Tag Archives: cannabis

The profile of the changing genes was comparable between tobacco and marijuana exposed cells

Exposure to tobacco smoke can also trigger an inflammatory response and induce oxidative stress through increased levels of reactive oxygen species. Persistent induction of these processes following repeated exposure contributes to loss of normal growth control mechanisms, which is a key step in cancer development. Our study supports many of these findings, with exposure to TSC inducing the expression of genes involved in xenobiotic metabolism , oxidative stress , and DNA damage response as evidenced by changes in the expression of genes involved in cell cycle arrest, protein unfolding,transcription regulation, and inflammation. These same pathways were also significantly affected following MSC exposure, indicating that, as expected, MSC impacts many of the same molecular processes and functions as TSC. Although the effects of the condensates were largely similar, dose–response analysis indicates that the MSC is substantially more potent than TSC, with BMDs that in many instances are an order of magnitude lower than those for TSC. In addition, the results also highlighted some differences in steroid biosynthesis , apoptosis and inflammation, which were more significantly affected following MSC exposure, and cell cycle , which was more affected following TSC exposure.IPA canonical pathways related to the metabolism of xenobiotics were significantly affected in both TSC and MSC exposed cells at both time points. These pathways included Xenobiotic Metabolism Signaling, Metabolism of Xenobiotics by CYP450,microgreen flood table and AHR Signaling. For both TSC and MSC, the number of genes that were significantly affected increased with increasing concentration and the greatest number of genes changing occurred at the 6 + 4 h time point.

Many of the genes that were differentially expressed in TSC exposed cells are among those that have been typically observed to be induced by cigarette smoke [e.g., Nqo1 , Esd , Hmox1 , Cyp1a1 and Cyp1b1. Moreover, the concentration response patterns support the assertion of initial metabolic responses , followed by responses to toxic insult and secondary metabolism. Similar concentration response trends were noted in our previous toxicogenomics analysis of three different TSCs. Although very few studies have been conducted with marijuana smoke, Roth et al. demonstrated the induction of cytochrome P450 genes following exposure of Hepa-1 cells to marijuana tar extracts. Furthermore, the authors showed that tar from marijuana cigarettes tends to be more effective than tar from tobacco at inducing Cyp1a1 gene expression. Since the cannabinoids present in marijuana are capable of acting through the aryl hydrocarbon receptor to induce cytochrome P450 enzymes , and Cyp1a1 is known to bioactivate procarcinogens such as PAHs , questions have been raised about the role of cannabinoids in augmenting the carcinogenic risk posed by marijuana smoke. The question becomes increasingly complex as the cannabinoids THC, CBD and CBN have also been shown to competitively inhibit Cyp1a1, potentially decreasing the production of carcinogens and curtailing negative consequences. In the present study, however, substantial differences in the expression profiles of cytochrome P450 genes between the two smoke types were not observed. The expression of Cyp1a1 following exposure to MSC was comparable to that following TSC exposure, and the micro-array results were supported by RT-PCR. One of the differences in the xenobiotic metabolism responses for the two condensate types is that Hsp90 and Rras2 were only upregulated following MSC exposure. Despite these findings, Hsp90 has been previously observed to be induced following cigarette smoke exposure , and mutations in genes from the Ras family are known to be associated with cigarette-induced cancers.The IPA Canonical Pathway most significantly affected by exposure to TSC was the NRF2-Mediated Oxidative Stress Response Pathway. In this pathway, the transcription factor Nrf2 is phosphorylated following exposure to reactive oxygen, and translocates to the nucleus where it binds to antioxidant response elements. It then activates the expression of detoxification and antioxidant genes that protect the cell against oxidative damage. Of the 192 genes in this pathway, 6–18 genes were perturbed by TSC at the various time points in a concentration dependent manner. The largest expression changes and number of genes were associated with the 6 h time point.

Nrf2-regulated antioxidant genes have been shown to play an important role in protection against the toxic effects of tobacco smoke. Iizuka et al. showed that neutrophilic lung inflammation was significantly enhanced in Nrf2-knockout mice following cigarette smoke exposure. In addition, emphysema was observed 8 and 16 weeks following cigarette smoke exposure in the knockout mice, whereas no pathological abnormalities were observed in wild-type mice. Similarly, Gebel et al. confirmed the protective nature of Nrf2 against the development of emphysema in cigarette smoke exposed wild type mice versus Nrf2 knockout mice, and further investigated the relationships between Nrf2 and inflammation and cell cycle arrest. Comandini et al. conducted a meta-analysis of eight genomic studies on the mechanisms of smoke-induced lung damage in healthy smokers, COPD smokers and non-smokers. They found the Nrf2-mediated oxidative stress response Pathway to be the most significantly altered pathway in healthy smokers compared to non-smokers. In contrast, the Nrf2 pathway was not significantly differentially expressed in COPD smokers, indicating that Nrf2-regulated genes play a key role in protecting against the toxic effects of TSC. The authors suggest that the response of Nrf2- regulated genes may potentially be used as a biomarker for COPD susceptibility. In the present study, we found that the NRF2-Mediated Oxidative Stress Response Pathway is also an important component of the toxicological response to MSC. IPA analyses identified it as one of the top five pathways for both time points and all concentrations of MSC, except for the lowest concentration at the 6 + 4 h time point. A comparison of the Nrf2 pathway at the 6 h time point for the highest exposure concentrations of TSC and MSC shows many similarities. The Nrf2 gene itself was up-regulated along with several basic leucine zipper family transcription factors such as Jun, Atf4, and Maff. In addition, several antioxidant and stress response proteins such as Nqo1, Prdx1, Hmox1, Sod, Txnrd1, Herpud1, Dnajb1/9 were up-regulated. Other studies have also noted that these genes are up-regulated following cigarette smoke exposure. 

However, a notable difference between the two condensates studied here is that Gclc and Gclm, the rate limiting enzymes in glutathione synthesis, were significantly upregulated by TSC , but were not statistically significantly affected in MSC exposed cells. Furthermore Gsta genes were up-regulated in TSC and Gstm genes were down-regulated in MSC exposed cells. These findings were further confirmed by the significant up-regulation of the Glutathione Metabolism Pathway in tobacco exposed cells a tall times and concentrations and the significant down-regulation of this pathway in marijuana exposed cells, particularly at the high concentration at the 6 + 4 h time point. These results suggest that exposure to MSC elicits more severe oxidative stress than exposure to TSC. The relative difference between the two condensates to mount an antioxidant defense may account for the greater cytotoxicity of MSC observed here and in our earlier genotoxicity study, where it appeared that the acute toxicity of MSC prevented the manifestation of micro-nucleus induction. The assertion regarding the relative severity of oxidative stress induced by MSC and TSC is supported by published results from other studies. In a previous study, Sarafian et al. examined reactive oxygen species production and reduced glutathione levels as indicators of oxidative damage following exposure to marijuana smoke. They showed that exposure of human endothelial cells to marijuana smoke resulted in an 80% increase in ROS over control levels, and these levels were as much as three times higher than those resulting from tobacco smoke. Moreover, intracellular glutathione levels following marijuana exposure were lower than for tobacco, and were reduced by 81% relative to controls. The authors argued that the products produced by the pyrolysis of the cannabinoids were likely responsible for the oxidative damage. The same authors also conducted preliminary studies with cultured lung alveolar macrophages from non-smokers and marijuana smokers, and found that marijuana smokers had lower levels of GSH than non-smokers, suggesting a decrease in GSH dependent oxidative defenses in habitual marijuana smokers.

