratory of the breeding and seed production department of the Institute of Bast Crops of the NAAS of Ukraine (Glukhiv, 2014–2019). The content of neutral substances [cannabinol (CBN), cannabidiol (CBD), tetrahydrocannabinol (THC)] and acids (CBDA, THCA) was determined by using a thin layer chromatography (TLC) and gas-liquid chromatography (GLC) methods. For the study, 11 monoecious varieties of C. sativa: ‘YuSO 1’, ‘YuSO 31’, ‘Hliana’, ‘Viktoriia’, ‘Hlesiia’, ‘Hlukhivskyi 46’, ‘Hlukhivski 51’, ‘Zoloto­niski 15’ and 3 introduced varieties: ‘Fedora 17’, ‘Felina 32’, ‘Futura 77’ (France) which differed among themselves in morphological, biological, genetic, ecological-geographical and economically valuable characteristics were selected.Results. Biological and morphological characteristics of modern monoecious varieties of C. sativa were investigated depending on the ecological and geographical zones of origin, conditions of the growing season and the stage of ontogenesis. The functional interdependence between the formation of specialized excretory structures and the content of cannabinoid substances in plants of different varieties of C. sa­tiva was revealed. The following promising varieties of C. sativa in a ration of ((THC + CBN) ≤ CBD) point the possibility to use them as the source material for creating the medicinal varie­ties of plants with a high content of CBD.Conclusions. Nowadays C. sativa is the main source of phyto material for industrial production of natural CBD. In recent years, high-yielding varieties of C. sativa have been created, which, together with high commercially valuable traits, did not contain psychoactive THC and had an insignificant amount of CBD. The results of the research indicated the features of the seasonal growth rhythm, development of the plants and the morphological and biological features of modern monoecious introduced and domestic varieties of C. sativa in the North-east region of Ukraine. The low correlation between the content of cannabinoid substances and their acids in varieties, which are cha­racterized by the low content of cannabinoid compounds was established. According to the developed own classification (THC + KBN): CBD, varieties with a predominant CBD content ‘Hlukhivskyi 46’, ‘Zolotoniski 15’, ‘Hliana’ which are of significant medicinal value were identified.
... The use of aluminum foil is a common Raman trick to avoid the fluorescence produced by the glass [12]. The focus was set on the trichome heads because they are the site of the cannabinoid production [13][14][15][16] and the THC content is expected to be the highest (15 to 25%) and consequently, there is a stronger spectral signal. This was especially important because uninformative spectra were obtained when randomly focusing and irradiating on any bulk marijuana sample. ...
... Since the capitate-stalked and bulbous trichomes of the marijuana contain high cannabinoid accumulation levels [13,14], they were the target structures to be located using a microscope as shown in Figure 2 [15]. Figure 3 shows the average Raman spectrum of marijuana. ...
Article
Full-text available
The Raman analysis of marijuana is challenging because of the sample’s easy photo-degradation caused by the laser intensity. In this study, optimization of collection parameters and laser focusing on marijuana trichome heads allowed collecting Raman spectra without damaging the samples. The Raman spectra of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) standard cannabinoids were compared with Raman spectra of five different types of marijuana: four Sativa varieties (Amnesia Haze, Amnesia Hy-Pro, Original Amnesia, and Y Griega) and one Indica variety (Black Domina). The results verified the presence of several common spectral bands that are useful for marijuana characterization. Results were corroborated by the quantum chemical simulated Raman spectra of their acid-form (tetrahydrocannabinol acid (THCA), cannabidiol acid (CBDA)) and decarboxylated cannabinoids (THC, CBD, and CBN). A chemometrics-assisted method based on Raman microscopy and OPLS-DA offered good classification among the different marijuana varieties allowing identification of the most significant spectral bands.
... Cannabinoid biosynthesis (Fig. 3.3) takes place in the glandular trichomes: capitate-stalked, capitate-sessile, and bulbous (Dayanandan and Kaufman 1976;Kim and Mahlberg 1997b;Turner et al. 1978). The capitate-stalked glandular trichomes contain the largest quantities of cannabinoids and are abundantly found on female inflorescences (pistillates) (Hammond and Mahlberg 1973;Happyana et al. 2013;Mahlberg and Kim 2004). The precursors of cannabinoids, olivetolic acid (OA) or alternatively divarinolic acid (DA), and geranyl diphosphate (GPP) originate from polyketide pathway and the plastidal 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, respectively (Gagne et al. 2012;Phillips et al. 2008). ...
... Within cannabis, there are many phenotypes; for example, while one has the ability to grow over a few meters high, another stay small. Furthermore, variations in glandular trichome densities have also been observed with regard to THCA content and ratios (Happyana et al. 2013). By combining various cannabis phenotypes, it is possible to breed a new strain that will be, for example, tall with high glandular trichome density and/or high THCA content. ...
Chapter
Cultivation and breeding of Cannabis sativa L. for medicinal purposes is of actual importance since in many countries the legalization allows its use by patients. So far, Cannabis sativa L. and its various strains, optimized for their cannabinoids, have mostly been bred and cultivated under illegal conditions to provide an illicit drug without integration of any pharmaceutical standards according to GAP. The situation has changed now, and many professional breeders introduced the rational concepts for growing cannabis mostly indoor under strict control of drug agencies and international pharmaceutical standards. Here, we report and summarize the actual status of analytics, phytochemistry, and phytoengineering of cannabinoid optimization. Special focus is set on legislation, pharmaceutical standardization, and basic concepts for industrial growing and breeding of cannabis plants.
... Найбільш поширеними канабіноїдними сполуками є тетрагідроканабінолова кислота (далі -ТГКК), канабідіолова кислота (далі -КБДК) і канабігеролова кислота (далі -КБГК). Відповідні їм біоактивні форми канабіноїдів -тетрагідроканабінол (далі -ТГК), канабідіол (далі -КБД) і канабігерол (далі -КБГ) -утворюються в результаті реакції декарбоксилування під впливом зовнішніх умов 8 . Загалом, у конопель виявлено близько 120-ти фітоканабіноїдів 9 , які останнім часом класифікують за їх хімічною структурою, загалом виділяючи 11 підкласів: сім типів КБГ; п'ять типів канабіхромену (далі -КБХ); п'ять типів КБД; основний психоактивний Δ 9 -ТГК у дев'яти різних формах, включаючи його прекурсор Δ 9 -ТГКК (кислотну форму), і Δ 8 -ТГК, який є найбільш стійким ізомером Δ 9 -ТГК, але на 20% менш активним; три типи канабіциклолу (далі -КБЛ), п'ять різних форм канабіелсоїну (далі -КБЕ); сім типів канабінолу (далі -КБН), який є кінцевим продуктом синтезу (окиснення) Δ 9 -ТГК; канабітріол (далі -КБТ); канабідіварін (далі -КБДВ); тетрагідроканабіварін (далі -ТГКВ); різний тип 10,11,12 . ...
... Загалом, у конопель виявлено близько 120-ти фітоканабіноїдів 9 , які останнім часом класифікують за їх хімічною структурою, загалом виділяючи 11 підкласів: сім типів КБГ; п'ять типів канабіхромену (далі -КБХ); п'ять типів КБД; основний психоактивний Δ 9 -ТГК у дев'яти різних формах, включаючи його прекурсор Δ 9 -ТГКК (кислотну форму), і Δ 8 -ТГК, який є найбільш стійким ізомером Δ 9 -ТГК, але на 20% менш активним; три типи канабіциклолу (далі -КБЛ), п'ять різних форм канабіелсоїну (далі -КБЕ); сім типів канабінолу (далі -КБН), який є кінцевим продуктом синтезу (окиснення) Δ 9 -ТГК; канабітріол (далі -КБТ); канабідіварін (далі -КБДВ); тетрагідроканабіварін (далі -ТГКВ); різний тип 10,11,12 . 5 8 Створені протягом останніх десятиліть сорти конопель хоча і характеризуються майже повною відсутністю психотропного ТГК (від 0,00 до 0,01%), однак популяції таких сортів дещо нестабільні і потребують постійного контролю та нейтралізації фізіологобіохімічної функції рослин до формування канабіноїдів селекційним шляхом на основі встановлення генетичних закономірностей даного явища. ...
Chapter
Проаналізовано особливості успадкування ознаки вмісту канабіноїдів гібридами конопель F1і F2, створеними у результаті схрещування рослин із різним рівнем її вираження. Установлено, що вміст канабіноїдних сполук успадковувався полігенно за ядерно-цитоплазматичним типом. Для створення непсихотропного селекційного матеріалу зі зниженим вмістом усіх канабіноїдів доцільно проводити добір у гібридів типу відсутність / відсутність, відсутність / мінімум і відсутність / максимум. За материнську форму слід добирати матеріал із нульовими показниками канабіноїдних сполук. У потомстві таких варіантів не лише був менший уміст цих речовин, а й у F2не виявлялися рослини з їх високим вмістом. Узагальнено літературні джерела щодо теорії генетичного контролю досліджуваної ознаки.
... Phytocannabinoids and terpenes accumulate in the secretory cavity of the glandular trichomes in C. Sativa [109,110], and are present in the highest quantity on the female flower of the plant. Male plants produce lower levels of phytocannabinoids [111]. ...
... Glandular trichomes consist of a saclike cavity packed with secretory vesicles known as glandular hairs. Glandular trichomes of C. sativa alter morphology and metabolite content during flower maturation, and phytocannabinoids/terpenes are found on the calyx and the underside of anthers of flowers, leaves and bracts [109]. Trichomes rupture due to environmental stress or damage (due to high temperatures and herbivorous Downloaded from http://portlandpress.com/neuronalsignal/article-pdf/doi/10.1042/NS20200080/918663/ns-2020-0080c.pdf by guest on 10 August 2021 consumption), resulting in the release of phytocannabinoids and terpenes as a noxious, sticky liquid on the plant surface. ...
Article
Full-text available
Cannabidiol (CBD), one of the primary non-euphoric components in the Cannabis sativa L. plant, has undergone clinical development over the last number of years as a therapeutic for patients with Lennox-Gastaut syndrome and Dravet syndromes. This phytocannabinoid demonstrates functional and pharmacological diversity, and research data indicate that CBD is a comparable antioxidant to common antioxidants. This review gathers the latest knowledge regarding the impact of CBD on oxidative signalling, with focus on the proclivity of CBD to regulate antioxidants and control the production of reactive oxygen species. CBD is considered an attractive therapeutic agent for neuroimmune disorders, and a body of literature indicates that CBD can regulate redox function at multiple levels, with a range of downstream effects on cells and tissues. However, pro-oxidant capacity of CBD has also been reported, and hence caution must be applied when considering CBD from a therapeutic standpoint. Such pro- and antioxidant functions of CBD may be cell- and model-dependent, and may also be influenced by CBD dose, the duration of CBD treatment and the underlying pathology.
