Can Trema micranthum (L.) Blume (Cannabaceae) become a new source of cannabinoids? A UHPLC- HRMS/MS analysis

Can Trema micranthum (L.) Blume (Cannabaceae) become a new source of cannabinoids? 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Blume (Cannabaceae) become a new source of cannabinoids? A UHPLC- HRMS/MS analysis Rayssa Ribeiro, Yasmin Cunha Silva, Ricardo Finotti, Gabriel Reis Alves Carneiro, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5125664/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Trema micranthum (Cannabaceae) has emerged as a promising new source of cannabinoids, including cannabidiol (CBD). Given the substantial medicinal demand for cannabinoids and the regulatory challenges associated with Cannabis sativa due to the presence of Δ 9 -tetrahydrocannabinol (THC), this study sought to explore the presence of CBD, THC, and their precursors, Δ 9 -tetrahydrocannabinolic acid A (THCA A) and cannabidiolic acid (CBDA), in various parts of Trema micranthum using UHPLC-HRMS/MS (Orbitrap). Extracts from fruits, leaves, inflorescences, and stems were obtained using a methanol/hexane (9:1, v/v) solvent mixture. UHPLC coupled with an Orbitrap mass spectrometer was employed for cannabinoid identification and quantification, with standard mixtures prepared in methanol. The extracts yielded significant quantities, such as 6.6%/g from leaves and 3%/g from fruits. Cannabinoids were detected in fruits, leaves, and inflorescences, with acidic forms (CBDA and THCA A) present in higher concentrations than their neutral counterparts. Notably, leaves contained 4.43 x 10⁻³ µg/g of CBD and 1.05 x 10⁻³ µg/g of THC. These findings, facilitated by high-resolution analytical methods, underscore the potential of Trema micranthum as an alternative source for cannabinoids, guiding future research in this area. Biological sciences/Chemical biology/Natural products Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Chemical biology Biological sciences/Drug discovery Biological sciences/Plant sciences Physical sciences/Chemistry Cannabaceae Trema micranthum Cannabinoids CBD/THC UHPLC-HRMS/MS Figures Figure 1 Figure 2 Figure 3 Introduction Cannabidiol (CBD) is a non-psychoactive cannabinoid with important therapeutic benefits. The recent FDA approval for formulation containing CBD, such as Epidiolex® used to treat seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, or tuberous sclerosis complex in patients, gave new impetus for research around the theme 1 . Despite the huge health importance and wellness and drug industries interest, the use of CBD formulation still faces several commercialization challenges. In many countries, CBD is regulated differently depending on its source, hemp or marijuana buds (varieties of Cannabis sativa L. – Cannabaceae Martinov). In addition, the content of Δ 9 -tetrahydrocannabinol (THC), the main psychoactive cannabinoid and responsible for several important pharmacological properties but also for recreational use, brings a lot of controverses to the discussion 2 . Cannabis sativa buds are commonly extracted with ethanol and, after the solvent evaporates, but before adding coconut oil (or similar) to produce the commercially material known as Cannabis oil, the extract needs to be activated. The carboxylic acid precursors of THC and CBD present in the extracts are mainly Δ 9 -tetrahydrocannabinoic acid (THCA) and cannabidiolic acid (CBDA), respectively. The extract needs to be heated to allows the decarboxylation process to produce the bioactives CBD and THC, when it is finally considered activated. However, this process can sometimes lead to the degradation or evaporation of other bioactive constituents, such as the volatile sesquiterpene caryophyllene. Sources of cannabinoids with natural high content of CBD is highly desired, without the need to activation and avoiding the presence of THC 3 , 4 . Besides the production issues and the social preconception against the use of Cannabis L., the main difficulties of CBD-based products commerce lie on the complex and often lack of a standardized regulatory background. As consequence, there is a clear concern about the product quality, safety, and labelling, leading to a framework of laws that can confuse consumers and complicate commercial operations, challenging international trade 5 , 6 . The legal issues related to the presence of THC are also a consistent justification for the search for new CBD-producing species. The main alternative for obtaining cannabinoids is the use of other species of Cannabaceae botanical family that can be easy to obtain and produce. Diversifying cannabinoid sources can address sustainability and environmental concerns associated with large-scale cultivation more efficiently or under less resource-intensive conditions. This approach can mitigate the