The Biosynthesis of Steroids Pathway was among the most significantly affected IPA Canonical Pathways for MSC exposed cells. This held true both when all of the significantly altered MSC genes were taken into account, and when only the genes unique to MSC were considered. The Biosynthesis of Steroids Pathway is a lipid metabolism pathway that controls the synthesis of cholesterol, which is an essential component of cell membranes and a precursor in the production of bile acids, steroid hormones,seedling grow rack and vitamin D. This pathway was significantly down-regulated for all concentrations of the MSC at both time points, and the number of genes that were significantly affected increased with increasing concentration. The greatest number of genes was affected at the 6 + 4 h time point, and these included Dhcr7, Fdft1, Fdps, Hmgcr, Idi1, Mvd, Mvk, Nqo1, Pmvk, Sc5dl, and Sqle. The majority ofthese genes are involved in the mevalonate and squalene synthesis portions of the pathway. Although no studies have been conducted to specifically investigate the effect of marijuana smoke on lipid metabolism and steroid biosynthesis, early investigations using rodent cells have shown that cannabinoids can affect lipid metabolism, and the effects include an increase in lipolysis in adipose tissue ,the inhibition of corticosteroidogenesis , and the reduced testosterone and progesterone production. The cannabinoid CBD has also been shown to affect cholesterol metabolism in human fibroblasts and aortic medial cells through the inhibition of cholesteryl ester formation. In the present study, HMG-CoA reductase , which is the rate-limiting enzyme for cholesterol synthesis, was notably down-regulated for the medium and high concentrations of MSC at both time points. Previous in vitro investigations with THC have shown that this cannabinoid reduces Hmgcr by 29% , whereas CBD had no effect on Hmgcr levels. When comparing TSC and MSC exposed cells, the Biosynthesis of Steroids Pathway was also significant for TSC, particularly for the 6 + 4 h time point.

However, only one to three genes were perturbed, depending on the concentration. These genes included Fdps, Ggps1, Nqo1, and Hmgcr. The LXR/RXR pathway, which is involved in the regulation of lipid metabolism and cholesterol to bile acid catabolism, was also significantly down-regulated at the 6 + 4 h time point in both MSC and TSC exposed cells. Of note in this pathway is Ldlr, which is the greatest down-regulated gene in MSC exposed cells. This gene was down-regulated 10 fold following the highest MSC exposure concentration but only 1.6 fold following the highest TSC exposure.Exposure to MSC but not TSC appears to have affected apoptosis pathways. Genes in the TWEAK Signaling, TNFR1 and TNFR2 Signaling Pathways were significantly up-regulated following exposure to MSC particularly at the 6 h time point. The up-regulation of these particular genes suggests that MSC inhibits apoptosis and may promote a TNF receptor mediated survival pathway.In a previous study, Sarafian et al. investigated the effects of marijuana smoke and tobacco smoke on apoptosis and necrosis in A549 lung tumor cells. They found that both tobacco and marijuana whole smoke inhibited Fasmediated apoptosis but promoted necrotic cell death. In addition, particulate phase smoke from marijuana was a more potent inhibitor of Fas-induced caspase-3 activity than tobacco. In a later study, the authors also noted the decreased expression of Bax and caspase-8 in human small airway epithelial cells exposed to THC, which they suggest could have accounted for the previously observed suppression in Fas-mediated apoptosis. Although apoptotic pathways were not significantly perturbed following TSC exposure in our present study, Sarafian et al. and other investigators of tobacco smoke effects have found this to be a commonly disrupted pathway. It is suspected that the gene expression fold change cutoff of 2 used in the present study likely prevented a number of apoptotic genes from being included in the analyses. Cursory analyses with a cutoff of 1.5 shows apoptotic pathways as being significant for TSC exposure as well. It is important to note that the marijuana used for this study was obtained from a contracted supplier that provides marijuana for therapeutic use in Canada. It is grown under strictly controlled and documented conditions. Although this study has only examined smoke condensate from a single lot of marijuana, the quality control measures would be expected to minimise differences between marijuana harvests.

Terpenes are usually classified by the number of isoprene units in the molecule

With the increase in production,advanced and standardized processing technologies,including drying,extraction and purification,are required to ensure the quality and safety of these products for food applications,as well as the processing efficiency and sustainability of hemp.Some terpenes,on the other hand,are considered generally regarded-as-safe substances,which are permitted to be added and have been used in foods.However,the incorporation of hemp terpenes in foods and preservation of terpenes during processing is still challenging,requiring further research.In recent years,many studies have been conducted to identify the functionalities and pharmaceutical values of hemp CBD and terpenes,as well as extraction technologies of CBD.However,there is currently not a comprehensive review that discusses the potential of hemp CBD and terpenes as future functional food ingredients and evaluates the available and potential processing technologies for hemp.Therefore,this article reviews the legal regulations,challenges in incorporating hemp CBD and terpenes in foods and potential solutions,as well as current research status and future prospects in processing technologies of hemp biomass.Through this review,we expect to draw a clearer image for food manufacturers and researchers on the potential of applying hemp CBD and terpenes as future functional food ingredients and provide information for standardizing and improving the processing technologies of hemp for efficient production of high quality and food-safe products.Future research needs are also identified in this article.In the hemp plant,cannabinoids mainly reside in the flowers and leaves.More specifically,they are primarily stored in the glandular and non-glandular epidermal appendages called trichomes,which are transparent glandular ‘hairs’ on the surface of inflorescence and leaves.

The cannabinoids are chemically bounded with the cell walls,secretory vesicles and fibrillar material in the secretory cavity of the disc cell.In this section,the physical properties and chemistry of CBD,cannabis growing system health benefits,potentials and associated challenges as future functional food ingredients are discussed.Chemically,cannabinoids are a type of molecules that contain terpenoids and alicyclic units.Currently,more than 120 types of cannabinoids have been found in hemp plants.Fig.3 shows the biosynthetic pathway of cannabinoids in hemp plant.The basic reaction in the cannabinoids synthesis involves the alkylation of the phenolic moiety of olivetolic acid with the terpenoid component of geranyl pyrophosphate ,which produces cannabigerolic acid ,the common precursor of all major cannabinoids in the hemp.CBGA can then convert into other cannabinoids such as tetrahydrocannabinolic acid ,cannabichromenic acid and cannabidiolic acid ,etc.,through the enzymatic catalytic reactions by THCA-synthase,CBCA-synthase and CBDA-synthase.It is worthy of mentioning that only the acidic cannabinoids exist naturally in hemp plants.According to their molecular structures,cannabinoids are non-polar,and thus have low solubility in aqueous solution.The boiling points of cannabinoids ranged from 157 °C to 220 °C,which is not volatile.However,CBD is thermally unstable and very sensitive to photolytic reaction and oxidation.The acidic form cannabinoids are not considered to be pharmacologically active since they do not affect the endocannabinoid system of human body in the same way as the neutral forms.Therefore,a decarboxylation process is usually needed to convert the CBDA to CBD,which involves the removal of a carboxyl group from the acidic molecule and the release of a CO2 under high temperature.The reaction mechanisms of the decarboxylation process are illustrated in Fig.3B.Recently,research has proven the functionalities of CBD,which include pain relief,reduction of anxiety/distress,nausea and promotion of relaxation.