... The others include cannabigerol (CBG), cannabichromene (CBC), (−)-Δ 8 -trans-tetrahydrocannabinols (Δ 8 -THC), cannabicyclol (CBLs), cannabielsoin (CBE), cannabinol (CBN), cannabinodiol (CBND), cannabitriol (CBT), and the miscellaneous cannabinoids [5,8]. Cannabinoids biosynthesis is primarily localised on the capitate-stalked trichomes mainly found on the flower and sugar leaf surface of the pistillate plant [9]. Disk-like structures formed on the trichome head synthesizes and secretes the cannabinoids which are then accumulated in the fibrillar matrix, the subcuticular wall, and the cuticle [9,10]. ...
... Cannabinoids biosynthesis is primarily localised on the capitate-stalked trichomes mainly found on the flower and sugar leaf surface of the pistillate plant [9]. Disk-like structures formed on the trichome head synthesizes and secretes the cannabinoids which are then accumulated in the fibrillar matrix, the subcuticular wall, and the cuticle [9,10]. Shortly after the recoveries of the cannabinoids, the cannabinoid receptors (CB1 and CB2) were identified and many endogenous ligands for the cannabinoid receptors were also recognised [11]. ...
Article
Full-text available
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.
... It is easily available in Eastern Asia especially in India and very easily found in our surroundings. C. sativa contains Cannabinol, Cannabinoid, and Cannabichromenic acid as its main phytochemical compounds [21], [22]. The main phytochemical constituents isolated from the leaves of C. sativahave been shown in Fig. 1. ...
Article
The leaves extract of the Cannabis sativaplant was used for the corrosion resistance of low carbon steel (LCS) in the acidic medium (0.5 M sulfuric acid) utilizing the weight-loss method, Tafel and EIS. The state of mixed inhibitor adsorption on the LCS surface is shown by potentiodynamic polarization. C. sativaachieved anextraordinary inhibition efficiency of 97.31% at 200 mg/L of inhibitor concentration. SEM and AFM were used to know about the thin layer which was formed on the surface of LCS for its protection from corrosion and the adsorption of inhibitor were shown by UV-vis. spectroscopic technique. FT-IR technique confirmed the existence of functional groups and the heteroatoms exhibit in the inhibitor. Adsorbanceby the inhibitory molecules on the LCS surface followed the Langmuir adsorption isotherm. Hypothetical investigations (computational) showed a very valuable report. All acquired outcomes ensure that C. sativaleaves extract can procedure an effectualpreventing layer and restrict the corrosion procedure. The results of the molecular dynamics (MD) simulations agree with the appointed inhibition efficiencies of the experimentally.
... Glandular trichomes are able to produce, store and often secrete exudates carrying a terrific plethora of chemo-diverse molecules, such as essential oils and oleoresins, phenols, glycerids and very complex terpenes. Outstanding examples of high economic value are the anti-malarial artemisinin in Artemisia annua [201], psychoactive cannabinoids in Cannabis sativa [202], and the psychotropic molecule salvinorin A in Salvia divinorum [203], but also acylsugars that are glycolipids consisting of a sugar core linked to straight or branched acylic chains [204]. To biosynthesize these complex molecules, plants require an incredible energetic effort. ...
Article
Full-text available
Plant specialized metabolites (SMs) play an important role in the interaction with the environment and are part of the plant defense response. These natural products are volatile, semi-volatile and non-volatile compounds produced from common building blocks deriving from primary metabolic pathways and rapidly evolved to allow a better adaptation of plants to environmental cues. Specialized metabolites include terpenes, flavonoids, alkaloids, glucosinolates, tan-nins, resins, etc. that can be used as phytochemicals, food additives, flavoring agents and pharmaceutical compounds. This review will be focused on Mediterranean crop plants as a source of SMs, with a special attention on the strategies that can be used to modulate their production, including abiotic stresses, interaction with beneficial soil microorganisms and novel genetic approaches.
... Stalked glandular trichomes are abundant on mature flowers (Turner et al., 1977;Potter, 2009;Small and Naraine, 2016), and they store cannabinoids in higher abundance than sessile trichomes (Turner et al., 1978). A study using laser capture microdissection sampling of different floral trichomes has been performed (Happyana et al., 2013), however, non-glandular hairs were incorrectly classified as 'capitate-sessile trichomes', and therefore did not compare the sessile glandular trichomes as defined by others (Hammond and Mahlberg, 1973;Potter, 2009) to stalked trichomes. In our own microcapillary sampling, sessile and stalked trichomes were both dominated by CBDA, as expected for a hemp cannabis variety, with marginal variations in other cannabinoids. ...
Article
The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile‐like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two‐photon microscopy technique of glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12‐16 secretory disc cells and strongly monoterpene‐dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed red‐shifted autofluorescence, eight secretory disc cells, and less monoterpene‐dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co‐expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly‐expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products. This article is protected by copyright. All rights reserved.
... Mean ± SEM of the numbers of experiments (n) are indicated throughout.Two types of trichomes are characteristic of hemp, that is, glandular and nonglandular ones. Among glandular trichomes three types are recognised in C. sativa: large capitate sessile, large capitate-stalked, and small bulbous trichomes(Happyana et al., 2013;Mahlberg & Kim, 2004).The bracteoles surrounding the female flowers of the two Cannabis biotypes showed two main types of trichomes, namely unicellular, nonglandular trichomes, appearing rigid and curved, with a slender point apex and glandular trichomes, with large heads as sites of resin production and storage(Figures 1a-d and 2a-c).Both varieties of Cannabis show bracteoles with numerous long, multicellular, capitate-stalked glandular trichomes(Figures 1b,d and ...
Article
The aim of this study was to compare the micro‐morphological features of two different non‐drug Cannabis sativa L. biotypes (Chinese accession G‐309 and one fibrante variety) and to evaluate the phytochemical profile as well as some biological properties of the essential oils (EOs) obtained by hydrodistillation of dried flowering tops. After a micro‐morphological evaluation by scanning electron microscopy, the phytochemical composition was analysed by GC–FID and GC–MS analyses. Antioxidant and anti‐acetylcholinesterase properties were investigated by several in vitro cell‐free assays, while neuroactive effects were evaluated on mouse cortical neuronal as well as human iPS cell‐derived central nervous system cells grown on MEA chips. Both EOs showed strong antioxidant properties mainly attributable to the high content of hydroxylated compounds as well as significant anti‐acetylcholinesterase activities (IC50 74.64 and 57.31 μg/ml for Chinese accession and fibrante variety, respectively). Furthermore, they showed a concentration‐dependent inhibition of spontaneous electrical activity of human and mouse neuronal networks, with the fibrante variety, which showed the best activity (MFR, IC50 0.71 and 10.60 μg/ml, respectively). The observed biological activities could be due to a synergic effect between terpenes and phytocannabinoids, although in vivo studies, which clarify the molecular mechanism, are still lacking.
... ence of external conditions. Decarboxylated derivativescannabichromen (CBC) and cannabinol (CBN) are found in small quantities [9]. About 120 phytocannabinoids have been identified in cannabis [10,11]. ...
Article
Full-text available
Purpose and objectives. To reveal peculiarities of the inheritance of the traits of cannabinoid compound presence and content upon self-pollination in monoecious hemp, to compare the pair correlation coefficients between the contents of major cannabinoid compounds in initial genotypes and self-pollinated lines, and to evaluate the effectiveness of self-pollination in hemp breeding. Material and methods. The study was carried out at the Institute of Bast Crops of the National Academy of Agricultural Sciences (Hlukhiv, Sumy Oblast, Ukraine) in 2009–2019. Inbred lines of industrial hemp varieties Hlukhivski 58, Hlesiia, Mykolaichyk, and Hlukhivski 46 belonging to the Central European eco-geographical type and variety Zolotoniski 15 belonging to the Southern eco-geographical type were taken as test objects. Self-pollination of plants (with and without cannabinoids) was carried out under individual agrotextile bags in a greenhouse. The offspring were grown in a nursery. Evaluation and analysis of cannabinoid compounds were conducted by thin-layer chromatography. In all the genotypes under study, the tetrahydrocannabinol (THC) content did not exceed 0.08% that is standard allowed by the current legislation of Ukraine. Data were statistically processed with calculating arithmetic mean, sampling mean error, pairwise correlation coefficients, and curvilinear regression. Results and discussion. Provided directional selection of initial plants that do not contain cannabidiol (CBD), THC or cannabinol (CBN), self-pollination reduced their contents to complete absence. Stabilization (homozygation) of the lines occurred in I2–I6 and was specific to a particular variety. Inbred lines of these generations are recommended to involve as parents in crossing. The ability to segregate CBD-, THC-, and CBN-free families as early as in I1. is a characteristic feature of the hemp varieties under investigation. There were strong positive correlations between the cannabinoid contents, in particular there was a strong positive correlation was found between the CBD and THC contents (r = 0.72–0.79 and 0.71–0.90, respectively), a strong or medium positive correlation between the CBD and CBN contents (0.68–0.80 and 0.67–0.82, respectively), a strong positive correlation between the THC and CBN contents (0.71–0.83 and 0.80–0.85, respectively) in I1–I3 Hlukhivski 58 and I1–I3 Zolotoniski 15. This makes selection for reduced contents of all cannabinoid compounds and easier, but at the same time significantly complicates breeding for increased CBD content with concurrent reduced THC content or increased contents of non-psychotropic cannabinoids. The correlations between the contents of cannabinoid compounds in inbred genotypes are weaker than those in the original breeding genotypes, with the coefficients ranging significantly, which allows using closely related reproduction in breeding aimed at reducing THC content or increasing non-psychotropic cannabinoid contents. Conclusions. Self-pollination is an effective method of determination of the hemp population stability in terms of the cannabinoid compound presence and contents and at the same time is a method of creating breeding genotypes with stable traits of cannabinoid absence or presence.
... Therefore, the decrease in the amount of cellulose and lignin in LV1 is one of the major factors responsible for the low concentration of phenols in LV1. In addition, the biosynthesis of unique cannabinoid compounds takes place mainly in the glandular trichomes in the leaves of the hemp plant [43]. This accounts for the presence of cannabinoids such as THC, CBD, etc. in LV1 and explains the major chemical composition differences when LV1 is compared to HR1 and RT1. ...
