We found that the expression levels of five genes were significantly up-regulated after GA3 treatment for 3 h.In addition,among these 39 CasWRKYs,the transcription levels of four CasWRKYs were continuously up-regulated in the stem barks under the GA3 stress.The above results suggest that these CasWRKY may be involved in the development of fibers under GA3 stress.Similarly,previous studies show that Arabidopsis WRKY transcription factors were involved in the formation of secondary cell walls and can significantly increase plant biomass,such as AtWRKY12.Interestingly,nine of the 39 CasWRKYs were not expressed at all-time points,indicating that they might be pseudogenes or may not be involved in expressed during this period.Previous studies have proved that gibberellin-mediated signal cascade regulations can respond to glucose and gibberellin signals and effectively promote cellulose synthesis.Overall,it is very meaningful to further study the effect of CasWRKY genes response to GA3 stress on fiber development and can promote our understanding of the regulation mechanisms of CasWRKY gene.Hemp is one of the oldest and the most controversial cultivated annual crops with high potential in the treatment of several disorders affecting 30–50 millions of people worldwide.Up to date,538 natural compounds have been identified from C.sativa,and among them,more than 100 are phytocannabinoids.These lipid-based compounds consist of seven main types of compounds,which are produced in different cultivars of C.sativa through distinct synthesis pathways using diverse enzymes and precursors.
Medicinal C.sativa contains high concentrations of the psychotropic cannabinoids,as 8- and 9-tetrahydrocannabinol,cannabis drying racks their acidic forms and cannabinol. 9-tetrahydrocannabinol has the strongest psychoactive effects,and itis generally referred as THC.In the fiber and seed types of C.sativa non-narcotic phytocannabinoids are accumulated in larger quantities,such as cannabidiol with its acid form named as cannabidiolic acid,cannabigerol,and cannabichromene which present in notable quantities in the leafy part of hemp.Industrial hemp is cultivated for the production of fiber for paper and textile,cellulose,and hemp seed oil for further applications in food,cosmetics and pharmaceutical industries.These industrial cultivars accumulate a minimal amount of 9- tetrahydrocannabinol therefore they are legally allowed for cultivation in the USA,Canada and in many European countries.During the harvesting and processing of hemp,a large amount of threshing waste is generated,which is a natural cannabinoid-source.It is estimated that up to 2 t ha−1 of dry threshing residue can be potentially collected and available for further processing.The non-psychoactive cannabinoids possess numerous beneficial medicinal and therapeutic properties.CBD and CBDA were shown to exert modulating-effects of the human endocannabinoid system; they possess analgesic,antibacterial,antiemetic,antiepileptic,anti-inflflammatory,anti-proliferative,antipsychotic properties.Therefore,natural pharmaceuticals containing cannabinoids are highly desirable as promising agents in treatments of a variety of central nervous system and peripheral disorders,depression,anxiety,sleep disorders,and epilepsy.The use of cannabinoids for the treatment of chronic neuropathic or cancer pain,nausea and vomiting also has shown pharmacological potential.Generally,losses on harvested crops were found substantial.
The dry matter based loss might reach 44% before reaching costumers.By reducing this amount,more sustainable land use and more efficient utilization of agro-food wastes are reliable.Phytocannabinoids present in the stalks,leaves of various C.sativa industrial hemp residues,therefore,the utilization of harvested hemp residues can be feasible with economic potential.For recovering these high valued cannabinoids,different techniques are available from traditional solvent extraction,pressurized liquid extraction,enzyme-assisted-,microwave and ultrasound extraction to supercritical fluid extraction.Applying carbon dioxide as a supercritical solvent in SFE,makes it an environmentally friendly extraction technique with the possibility of recycling the extraction solvent resulting in solvent-residue free extracts rich in lipophilic compounds.Supercritical carbon dioxide extraction has been already applied for aroma seed oil and cannabinoid extraction from medicinal C.sativa L.,as well as from threshing residue and hemp dust of fiber production.The solubility of some pure cannabinoids was determined in supercritical carbon dioxide at certain temperature- and pressure ranges by Perrotin-Brunel et al..The solubility of these four cannabinoids in scCO2 increases at 326K in the following order: 9-THC < CBG < CBD < CBN and is mainly pressure and less temperature-dependent.The effects of extraction pressure and temperature on the yield were investigated by Rovetto and Aieta,Gallo-Molina et al. and by our group.For cannabinoids extraction from C.sativa L.low extraction temperature was more favorable.Ethanol cosolvent enhanced the extraction process of THC from the inflorescences,buds and leaves of medicinal C.sativa L.by increasing the overall yield.Extracts with high THC content were obtained at 33 MPa,60 ◦C with 2% co-solvent concentration.Regarding industrial application,two patented processes exist for obtaining THC and CBD rich hemp extracts using supercritical carbon dioxide extraction.WO2004066277 patent claims include procedures applying sub- and supercritical carbon dioxide between 6−60 MPa pressure-,and 10−35 ◦C temperature ranges with 1000−1500 kg/h mass flow of CO2.