overreliance on a single species, thereby reducing the risks associated with crop failure due to pests, diseases, or changing climatic conditions. A reasonable outcome is the stimulus to innovation in agriculture and biotechnology. Such advancements would not only benefit the pharmaceutical industry, but also have implications for other sectors, including food, cosmetics, and wellness products 7 , 8 . The genus Trema Lour (Cannabaceae) is pantropical with nearly 20 species, six of them being cited for Americas 9 . Trema micranthum L. (Blume), synonym Trema micrantha , is the one traditionally registered for Brazil 10 . It is a shrub present as ruderal species, native from tropical and subtropical America, occurring naturally at all the Brazilian biomes and in diverse vegetational forest types (ever-green, semi-decidual and gallery forests) 11 , 12 . Phylogenetically, Trema-Parasponia group is a sister group of Humulus-Cannabis group, been diverged nearly from 58.5 to 46.3 Ma, between the late Paleogene and the early Neogene 13 . Other species of the previous cited groups are known cannabinoid producers as Cannabis sativa L. and Trema orientale (L) Blume 14 . Recently, glandular trichomes of T. micranthum flowers and leaves were described as cannabinoid secreting structures 15 . Its popular uses are described in Bolivia, to cough and wounds, and in Mexico, where traditional use includes timber products for house construction (wood for wall and roof support) and Mexican popular medicine with leaves used in baths for skin injuries 16 . Previous studies with T. micranthum extracts showed antioxidant properties at 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase assays 17 and some antimicrobial activities in vitro (62%) and in vivo (81% at 100 mg/kg) 18 . Ether and ethanol extracts of T. micranthum also presented in vivo (mice and rats) anti-inflammatory and antiarthritic activities, with inhibition of carrageenin-induced oedema and adjuvant arthritis, and this effect was comparable to that of indomethacin 19 . The ethanolic extract also showed hypoglycaemic activity in acute treatment in rats (250 and 1000 mg/kg doses) 20 . The consumption of T. micranthum by animals has been reported to cause several toxic effects, including hepatocellular necrosis in cattle, sheep, and goats and oedema, and cerebral haemorrhage in horses 22 . The exact mechanism of action and the substance responsible for the toxicity remain unidentified. A recent publication reported the analyses of leaves, branches, fruits, and inflorescences of the methanolic extracts obtained from T. micranthum using HPLC-DAD and GC-MS. Despite these traditional tools employed in natural products research, no cannabinoids were detected 21 . However, anecdotic information from different sources creates the hypotheses of the production of CBD without THC formation. To the best of our knowledge, only two studies have accurately described the chemical composition of Trema micranthum leaves with the identification of the ubiquitous sterols β-sitosterol and 3β- O -β-D-glucopyranosyl sitosterol, the triterpenes ursolic acid and 2α,3β-dihydroxyurs-12-en-28-oic acid, the amide paprazine ((2E)-3-(4-hydroxyphenyl)- N -[2-(4-hydroxyphenyl)ethyl]prop-2-enamide) and the flavonoids vitexin (8-glucopyranosyl-4,5,7-trihydroxyflavone) 23 and also vitexin (or isovitexin) and orientin (or isoorientin) arabinosides (or xylosides) derivatives 24 . The lack of a deep investigation relative to the molecular composition of Trema micranthum opens the perspective of contributions using the “State of the Art” in organic analytes identification, using high quality approaches already well tested in different fields such as analytical toxicology and food residue analysis. The present study intends to perform a UHPLC-HRMS/MS (Orbitrap) search for the cannabinoids CBD and THC and their main precursors, THCA and CBDA A, at leaves, inflorescences, fruits, and branches of Trema micranthum extracts to verify their possible use as an alternative to Cannabis and source of CBD. Methods Chemicals and reagents For sample preparation, methanol and n-hexane, both GC grade, were acquired from Tedia (Fairfield, OH, USA). CBD and THC standards were provided by AB Científica Ltda (Espírito Santo, Brazil). CBDA standards were supplied by LGC group (North Charleston, United States). THCA A was provided Sigma-Aldrich (Darmstadt, Germany). For LC-HRMS/MS analysis, methanol HPLC grade, formic acid 98%-100%, and ammonium formiate were provided by Tedia (Fairfield, OH, USA), Merck (Darmstadt, Germany) and Spectrum Chemical (Gardena, CA, USA), respectively. Ultrapure water (18.2 MΩ.cm) was obtained from a Millipore Milli-Q purification system (Billerica, MA, USA). Extraction process Twigs, leaves, inflorescences, and fruits were collected from an individual