In addition,since CBD is not psychoactive,it does not result in addictive effect as caused by THC.At present,there is an increasing prevalence of lifestyle induced chronic diseases,and consumers are pursuing healthier diets,seeking more functional foods reinforced with functional ingredients.Currently,the global health and wellness market is at $1.5 trillion,and is projected to increase annually at 5%–10%.In 2019,the global functional food market was $250 million,and was projected to $440 billion by 2022.Therefore,there is a high potential in this rising trend to add hemp CBD in foods or beverages as future functional food ingredients.However,there are several challenges that are limiting the development of CBD-containing foods,which involves legal regulations,consumer awareness,and technological deficiencies.Legal regulations and policies are one of the most important factors in directing the developments of markets and products.Currently,regulations on the hemp CBD varies country by country,and CBD is in the ‘grey zone’,of which the legality is not clearly defined.In the United States,the 2018 Farm Bill removed industrial hemp from the definition of marijuana,which allowed more research to be done on hemp.However,it is still prohibited in federal level to add CBD to any foods,beverages or nutrient supplements.FDA has not waived it from the illegal ingredients list due to safety concerns of the non-approved CBD products and the fact that CBD was first studied as a drug.In fact,in 2018,FDA approved the first and by far the only medicine containing CBD for the treatment of seizures associated with two rare and severe forms of epilepsy,Lennox-Gastaut syndrome and Dravet syndrome.Even though it is still not approved in federal level in the US,some food products are already in the markets in some states as they deregulated the CBD,such as tea,coffee and chocolate,etc..In Canada,cannabis is nationally legalized,but phyto-cannabinoids such as CBD is in the Prescription Drug List and cannot be legally sold in self-care natural health products or cosmetics.

Canadian Health Food Association has recently released a report,which emphasized the potential positive economic impacts of permitting CBD in NHPs,foods and beverages for the economic recovery.In Europe Union ,CBD was catalogued into ‘novel foods’ in January 2019,and the EU has required extensive testing and authorization of CBD products from food safety authorities.In addition,different countries have different standards in legalizing the THC level in the products.China was projected to be largest producer and consumer of hemp CBD,given the long cultivating traditional and production area,however,recently a national level regulation forbids any use of CBD in foods or cosmetics.The strict regulations have a significant impact on the development of the hemp industry,and the major food manufacturers are conservative in entering the CBD market.In fact,due to these strict regulations and the influence of pandemic on the global logistics,the hemp market and product development process have been significantly impacted.The hemp biomass and CBD are oversupplied in the market,and the price of CBD has hit the rock bottom in the first half of year 2021.The enthusiasm of farmers in cultivating hemp is cooling down.However,we envision that this ‘downturn’ could be temporary.With more research works done and more scientific evidence published proving the health benefits and safety of CBD,the regulations shall be more relaxed and standardized,which will be a strong booster for both the CBD-food market and in return benefit more related research to be conducted.In most cultures,the words ‘hemp’ and ‘cannabis’ are associated with ‘drugs’ and ‘illegal’,and thus are repelled.The word ‘CBD’ is still unfamiliar to most people in the world,not to mention its health benefits.Therefore,to promote the use of CBD as functional food ingredients,educating the consumers about the health benefits,safety,and its distinction from ‘drug’ is as important as the relaxing of legal regulations.At the same time,it is critical to find the right market position and target consumer groups.Currently,regular CBD consumers are buying CBD to obtain a functionality or health benefit,and usually taking it as a dietary supplement/medicine,which only represent a niche market.It is important to identify the types of foods that are compatible with CBD.Incorporating CBD into staple foods and beverages may potentially expand the consumer base,which is currently challenging and require more extensive research.The consumer needs for the CBD reinforced foods are another important factor to be considered in the product developments.For example,an afternoon pick-me-up ‘CBD-containing coffee’ or a sports drink need to be fast-responding,meanwhile it should not cause any adverse side effect to the human health.

Additionally,the food products need to be shelf-stable and maintain the activity of CBD under normal storage conditions,particularly given the fact that CBD is not a very stable substance.Besides the regulation and consumers aspects,there are currently some technological challenges that need to be resolved to add hemp CBD in foods and beverages.The types of products that can be reinforced with CBD are dependent on the physical and chemical properties of CBD.As discussed above,CBD is a highly non-polar molecule,meaning it has low aqueous solubility.Therefore,it is difficult to incorporate it into most aqueous-based food and beverage systems.Besides,recent studies have shown that the bio-accessibility of CBD is relatively poor in animal and human bodies,which may be due to the low cell penetration rate.In addition,CBD has relatively poor stability,which is affected by various factors such as temperature,humidity,pH,oxygen,light,and the solvent types,etc..Its instability suggests that CBD is vulnerable to deteriorate when it is inappropriately added to food systems or exposed under stress conditions.The oral ingestion of cannabinoids usually results in long lasting and slow effects,hydroponic rack system which is not desirable for the fast-responding needs,and is identified as an area of improvement.Currently,studies on the solubility and stability in various food and beverage systems are not well reported.The studies on the bioaccessbility of CBD in human body are also scarce.More extensive research in these aspects is urgently needed to address these challenges.The outcomes will provide scientific evidence to support the feasibility of adding CBD into foods and further prove their health benefits.Although the most of CBD-containing foods are considered illegal at present,the future can still be bright and optimistic.The 2018 Farm Bill of the U.S.opened the gate of industrial hemp to industries and researchers.Following that,more and more capital investments are putting into the hemp industry and scientific research.According to statistics,the global market size of hemp was $5.73 billion,and was expected to grow to $27.72 billion by 2028.This allows more universities and research institutions to conduct necessary research,which in return will be beneficial for the development of CBD-containing foods.Although current regulations prohibit the use of CBD as food ingredients,we envision that some regulations will ease up in the near future.This will depend on more scientific studies to be conducted and evidence that proves the health benefits and determines any side effects of CBD to human beings.In fact,United Nations has recently removed cannabis from its most rigid drug control list,the ‘Schedule IV of the 1961 Single Convention on Narcotic Drugs’,which was a huge win for the hemp industry.More organized and standardized regulations all over the world will also be helpful for the development of CBD-containing food products.In technological aspects,some studies have been conducted to improve the solubility,stability and bio-accessbility of CBD in medicine or food systems.Encapsulation,emulsification and microfluidization are being used to incorporate various bio-active compounds into food and beverage systems in the industry and offer potential solutions to the development of CBD-containing foods.