Article
Full-text available
This study assessed the pyrolysis liquids obtained by slow pyrolysis of industrial hemp leaves, hurds, and roots. The liquids recovered between a pyrolysis temperature of 275–350 °C, at two condensation temperatures 130 °C and 70 °C, were analyzed. Aqueous and bio-oil pyrolysis liquids were produced and analyzed by proton nuclear magnetic resonance (NMR), gas chromatography–mass spectrometry (GC-MS), and atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry (APPI FT-ICR MS). NMR revealed quantitative concentrations of the most abundant compounds in the aqueous fractions and compound groups in the oily fractions. In the aqueous fractions, the concentration range of acetic acid was 50–241 gL−1, methanol 2–30 gL−1, propanoic acid 5–20 gL−1, and 1-hydroxybutan-2-one 2 gL−1. GC-MS was used to compare the compositions of the volatile compounds and APPI FT-ICR MS was utilized to determine the most abundant higher molecular weight compounds. The different obtained pyrolysis liquids (aqueous and oily) had various volatile and nonvolatile compounds such as acetic acid, 2,6-dimethoxyphenol, 2-methoxyphenol, and cannabidiol. This study provides a detailed understanding of the chemical composition of pyrolysis liquids from different parts of the industrial hemp plant and assesses their possible economic potential.
... At least there are 3 types of trichomes in Cannabis, including capitate-stalked, capitate-sessile, and bulbous trichomes. Based on our previous works, capitate-stalked trichomes contain more cannabinoids compare to the others [8]. ...
Article
Full-text available
Trichomes of Cannabis sativa are the main tissue for synthesizing and storing cannabinoids, the most interesting compounds in this plant. In this report, metabolic changes in the trichomes of C. sativa var. bedrobinol were investigated by 1H-NMR-based metabolomics over the flowering session. Three cannabinoids, including Δ9-tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA), and Δ9-tetrahydrocannabinol (THC), were successfully identified in the chloroform extracts of the Cannabis trichomes. Meanwhile, 20 non-cannabinoid compounds, including sugars, amino acids, and other acidic constituents, were detected in the water extracts. Metabolic changes of the Cannabis trichomes during the monitoring time were successfully revealed using the models of partial least squares discriminant analysis (PLSDA) and 1H-NMR quantitative analysis. Score plots of the PLSDA models classified metabolomes based on the harvest time. Discriminant metabolites for the differentiation were detected in the loading plots of the models. THCA was found as an important discriminant compound in the chloroform extracts, while all quantified water-soluble compounds were detected, contributing to the metabolic changes of the water extracts. The obtained results shed more light on the biosynthesis of metabolites in the Cannabis trichomes over the flowering season.
... The hemp essential oil is produced in the capitate trichomes that are particularly abundant in inflorescences and, to a minor extent, in leaves (Happyana et al., 2013). The main volatile components can be divided into three groups depending on the cultivar, plant organ, storage, processing and extraction technique: i.e. monoterpenes including αpinene, myrcene and terpinolene, sesquiterpenes such as (E)-caryophyllene, α-humulene and caryophyllene, and cannabinoids with cannabidiol (CBD) as the predominant compound, whereas δ-9-tetrahydrocannabinol (THC) is missing or occurs at trace levels (Mead, 2017;Benelli et al., 2018a, b;Bertoli et al., 2010). ...
Article
The increase of cultivation of industrial hemp (Cannabis sativa L.) all over the world offers new opportunities for the industry to manufacture innovative products from this multipurpose crop. In this regard, the hemp essential oil represents a niche product with potential interest for the pharmaceutical, nutraceutical, cosmeceutical and agrochemical companies. On this basis, in the present work we used the microwave-assisted extraction (MAE) to get an essential oil enriched in bioactive compounds, especially cannabidiol (CBD), from the dry inflorescences of the Italian variety CS (Carmagnola Selezionata). For this purpose, the operative conditions to increase the essential oil yield and CBD concentration in terms of microwave irradiation power (W/g), extraction time (min) and water added to the plant matrix after moistening (%), were optimized using a central composite design (CCD) approach using a Milestone ETHOS X device. The conventional hydrodistillation (HD) performed for 240 min was used for comparative purposes. The qualitative compositions of essential oils obtained by MAE and HD were analysed by GC-MS, whereas the quantitative detection of CBD and main terpenoids (α-pinene, β-pinene, myrcene, limonene, terpinolene, (E)-caryophyllene, α-humulene and caryophyllene oxide) was achieved by GC-FID. Furthermore, the enantiomeric distribution of the chiral constituents (α-pinene, β-pinene, limonene, (E)-caryophyllene and caryophyllene oxide) was determined using chiral chromatography. Results showed that the MAE treatment, using high irradiation power and relatively long extraction times, increased significantly the content of CBD in the essential oil while maintaining high oil yield values when compared with conventional HD. The enantiomeric excess of three chiral monoterpenes (α-pinene, β-pinene and limonene) was determined, with the (+)-enantiomers being predominant, whereas (E)-caryophyllene and caryophyllene oxide were enantiomerically pure. In conclusion, the MAE was successfully applied to hemp dry inflorescences in order to obtain a CBD-rich essential oil which may be exploited in several industrial applications.
... Investigations show that GTs and NGTs are widely distributed in plant species, such as Medicago sativa [35], Humulus lupulus [36], Cannabis sativa [37], Lycopersicum esculentum [38], Nicotiana tabacum [39], etc. Non-glandular trichomes form the first barrier preventing the invasion of herbivores and pathogens [40]. Glandular trichomes are variously shaped and have different functions in terms of their biological activities. ...
Article
Full-text available
Opisthopappus taihangensis (Ling) Shih, a wild relative germplasm of chrysanthemum, releases a completely different fragrance from chrysanthemum species. We aimed to identify the volatile compounds of the leaves of O. taihangensis and four other Chrysanthemum species using headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry (HS-SPME-GC/MS). In total, 70 compounds were detected, and terpenoids accounted for the largest percentage in these five species. Many specific compounds were only emitted from O. taihangensis and not from the other four species. In particular, 1,8-cineole could be responsible for the special leaf fragrance of O. taihangensis as it accounted for the largest proportion of the compounds in O. taihangensis but a small or no proportion at all in other species. The glandular trichomes (GTs) in the leaves are the main organs responsible for the emission of volatiles. To explore the relationship between the emissions and the density of the GTs on the leaf epidermis, the shape and density of the GTs were observed and calculated, respectively. The results showed that the trichomes have two shapes in these leaves: T-shaped non-glandular trichomes and capitate trichomes. Histochemical staining analyses indicated that terpenoids are mainly emitted from capitate glandular trichomes. Correlation analysis showed that the volatile amount of terpenoids is highly related to the density of capitate trichomes. In O. taihangensis, the terpenoids content and density of capitate trichomes are the highest. We identified the diversity of leaf volatiles from O. taihangensis and four other Chrysanthemum species and found a possible relationship between the content of volatile compounds and the density of capitate trichomes, which explained the cause of the fragrance of O. taihangensis leaves.
... Moreover, GT-specific natural products have been of great interest due to their pharmaceutical value. Well-known examples include artemisinin, an antimalarial agent produced by the GTs of Artemisia annua (Lu et al., 2013), Δ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD), psychoactive and anti-convulsant agents stored in the GTs of Cannabis sativa (Happyana et al., 2013). ...
Article
The Lamiaceae plant Ajuga forrestii Diels is a traditional Chinese herbal medicine with abundant glandular trichomes (GTs), but their chemistry and biological functions remain uninvestigated. Here, a panel of six highly functionalized neo-clerodane diterpenoids was localized to the peltate GTs of A. forrestii using laser microdissection coupled with HPLC analysis, indicating that the GTs of A. forrestii are an excellent material for the elucidation of the yet unclear biosynthetic pathway of natural neo-clerodane diterpenoids. In addition, four undescribed neo-clerodane diterpenoids with an acyclic C-9 side chain including two pairs of 1:1 mixture of inseparable diastereomers, ajuforrestins D-G, were isolated from the fresh leaves of A. forrestii together with six known compounds. The structures of the undescribed compounds were elucidated by spectroscopic (including 1D and 2D NMR and HR-ESI-MS) analyses. Biological assays indicated that the major GT compound ajugacumbin B and undescribed ajuforrestins D/E showed antifeedant activity against Helicoverpa armigera, suggesting that neo-clerodanes in A. forrestii should be involved in plant defence against insects. Moreover, the abietane diterpenoid ajuforrestin B exhibited significant anti-inflammatory activity on the secretion of interleukin-2 (IL-2) and cytotoxicity against three cancer cell lines, NCI-H1975, HepG2 and MCF-7, suggesting that ajuforrestin B could positively contribute to the therapeutic effects of this traditional Chinese medicine.
... In C. sativa, phytocannabinoids are stored in glandular trichomes, located all over the aerial part of the plant, so root surface and root tissues do not keep phytocannabinoid. Female flowers possess a high density of phytocannabinoid [22,23]. Glandular trichomes have balloon shaped secretory vessicle which store cannabinoid. ...
Article
Full-text available
Phytocannabinoids are a structurally diverse class of bioactive naturally occurring compounds found in angiosperms, fungi, and liverworts and produced in several plant organs such as the flower and glandular trichrome of Cannabis sativa, the scales in Rhododendron, and oil bodies of liverworts such as Radula species; they show a diverse role in humans and plants. Moreover, phytocannabinoids are prenylated polyketides, i.e., terpenophenolics, which are derived from isoprenoid and fatty acid precursors. Additionally, targeted productions of active phytocannabinoids have beneficial properties via the genes involved and their expression in a heterologous host. Bioactive compounds show a remarkable non-hallucinogenic biological property that is determined by the variable nature of the side chain and prenyl group defined by the enzymes involved in their biosynthesis. Phytocannabinoids possess therapeutic, antibacterial, and antimicrobial properties; thus, they are used in treating several human diseases. This review gives the latest knowledge on their role in the amelioration of abiotic (heat, cold, and radiation) stress in plants. It also aims to provide synthetic and biotechnological approaches based on combinatorial biochemical and protein engineering to synthesize phytocannabinoids with enhanced properties.
... [12] These are hair-like structures which are abundant on the epidermal surfaces of floral tissues of female inflorescences. [13] Unisexual female inflorescences are highly branched compound racemes that are comprised of monophotometric structures consisting of an axillary shoot, solitary pistillate flowers, subtending bracts, and reduced leaves. [14] In contrast, male inflorescences have sparse leaf coverage and consist largely of pendulous panicles. ...
Article
Cannabis is a mostly dioecious multi-use flowering plant genus. Sexual dimorphism is an important characteristic in Cannabis-based commercial production systems, which has consequences for fibre, seed, and the yield of secondary metabolites, such as phytocannabinoid and terpenes for therapeutic uses. Beyond the obvious morphological differences between male and female plants, metabolic variation among dioecious flowers is largely undefined. Here, we report a pilot metabolomic study comparing staminate (male) and pistillate (female) unisexual flowers. Enrichment of the α-linolenic acid pathway and consensus evaluation of the jasmonic acid (JA) related compound 12-oxo-phytodienoicacid (OPDA) among differentially abundant metabolites suggests that oxylipin signalling is associated with secondary metabolism and sex expression in female flowers. Several putative phytocannabinoid-like compounds were observed to be upregulated in female flowers, but full identification was not possible due to the limitation of available databases. Targeted analysis of 14 phytocannabinoids using certified reference standards (cannabidiolic acid (CBDA), cannabidiol (CBD), Δ9-tetrahydrocannabinolic acid A (Δ9-THCAA), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabinolic acid (CBNA), cannabinol (CBN), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), tetrahydrocannabivarinic acid (THCVA), and tetrahydrocannabivarin (THCV)) showed a higher total phytocannabinoid content in female flowers compared with the male flowers, as expected. In summary, the development of a phytocannabinoid-specific accurate-mass MSn fragmentation spectral library and gene pool representative metabolome has the potential to improve small molecule compound annotation and accelerate understanding of metabolic variation underlying phenotypic diversity in Cannabis.