of Trema micranthum found at Parque Nacional da Tijuca, close to the Pedra da Gávea, Rio de Janeiro-RJ, Brazil, on July 10th, 2024, with a voucher deposited at RB 648300 Herbarium. his work was registered in the National Management System of Genetic Heritage and Associated Traditional Knowledge (SISGEN) under number xx. Ten grams of plant parts fresh, including branches, leaves, and inflorescences, were extracted using a methanol/hexane solvent mixture in a 9:1 (v/v) ratio. One gram of macerated fruits fresh was weighed, and the same solvent mixture was used for extraction. The plant materials immersed in the solvents were subjected to extraction under agitation in a shaker at 150 rpm for 12 hours. The resulting organic extract was centrifuged at 4000 rpm for 20 minutes and evaporated under nitrogen flow at 40°C. The dry residue was reconstituted with methanol to obtain a final concentration of 10 µg/mL. The extracts were vortexed for 30 seconds, 1.5 mL was transferred to an Eppendorf tube, and centrifuged at 13000 rpm for 15 minutes at 15°C. The supernatant was transferred to a vial and injected into LC-HRMS. UHPLC-HRMS Instrumentation and analysis A Dionex Ultimate 3000 ultra-high performance liquid chromatography (UHPLC) system coupled to a QExactive Plus hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ionization (ESI) source was used. Separation was performed in a reversed-phase column (kinetex 2.6 µm PS C18, 100 Å, 100 mm x 2,1 mm), at 40°C, constant flow rate of 300 µL/min and injection volume of 5 µL. A gradient chromatographic run started at 5% of mobile phase B (methanol with 0.1% formic acid) and 95% of mobile phase A (water with 5 mM ammonium formiate and 0.1% formic acid). Mobile phase B was increased to 10% at 1.0 minute, then to 25% at 2 minutes, and then 90% at 10 minutes. After reaching 100% of B at 14 minutes and maintaining this ratio until 16 minutes, the initial chromatographic condition was restored from 16.1 to 20.0 minutes. The LC effluent was pumped to the mass spectrometer operating in a positive and negative ESI mode, calibrated daily with a manufacturer's calibration solution (Thermo Fisher Scientific, Bremen, Germany). ESI parameters were further optimized, with the final setup: spray voltage of 2.9 kV, S-lens voltage of 80 V, the capillary temperature of 380°C, auxiliar gas heater temperature of 350°C, nitrogen sheath, auxiliary, and sweep gas were set at 30, 10, and 1 arbitrary unit, respectively. Full-scan data were acquired in a range of m/z 70–1050 at a resolution of 70,000 full width at half maximum (FWHM), automatic gain control (AGC) of 1 x 10 6 and maximum injection time (IT) of 100 ms. Targeted mass spectrometry-based approaches were performed using the parallel reaction monitoring technique (PRM), the precursor ions were fixed at a resolution of 17,500 full width at half maximum (FWHM), automatic gain control (AGC) of 1 x 10 6 , maximum injection time (IT) of 100 ms and quadrupole isolation window of m/z 2. In the Full MS approach, the target exact mass was m/z 315.23184 ([M - H] + ) to CBD and THC, and m/z 357.20713 ([M - H] − ) to CBDA and THCA A. The detection limit established by PRM was 5.2 x10 − 4 µg/g for CBD and 3.7 x10 − 4 µg/g for THC, 1.4 x10 − 3 µg/g for CBDA and 1.0 x10 − 3 µg/g for THCA A per gram of extract. The estimated limit of quantification established by PRM was 1.7 x10 − 3 µg/g for CBD and 1.2 x10 − 3 µg/g for THC, 4.7 x10 − 3 µg/g for CBDA and 3.5 x10 − 3 µg/g for THCA A per gram of extract. In the PRM approach, precursor ions of m/z 315.23184 [M - H] + (CBD and THC), and m/z 357.20713 [M - H] − (CBDA and THCA A) were fragmented in a higher energy collisional dissociation (HCD) cell with (N)CE of 40%. To detect and quantify CBD, CBDA, THC and THCA A in the plant parts, the standard mixtures were prepared in methanol at a concentration of 3.16 x 10 − 1 µg/g. Data was acquired and processed using Thermo ScientificTM TraceFinderTM 4.1 software (Thermo Fisher Scientific, Austin, TX, USA), with a ± 6 ppm mass tolerance. Results and Discussion Phytocannabinoids are usually biosynthesized as acids. Neutral forms are produced through non-enzymatic decarboxylation. Cannabigerolic acid (CBGA) is the bioprecursor of other acidic cannabinoids in the plant, such as Δ 9 -tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) 25 . These substances give rise to their respective neutral forms, THC and CBD. Sequentially, isomerization and oxidation products are formed 3 , 4 . In the present study, to confirm the presence and estimate the concentration of the cannabinoids CBD, THC, CBDA and THCA A in Trema micranthum , its fruits, inflorescences, branches and leaves (Fig. 1 ) were analysed. The extracts were obtained following a solvent system that was suggested based on a huge literature search and analysis (results