Distribution of cell-wall polysaccharides and proteins during growth of the hemp hypocotyl.

MAIN CONCLUSION: The immuno-ultrastructural investigation localized cell-wall polysaccharides of bast fibers during hemp hypocotyl growth. Moreover, for the first time, the localization of a peroxidase and laccase is provided in textile hemp. In the hypocotyl of textile hemp, elongation and girth increase are separated in time. This organ is therefore ideal for time-course analyses. Here, we follow the ultrastructural rearrangement of cell-wall components during the development of the hemp hypocotyl. An expression analysis of genes involved in the biosynthesis of cellulose, the chief polysaccharide of bast fiber cell walls and xylan, the main hemicellulose of secondary cell walls, is also provided. The analysis shows a higher expression of cellulose and xylan-related genes at 15 and 20 days after sowing, as compared to 9 days. In the young hypocotyl, the cell walls of bast fibers show cellulose microfibrils that are not yet compacted to form a mature G-layer. Crystalline cellulose is detected abundantly in the S1-layer, together with unsubstituted/low-substituted xylan and, to a lesser extent, in the G-layer. The LM5 galactan epitope is confined to the walls of parenchymatic cells. LM6-specific arabinans are detected at the interface between the cytoplasm and the gelatinous cell wall of bast fibers. The class III peroxidase antibody shows localization in the G-layer only at older developmental stages. The laccase antibody shows a distinctive labelling of the G-layer region closest to the S1-layer; the signal becomes more homogeneous as the hypocotyl matures. The data provide important insights on the cell wall distribution of polysaccharide and protein components in bast fibers during the hypocotyl growth of textile hemp.

Extraction Method and Analysis of Cannabinoids in Cannabis Olive Oil Preparations.

Recently, an increasing number of pharmacists had to supply medicinal products based on Cannabis sativa L. (Cannabaceae), prescribed by physicians to individual patients. Cannabis olive oil preparation is the first choice as a concentrated extract of cannabinoids, even though standardized operative conditions for obtaining it are still not available. In this work, the impact of temperature and extraction time on the concentration of active principles was studied to harmonize the different compounding methods, optimize the extraction process, and reduce the variability among preparations. Moreover, starting from the cannabis inflorescence, the effect of temperature on tetrahydrocannabinolic acid decarboxylation was evaluated. For the analysis, a GC/MS method, as suggested by the Italian Ministry of Health, and a GC/flame ionization detection method were developed, validated, and compared.

The molecular structure within dislocations in Cannabis sativa fibres studied by polarised Raman microspectroscopy.

Polarised Raman micrsospectroscopy was employed to study the molecular structure within dislocations (slip planes) in the cell walls of Hemp fibre cells (Cannabis sativa (L.)). It was found that the cellulose microfibrils within dislocations have a different orientation than in the surrounding cell wall, and that the cellulose in the transition zones between a large dislocation and the surrounding wall may have yet another orientation. Furthermore, cellulose orientation seemed to be less uniform within dislocations than in the surrounding cell wall.

Archaeobotanical evidence of the use of medicinal cannabis in a secular context unearthed from south China.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of the first plants used by ancient people, cannabis has been used for medicinal purposes for thousands of years. The long history of medicinal cannabis use contrasts with the paucity of archaeobotanical records. Moreover, physical evidence of medicinal cannabis use in a secular context is much rarer than evidence of medicinal cannabis use in religious or ritual activities, which impedes our understanding of the history of medicinal cannabis use. AIM OF THE STUDY: This study aims to provide archaeobotanical evidence of medicinal cannabis use and analyse the specific medicinal usage of cannabis in a secular context in ancient times. MATERIALS AND METHODS: Plant remains were collected from the Laoguanshan Cemetery of the Han Dynasty in Chengdu, South China, with the archaeological flotation process and were identified based on morphological and anatomical characteristics. The examination of the medicinal significance of the remains relied on the investigation of the documentation on unearthed medical bamboo slips, the diseases of the tomb occupants, the cemetery's cultural background and Chinese historical records. RESULTS: The botanical remains were accurately identified as cannabis. More than 120 thousand fruits were found, which represents the largest amount of cannabis fruit remains that have been statistically analysed from any cemetery in the world thus far. The cannabis fruits are suspected to have been used for medical purposes in a secular context and were most likely used to stop severe bleeding of the uterus and treat lumbago and/or arthralgia. CONCLUSIONS: The cannabis fruit remains reported here likely represent the first physical evidence of medicinal cannabis use for the treatment of metrorrhagia, severe lumbago, and/or arthralgia. This study emphasizes the importance of the evidence of the diseases suffered by the occupants of the tomb in determining the medicinal use of cannabis in a secular context and contributes to a comprehensive understanding of the ancient history of medicinal cannabis.

Valorization of CBD-hemp through distillation to provide essential oil and improved cannabinoids profile.