... Still, it remains one of the few techniques capable of elucidating the chemical structure and stereochemistry of an unknown compound [99]. Besides its use as a qualitative technique, NMR is an accurate and reliable quantitative platform for the analysis of cannabinoids in a very short analysis time, especially with the latest highly sensitive probes [100][101][102][103]. Notwithstanding these great advantages, this technique is not very common due to the high instrumental costs (purchase and maintenance) and the need of highly qualified personnel. ...
Article
The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist. Graphical abstract
... Cannabinoid profile of HSO. LC-MS/HPLC-UV and GC/GC-MS have been typically used for identification and quantification of cannabinoids in hempseed oil [34][35][36] . Using LC-MS and comparing with a mixture of cannabinoids standards in this study, seven cannabinoid compounds including Cannabidivarin (CBDV), CBDA, CBD, Cannabigerol (CBG), and small amounts of Tetrahydrocannabivarin (THCV), THCA and THC (<0.2%) were detected ( Table 2). ...
Article
Full-text available
Abstract The seed of the hemp plant (Cannabis sativa L.) has been revered as a nutritional resource in Old World Cultures. This has been confirmed by contemporary science wherein hempseed oil (HSO) was found to exhibit a desirable ratio of omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) considered optimal for human nutrition. HSO also contains gamma-linoleic acid (GLA) and non-psychoactive cannabinoids, which further contribute to its’ potential bioactive properties. Herein, we present the kinetics of the thermal stability of these nutraceutical compounds in HSO, in the presence of various antioxidants (e.g. butylated hydroxytoluene, alpha-tocopherol, and ascorbyl palmitate). We focussed on oxidative changes in fatty acid profile and acidic cannabinoid stability when HSO was heated at different temperatures (25 °C to 85 °C) for upto 24 h. The fatty acid composition was evaluated using both GC/MS and 1H-NMR, and the cannabinoids profile of HSO was obtained using both HPLC-UV and HPLC/MS methods. The predicted half-life (DT50) for omega-6 and omega-3 PUFAs in HSO at 25 °C was about 3 and 5 days, respectively; while that at 85 °C was about 7 and 5 hours respectively, with respective activation energies (Ea) being 54.78 ± 2.36 and 45.02 ± 2.87 kJ/mol. Analysis of the conjugated diene hydroperoxides (CDH) and p-Anisidine value (p-AV) revealed that the addition of antioxidants significantly (p
... The secretory structures of Cannabaceae, especially of Cannabis sativa L., have aroused the interest of researchers since a long time ago because they are responsible for the production of a large amount of secondary metabolites of medicinal importance (Furr and Mahlberg 1981;Mahlberg 1991, 1997;Williamson and Evans 2000;Happyana et al. 2013). The latex of C. sativa, for example, is rich in cannabinoids and alkaloids (Furr and Mahlberg 1981), substances that have the medicinal potential to relieve symptoms related to the treatment of cancer, AIDS, and sclerosis (Ashton 2001;Honório et al. 2006;Hill et al. 2010). ...
Article
Full-text available
Cannabaceae is a known family because of the production of cannabinoids in laticifers and glandular trichomes of Cannabis sativa. Laticifers are latex-secreting structures, which in Cannabaceae were identified only in C. sativa and Humulus lupulus. This study aimed to expand the knowledge of laticifers in Cannabaceae by checking their structural type and distribution, and the main classes of substances in the latex of Celtis pubescens, Pteroceltis tatarinowii, and Trema micrantha. Such information is also updated for C. sativa. Samples of shoot apices, stems, leaves, and flowers were processed for anatomical, histochemical, ultrastructural, and cytochemical analyses. Laticifers are articulated unbranched in all species instead of non-articulated as previously described for the family. They occur in all sampled organs. They are thick-walled, multinucleate, with a large vacuole and a peripheral cytoplasm. The cytoplasm is rich in mitochondria, endoplasmic reticulum, dictyosomes, ribosomes, and plastids containing starch grains and oil drops. Pectinase and cellulase activities were detected in the laticifer wall and vacuole, confirming its articulated origin, described by first time in the family. These enzymes promote the complete dissolution of the laticifer terminal walls. The latex contains proteins, lipids, and polysaccharides in addition to phenolics (C. sativa) and terpenes (C. pubescens, T. micrantha). The presence of laticifers with similar distribution and morphology supports the recent insertion of Celtis, Pteroceltis, and Trema in Cannabaceae. The articulated type of laticifer found in Cannabaceae, Moraceae, and Urticaceae indicates that the separation of these families by having distinct laticifer types should be reviewed.
... Glandular trichomes are widely distributed in the flowers of the studied species Cannabaceae (this study, see Table 2), except for Humulus lupulus, in which this type of trichome is restricted to the flower subtending bract (Sugiyama, Oda & Kurosaki, 2006). The glandular trichomes of Cannabis sativa have been exhaustively studied, so that their morphology (Hammond & Mahlberg, 1973, 1977Dayanandan & Kaufman, 1976;Gangadhara & Inamdar, 1977), ultrastructure (Hammond & Mahlberg, 1978) and the chemical composition of the exudate (Turner, Hemphill & Mahlberg, 1980, 1981Happyana et al., 2013) are much discussed topics in the literature. Our study extends the knowledge of their distribution to the anther connective. ...
Preprint
Full-text available
Species of Cannabaceae are wind pollinated, have inconspicuous and reduced flowers that are pistillate, staminate and apparently perfect on the same individual or on different individuals, with a single-whorled perianth and a pseudomonomerous gynoecium. Our objective is to understand the developmental processes that lead to such a reduced flower morphology and polygamy in Cannabis sativa, Celtis iguanaea and Trema micrantha. Floral buds and flowers were processed for surface, histological examinations and 3D reconstructions of vasculature. The single-whorled perianth is interpreted as a calyx because the organs are robust, have a broad base, an acute apex and quincuncial aestivation and are opposite the stamens. Petals are absent from inception. The dicliny is established at different development stages: stamens or carpels are absent from inception (Cannabis sativa), initiated and aborted during early (Trema micrantha, before sporo/gametogenesis) or late (Celtis iguanaea, after sporo/gametogenesis) development. Furthermore, in all species studied the carpels are congenitally united and the pseudomonomerous nature of the gynoecium is confirmed. Glandular trichomes are distributed on the bracts, sepals, anther connective and receptacle. Special floral features shared by species of Cannabaceae include precocious ovule development and sepals that are each vascularized by one bundle. The reduced flowers of Cannabaceae are the result of the absence from inception and/or abortion of organs and even of a whole whorl at different developmental stages, which were probably selected in response to pressures exerted by the similar pollination mechanism.
... THCA-B (6) was discovered four years later by Raphael Mechoulam at the University of Jerusalem (Mechoulam et al. 1969). THCA synthase is expressed primarily in glandular trichomes, where THCA is both formed and stored (Happyana et al. 2013). The most likely explanation for plants producing precannabinoids here lies in their cytotoxicity, as both 4 and THCA cause cell death in cultures of Cannabis as well as insect cells (e.g., Spodoptera frugiperda). ...
Article
Full-text available
Cannabis spp. are some of the most controversial medicinal plants in the world. They contain great amounts of biologically active secondary metabolites, including the typical phenolic compounds called cannabinoids. Because of their low toxicity and complex biological activities, cannabinoids can be useful in the therapy of various diseases, but adverse psychological effects (of Δ9-THC in particular) raise concerns. This review summarizes the current knowledge of selected active C. indica compounds and their therapeutic potential. We summarize the main compounds contained in cannabis, the mechanisms of their effects, and their potential therapeutic applications. Further, we mention some of the clinical tests used to evaluate the efficacy of cannabinoids in therapy.
... While C. sativa is often cultivated for its phytocannabinoids (i.e., secondary metabolites primarily concentrated in floral tissue that provide various medicinal and intoxicating effects), the plant can also be a source of oilseeds and fibers in its primary stems (Small and Marcus, 2002). The majority of the plant, particularly the upper surface of leaves and flowers, are coated with trichomes (Happyana et al., 2013;Spitzer-Rimon et al., 2019) that produce cannabinoid precursors and ultimately cannabinoids (De Backer et al., 2012;Burgel et al., 2020). Unpollinated pistillate flowers (i.e., sensimilla) are the portion of the plant harvested for recreational and biopharmaceutical use as these inflorescences contain up to 10 times more cannabinoids in them than vegetative tissue . ...
Article
Full-text available
Cannabis sativa L. is an annual, short-day plant, such that long-day lighting promotes vegetative growth while short-day lighting induces flowering. To date, there has been no substantial investigation on how the switch between these photoperiods influences yield of C. sativa despite the tight correlation that plant size and floral biomass have with the timing of photoperiod switches in indoor growing facilities worldwide. Moreover, there are only casual predictions around how the timing of the photoperiodic switch may affect the production of secondary metabolites, like cannabinoids. Here we use a meta-analytic approach to determine when growers should switch photoperiods to optimize C. sativa floral biomass and cannabinoid content. To this end, we searched through ISI Web of Science for peer-reviewed publications of C. sativa that reported experimental photoperiod durations and results containing cannabinoid concentrations and/or floral biomass, then from 26 studies, we estimated the relationship between photoperiod and yield using quantile regression. Floral biomass was maximized when the long daylength photoperiod was minimized (i.e., 14 days), while THC and CBD potency was maximized under long day length photoperiod for ~42 and 49–50 days, respectively. Our work reveals a yield trade-off in C. sativa between cannabinoid concentration and floral biomass where more time spent under long-day lighting maximizes cannabinoid content and less time spent under long-day lighting maximizes floral biomass. Growers should carefully consider the length of long-day lighting exposure as it can be used as a tool to maximize desired yield outcomes.
... Methods for the identification of marijuana include: botanical identification, microscopical examination of leaves (58), chemical screening tests (57)(58)(59), THC identification through biochemical methods (60), and the use of molecular sequencing to identify DNA sequence homology to reference marijuana samples (61)(62)(63). Besides that, there are some unusual techniques used for this proposital, such as Nuclear Magnetic Resonance and electronic nose systems (51,54) The genetic analysis provides the opportunity to link products on the basis of their genetic profiles, which could be useful from an investigative point of view, e.g. to link producers, traffickers and consumers (9). The main technique used is the PCR for analysis of SNPs (15,28), Inter-Simple Sequence Repeat (ISSR) (49,61), STR (45,62) or specific genes (16,63). ...