Micro-encapsulation is one possibility to improve the solubility and reduce the oxidative susceptibility of sensitive bio-active compounds,where the hydrophobic molecule is encapsulated and dispersed in aqueous phase.Feng et al.applied sesame oil as a delivery system for CBD in medicines and revealed that adding of medium-chain triglyceride improved the solubility of CBD and superior bio-availability.Lange and Schilderink found that micro-encapsulated CBD had higher CBD bio-accessibility compared to CBD-isolate without micro-encapsulation.Mozaffari et al.discovered that the CBD bio-accessbility was improved with the incorporation into a food system made of olive oil and baby food,which might be attributed to the improved efficiency of micelle forming from hydrolyzed lipids.Nanotechnology has already been applied in drug delivery systems and can be another promising solution.Durán-Lobato et al.developed cannabinoid-loaded lipid nanoparticles through solvent-emulsion evaporation and improved the stability of the products.With more relaxed regulations and more scientific research being conducted,it is envisioned that the technological challenges in the development of CBD edibles may be overcome in the future.Terpenes are another type of bio-active compounds and secondary metabolites found in hemp,which are gaining increasing interest in recent years.There are currently more than 120 terpenes already identified from hemp.The most distinct characteristic of terpenes is the smell,which depicts the unique aromatic characteristics of hemp plant and is used by the plant to repel and defense against herbivores,attract pollinators,and inhibit the microbial growth.Depending on the variety of hemp,the typical terpene contents in hemp range from 0.125% to 0.278% weight in leaves and 1.283% to 2.141% in the inflorescence on a dry basis.Although hemp is abundant in terpenes,it is not the only source.In fact,terpenes are found in lots of fruits,vegetables and herbs.Chemically,terpenes are hydrocarbons consisting of small isoprene units linked to one another to form chains.The biosynthetic pathway of terpenes is shown in Fig.4.Interestingly,terpenes and cannabinoids share a common precursor,which is the GPP,a 10-carbon molecule.As a result,the contents of terpenes and cannabinoids in the hemp are usually positively correlated.Monoterpenes are composed of two isoprenes such as pinene,limonene,myrcene etc..Sesquiterpenes consist of 3 isoprene units,such as humulene,farnesol and caryophyllene.More complex terpene molecules include triterpenes that are made of 6 isoprene units,and polyterpenes.As shown in Fig.4A,the monoterpenes and sesquiterpenes are the major terpene species found in the hemp.

The collapse of the fibers was explained by the significant changes in their chemical composition

The influence of the treatments with Na2CO3 and BTCA on the shives SHI-C and SHI-BTCA microstructure is clearly visible on the nano-CT scans.The removal of a part of the hemicelluloses in the case of sample SHI-C led to the partial deconstruction of the woody fibers cell wall,and general disorganization of the cells within the tissue,which is directly observed through the misorientation and waviness of the woody rays.The fiber walls were also strongly affected by grafting ,the woody rays degraded,the vessels distorted,and the tissues got fully disorganized as shown in Fig.3c.Indeed,the cellulose compartment seems the most affected,with a strong decrease,whereas the lignin content was increased,reflecting an enrichment effect.One can note that the pectin content is also higher in the BTCA sample,suggesting a selective chemical targeting of the treatment on cellulose polymers Fig.3.also shows EDX spectra of SHI-W,SHI-C,and SHI-BTCA samples before copper adsorption,highlighting the presence of sodium on the surface of hemp shives after Na2CO3 treatment and BTCA grafting.Fig.4 presents the X-ray nano-CT images and EDX spectra of the raw and modified hemp shives after copper adsorption.No significant effect of the exposition to copper on the shives’ microstructure can be detected but a major change in the EDX spectra can be observed.The data point out the presence of peaks that were not observed before the shives immersion in the copper salt solution,flood and drain tray which was assigned to copper.It is worthy to remind that EDX is a technique for surface characterization with limited penetration in matter thickness.

To better identify the spatial localization of the copper in the whole shives’ volume,a density-based segmentation of the 3D computed nanotomography images was done.The obtained images are proposed in Fig.5.The figures 5 b and d show the typical microstructure of hemp shives where the vessels are isolated or grouped by two or three,rarely by more,and then they deform one another.The vessels have a quite thin cell wall and a diameter of approximately 50 to 150 μm.They are surrounded by relatively thick-walled woody fibers with diameters of only a few μm and an irregularly polygonal section with a rounded cavity.It is evident that copper is adsorbed on all the free surfaces of the SHI-WCu sample,meaning shives external surface but also on the internal surface of the cells’ lumen.The situation is something different in the case of SHI-CCu shives where it is possible to observe that the copper is not only located on the internal surface of the cell wall bordering the lumen but also in the wall itself.This is attributed to the partial removal of the hemicelluloses in the cell wall induced by the treatment with sodium carbonate and by the micro- and nano-porosities created in the cell wall in which the aqueous solution can diffuse.Furthermore,the copper is massively adsorbed on the internal surface of the vessels’ wall bordering the lumen of the sample SHIBTCACu.The BTCA presence allowed more copper to be absorbed in these areas when compared to raw shives,which is in accordance with the results presented in Fig.1.The detailed analysis of EDX and nano-CT results showed that these two techniques were complementary in revealing the mechanism of copper adsorption on materials.In the EDX spectra ,we can observe an important decrease in the sodium peak and its replacement by copper cations,suggesting the presence of interaction by ion-exchange.In addition,some copper agglomerates are also observed in the internal part of the wall of some of the vessels,suggesting micro-precipitation during the adsorption onto the SHI-BTCA sample.So,the results point out that,even if the two treatments performed similarly in terms of copper removal,the spatial localization of copper and the adsorption mechanisms were significantly different.

FTIR and Raman spectroscopy were further used for the characterization of the shives before and after copper adsorption.The main IR and Raman absorption bands and their assignments according to the literature are summarized in Table 2 and Table 3,respectively.These bands were generally attributed to the three main shives components: cellulose,hemicelluloses,and lignin.The comparison of SHI-W and SHI-C samples shows an important difference in their IR spectra.Namely,the bands at 1730 cm− 1 and 1250 cm− 1 assigned to the stretching of unconjugated C=O groups present in hemicelluloses disappeared after the activation with sodium carbonate.This is in agreement with the data of the chemical composition and is also observed after the partial hemicellulose removal from jute fibers using alkaline treatment.When the FTIR spectrum of the SHI-BTCA sample was observed,the bands in the region 1500-1800 cm− 1 in particular those of the carboxyl and carboxylate groups ,are more intense than in the case of the two other samples which is ascribed to the esterification reaction.In addition,the comparison of SHI-W and SHI-BTCA samples also shows an important increase of the band at 1730 cm− 1 and a decrease of the band at 1250 cm− 1 corresponding to the elimination of hemicelluloses enhancing the lignin content in the material.The interpretation of Raman data indicated that the SHI-W and SHI-C spectra were similar with little differences in the intensity of the bands.On contrary,the Raman spectrum of the SHI-BTCA sample showed an important change in the intensity of the ester band at 1743 cm− 1.The comparison between the FTIR and Raman spectra before and after copper adsorption for the studied samples is shown in Fig.6.After copper adsorption,no changes were observed in the IR spectra of SHIWCu and SHI-CCu,while the spectrum of SHI-BTCACu showed an important change at 1590 cm− 1.Indeed,this band underwent a profound change in both intensity and width,and shifted to 1610 cm− 1.This data reflected the implication of the carboxylate groups on the copper removal through complexation and/or ion-exchange.By comparing the samples’ Raman spectra,we observed that the alkaline activation resulted in the disappearance of the ester band at 1743 cm− 1.The addition of copper contributed to obtain samples with a very high sensitivity to burning,resulting in spectra with enhanced background noise.