Hemp (Cannabis sativa L.) synthesizes and accumulates a number of secondary metabolites such as terpenes and cannabinoids. They are mostly deposited as resin into the glandular trichomes occurring on the leaves and, to a major extent, on the flower bracts. In the last few years, hemp for production of high-value chemicals became a major commodity in the U.S. and across the world. The hypothesis was that hemp biomass valorization can be achieved through distillation and procurement of two high-value products: the essential oil (EO) and cannabinoids. Furthermore, the secondary hypothesis was that the distillation process will decarboxylate cannabinoids hence improving cannabinoid composition of extracted hemp biomass. Therefore, this study elucidated the effect of steam distillation on changes in the content and compositional profile of cannabinoids in the extracted biomass. Certified organic CBD-hemp strains (chemovars, varieties) Red Bordeaux, Cherry Wine and Umpqua (flowers and some upper leaves) and a T&H strain that included chopped whole-plant biomass, were subjected to steam distillation, and the EO and cannabinoids profile were analyzed by gas chromatography-mass spectrometry (GC-MS) and HPLC, respectively. The distillation of hemp resulted in apparent decarboxylation and conversion of cannabinoids in the distilled biomass. The study demonstrated a simple method for valorization of CBD-hemp through the production of two high-value chemicals, i.e. EO and cannabinoids with improved profile through the conversion of cannabidiolic acid (CBD-A) into cannabidiol (CBD), cannabichromenic acid (CBC-A) into cannabichromene (CBC), cannabidivarinic acid (CBDV-A) into cannabidivarin (CBDV), cannabigerolic acid (CBG-A) into cannabigerol (CBG), and δ-9-tetrahydrocannabinolic acid (THC-A) into δ-9-tetrahydrocannabinol (THC). In addition, the distilled biomass contained CBN while the non-distilled did not. Distillation improved the cannabinoids profile; e.g. the distilled hemp biomass had 3.4 times higher CBD in variety Red Bordeaux, 5.6 times in Cherry Wine, 9 times in variety Umpqua, and 6 times in T&H compared to the original non-distilled samples, respectively. Most of the cannabinoids remained in the distilled biomass and small amounts of CBD were transferred to the EO. The CBD concentration in the EO was as follows: 5.3% in the EO of Umpqua, 0.15% in the EO of Cherry Wine and Red Bordeaux and 0.06% in the EO of T&H. The main 3 EO constituents were similar but in different ratio; myrcene (23.2%), (E)-caryophyllene (16.7%) and selina-3,7(11)-diene (9.6%) in Cherry Wine; (E)-caryophyllene (~ 20%), myrcene (16.6%), selina-3,7(11)-diene (9.6%), α-humulene (8.0%) in Red Bordeaux; (E)-caryophyllene (18.2%) guaiol (7.0%), 10-epi-γ-eudesmol (6.9%) in Umpqua; and (E)-caryophyllene (30.5%), α-humulene (9.1%), and (E)-α-bisabolene (6.5%) in T&H. In addition, distillation reduced total THC in the distilled biomass. Scanning electron microscopy (SEM) analyses revealed that most of the glandular trichomes in the distilled biomass were not disturbed (remained intact); that suggest a possibility for terpenes evaporation through the epidermal membrane covering the glandular trichomes leaving the cannabinoids in the trichomes. This explained the fact that distillation resulted in terpene extraction while the cannabinoids remained in the distilled material.

Identification of the aquaporin gene family in Cannabis sativa and evidence for the accumulation of silicon in its tissues.

Cannabis sativa is an economically important crop providing bast fibres for the textile and biocomposite sector. Length is a fundamental characteristic determining the properties of bast fibres. Aquaporins, channel-forming proteins facilitating the passage of water, urea, as well as elements such as boron and silicon, are known to play a role in the control of fibre length in other species, like cotton. By mining the available genome, we here identify, for the first time, the aquaporin gene family of C. sativa. The analysis of published RNA-Seq data and targeted qPCR on a textile variety reveal an organ-specific expression of aquaporin genes. Computational analyses, including homology-based search, phylogeny and protein modelling, identify two NOD26-like intrinsic proteins harbouring the Gly-Ser-Gly-Arg (GSGR) aromatic/Arg selectivity filter and 108 amino acid NPA (Asn-Pro-Ala) spacing, features reported to be associated with silicon permeability. SIMS nano-analysis and silica extraction coupled to fluorescence microscopy performed on hemp plantlets reveal the presence of silicon in the bast fibres of the hypocotyl and in leaves. The accumulation of silica in the distal cell walls of bast fibres and in the basal cells of leaf trichomes is indicative of a mechanical role.

Structural changes of plant residues during decomposition in a compost environment.