Article
Full-text available
Background: Cannabis has been the most widely used illicit drug worldwide throughout many years. Reports from different countries indicate that the potency of cannabis preparation has been increasing, as well as the ratio of tetrahydrocannabinol/cannabidiol has been changing. The high consumption along with the changing chemical profile of the drug has led increasingly to the interest in researching the cannabis plant. Methods: This article reviews available literature on the analytical methods currently used for the detection and quantification of cannabinoids in cannabis plant. The papers were screened by two researchers independently and following a pre-specified protocol. Results and Discussion: The systematic review of the literature allowed to include 42 citations on cannabis plant analysis. Conclusions: The analytical methods for cannabis material published in the included articles of this systematic review showed a lack of relevant information of the development of methods on GC and LC analysis and the limits of detection and quantification of mass detectors.
... Although CBD is present in both male and female plants, most are found in resin glands on trichomes of the female flower buds (Mahlberg and Kim, 2004). The terpenophenolic compounds are secreted from head cells of trichome glands, specifically from the capitate-stalked glandular hairs (Happyana et al., 2013). Female flowers require mild temperatures and high nitrogen concentrations and light intensity. ...
Article
Full-text available
Forensic laboratories are required to have analytical tools to confidently differentiate illegal substances such as marijuana from legal products (i.e., industrial hemp). The Achilles heel of industrial hemp is its association with marijuana. Industrial hemp from the Cannabis sativa L. plant is reported to be one of the strongest natural multipurpose fibers on earth. The Cannabis plant is a vigorous annual crop broadly separated into two classes: industrial hemp and marijuana. Up until the eighteenth century, hemp was one of the major fibers in the United States. The decline of its cultivation and applications is largely due to burgeoning manufacture of synthetic fibers. Traditional composite materials such as concrete, fiberglass insulation, and lumber are environmentally unfavorable. Industrial hemp exhibits environmental sustainability, low maintenance, and high local and national economic impacts. The 2018 Farm Bill made way for the legalization of hemp by categorizing it as an ordinary agricultural commodity. Unlike marijuana, hemp contains less than 0.3% of the cannabinoid, Δ9-tetrahydrocannabinol, the psychoactive compound which gives users psychotropic effects and confers illegality in some locations. On the other hand, industrial hemp contains cannabidiol found in the resinous flower of Cannabis and is purported to have multiple advantageous uses. There is a paucity of investigations of the identity, microbial diversity, and biochemical characterizations of industrial hemp. This review provides background on important topics regarding hemp and the quantification of total tetrahydrocannabinol in hemp products. It will also serve as an overview of emergent microbiological studies regarding hemp inflorescences. Further, we examine challenges in using forensic analytical methodologies tasked to distinguish legal fiber-type material from illegal drug-types.
... These include growth conditions such as humidity, light quality and intensity, CO 2 concentration and mineral nutrition (Chandra et al., 2008;Chandra et al., 2017;Bernstein et al., 2019a). The tissue type is also an important factor as within the plant there is a locationand organ-specific distribution of the active secondary metabolites (Happyana et al., 2013;Bernstein et al., 2019a;Bernstein et al., 2019b). Phytocannabinoids are synthesized in glandular trichomes that are located in the highest density on the inflorescences of unfertilized female plants (Lipson Feder et al., 2021), and their accumulation varies in the different aerial parts (flowers, fan leaves, inflorescence leaves, stalk and stem). ...
Article
Full-text available
Medical Cannabis and its major cannabinoids (−)-trans-Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are gaining momentum for various medical purposes as their therapeutic qualities are becoming better established. However, studies regarding their efficacy are oftentimes inconclusive. This is chiefly because Cannabis is a versatile plant rather than a single drug and its effects do not depend only on the amount of THC and CBD. Hundreds of Cannabis cultivars and hybrids exist worldwide, each with a unique and distinct chemical profile. Most studies focus on THC and CBD, but these are just two of over 140 phytocannabinoids found in the plant in addition to a milieu of terpenoids, flavonoids and other compounds with potential therapeutic activities. Different plants contain a very different array of these metabolites in varying relative ratios, and it is the interplay between these molecules from the plant and the endocannabinoid system in the body that determines the ultimate therapeutic response and associated adverse effects. Here, we discuss how phytocannabinoid profiles differ between plants depending on the chemovar types, review the major factors that affect secondary metabolite accumulation in the plant including the genotype, growth conditions, processing, storage and the delivery route; and highlight how these factors make Cannabis treatment highly complex.
... C. sativa accumulates phytocannabinoids and terpenes in glandular trichomes located all over the aerial parts of the plant and in highest density on the female flowers [7,23]. No glandular trichomes are found on the root surfaces, and the root tissue therefore does not accumulate phytocannabinoids [24]. ...
Article
Full-text available
Phytocannabinoids are bioactive natural products found in some flowering plants, liverworts, and fungi that can be beneficial for the treatment of human ailments such as pain, anxiety, and cachexia. Targeted biosynthesis of cannabinoids with desirable properties requires identification of the underlying genes and their expression in a suitable heterologous host. We provide an overview of the structural classification of phytocannabinoids based on their decorated resorcinol core and the bioactivities of naturally occurring cannabinoids, and we review current knowledge of phytocannabinoid biosynthesis in Cannabis, Rhododendron, and Radula species. We also highlight the potential in planta roles of phytocannabinoids and the opportunity for synthetic biology approaches based on combinatorial biochemistry and protein engineering to produce cannabinoid derivatives with improved properties.
Article
This chapter discusses the ethnopharmacological properties, phytochemistry, and culture conditions of the Cannabis species. Cannabis sativa L. (Fam. – Cannabaceae) is an annual herbaceous plant and indigenous to Central Asia and India subcontinent. The phytochemicals in this plant species are secreted by the glandular trichomes found on the calyx and bracts of flowers. The Cannabis plant is used in the treatment of nausea and vomiting during chemotherapy. The toxicity levels of glutamate have been reduced significantly by cannabidiol and by antioxidant compounds because several cannabinoids demonstrate antioxidant effects. Callus cultures have been established in C. sativa by using the explants of seedlings and flowers. The fast synthesis of cannabinoids has been observed in light, while slow response was achieved in darkness. In suspension cultures, the cannabinoids could not be synthesized due to lack of polyketide synthase activity.
Article
The butyl homologues of Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabutol (Δ9-THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) inflorescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute configurations of the isolated cannabinoids were confirmed by comparison with the spectroscopic data of the respective compounds obtained by stereoselective synthesis. The butyl homologue of Δ9-THC, Δ9-THCB, showed an affinity for the human CB1 (Ki = 15 nM) and CB2 receptors (Ki = 51 nM) comparable to that of (-)-trans-Δ9-THC. Docking studies suggested the key bonds responsible for THC-like binding affinity for the CB1 receptor. The formalin test in vivo was performed on Δ9-THCB in order to reveal possible analgesic and anti-inflammatory properties. The tetrad test in mice showed a partial agonistic activity of Δ9-THCB toward the CB1 receptor.
Article
Full-text available
Cannabis (Cannabis sativa L.) is a complex, polymorphic plant species, which produces a vast array of bioactive metabolites, the two major chemical groups being cannabinoids and terpenoids. Nonetheless, the psychoactive cannabinoid tetrahydrocannabinol (Δ 9 -THC) and the non-psychoactive cannabidiol (CBD), are the two major cannabinoids that have monopolized the research interest. Currently, more than 600 Cannabis varieties are commercially available, providing access to a multitude of potent extracts with complex compositions, whose genetics are largely inconclusive. Recently introduced legislation on Cannabis cultivation in many countries represents a great opportunity, but at the same time, a great challenge for Cannabis research and development (R&D) toward applications in the pharmaceutical, food, cosmetics, and agrochemical industries. Based on its versatility and unique capabilities in the deconvolution of the metabolite composition of complex matrices, metabolomics represents an ideal bioanalytical tool that could greatly assist and accelerate Cannabis R&D. Among others, Cannabis metabolomics or cannabinomics can be applied in the taxonomy of Cannabis varieties in chemovars, the research on the discovery and assessment of new Cannabis-based sources of bioactivity in medicine, the development of new food products, and the optimization of its cultivation, aiming for improvements in yield and potency. Although Cannabis research is still in its infancy, it is highly foreseen that the employment of advanced metabolomics will provide insights that could assist the sector to face the aforementioned challenges. Within this context, here, the current state-of-the-art and conceptual aspects of cannabinomics are presented.
Article
Glandular trichomes are epidermal outgrowths that are the site of biosynthesis and storage of large quantities of specialized metabolites. Besides their role in the protection of plants against biotic and abiotic stresses, they have attracted interest due to the importance of the compounds they produce for human use, for example as pharmaceuticals, flavor and fragrance ingredients or pesticides. Here, we review what novel concepts investigations on glandular trichomes have brought to the field of specialized metabolism, in particular with respect to chemical and enzymatic diversity. Furthermore, understanding the metabolic network underlying the high productivity of glandular trichomes, as well as the transport and storage of metabolites, represent the next challenges in the field. Another emerging area is the development of glandular trichomes. Studies in some model species, essentially tomato, tobacco and Artemisia, are now providing the first molecular clues, but many open questions remain: how is the distribution and density of different trichome types on the leaf surface controlled? When is the decision for an epidermal cell to differentiate into one type of trichome or another taken? Recent advances in gene editing make it now possible to address these questions and promise exciting discoveries in the near future.
Article
Plants of Cannabis sativa L. (Cannabaceae) produce an array of more than 160 isoprenylated resorcinyl polyketides, commonly referred to as phytocannabinoids. These compounds represent molecules of therapeutic importance due to their modulation of the human endocannabinoid system (ECS). While understanding of the biosynthesis of the major phytocannabinoids Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) has grown rapidly in recent years, the biosynthetic origin and genetic regulation of many potentially therapeutically relevant minor phytocannabinoids remains unknown, which limits the development of chemotypically elite varieties of C. sativa. This review provides an up-to-date inventory of unusual phytocannabinoids which exhibit cannabimimetic-like activities and proposes putative metabolic origins. Metabolic branch points exploitable for combinatorial biosynthesis and engineering of phytocannabinoids with augmented therapeutic activities are also described, as is the role of phytocannabinoid remodelling to accelerate therapeutic portfolio expansion in C. sativa.