This is the reason why we had to work at 1% laser to avoid such noisy phenomena.However,it was difficult to explain the difference in the ratio between polysaccharides and lignin.In this case,we observed more lignin signals in samples containing copper.SHIBTCA gave very fluorescent samples,around 60,000 counts in comparison to 10,000 counts or less for SHI-C.For this sample,we also worked with a 1% laser both to avoid the detector’s ’ saturation and to burn the copper containing sample.The Raman spectrum for SHI-BTCACu also showed the involvement of the ester group in the removal of copper ions,confirming the presence of various interactions between them.Finally,the Raman spectra of samples after copper adsorption also revealed two bands at 1420 cm− 1 corresponding to carboxylate group and at 885 cm− 1 ,in agreement with IR data.Fig.7 compares the XPS wide-spectra of the SHI-W,SHI-C,and SHIBTCA samples before and after copper adsorption.The spectra obtained before copper adsorption are very similar showing the presence of oxygen and carbon C1s at their surfaces.Nevertheless,hydroponic tables canada the sample SHI-BTCA has an additional peak corresponding to the presence of sodium localized near the surface at 1072 eV.The presence of this element is due to the conversion of the carboxylic groups into a carboxylate by immersing the BTCA treated shives in a sodium hydrogen carbonate solution.The 1s level of carbon allows access to the different oxygen functions.The decomposition of level 1s of carbon is significantly different for sample SHI-BTCA with a strong decrease in C-O / C-OH bonds in favour of carboxylic functions.These results are in agreement with the data of the chemical composition.After copper adsorption,Fig.7 clearly indicates the disappearance of the sodium signal in the SHI-BTCA spectrum,due to the ion-exchange with copper cations.Fig.8 shows the main and satellite peaks of Cu 2p3/2 and Cu 2p1/2 after copper adsorption onto three studied samples,while Table 4 reports atomic concentrations of detected elements before and after adsorption.All three samples showed XPS peaks around 933 eV and 953 eV characteristic for Cu 2p3/2 and Cu 2p1/2,respectively.

The XPS analysis of the Cu 2p peaks showed that this element was not in the same oxidation state in all studied samples.Indeed,the detection of satellites on the 2p level of copper for the SHI-BTCACu sample makes it possible to deduce also the presence of copper at the oxidation number for 60%.These satellites are associated with the peak at 934.66 eV of the 2p3/2 level which corresponds to copper in the form of Cu2 [33].In the case of SHI-CCu and SHI-WCu,the detected copper was only in the I oxidation state.Indeed,there are no satellites,the 2p3/2 level showed only one component at about 933 eV.The measurement of the kinetic energy of the Auger transition of copper L3M45M45 at approximately 913.7 eV allowed us to calculate a modified Auger parameter value at 1846.7 eV close to the species Cu2O.This shows that the reactions involved in the removal of copper were different for each hemp sample.In order to know the rate of adsorbed copper on the surface,the atomic percentage of copper was calculated from the area of the Cu 2p peaks.SHI-BTCACu sample had the highest ratio Cu/C = 8.3,followed by SHI-CCu which ratio is 1.5.SHI-WCu sample had a very low Cu/C ratio of 0.4.These results agree with those obtained with other characterization techniques,notably on the fact that copper is well absorbed at the surface for SHI-BTCACu,which gives it this green colour.The less important Cu/C ratio for SHI-CCu confirms that,in this case,the mechanism of copper adsorption is different from those of SHI-BTCACu.Namely,the main interaction between copper and sodium carbonate activated shives is diffusion inside the particles.XANES spectroscopy provides quantitative insight into the oxidation states of copper present in materials.The XANES spectra of SHI-CCu and SHI-BTACCu display the same shape with a pre-edge at 8977.5 eV,the edge at 8991 eV,the maximum of the white line at 8996.5 eV,and a broad oscillation centered on 9052 eV.The absence of peak around 8983 eV,as found in Cu2O,shows that Cu was not detected and that Cu oxidation did not change during the Cu adsorption,it was kept as Cu.CuSO4,5H2O spectrum shows specific features such as a shoulder at 8990.0 eV and an oscillation at 9008.0 eV that are not present in hemp samples’ spectra.Moreover,the white line of CuSO4,5H2O spectra is slightly shifted toward lower energies and is more symmetric compared to that in hemp samples’ spectra.

These differences indicate that after adsorption onto hemp,Cu did not correspond to the crystalline form of CuSO4,5H2O.Furthermore,the hemp samples’ spectra are quite different from those of CuO and Cu2,corresponding to other Cu-bearing precipitates.The spectra of hemp samples are similar to the ones of Cu2+ measured in liquid phase; CuSO4 but also Cu2,another Cu complex,both when recorded in a liquid phase.The results obtained by XANES and those obtained by XPS or both Cu and Cu seems to differ.However,they are compatible and complementary as XANES at Cu K-edge sounded the bulk sample whereas XPS sounded the first atomic layers on the surface of the sample.XANES spectroscopy evidenced that most of the Cu atoms are present in Cu form in both SHI-CCu and SHI-BTCACu samples and XPS spectroscopy indicated that the external surfaces of SHI-WCu and SHI-CCu are coated by Cu2O and that the external surface of SHIBTCACu is coated by a mixture of Cu2O and Cu2,explaining this characteristic blue color.An increased interest in the acceptance of novel foods,that is,foods to which a consumer has not been previously exposed ,is borne of an increasing global population that should reach nine billion by the year 2050.Globalisation has resulted in increased exposure of the world’s population to foods from other cultures,and increased multicultural culinary experiences have altered normative perceptions for many.The increasing global population presents a challenge to food security as many traditional food production techniques will become environmentally unsustainable at the levels required to meet world food demands.Hemp foods might suitably address many food security issues.The hemp plant,Cannabis sativa,from which hemp foods are produced is environmentally sustainable due to a reported low water need and natural pest resistance ,can be economically lucrative with high yields and shorter growth cycles compared with many traditional crops ,and has many nutritional benefits.Nutritional benefits of hemp include being high in levels of plant protein,high in dietary fibre,a rich source of Omega 3 and 6,and contains all of the amino acids essential to human life.

The separated hull and heart fractions were ground prior to carbohydrate analysis

Another benefit of hemp seeds is that they contain proteins that are rich in several essential amino acids including arginine and the sulfur-rich amino acids methionine and cysteine.Hemp contains other known beneficial and nutraceutical compounds but also some antinutrients including phytate and trypsin inhibitor that can impact on nutrient uptake especially of minerals and amino acids.However rat model studies confirm the high bio-availability of hemp proteins suggesting that the anti-nutrients may not be a major concern.A disadvantage of one of the earliest seed cultivars,FINOLA,is that the seed is about 50% smaller than other seed varieties.Comparisons between different lines are usually based on thousand seed weight.A recent comparison of 33 lines showed TSW ranged from approximately 7.5 g–23 g,with FINOLA at around 12 g,which is at the lower end of the expected range of 12–15 g for FINOLA.Seed size is not the only important consideration for seed varieties.A higher proportion of heart to hull tissue ,here called heart %,is desirable since it is the heart that attracts a higher value margin.A study of five hemp cultivars in Romania showed that heart % varied from 59.0% to 69.5%.However,current data on the heart % trait is limiting with very few published studies.Hemp hearts,also known as dehulled seed have more protein and more “digestible” fiber than whole seed and seed meal ,based on analysis of a “typical” variety.Interpretation of fiber data in food products can be difficult,due to the use of different terminology and methods.Scientific papers often report both neutral detergent fiber and acid detergent fiber ,where NDF includes cellulose,‘hemicellulose’,and lignin whereas ADF only includes cellulose and lignin,thus providing a measure of non-fermentable fiber.One disadvantage of NDF and ADF is that they underestimate the amount of total fiber,as the methods may not recover all of particular components such as pectins and gums,which can be some of the healthier fermentable fibers.However,this information is useful because it provides an indirect way to determine lignin content,trimming tray for weed and high levels of lignin can negatively affect palatability.