The US patent has a focus on the pain relief of cannabis extracts containing THC and CBD in different ratios referring to the content of WO2004066277 patent.In both patents,medicinal hemp with a high initial concentration of THC and CBD were extracted after a heating step in which the cannabinoid-acids had converted to THC and CBD with decarboxylation at 100−150 ◦C.After extraction with sub- or supercritical CO2,winterization was described to precipitate the waxy compounds.The end-products of these processes were THC-rich extracts which required numerous izolation steps and extended use of organic solvents.This present study aimed to compare,quantify and fractionate non-psychoactive CBD rich extracts from six different industrial hemp threshing residues harvested at different times.Sub- and supercritical carbon dioxide was used as extraction solvent,in specific experiments ethanol cosolvent was added to increase the polarity of the solvent phase.Furthermore,the optimization of the extraction process obtaining cannabinoid-rich extract at the highest yield using fractionated separation in a pilot plant extraction plant was also a scope of this study.The threshing residues of C.sativa cultivar Komploti and Felina 32 were used in this study containing the stalks and leaves of the dry plant material.The harvesting years and the suppliers varied,but all samples contained less than 0.2% THC,which was officially certified.The year of harvest,the moisture contents,the average particle sizes and the uniformity factors of the hemp residues are collected in Table 1.The samples were stored in dark,low humidity,well-ventilated storage area below 18 ◦C.The data was evaluated by applying the Rosin-RammlerBennet distribution model using Statistica software calculating the characteristic particle size and the uniformity for each ground samples.Sample 1 was a Felina 32 variety hemp residue harvested in 2014.It was received in a pelleted form and had 0.522% ± 0.16% NPC and 0.010% ± 0.006% PC content.Sample 2 and 3 were from the same Kompolti variety harvested in 2014 and 2016,respectively.The NPC content of Sample 2 was 0.945% ± 0.003% while its cumulative PC content was 0.029% ± 0.003%.Sample 3 contained 1.833% ± 0.016% NPCs and 0.060 ± 0.002% PCs,respectively.Sample 4 was a ground,fiber-type hemp residue having 0.024% ± 0.002% NPCs and 0.009% ± 0.011% PCs.
Sample 5 was also a fiber-type hemp residue containing the stalks and leaves of the plant.As the average of all measurements performed the total NPC content was estimated as 1.049% ± 0.228% and the total PC content as 0.044% ± 0.014%.Sample 6 was a fiber-type hemp residue,ground leaves and stalks harvested in 2017 and had 1.611% ± 0.052% NPC and 0.124% ± 0.039% PC content.All samples except Sample 1 were ground by a Fritsch 15 cutting mill using a 1 mm hole size sieve before the extraction.All other chemicals and solvents were analytical and HPLCgrade and purchased from Sigma-Aldrich.Liquid,food-grade CO2 was purchased from Linde Ltd.Hungary with a purity of 99.99%.For the quantification of cannabinoids analytical standards were purchased from THC Pharm GMBH,Sigma-Aldrich and LGC,the uncertainties of the certified concentrations were ≤1%.The high-pressure extractions using CO2 solvent in sub- and in supercritical states were performed in a pilot plant apparatus with one 5 L extraction vessel and two 1 L separation vessels in series.The carbon dioxide was carried with a high-pressure liquid pump,when needed the cosolvent was added by another liquid pump.The extraction pressure and the flow were maintained constant using a back pressure regulator..500 g of hemp threshing residue was weighted into the stainless steel extraction basket,which was loaded into the jacketed extraction vessel.The flow rate of supercritical solvent was set at 7 kg/h in all experiments.In the cosolvent enhanced experiments,ethanol was mixed into the scCO2 in 10% and fed onto the plant material.The extraction pressure was varied between 8–45 MPa,while the extraction temperature was set at 27 ◦C in sub-critical extraction and at 45 ◦C in all other experiments.The extract was collected either step-wise from the first separator,which was set at 4 MPa and 40 ◦C when the extract was not fractionated,or from both separators operated at different conditions.In other experiments with fractionation separation,the first separator