not presented) as the most efficient to extract cannabinoids: maceration on methanol/hexane 9:1 26 . The yield obtained from the extracts is present in Table 1 , resulting in some very interesting amounts, such as 6.6% at the leaves, that could allow sustainable production. Table 1 Yield of Trema micranthum extracts. Extracts of fresh Trema micranthum Fruits Inflorescences Leaves Branches Plant material (g) 1.00 11.04 10.37 10.18 Extracts (g) 0.03 0.34 0.68 0.18 Extract yield (%) 3% 3.1% 6.6% 1.8% Given that recently published papers have unsuccessfully searched for cannabinoids in this species, a brief discussion of the methods typically used is relevant. From the analytical point of view of the United Nations Office on Drugs and Crime, a combination of colorimetric tests, thin-layer chromatography, and physical (macroscopic and microscopic) examination is considered an acceptable minimum analytical approach for identification of cannabinoids in plant material 27 . One of the colorimetric tests used in Brazil, specifically for marijuana samples, is the Fast Blue BB or B Salt. This assay is based on a colorimetric reaction attributed to the phenolic nature of the chemical structures of cannabinoids. Regarding to colorimetric tests, in addition to sensitivity, there is a lack of specificity. In the case described, phenolic compounds present in plant extracts would potentially yield a false positive result in the test 28 . Gas chromatography coupled with flame ionization detector (GC-FID) or coupled with mass spectrometry, as well as liquid chromatography coupled with mass spectrometry with single analyzer or in tandem and any other combinations could be used. The selection of the appropriate method depends on the purpose of the analysis 29 . Gas chromatography is considered one of the most used techniques in studying the chemical profile of Cannabis and its products. However, the principle of this technique assumes the sample aliquot is heated in the vaporization chamber, leading a high potential of degradation of polar groups. Acid carboxylic groups seem to be particular sensitive to this phenomenon, resulting in the loss of the acid moiety. Consequently, acidic compounds are converted to neutral ones, often incompletely 30 . To prevent decarboxylation, derivatization reactions are used beforehand. Derivatization reactions often do not achieve complete conversion and can introduce an additional analytical step, potentially leading to systematic errors. The introduction of electrospray ionization changes the paradigm of polar analytes. At liquid chromatography, DAD detector depends of the presence of a chromophore group, presenting low specificity to target analysis, especially in the presence of complex matrix. Based on that, LC-MS allows direct determination of neutral and acidic cannabinoid species is possible 31 . Indeed, the used of ultra-high performance liquid chromatography coupled with high resolution mass spectrometry analysis allow a completely different perspective of specificity and sensitivity, when compared with colorimetric assays, TLC, or even to detector as diode array. The ability to identify an analyte depends on the analytical method, and in this research, we refined the approach to enable cannabinoid detection in compliance with the high analytical standards set by the World Anti-Doping Agency (WADA). The identification of an analyte using chromatography coupled with mass spectrometry is based upon a comparison of the Retention Time (RT) and Relative Abundances of the Diagnostic Ions (RADI) of the analyte detected in the sample with those in a reference specimen analysed in the same analytical sequence. The principles applied at the present study were the use of the retention time of the analyte’s chromatographic peak in the sample that shall not differ by more than one percent (%) or ± 0.1 minutes, from that of the same analyte in a standard. A maximum ΔRT is set at the FWHM of the reference peak in the standard. Similarly, the MS criteria for identification applied were at least two diagnostic ions, the signal-to-noise ratio of all diagnostic ions shall be greater than three to one. The greater one was used in the diagnostic. The abundance of Diagnostic Ions shall be determined from the peak area or height in the integrated selected ion chromatograms. The relative abundance of the Diagnostic Ions did not differ by more than (≤) the amount from the corresponding relative abundances of the same ions acquired from the sample 27 . In this project, a chromatographic system based on a column with stationary phase particles with a diameter of 2.5 µm or smaller achieved high separation efficiency and resolution power achieved. The coupling with a high-resolution mass spectrometer working at 70,000 full widths at half maximum (FWHM) deliver an