The degradation of plant material during composting was investigated qualitatively by scanning electron microscopy (SEM) and quantitatively by chemical methods. Decomposition of Miscanthus (Miscanthus oogiformis L.), hemp (Cannabis sativa L.) and wheat (Triticum aestivum L.) straw was observed by placing litterbags containing these materials in compost piles. Hemp and Miscanthus straw were more stable than wheat straw, but the two materials differed in the way they were degraded despite similar chemical compositions. Hemp straw was broken down in more flexible structures compared to the rigid breakdown of Miscanthus straw. It was concluded that the anatomical arrangement of the tissue is just as important as the content of recalcitrant compounds in determining decomposition rate. Thus, when using composted plant materials as growing medium, the choice of material must depend not only on nutritional quality but also on structural quality. This study indicated that hemp material might be a good structural component in a compost to be used as a growing medium.

A new insight into Cannabis sativa (Cannabaceae) utilization from 2500-year-old Yanghai Tombs, Xinjiang, China.

A cache of shoots, leaves and fruits dated by (14)C at 2500 years B.P. were unearthed in the Yanghai Tombs, Turpan District in Xinjiang, China. By comparing the morphological and anatomical characteristics of the plant remains found in the tomb and specimens of modern plants, it is shown that the remains belong to Cannabis. Based on the shamanistic background of the deceased man and ancient customs, it is assumed that the Cannabis was utilized for ritual/medicinal purposes.

Morphology and structure of hemp fibre after bioscouring.

Bioscouring of hemp (Cannabis Sativa L) using pectate lyase (EC 4.2.2.2), Scourzyme L, was performed at 55 degrees C and pH 8.5 in a nonagitated system. The enzyme concentration, treatment time and substrate concentration were varied to obtain the kinetic constants, K(m) and V(m). Greater enzyme concentration and a longer treatment improved the removal of the low methoxy pectin component as indicated by UV spectroscopy. Removal of pectate caused no crystalline transformation in the fibres, except for a slight decline in the crystallinity order index analysed by Fourier Transform infrared spectroscopy and wide angle X-ray diffraction. This corresponded well with the single fibre bundle tensile mechanical properties test. Smooth surfaces and separated fibres observed using SEM images were evidence of successful treatment, supported by weight loss at low temperature of a pectic substance. After treatment, the pectin substance was no longer observed during thermogravimetry. An increase in surface area and pore size after scouring were further evidence of modification.

Plastid development in disc cells of glandular trichomes of Cannabis (Cannabaceae).

Plastids in lipophilic glandular trichomes of chemically fixed (CF) and high pressure cryofixed-cryosubstituted (HPC-CS) bracteal tissues of Cannabis were examined by transmission electron microscopy. In CF preparations, plastids in disc cells prior to secretory cavity formation possessed several lobed and dilated thylakoid-like features. In glands with secretory cavities, thylakoid-like features aggregated to form reticulate bodies that distended regions of the elongated plastids. Electron-gray inclusions evident on the plastid surface appeared continuous with the reticulate body. Inclusions of similar electron density also appeared in the cell cytoplasm, along the plasma membrane, between the plasma membrane and cell wall facing the cavity, and in the secretory cavity in both CF and HPC-CS preparations. The bilayer structure of membranes of the plastid envelope was evident in HPC-CS but not in CF preparations. In HPC-CS preparations, secretions were evident on the plastid surface and were continuous with those in the plastid through pores in the envelope. This study supports an interpretation that these specialized plastids, lipoplasts, synthesize secretions that are transported through the plasma membrane and cell wall to subsequently accumulate in the secretory cavity.

Secretory vesicle formation in the secretory cavity of glandular trichomes of Cannabis sativa L. (Cannabaceae).

The disc cell wall facing the secretory cavity in lipophilic glands of Cannabis was studied for origin and distribution of hyaline areas, secretory vesicles, fibrillar matrix and particulate material. Secretions evident as light areas in the disc cell cytoplasm pass through modified regions in the plasma membrane and appear as hyaline areas in the cell wall. Hyaline areas, surrounded with a filamentous outline, accumulate near the wall surface facing the secretory cavity where they fuse to form enlarged hyaline areas. Fibrillar matrix is related to and may originate from the dense outer layer of the plasma membrane. This matrix becomes distributed throughout the wall material and contributes in part to the composition of the surface feature of secretory vesicles. Thickening of the cell wall is associated with secretions from the disc cells that facilitates movement of hyaline areas, fibrillar matrix and other possible secretions through the wall to form secretory vesicles and intervesicular materials in the secretory cavity. The outer wall of disc cells in aggregate forms the basilar wall surface of the secretory cavity which facilitates the organization of secretory vesicles that fill the secretory cavity.

Histochemical detection of hemp trichomes and their correlation with the THC content.

Authors investigated some fiber and some hashish hemp sorts concerning their trichomes on the leaves. Histochemical reactions were developed using the Fast Blue Salt (FBB) reagent applied so far only thin layer chromatography. Glandular hairs were found giving positive reactions due to cannabinoids contained by the cells. The electron microscopic features were studied and the cannabinoid content was measured with GC. A correlation was found between the number of typical glandular hairs and cannabinoid content.