The nature of the non-cellulosic polysaccharides that contribute to dietary fiber in hemp has not yet been studied.One review suggests that hemp seed contain 25% starch,however the two cited references report soluble fiber and not starch.Since “detailed chemical characterization of dietary fiber is crucial to explain its effect on health” ,there is a clear knowledge gap of the nature of the polysaccharides in hemp lines grown for food.Different cell wall polysaccharides have remarkably different functional properties and these in turn influence fermentability,nutrient bio-availability and composition of gut microbes.The role of complex carbohydrates in microbiome diversity and human health is becoming clearer,and many positive effects relate to the fermentation products or short chain fatty acids such as butyrate,acetate and lactate.It has long been established that plant polysaccharides can differ in length of backbone,number and distribution of side branches and the form of each monosaccharide and pyranosyl and the linkages between them.Even within the same general class the structures can be markedly different.For example,heteroxylans from different species of Plantago have different proportions of unsubstituted xylan and linkages along the xylan backbone.It is now understood that microbes exist in cooperative metabolic networks where selected bacterial species initiate degradation and other species continue fermenting the partially degraded polysaccharides,thus supporting microbial biodiversity and a healthy colon.To fill the knowledge gap on the nature of hemp polysaccharides,we analyzed the composition of 20 different industrial hemp varieties and breeding lines that are currently available in Australia.We report analysis of 1000 seed weight,heart %,% nitrogen ,lipid profiles,lignin,phytate,and a detailed analysis of the carbohydrates in hemp heart and hull fractions including cellulose,starch,monosaccharide composition of complex carbohydrates,and soluble sugars.In addition,we use specific antibodies to investigate the distribution of plant polysaccharides in hull and heart fractions,thus providing critical information on the distribution of non-cellulosic polysaccharides in hemp seed.One hundred seed were counted out and weighed,then multiplied by 10 to provide 1000 seed weight.A single replicate of 100 seed was selected for further analysis.Hull and heart tissues were separated using a fine spatula and the separated fractions weighed.

Hull fractions were ground in a retsch mill for 30 s,and the softer heart fractions were ground in liquid nitrogen and air dried prior to analysis.These ground fractions were used for quantification of cellulose and soluble sugars and monosaccharide analysis ,lignin and starch.This research is the first detailed study of complex carbohydrates in hemp seed using a combination of chemical analysis and immunolabelling of tissue sections from a selected set of germplasm mainly sourced from Ecofibre’s seed bank.The focus of this study was on end-user/health traits where diversity of dietary complex carbohydrates is positively correlated with microbiome diversity and improved human health outcomes.The new findings on carbohydrate composition are combined with analysis of heart %,1000 seed weight,lipid composition,total protein,lignin and phytate,thus providing a foundation for selection of plant lines with improved human health attributes for expanding the hemp industry in Australia and internationally.Coefficient of variation % is a useful parameter for determining the characteristics that vary most between lines.Since the batches of seed used in this study were sourced from different regions of Australia,it is not possible to tell if the source of variation is from genotype or environment or an interaction of both.However,CV information is still useful for prioritising hemp lines for future field trials,and traits for subsequent analysis,and for identifying traits with a strong genetic component suitable for targeting in breeding programs.In the following discussion,we make the assumption that traits with a high CV have a strong genetic component,but obviously this will need to be tested in the future.There is some support for this assumption based on a hemp field trial from Canada that evaluated 11 cultivars over two years in seven different environments.Analysis of field trial data revealed that most of the traits evaluated had statistically significant genetic ,environment and G x E components,including seed protein,seed yield,plant height,biomass yield,biomass cellulose and biomass hemicellulose.

However,for the 11 varieties studied there was not a significant genetic component for biomass lignin and seed oil,but both had significant G x E components,and seed oil also had a significant environmental component.In our study,heart %,seed lignin and total lipids had a CV of <10% indicating low variability,whereas,many traits had intermediate CV ,including crystalline cellulose,hull monosaccharides ,non-resistant starch,phytate,the essential omega-3 fatty acid ALA.A few traits had high CV ,including 1000 seed weight,GLA,soluble sucrose and soluble raffinose.This information can be used to select a smaller number of appropriate lines for statistical validation of each desired trait,and used by breeders to select lines for future field trials to determine the extent of the genetic and environmental components of each trait.There is high variation in 1000 seed weight.Thousand seed weight provides an approximation of seed size,as generally the bigger the seed,the heavier it will be,although variation in the composition and ratio of heart % means that this correlation may not always hold.Furthermore,seed size does not usually correlate with overall seed yield which is key for profitability,since yield is heavily influenced by inflorescence architecture and shattering resistance.Higher heart % provides an opportunity to increase profitability,through increases in higher value hemp heart products.The heart % trait has not been widely reported for hemp,with only one published study to our knowledge,where heart % varied from 59.0% to 69.5%.Twelve of the 20 lines analyzed in this study had heart % greater than 59%.This does not guarantee that these lines are high yielding,profitable lines for current use,but rather they are potential lines for breeding,if the heart % trait has a strong genetic component.For other nuts,such as Macadamia,high heart % is called kernel recovery,and is known to be influenced by the genotype of the pollen donor.Kernel recovery in Macadamia is also sensitive to mild drought stress during nut development.To our knowledge there have not been any studies in C.sativa on what factors affect kernel recovery ,but we hypothesize that pollen donor and/or stress could be important,and should be explored in future to determine genotype by environment effects on this trait.Hemp hulls contain crystalline cellulose as the major polysaccharide ,with xylan as the next most abundant polysaccharide.The identification of xylan as a major complex carbohydrate in hemp hulls is based on monosaccharide analysis and immunolocalization analysis which demonstrates the polysaccharide is present as an unsubstituted xylan and not arabinoxylan.

The variability in hull xylose content is of significant interest to the food industry for adding value to agricultural wastes.Xylan is a source for the production of xylan oligosaccharides for prebiotics,which are non-digestible food ingredients that promote growth of bacteria in the colon and can improve health.Xylan oligosaccharides have been produced from peanut shells.The two hemp lines with the highest levels of xylose in their hulls,Frog-1 and Yunma-1 ,are likely to have higher levels of xylan than peanut shells,assuming that all the xylose is hemp is found in the xylan as suggested by immunolocalization.Hemp hulls and hearts are low in soluble sugars,with hemp hearts generally containing more soluble sugars than the hull fraction.The new data reported here is mostly consistent with a previous study that showed that sucrose was the most abundant soluble sugar.The major difference between the earlier study and this study is that they did not detect any raffinose in whole hemp seeds,whereas here raffinose was found in all 20 heart samples.This difference is puzzling since the 2018 study was able to detect raffinose in other food samples,weed trimming tray however it is not of great importance given the low levels <0.5% w/w observed in the 20 lines analyzed here.An important finding from the current study is that starch is not a major component of hemp seed,based on the trace levels of glucose in the polysaccharide fraction and <2% total starch in the six lines chosen for starch quantification.This contrasts with information in a recent review where the authors state that hemp seeds contain 25% starch.The results presented here show that the dietary fiber is not starch but predominantly xylan and pectin in hulls,with a small amount of xyloglucan and pectin in hemp hearts.This is important because it suggests that hemp will have a low glycemic index and contains a range of complex carbohydrates that could be fermented in the gut to varying degrees.This could be readily tested in future by including hemp as a protein source in human diets,because SCFAs,the products of gut fermentation,are readily detected in stools making them convenient biomarkers.Other studies adding whole seeds to human diet plans show that increases in SCFAs can be achieved in relatively short time frames.