Article
Aim. To investigate hemp (Cannabis sativa L.) collection accessions of different genetic and eco-geographical origin in terms of major cannabinoid compound contents – cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabigerol (CBG), to establish chemotypes of the accessions and correlations between cannabinoid contents. Results and Discussion. Fifty five hemp accessions were analyzed by gas-liquid chromatography. The identified cannabinoid compounds showed variability. The CBD content ranged from 0.0052 to 1.7251 %; the THC content - from 0.0000 (complete absence) to 0.0775 % (does not exceed the law-allowable level); and the CBG content - from 0.0000 (complete absence) to 0.8892 %. Twenty-one accessions (38 %), ВО203327, ЮС 12, UF0600047, Odnodomni 8, ВО78142, ВО5799, UF0600145, UF0600146, ВО2539, UF0600183, YuS 58, Malo-Perevozskaya, Hibryd 3, Yuzhnaya Bolshe-Pisarevskaya, Almetyevskaya, UF0600253 et al. were distinguished due to high CBD content. Nine accessions (16.4 %), YuSO 31, Hlukhivski 51, Hliana, Kirovskaya К-314, Mykolaichyk, Artemida, Harmoniia, Mih 2 and Hlukhivski 85 had no THC at al. Three accessions (5.4 % of the total), UF0600253, Mozdokskaya and VIK CBN, were distinguished because of high CBG content. Conclusions. The accessions distinguished were recommended for use in practical breeding, in particular for developing medicinal varieties with a high content of CBD and/or wiithout CBG or THC. The analyzed accessions belonged to 3 hemp сhemotypes: III (36 accessions or 65.5 %), IV (1 accession or 1.8 %) and V (18 accessions or 32.7 % of the total). Strong correlations between CBD and THC and no relationships between CBG and other cannabinoid compounds have been established. Chemotype III accessions showed weaker correlations compared with chemotype V. This pattern should be taken into account, when selecting starting forms for breeding.
Article
Despite being a controversial crop, Cannabis sativa L. has a long history of cultivation throughout the world. Following recent legalisation in Canada, it is emerging as an important plant for both medicinal and recreational purposes. Recent progress in genome sequencing of both cannabis and hemp varieties allows for systematic analysis of genes coding for enzymes involved in the cannabinoid biosynthesis pathway. Single nucleotide polymorphisms in the coding regions of cannabinoid synthases play important role in determining plant chemotype. Deep understanding of how these variants affect enzymes activity and accumulation of cannabinoids will allow breeding of novel cultivars with desirable cannabinoid profile. Here we present a short overview of the major cannabinoid synthases and present the data on the analysis of their genetic variants and their effect on cannabinoid content using several in-house sequenced Cannabis cultivars.
Chapter
Cannabis (Cannabis sativa L.) growers worldwide lack reliable and research-based information about precision management practices (PMP) of cannabis. The history, legal framework, and PMP for cultivation of cannabis have been reviewed with special emphasis on water management, nutrient management, and disease control for optimum cannabis production. The aim is to provide guidelines for precision farming of cannabis to meet fibrous and medicinal needs of the humankind. Therefore, the scope of this chapter is for the potential of hemp cultivation to meet industry needs of fiber and medicine. Methods of irrigation scheduling, nutrient applications, and keeping greenhouse hygienically clean for disease-free (i.e., powdery mildew) hemp production are discussed. Reviewed and recommended application rates of irrigation and nutrients, and environment controls have been tabulated. Chemical, biological, and physical controls of PM control and crop input requirements for disease-free cultivation of hemp are presented.
Article
Differences in cannabis (Cannabis sativa L.) plant chemistry between strains are influenced by genetics, plant growth and development, in addition to environmental conditions such as abiotic and biotic stress. Resulting secondary metabolite profiles are further altered post-harvest by drying and extraction, all of which present sizable challenges to industrial scale producers of pharmaceutical grade products in Canada and elsewhere. Consistent quantity and quality of cannabis extracts, demonstrated by preferred cannabinoid ratios and other secondary metabolites present are important, particularly as the list of therapeutic uses for medical cannabis is expanding. As more countries contemplate the legalization and licensure of medical cannabis, the number of cannabis extraction and testing laboratories is increasing to keep up with demand. However, it is not always known what standards are adhered to, resulting in numerous non-validated methods. In this review, a summary of cannabis chemistry and biosynthesis of secondary compounds is provided, and post-harvest processing practices occurring along the cannabis product value chain that might affect cannabis phytochemistry, potency and volatility are presented. An emphasis is placed on improved drying and extraction methods for plant material suitable for the cannabis industry. Finally, new approaches to secondary metabolite profiling for cannabis products are compared.
Article
Full-text available
To create an industrial hemp variety of the Central European ecological and geographical type with a high cannabigerol content and universal application. Methods. Breeding (self-pollination, creation of artificial populations, selection), field, biochemical (thin-layer and gas-liquid chromatography of cannabinoid compounds), instrumental and technological assessment of fibre quality, and statistical methods. Results. Variety ‘Vik 2020’ was obtained as a result of creation of artificial populations. The plants are characterized by higher content of cannabigerol (1.034 ± 0.0323%), and almost zero of other secondary metabolites, such as cannabidivarin, cannabidiol, cannabichromene and psychotropic tetrahydrocannabinol (0.003 ± 0.0011; 0.018 ± 0.0080; 0.012 ± 0.0027, and 0.005 ± 0.0012%, respectively). The t rait of cannabigerol content is quite stable within the population and is not correlated with the trait of tetrahydrocannabinol content (r = -0.23). TLC showed that cannabigerol accumulated mainly in the form of cannabigerolic acid and to a lesser extent as a neutral compound, which is consistent with the theory that this substance is a precursor for the synthesis of other cannabinoids. According to the results of the competitive variety test, when growing to obtain fibre and seeds, the variety features short height, specifically significantly lower total (206.4 cm) and technical stem length (135.6 cm) compared to the standard variety, significantly higher inflorescence length (70.8 cm), which determine the formation of the significant yield of biomass suitable for pharmaceutical use and high seed yield (0.98 t/ha). The yield of total fibre was the same as in the standard variety (29.0%), but its quality and technological value for primary processing were higher. The variety had a homogeneous sex structure, resistance to bioltic and abiotic environmental factors. Plants reached biological maturity in 116 days (BBCH 89). This cultivar is recommended for obtaining seeds, quality fiber and potentially cannabigerol (on condition of changes in legislation). Conclusions. The efficiency of using self-pollinating lines in breeding with their subsequent combining into a synthetic population and improving selection was proved by the case of a new variety of industrial hemp ‘Vik 2020’, characterized by an increased content of cannabigerol and the absence of psychotropic properties
Article
Introduction: Cannabis is a valuable plant, cultivated by humans for millennia. However, it has only been in the past several decades that biologists have begun to clarify the interesting Cannabis biosynthesis details, especially the production of its fascinating natural products termed acidic cannabinoids. Discussion: Acidic cannabinoids can experience a common organic chemistry reaction known as decarboxylation, transforming them into structural analogues referred to as neutral cannabinoids with far different pharmacology. This review addresses acidic and neutral cannabinoid structural pairs, when and where acidic cannabinoid decarboxylation occurs, the kinetics and mechanism of the decarboxylation reaction as well as possible future directions for this topic. Conclusions: Acidic cannabinoid decarboxylation is a unique transformation that has been increasingly investigated over the past several decades. Understanding how acidic cannabinoid decarboxylation occurs naturally as well as how it can be promoted or prevented during harvesting or storage is important for the various stakeholders in Cannabis cultivation.
Article
Cembranoids are a class of glandular trichome exudates with a variety of structural variations and biological activities, and are found in high quantities in tobacco (Nicotiana tabacum L.). However, few studies have conducted preparative separation and examined the activities of cembranoids other than (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (α-CBT-diol, 1) and (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT-diol, 2); thus, the structure-activity relationship (SAR) of cembranoids remains unclear. Here, to deduce the SAR of cembranoids, raw cembranoid extract was systematically separated through an activity-oriented approach with preparative reversed-phase liquid chromatography. Each fraction was identified, and their antifungal, insecticidal, and cytotoxic bioactivities were evaluated. Composition analysis and activity detection of primary fractions (Fr.1–Fr.10) revealed that cembranoid activity might be influenced by the number of hydroxyl groups and degree of unsaturation (macrocycle and double bonds). Sub-fraction analysis showed that the antifungal activity of cembranoids was related to the number of hydroxyl groups and double bonds, and that their cytotoxic activity was affected by the type and position of substituents. Furthermore, all four factors could influence insecticidal activity. By conducting in-depth studies of single cembranoids, their SARs were summarized. The existence of Δ11,12/Δ7,8 and hydroxylation at C-6/C-4 contributed to the antifungal activity of cembranoids. Variation in the type and position of substituents also significantly affected antifungal activity, of which Δ11,12 was the most influential factor. Additionally, the change in substituent configuration at C-4 and C-12 had no significant effect on antifungal activity. The insecticidal activity of cembranoids benefited from the existence of 4-hydroxyl, which greatly enhanced their binding affinity; however, functional groups substituted at other positions decreased insecticidal activity. Cytotoxic activity was positively correlated with α-configuration at C-4 and hydroxylation at C-6 but inversely correlated with 4-methoxy substitution. Among them, 6-hydroxyl formed the core; the α-/β-configuration at C-12 and 4-/12-substitution of cembranoids did not affect their insecticidal activity. Insecticidal activity might play a key role by restraining acetylcholinesterase activity through hydrophobic interactions between the cembranoid carbon skeleton and residues located at the acetylcholinesterase binding pocket. These findings may provide a foundation for the development of novel cembranoid-based pesticides and medicines.
Article
Full-text available
Cannabis sativa L. has been one of the oldest medicinal plants cultivated since 10,000 years for several agricultural and industrial applications. However, the plant became controversial due 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 an 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 an herbal medicine to latest potential in COVID, 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.
Article
Full-text available
Analysis of hemp collection samples based on the content of minor (rare) non-psychotropic cannabinoids, such as cannabichromene (CBC), cannabidivarin (CBDV), and cannabinol (CBN); determination of correlation relationships between them and common compounds; selection of valuable breeding genotypes. Methods. Field, biochemical (gas chromatography of cannabinoid compounds), and statistical (pair, partial, and multiple linear correlations). Results. Quantitative analysis of 210 samp­les of various ecological-geographical and genetic origin (local and wild forms, self-filing lines, hybrids, varieties, synthetic populations, polyploids) with a tetrahydrocannabinol (THC) content of less than 0.08% in dried plants showed the level of manifestation of the trait from its absence within the sensitivity of the gas chromatograph up to 0.6838% CBC, 0.1719% CBC and 0.3274% CBN. In the studied hemp samples, a medium negative relationship was found between the signs of the CBC and cannabidiol (CBD) contents (r = –0.53), a weak negative relationship between CBC and CBDV contents (r = –0.35), medium positive relationships between the signs of CBC and THC contents (r = 0.57) and CBC and CBN contents (r = 0.59). A medium positive correlation (r = 0.57) was found between the signs of CBDV and CBD contents, while CBN had a strong positive relationship with THC (r = 0.82). There is almost no correlation between cannabigerol (CBG) and the minor cannabinoids under study. The biosynthesis of minor cannabinoid compounds is quite complex. Signs manifestation is affected by many genetic and external factors. Partial correlation coefficients (given that one of the three signs is eliminated) and multiple correlation coefficients (given that the relationship of one sign is determined and two other signs are combined) give grounds to state that the gene for CBCA-synthase affects the production of CBD and, in particular THC. Conclusions. The closeness of the linear relationships between minor cannabinoids and common components allows selecting valuable hemp samples with a high content of one or several compounds under the absence or low content of psychotropic THC.