was operated at higher pressure and the second one at 4 MPa,while the extraction pressure was set at 35 MPa and temperature at 40 ◦C.The extractions ran in batches; therefore,the CO2 was not recycled.The extraction was carried on until the amount of extract collected for 1 h decreased under 0.1% of the raw material.During the supercritical carbon dioxide extraction,water was co-extracted,sequential decanted,and the crude extract was collected and stored.The crude extracts were weighted,and the yield was calculated as g extract / 100 g dry material.The experiments with ethanol cosolvent were carried out at 30 MPa extraction pressure and 45 ◦C extraction temperature using only one separator.The extract contained ethanol,which was evaporated by vacuum-evaporator.For comparison,solvent extractions with 96% ethanol,npentane,acetonitrile in laboratory Soxhlet extraction apparatus were carried out on the raw,ground plant materials.After the solvent was evaporated by vacuum-evaporator,cannabis grow tray the extracts were weighted.The solvent-free extract samples were stored under nitrogen in a refrigerator until further analysis.The extraction yields varied between 2.48 g/100 g dry material and 9.24 g/100 g d.m.depending mainly on the origin of raw material,and the applied process parameters.These results correspond well with data found in the literature.Higher yields were achieved by conventional solvent extraction especially using 96% ethanol as solvent,but these extracts contained more unwanted compounds with phytocannabinoids in similar or in lower concentrations.
Kitryte et al.investigated the effect of extraction pressure and temperature of supercritical CO2 extraction on the yield from hemp threshing residue remaining after harvesting and cleaning of industrial fibertype hemp seeds using a laboratory scale high pressure apparatus.The optimal condition was reported to be 46.5 MPa and 70 ◦C resulting an extraction yield of 8.3%.From the dust of industrial hemp processing waste,the maximal extraction yield was low reported by Attard et al.The effect of extraction pressure and temperature on the extraction yield was also studied and the 35 MPa extraction pressure and 50 ◦C extraction temperature of supercritical carbon dioxide extraction was found to be yielding CBD-rich extract from hemp dust.However,our research group had further examined the effect of extraction pressure on the yield of scCO2 extraction of three hemp threshing residue,and it was found that elevating the extraction pressure from 35 to 45 MPa at 45 ◦C extraction temperature the yield did not increased significantly while the yields of cannabinoids were also the highest at 35 MPa and 45 ◦C.The density of supercritical CO2 does not change dramatically at this pressure range therefore the slight increase in solubility was not be prevailed during extraction.The cannabinoids were extracted in the highest yields at 35 MPa and 45 ◦C.In the literature,solubility data can be found on cannabinoid pure compounds only up to 25.1 MPa,while other optimization results are only reported on the effect of pressure and ethanol cosolvent on the THC concentration,and the effect of extraction pressure and temperature was investigated on yields of extraction,CBD and CBDA but the experiments were carried outin a laboratory size high-pressure apparatus.Extraction with subcritical carbon dioxide at 8 MPa and 27 ◦C extraction pressure and the temperature was also investigated on Sample 3 and 5,respectively.The extraction yields were 2.42 and 2.48% for Sample 3 and 5 obtaining a light green,highly scented light oil in both cases.The extraction curves are on Fig.2.In the case of Sample 5 the subcritical extraction was followed by a subsequent extraction at elevated pressure to recover the all extractable matter from the plant material.This twostepextractionresultedin4.23%yield,whichcorresponds well with the average yield of scCO2 extraction of Sample 5 at 45 MPa and 45 ◦C.The compositions of extracts of subcritical- and supercritical CO2 extractions revealed that 66% of all NPCs and 67% of all PCs were quantified in the extracts obtained at low pressure and temperature.During the subcritical extraction,the concentration profiles of neutral- and acid forms of CBD and THC was also obtained.Changes in the concentration profiles of the extracts along the extraction are plotted in Figs.3 and 4.