The fate of the different cell wall polysaccharides is an area of active research,for example,xyloglucans from cranberry promote the growth of SCFA producing strains of Bifidobacterium longum.Furthermore a phenol-free xyloglucan derived oligosaccharide fraction from cranberry hulls reduces biofilm formation by strains of E.coli that cause urinary tract infections.Pectins are also substrates for gut bacteria,and although present in small amounts in hemp,they should contribute to microbiome diversity,especially as their fine-structure may be different to commonly consumed pectins such as citrus pectin.The fine-structure of the hemp carbohydrates remains to be determined,and xylan would be the obvious first target as it is the major noncellulosic polysaccharide in hemp.The hull should not be overlooked as a food source.It can be milled for example and used as a high-fiber ingredient,or better still,more emphasis could be placed on developing food products that use the whole seed.In addition to providing the best human health outcomes,use of whole seeds may have the benefit of reducing manufacturing costs and waste products generated through the dehulling process.Whole seed products would also contain lignin ,which can be perceived as a negative component.However,rather than being an inert compound,lignin is now thought to contain beneficial antioxidant activity.The mean value of lignin in hemp hulls observed in this current study was 17.6  1.0 ,which is higher than a prior study that reported a mean value of 11.2.However,the values are comparable given that our study used hemp hulls,not whole seed.The other major components of hemp hearts are proteins and lipids.The study conducted by House et al.,used a rat bio-assay to calculate protein digestibility-corrected amino acid score measurements.Importantly they showed that hemp protein had PDCAAS value equal to or greater than certain grains,nuts,and some pulses.specifically whole hemp seeds have a higher PDCAAS value than almonds and whole wheat,and hemp hearts have a higher score than lentils,pinto beans and rolled oats.

Stem elongation and fiber development are associated with elevated levels of gibberellins

Without shattering,immature seeds would all fully mature,increasing yield by up to 15%.Thus,further domestication of non-shattering cultivars could greatly improve yield via a multifold mechanism,preventing harvesting loss and permitting all seeds to reach maturity.Seed traits that expand market options will also be valuable.For example,there has been little research investigating the differences in hemp seed flavors.Taste tests in our lab identified varieties with weak to strong flavors of hazelnut or walnut ,as well as one with a mild flavor.More work has been done on altering seed oil composition,although hemp seeds already possess valuable v-3 characteristics.Hemp seed oil is 85% polyunsaturated fatty acids with 60% and 24% being v-6 and v-3 fats,respectively.Further increases in v-3 fatty acid levels might foster the favorability of hemp seed for human and animal dietary needs.Overall,different tastes and oil compositions would expand the use of hemp seed in human and animal food products.Hemp stalks contain two key fractions,the bast fiber and hurd.To separate bast fibers from the inner hurd,the stalks must undergo a process called ‘retting’.Retting relies on the diverse microbial populations in the environment to break down pectin and other components that bind the fibers to the hurd tissue.Crop maturity at harvest,retting method,environmental conditions,as well as the nature of the bacterial and fungal populations,are factors that impact retting.Harvesting the crop at initiation of flowering improves fiber yield,strength,and quality.Continuing studies on the biodiversity,relationships,and functions of microbial communities will improve our understanding of the retting process and augment the consistency of obtaining high quality products.Retting methods,ebb and flow primarily dew- and water-retting,pose drawbacks,including inconsistent fiber strength and quality,and polluted wastewater,respectively.

Development of varieties having bast fiber with higher cellulose content as well as lower pectin and lignin cross-linkages may decrease the retting requirements,thus improving fiber strength and quality while saving time and labor.Hemp produces a diverse array of nonintoxicating phytocannabinoids,terpenes,and phenolic compounds with potential pharmaceutical values as drugs or supplements.The biosynthesis of terpenophenolic phytocannabinoids in Cannabis is well understood,albeit,several early steps in the pathway remain to be characterized.Understanding the regulation of phytocannabinoid biosynthesis is vital to development of varieties that are optimized for production of desirable metabolites while maintaining low levels of THC.Little is known about the endogenous and environmental regulation of phytocannabinoids.Abscisic acid,ethylene,and gibberellic acid modulate the production of phytocannabinoids.However;at present,factors controlling the epigenetic,transcriptional,and post-transcriptional regulation of phytocannabinoid biosynthesis remain uncharacterized.Hemp trichomes are classified into bulbous,capitate-sessile,capitate-stalked,and nonglandular types.Phytocannabinoid production and accumulation are localized to the capitate-stalked glandular trichomes.Increased production of phytocannabinoids in marijuana is,at least partially,due to the presence of larger glandular trichomes.Elucidating hormonal and other signaling cascades that regulate the development and size of specific types of hemp trichomes will also be important in maximizing phytocannabinoid production in hemp.The effects of agronomic practices and nutrients on phytocannabinoid production also need to be investigated.Anecdotal claims from marijuana growers suggest that pollination of Cannabis flowers lowers phytocannabinoid yield,consistent with decreases in essential oil levels.Further studies to evaluate this concern are essential to maximize the production of CBD and other desired phytocannabinoids.Germplasm collections are a fundamental source of genetic and phenotypic diversity for plant breeding and research.Currently,access to and utility of accession collections remain limited due to the lack of a core Cannabis germplasm collection.

As THC levels may limit germplasm utility in many regions,accessions with <0.3% THC should be used to form a hemp-only germplasm core collection.Establishment of a core collection encompassing the range of hemp genetic and phenotypic diversity would increase the utility of germplasm resources and be invaluable for breeding and genetic analyses.Comparisons of accessions present in existing collections are needed to help establish such a core collection.Similarly,centralized and curated collections of hemp mutants are not available.The development of mutant germplasm collections will provide a rich source of genetic variation for studying gene function and improved traits for breeding.Hemp is a dioecious plant with female and male hemp plants being valued differently depending upon the products.For phytocannabinoid production,a pure female population is most desirable.As a seed crop,a female predominant population,with a limited number of male plants for pollination,or a monoecious variety,is most desirable to maximize yield.For fiber production,males and females are both utilized,although males are preferred.Therefore,a major goal of hemp growers and breeders is to quickly and easily determine or manipulate the sex of plants,preferably prior to planting.Sex in hemp is genetically determined by a pair of heteromorphic sex chromosomes; females have an XX chromosome pair whereas males have XY.However,environmental conditions and phytohormones can affect sexual phenotype,suggesting other overriding regulatory mechanisms are involved in determining sex in hemp.Monoecious cultivars possess XX sex chromosomes,but they produce flower clusters with male flowers at the bottom and females towards the top of each inflorescence.Notably,male flowers occur as the plant transitions from rapid growth to flowering.In hemp,gibberellins are associated with plant masculinity and greater fiber number,length,and diameter.Thus,a concentration gradient of gibberellin and other hormones may dictate inflorescence sex.Genetic markers have been developed to differentiate sex in hemp plants; however,such a method is not viable for commercial plantings.Recently,quantitative trait loci were identified for sex expression in dioecious and monoecious hemp.