Article
Zusammenfassung Hintergrund Cannabis ist weltweit immer noch die am häufigsten konsumierte illegale Droge, aber auch der Einsatz von Medizinalcannabis oder auch als Lebens‑/Nahrungsergänzungsmittel steigt stetig. Somit sind Kenntnisse über diese verschiedenen Produkte und die Komplexität der rechtlichen Einordnung von Cannabis für die Rechtsmedizin und die forensische Toxikologie von großer Relevanz. Fragestellung Ziel der Arbeit ist es, einen Überblick über aktuelle Trends des Cannabiskonsums zu geben und hierbei die verschiedenen Cannabisprodukte darzustellen sowie diese rechtlich einzuordnen. Material und Methode Für diese Übersichtarbeit wurde eine Literaturrecherche zu den verschiedenen Cannabisprodukten und ihrer rechtlichen Einordnung durchgeführt. Ergebnisse Beim Konsum von Tetrahydrocannabinol(THC)-reichem Cannabis zu Rauschzwecken ist ein Trend hin zu immer höheren THC-Gehalten im Pflanzenmaterial und zusätzlich zu intensiven Konsumformen wie dem „dabbing“ von Butan-Haschisch-Öl zu erkennen. Seit der Betäubungsmittelgesetzesänderung vom 10.03.2017 werden auch Cannabisblüten und -extrakte mit unterschiedlichen THC- bzw. Cannabidiol(CBD)-Gehalten auf Betäubungsmittelrezept stark zunehmend verordnet, einhergehend mit einer Steigerung der THC-Höchstverschreibungsmenge. Ein weiterer Trend besteht in dem Konsum von sogenannten CBD-Lifestyle-Produkten, die als Arzneimittel seit 2016 verschreibungspflichtig sind und als Lebensmittel nach Novel Food-Verordnung jeweils einzeln zu prüfen sind, ob sie als zulassungsbedürftiges neuartiges Lebensmittel einzustufen sind. Schlussfolgerungen Insgesamt ergibt sich für die forensisch-chemische sowie forensisch-toxikologische Beurteilung eine zunehmende Komplexität der potenziell konsumierten THC-haltigen Produkte, deren wissenschaftliche Untersuchung sowohl zur möglichen Differenzierbarkeit des Cannabismaterials als auch von biologischen Matrices nach Konsum verschiedener Cannabisprodukte notwendig macht.
Article
Introduction: Cannabis is an annual dioecious plant, which shares its origins with the inception of the first agricultural human societies in Asia. Over the course of time different parts of the plant have been utilized for therapeutic and recreational purposes. Linnaeus was the first person to describe Cannabis as Cannabis sativa (C.sativa). Numerous bioactive phytochemicals are extracted from C. sativa that signal for medicinal development. Methods:The review aims to provide a different perspective of the ethnobotanical, taxonomy and chemical aspects from the ancient times of C. sativa. The study was conducted with the review of scientific papers from Pubmed, Scopus, Wiley Online Library, Springer, Elseveir, Science Direct, Taylor Francis and online textbooks of C. sativa. Results: C. sativa has its origin from Asia. It has traditional spiritual, household and therapeutic uses. Cannabis is a monotypic genera with three different varieties: C sativa var. sativa, C sativa var. indica, C sativa var. ruderalis. A total of 565 chemicals (120 cannabinoids and 445 non cannabinoids) have been recorded in Cannabis. Conclusions: Cannabis is an ethnobotanical rich and phytochemical significant therapeutic plant. Because of lack of scientific research, the taxonomic aspects are still hidden. This study recommends exploratory study on ethnobotanical, taxonomical and phytochemicals of Nepalese Cannabis.
Article
Cannabinoids (CBDs) have been traditionally used as a folk medicine. Recently, they have been found to exhibit a high pharmacological potential. However, they are addicted and are often abused by drug users, thereby, becoming a threat to public safety. CBDs and their metabolites are usually found in trace levels in plants or in biological matrices and, are therefore not easy to be detected. Advances have been made toward accurately analyzing CBDs in plants or in biological matrices. This review aims at elucidating on the consumption of CBDs as well as its adverse effects and to provide a comprehensive overview of CBD pretreatment and detection methods. Moreover, novel pretreatment methods such as microextraction, Quick Easy Cheap Effective Rugged Safe and online technology as well as novel analytic methods such as ion-mobility mass spectrometry, application of high resolution mass spectrometry in nontarget screening are summarized. In addition, we discuss and compare the strengths and weaknesses of different methods and suggest their future prospect.
Article
Full-text available
The diversity of non-glandular and glandular hairs of Cannabis sativa L. (marihuana) are described by scanning electron microscopy. The non-glandular hairs are of two major types, as distinguished by size differences and locations, and all of them are highly silicified. The presence of silica as well as cystoliths of calcium carbonate help in the identification of marihuana even in its ash residues. X-ray microanalyses of Cannabis hairs are compared with those of Humulus lupulus and Lantana camera, whose hairs have been considered to resemble those of marihuana. Glandular hairs are found to be of two major categories. One group consists of glands whose heads are generally made up of eight cells and the other group whose heads are generally made up of two cells but never more than four cells. All glands of both categories are stalked. Some glands of the first category are massively stalked and these are restricted solely to anthers and bracts of staminate and pistillate plants. The massive stalk is considered to be made up of epidermal and hypodermal cells that have grown in response to some stimulation during anthesis. Fine details of the shoot system of Cannabis, such as cuticular ridges on epidermal cells, warty protuberances on non-glandular hairs, and surface views of glands in developing stages are also reported. Glandular hairs on the bracts of Humulus lupulus resemble those of Cannabis.
Chapter
Full-text available
Cannabinoids and in particular the main psychoactive Δ9-THC are promising substances for the development of new drugs and are of high importance in biomedicine and pharmacy. This review gives an overview of the chemical properties of Δ9-THC, its synthesis on industrial scale, and the synthesis of important metabolites. The biosynthesis of cannabinoids in Cannabis sativa is extensively described in addition to strategies for optimization of this plant for cannabinoid employment in medicine. The metabolism of Δ9-THC in humans is shown and, based on this, analytical procedures for cannabinoids and their metabolites in human forensic samples as well as in C.sativa will be discussed. Furthermore, some aspects of medicinal indications for Δ9-THC and its ways of administration are described. Finally, some synthetic cannabinoids and their importance in research and medicine are delineated.
Article
Full-text available
Cannabis sativa is an interesting crop for several industrial uses, but the legislations in Europe and USA require a tight control of cannabinoid type and content for cultivation and subsidies release. Therefore, cannabinoid survey by gas chromatography of materials under selection is an important step in hemp breeding. In this paper, a number of Cannabis accessions were examined for their cannabinoid composition. Their absolute and relative content was examined, and results are discussed in the light of both the current genetic model for cannabinoid’s inheritance, and the legislation’s requirements. In addition, the effectiveness of two different types of markers associated to the locus determining the chemotype in Cannabis was evaluated and discussed, as possible tools in marker-assisted selection in hemp, but also for possible applications in the forensic and pharmaceutical fields.
Article
Full-text available
Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored. We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of Δ9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp. The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.
Article
Full-text available
We performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. It had previously been shown that HESC lines had significantly higher non-CG methylation than differentiated cells, and we therefore asked whether these sites were conserved across cell lines. We find that heavily methylated non-CG sites are strongly conserved, especially when found within the motif TACAG. They are enriched in splice sites and are more methylated than other non-CG sites in genes. We next studied the relationship between allele-specific expression and allele-specific methylation. By combining bisulfite sequencing and whole transcriptome shotgun sequencing (RNA-seq) data we identified 1,020 genes that show allele-specific expression, and 14% of CG sites genome-wide have allele-specific methylation. Finally, we asked whether the methylation state of transcription factor binding sites affects the binding of transcription factors. We identified variations in methylation levels at binding sites and found that for several transcription factors the correlation between the methylation at binding sites and gene expression is generally stronger than in the neighboring sequences. These results suggest a possible but as yet unknown functional role for the highly methylated conserved non-CG sites in the regulation of HESCs. We also identified a novel set of genes that are likely transcriptionally regulated by methylation in an allele-specific manner. The analysis of transcription factor binding sites suggests that the methylation state of cis-regulatory elements impacts the ability of factors to bind and regulate transcription.
Article
Full-text available
RNA isolated from the glands of a Delta(9)-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.
Article
Full-text available
We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid.
Article
Full-text available
Δ1-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Δ1-tetrahydrocannabinol. We cloned a novel cDNA (GenBank™ accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.
Article
Full-text available
Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription-PCR (RT-PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme.
Article
Full-text available
Laser-assisted microdissection (LAM) is a powerful tool for isolating specific tissues, cell types and even organelles from sectioned biological specimen in a manner conducive to the extraction of RNA, DNA or protein. LAM, which is an established technique in many areas of biology, has now been successfully adapted for use with plant tissues. Here, we provide an overview of the processes involved in conducting a successful LAM study in plants and review recent developments that have made this technique even more desirable. We also discuss how the technology might be exploited to answer some pertinent questions in plant biology.
Article
Full-text available
Plant Methods is a new journal for plant biologists, specialising in the rapid publication of peer-reviewed articles with a focus on technological innovation in the plant sciences. The aim of Plant Methods is to stimulate the development and adoption of new and improved techniques and research tools in plant biology. We hope to promote more consistent standards in the plant sciences, and make readily accessible laboratory and computer-based research tools available to the whole community. This will be achieved by publishing Research articles, Methodology papers and Reviews using the BioMed Central Open Access publishing model. The journal is supported by a prestigious editorial board, whose members all recognise the importance of technological innovation as a driver for basic science.
Article
Full-text available
Laser microdissection is a useful tool for collecting tissue-specific samples or even single cells from animal and plant tissue sections. This technique has been successfully employed to study cell type-specific expression at the RNA, and more recently also at the protein level. However, metabolites were not amenable to analysis after laser microdissection, due to the procedures routinely applied for sample preparation. Using standard tissue fixation and embedding protocols to prepare histological sections, metabolites are either efficiently extracted by dehydrating solvents, or washed out by embedding agents. In this study, we used cryosectioning as an alternative method that preserves sufficient cellular structure while minimizing metabolite loss by excluding any solute exchange steps. Using this pre-treatment procedure, Arabidopsis thaliana stem sections were prepared for laser microdissection of vascular bundles. Collected samples were subsequently analyzed by gas chromatography-time of flight mass spectrometry (GC-TOF MS) to obtain metabolite profiles. From 100 collected vascular bundles (approximately 5,000 cells), 68 metabolites could be identified. More than half of the identified metabolites could be shown to be enriched or depleted in vascular bundles as compared to the surrounding tissues. This study uses the example of vascular bundles to demonstrate for the first time that it is possible to analyze a comprehensive set of metabolites from laser microdissected samples at a tissue-specific level, given that a suitable sample preparation procedure is used.