Cloning of the responsible genes from these QTLs will greatly improve our understanding of genetic control of sex in hemp.Identification of genes present on the sex chromosomes,especially outside the pseudoautosomal recombinant region,will be critical for understanding sex-linked traits.Continued development of molecular markers is needed to improve QTL mapping resolution and for marker-assisted selection of desirable traits in breeding programs.The organic food market is a key player in promoting hemp food and CBD products.As such,widespread public acceptance of transgenic hemp is unlikely.It also remains unknown whether the public will welcome hemp modified using gene-editing techniques,which lack non-plant or plant-pest DNA sequences.Thus,many improvements to hemp will probably be accomplished using traditional breeding methods.However; for research purposes,the development of applicable molecular biology techniques is imperative to further study the molecular mechanisms that determine important traits in hemp.Publication of a draft-quality Cannabis genome and other genetic studies have shed some light into the difference between marijuana and hemp.The Cannabis draft genomeAppendix Aiii has been compared with draft genome sequences of its closest relative common hop as well as more distant species including bread nut and mulberry.Recently,low coverage whole-genome sequencing and genotyping-by-sequencing have been performed on 54 and 325 distinct cultivars,respectively.However; with only raw data files available,the lack of websites with easy to-use graphical user interfaces for data analyses limits the utility of these draft-level genome sequences.Transcriptome assemblies are also available ,but are primarily targeted toward understanding phytocannabinoid metabolism.Comparison of marijuana and hemp indicates that the expression of phytocannabinoid biosynthetic genes is higher in marijuana,suggesting that transcriptional regulation of the pathway may be one factor controlling cannabinoid production.Recently,a transcriptome was generated for hemp bast fibers at different growth stages,providing insight into fiber development.The evolution of genetic differences between seed/oil,fiber,and dual-purpose cultivars is less studied.In-depth genetic comparisons of diverse seed/oil,fiber,and phytocannabinoid cultivars are needed to identify the specific genes and mechanisms controlling important yield traits.

To attain the full benefit of these and other studies across species,the genome sequence needs to be improved beyond draft quality,and websites with user-friendly graphical user interfaces must be developed.To characterize hemp gene functions,methods to manipulate gene expression are urgently needed.Protocols for developing transformed hairy roots and cell suspension cultures are available,but the utility of both methods is limited since neither tissue produces seed,fiber,or phytocannabinoids.A whole-plant regeneration protocol has been developed for marijuana,suggesting that the development of transgenic hemp plants is feasible.Virus-induced gene silencing methods would also prove useful for studying gene function,but thus far have been unsuccessful.Alternatively,isolation of mutants from chemical mutagenesis screens is possible,but extremely difficult due to the anemophilous and dioecious nature of hemp.Currently,exploitation of the natural genetic diversity present within hemp may be the most straightforward way to study gene functions.In the present study,salivary biomarkers for gastric ulceration in pigs were explored,and significant differences in oxylipin levels were observed between healthy pigs and pigs with gastric ulcers.The results suggest that reduced levels of oxylipins deriving from linoleic acid might function as biomarkers for gastric ulcers in pigs.The potential of oxylipins as biomarkers for early diagnosis of gastric ulceration and thereby as a tool to prevent severe gastric ulcer development would be relevant investigating.Moreover,hemp seed hulls might reduce the incidence of severe gastric ulcers in pigs.Ulceration of the stomach is a common disease in pig production worldwide and is associated with economic losses as well as animal health and welfare issues.Severe ulcers often lead to weight loss,and bleeding ulcers may lead to spontaneous death.Thus,prevention of gastric ulcers is important to improve animal production as well as animal health and welfare.The structure of the feed is known as a major risk factor for the development of gastric ulcers.Several studies have observed an association between fine-pelleted feed and incidences of gastric ulcers,presumably due to a more fluid stomach content,dry racks which increases the risk of the acidic chyme of the more distal regions coming in contact with the non-glandular part of the stomach.Thus,feeding ingredients with a high swelling capacity is expected to result in a more solid stomach content and thereby reduce the risk of developing gastric ulcers; however,there is a gap in knowledge regarding potential dietary treatments.Hemp is an old culture plant which is easy to cultivate and manage and is known as an important source of fibre as well as a medical product due to its high content of bio-active compounds with antioxidant properties.Different hemp feed products exist,including hemp seed cake and hemp seed hulls.Hemp seed cake is a by-product after oil extraction and has a high protein content as well as some fibre,whereas hemp seed hulls are the outer shell of the seed with a high fibre content.Studies indicate that hemp might protect the gastric mucosa by inhibition of gastric acid secretion through cannabinoid receptors in the gastrointestinal tract including the stomach.In addition,the potential swelling capacity of hemp seed hulls may result in a more solid stomach content.

Clinical diagnostics of gastric ulceration in pigs is difficult since gastric ulcers are often sub-clinical with only the most severe ulcers resulting in the development of clinical signs,such as anorexia,anaemia or sudden death.Therefore,an efficient non-invasive method is required to be able to detect early stages of gastric ulceration in pigs,allowing intervention with preventive measures,e.g.by regulating the particle size of the feed,to reduce the economic losses as well as animal health and welfare issues.Saliva is a bio-specimen that has received attention for detection of disease biomarkers,as it has shown to contain compounds that are associated with certain diseases.In addition,collection of saliva is simple and noninvasive.Salivary glands with high permeability are surrounded by capillaries allowing exchange of molecules.Hence,saliva contains a variety of locally synthesised compounds as well as systemic compounds that might act as biomarkers.Studies have indicated that several factors present in saliva protect the oesophagus,including epithelial growth factors,mucins and prostaglandins.The maintenance of oesophageal health is to a large degree dependent on saliva ,indicating that saliva might be a potential biospecimen for identifying possible biomarkers that reflect the health status of the upper part of the gastrointestinal tract,including pars oesophagea.Since gastric ulcers in pigs are mostly located in the region of pars oesophagea,saliva might contain metabolites that indicate the presence of gastric ulcers.Non-targeted metabolomics is an analytical technique used to study a wide range of small molecules in biospecimens,making it possible to discover metabolites that discriminate across phenotypes,including health status.In this study,the saliva metabolome was explored for potential biomarkers associated with the presence of gastric ulceration in pigs.In addition,the effect of hemp on the incidence of gastric ulcers was examined.It is hypothesised that saliva contains metabolites that can be used as biomarkers for gastric ulceration.Furthermore,it is hypothesised that hemp seed cake and hemp seed hulls reduce the incidence of gastric ulcers via anti-inflammatory effects and by conferring more solidness to the gastric content,respectively.The pigs were provided dry feed ad libitum in single-space feeders and were offered the experimental diets from approximately 30 kg BW and until slaughter weight.One dry-feed dispenser with water supply and one drinking trough were available per pen.All pigs had permanent free access to water.Four experimental diets were formulated: meal feed without addition of hemp seed products ; pelleted feed without addition of hemp seed products ; pelleted feed added 4% hemp seed cake ; pelleted feed added 4% hemp seed hulls.Ingredient composition of the experimental diets is shown in Table 1.