Article
Full-text available
Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.
Article
Full-text available
Cannabis as a medicine was used before the Christian era in Asia, mainly in India. The introduction of cannabis in the Western medicine occurred in the midst of the 19th century, reaching the climax in the last decade of that century, with the availability and usage of cannabis extracts or tinctures. In the first decades of the 20th century, the Western medical use of cannabis significantly decreased largely due to difficulties to obtain consistent results from batches of plant material of different potencies. The identification of the chemical structure of cannabis components and the possibility of obtaining its pure constituents were related to a significant increase in scientific interest in such plant, since 1965. This interest was renewed in the 1990's with the description of cannabinoid receptors and the identification of an endogenous cannabinoid system in the brain. A new and more consistent cycle of the use of cannabis derivatives as medication begins, since treatment effectiveness and safety started to be scientifically proven.
Article
Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis sativa L., which included both drug and fiber phenotypes, were investigated by gas-liquid chromatographic analyses. Capitate-stalked glands were selectively harvested from vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspicuously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis represent a dynamic system within the cannabinoid synthesizing activities of this plant.
Article
Trichome density and type and cannabinoid content of leaves and bracts were quantitated during organ ontogeny for three clones of Cannabis sativa L. Trichome initiation and development were found to occur throughout leaf and bract ontogeny. On leaves, bulbous glands were more abundant than capitate-sessile glands for all clones, although differences in density for each gland type were evident between clones. On pistillate bracts, capitate-sessile glands were more abundant than the bulbous form on all clones, and both types decreased in relative density during bract ontogeny for each clone. The capitate-stalked gland, present on bracts but absent from vegetative leaves, increased in density during bract ontogeny. The capitate-stalked gland appeared to be initiated later than bulbous or capitate-sessile glands during bract development and on one clone it was first found midway in bract ontogeny. Nonglandular trichomes decreased in density during organ ontogeny, but the densities differed between leaves and bracts and also between clones. Specific regulatory mechanisms appear to exist to control the development of each trichome type independently. In addition, control of trichome density seems to be related to the plant organ and clone on which the gland type is located. Cannabinoid synthesis occurs throughout organ development and is selectively regulated in each organ. Typically, cannabinoid synthesis occurred at an increasing rate during bract development, whereas in developing leaves synthesis occurred at a decreasing rate. Cannabinoid content on a dry weight basis was generally greater for bracts than leaves. Analyses of leaves indicate that other tissues in addition to glands may contain cannabinoids, while for bracts the gland population can accommodate the cannabinoid content for this organ. The functional significance of trichomes and cannabinoids in relation to evolution is discussed.
Article
Total synthesis of biologically interesting (?)-cannabichromene, (?)-cannabichromenic acid, and (?)-daurichromenic acid is described. The key step in the synthetic strategy involves the formation of benzopyrans by ethylenediamine diacetate-catalyzed reactions of resorcinols with \alpha,\beta-unsaturated aldehydes.
Article
As part of our program to study the phytochemistry of high potency Cannabis sativa L. [1,2], seven new non-cannabinoid constituents were isolated, namely 5-acetoxy-6-geranyl-3-n-pentyl-1,4-benzoquinone (1), 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), 4,7-dimethoxy-1,2,5-trihydroxyphenanthrene (4), α-cannabispiranol (5), cannflavin C (6) and β-sitosteryl-3-O-β-D-glucopyranoside-2'-O-palmitate (7). In addition, four known compounds, chrysoeriol (8), 6-prenyl-apigenin (9), cannflavin A (10) and β-acetyl cannabispiranol (11), were identified, with 8 and 9 being reported for the first time form cannabis. The antimicrobial, antileishmanial, antimalarial and anti-oxidant activities of these isolates were evaluated. Furthermore the analgesic activity of 2, 3 and 4 was evaluated in mice using the hot-plate and tail-flick nociception models. Acknowledgements: The project described was supported by Grant Number 5P20RR021929–02 from the National Center for Research Resources and by the National Institute on Drug Abuse, contract # N01DA-5–7746. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. We are grateful to Dr. Bharathi Avula for assistance with the HR-ESI-MS, and to Dr. Melissa Jacob, Ms. Marsha Wright and Dr. Babu Tekwani for conducting the antimicrobial and antiprotozoal testing. References: 1. Radwan, M.M., et al. (2008). Planta Med. 74: 267–272. 2. Ahmed, S.A., et al. (2008)J. Nat. Prod. (In press).
Article
Cannabis sativa L., one of the oldest plants known in medicine, is the most widely used illicit drug in the world today. A total of almost 500 natural constituents have been isolated and/or identified from cannabis [1], with Δ⁹-THC the main biologically active component [2]. The availability of high potency marijuana on the illicit market with unprecedented Δ⁹-THC concentrations (> 20% by dry weight)[3] has renewed our interest in the discovery of new constituents from cannabis. Phytochemical investigation of a high potency variety of C sativa L. resulted in the isolation of six new metabolites, (±)-6,7-trans-epoxycannabigerolic acid (1), (±)-6,7-cis-epoxycannabigerolic acid (2), (±)-6,7-cis-epoxycannabigerol (3), (±)-6,7-trans-epoxycannabigerol (4), 5ʹ-methyl-4-pentylbiphenyl-2,2ʹ,6-triol (5), and 7-methoxycannabispirone (5), along with seven known compounds (cannabigerolic acid, 5ʹ-methoxycannabigerolic acid, cannabispirone, β-cannabispiranol, dehydrocannabifuran, cannaflavin B and cannabigerol). The antimicrobial and antileishmanial activities were investigated. Acknowledgements: This work is supported by the Center of Research Excellence in Natural Products Neuroscience, The University of Mississippi, contract # 1P20RR021929-01, and by the National Institute on Drug Abuse, contract # N01DA-5-7746. We are grateful to Dr. Bharathi Avula for assistance with the HR-ESI-MS, and to Dr. Melissa Jacob and Ms. Marsha Wright for conducting the antimicrobial testing. References: [1] Grotenhermen F, Russo E, In Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic Potential. Grotenhermen F, Russo E, Ed.; The Haworth Press, Inc.: Binghamton, New York, 2002; Definitions and Explanations, pp. xxvii–xxxi. [2] Clarke RC, Watson DP, In Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic Potential. Grotenhermen F, Russo E, Ed.; The Haworth Press, Inc.: Binghamton, New York, 2002; Chapter 1 – Botany of Natural Cannabis Medicines, p.3–14.
Article
Development of the secretory cavity and formation of the subcuticular wall of glandular trichomes in Cannabis sativa L. was examined by transmission electron microscopy. The secretory cavity originated at the wall-cuticle interface in the peripheral wall of the discoid secretory cells. During the presecretory phase in development of the glandular trichome, the peripheral wall of the disc cells became laminated into a dense inner zone adjacent to the plasma membrane and a less dense outer zone subjacent to the cuticle. Loosening of wall matrix in the outer zone initiated a secretory cavity among fibrous wall materials. Membrane-bound hyaline areas, compressed in shape, arose in the wall matrix. They appeared first in the outer and subsequently in the inner zone of the wall. The membrane of the vesicles, and associated dense particles attached to the membrane, arose from the wall matrix. Hyaline areas, often with a conspicuous electron-dense content, were released into the secretory cavity where they formed rounded secretory vesicles. Fibrous wall material released from the surface of the disc cells became distributed throughout the secretory cavity among the numerous secretory vesicles. This wall material was incorporated into the developing subcuticular wall that increased five-fold in thickness during enlargement of the secretory cavity. The presence of a subcuticular wall in the cavity of Cannabis trichomes, as contrasted to the absence of this wall in described trichomes of other plants, supports a polyphyletic interpretation of the evolution of the secretory cavity in glandular trichomes among angiosperms.
Article
Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis sativa L., which included both drug and fiber phenotypes, were investigated by gas-liquid chromatographic analyses. Capitate-stalked glands were selectively harvested from vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspic­ uously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis represent a dynamic system within the cannabinoid synthesizing activities of this plant. GLANDULAR trichomes are prominent features on the shoot system of Cannabis saliva L. Sev­ eral studies using histochemical and analytical procedures
Article
Twelve new cannabinoid esters, together with three known cannabinoid acids and Δ9-THC, were isolated from a high potency variety of Cannabis sativa L [1,2]. The structures were determined by extensive spectral analysis to be: β-fenchyl-Δ9-THCA ester (1), α-fenchyl-Δ9-THCA ester (2), bornyl-Δ9-THCA ester (3), epi-bornyl-Δ9-THCA ester (4), α-terpenyl-Δ9-THCA ester (5), 4-terpenyl-Δ9-THCA ester (6), α-cadinyl-Δ9-THCA ester (7), γ-eudesmyl-Δ9-THCA ester (8), inseparable mixture of two sesquiterpenyl-Δ9-THCA esters (9), α-cadinyl-CBGA ester (10), γ-eudesmyl-CBGA ester (11), 4-terpenyl-CBNA ester (12), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid A (Δ9-THCA), cannabinolic acid A (CBNA) and cannabigerolic acid (CBGA). CB-1 receptor assays [3–6] indicated that these esters, as well as the parent Δ9-THCA, are not active compared to Δ9-THC. Acknowledgements: This work is supported by the National Institute on Drug Abuse (contract # N01DA-5-7746) and by the Center of Research Excellence in Natural Products Neuroscience, The University of Mississippi (contract # 1P20RR021929-01). We are grateful to Dr. Bharathi Avula for assistance with the HR-ESI-MS, and to Dr. Melissa Jacob and Ms. Marsha Wright for conducting the antimicrobial testing. References: [1] ElSohly MA, et al. (2000) Journal of Forensic Science 45: 24–30. [2] ElSohly MA, Slade D (2005) Life Sciences 78: 539–548. [3] Devane WA, (1988) Molecular Pharmacology 34: 605–613. [4] Munro S, et al. (1993) Nature 365: 61–65. [5] Barth F (2005) Annual Reports in Medicinal Chemistry 40: 103–118. [6] Ashton JC, Giass M (2007) Current Neuropharmacology 5: 73–80.
Article
Synthetic (−)-Δ9-trans-tetrahydrocannabinol impregnated on placebo Cannabis decomposed only 6.3% after being stored for 1 year at −18°. Storage at 5° and room temperature under various conditions led to severe decomposition. The amount of cannabinol observed when (−)-Δ9-trans-tetrahydrocannabinol decomposed indicates that cannabinol is not the only decomposition product.