analytical capability unmatched when compared with another works dedicated to identification of the component in Threma micranthum . This approach allowed us to identify previously undetected cannabinoids in this species. The compounds CBD and THC are constitutional isomers, meaning the same exact mass. The same can be said to their respective acid precursors, CBDA and THCA. Considering these structural similarities, the observation of similar mass spectrometry fragmentation profiles was already expected. The main MS outcomes must be recognized as a critical key to eliminating the impact of potential interferents from the matrix, while complementary and refined analytical strategy could be used to guarantee the trustiness of the conclusions. The comparison of chromatography and MS data with the ones obtained using reference material gives the analytical capability of the confident identification of the analytes. As observed in Fig. 2 , the conditions of the developed UHPLC method for the reference material allowed excellent separation of the analytes, with the well-resolved peaks of CBD and THC eluting in less than 14 minutes of chromatographic run time (Fig. 2 a). The same quality in separation can be observed for CBDA and THCA A (Fig. 3 a). After confirming the retention times with reference material under UHPLC conditions, the identification of CBD, THC, CBDA and THCA in the samples was performed by exact mass and retention time comparison (Fig. 2 b, c, d and Fig. 3 b, c, d). In Brazil, the commercialization of CBD and THC products is regulated by ANVISA, an official agency similar to the US FDA. CBD-based products, with concentrations ranging from 2500 µg/g to 200 x 103 µg/g of the extract, and without the presence of THC, are the most recommended for therapeutic purposes. Generally, the absence of THC is considered to be very low concentrations, or below 0.2%. At these THC concentrations, CBD-based products are permitted with a specific medical prescription for patients in terminal states or where there is no therapeutic alternative 32 . These products can be found in various pharmaceutical forms, such as oils, sprays, creams and ointments. Oils are the most commonly used and are obtained by extraction, presenting greater efficacy and a wider therapeutic window than synthetic products. This is because all the substances present in the plant act synergistically (entourage effect). Clinically, they are used to treat conditions such as epilepsy, chronic pain, anxiety and other neurological conditions. For quantitative analysis of the cannabinoids at the extracts, Parallel Reaction Monitoring (PRM) was applied (Table 2 ). PRM is a targeted method of high-resolution mass spectrometry quantification in which a full scan of each precursor ion transition is monitored, and all fragments derived from the precursor ion are monitored. Collision-induced dissociation (CID) experiments were performed to identify specific fragments for the analytes and corroborate previously published data in the literature 33 , 34 . As commented above, CBD and THC present similar fragmentation profiles. A neutral loss of 56 Da ( m/z 315 → m/z 259) represents the cleavage of the cannabinoid molecule's side chain (butyl group, C 4 H 8 ), resulting in the fragment [C 17 H 22 O 2 + H] + . A neutral loss of 122 Da ( m/z 315 → m/z 193) represents an ether function and a terpene ring cleavage. A neutral loss of 180 Da ( m/z 315 → m/z 135) is also observed. Table 2 Monitored compounds, their molecular formulas, mass transitions and (N)CE applied. Compound Molecular formula [M] Retention time (min) Precursor ion ( m/z ) [M-H] + or [M-H] − (N)CE (%) Product ion ( m/z ) [M-H] + or [M-H] − CBD C 21 H 30 O 2 12.52 315.2318 40 193.1223 259.1691 135.1168 THC C 21 H 30 O 2 13.26 315.2318 40 193.1223 259.1691 135.1168 CBDA C 22 H 30 O 4 12.68 357.2 40 339.1970 311.2022 245.15494 271.1345 THCA C 22 H 30 O 4 14.12 357.2 40 313.2177 245.1545 For the CBDA, the suggested fragmentations begin with the transition from m/z 357 to m/z 339, represented by the neutral loss of 18 Da (H 2 O), resulting in the fragment m/z 339. Next, the transition from m/z 339 to m/z 311 involves the neutral loss of 28 Da (CO), forming the fragment m/z 311, which then loses 40 Da to form m/z 271. For THCA, the initial transition from m/z 357 to m/z 313 was observed, which can be related to the decarboxylation process, involving the neutral loss of 44 Da (CO 2 ), resulting in the formation of the deprotonated THC molecule ( m/z 313). Next, the transition from m/z 313 to m/z 245 involves the neutral loss of 68 Da (C 5 H 8 ). Estimates of cannabinoid concentrations mapped in the present study in T. micranthum are shown in Table 3 . Table 3 Estimated concentration of CBD, THC, CBDA and THCA in Trema micranthum extracts Cannabinoids (µg.g − 1 ) CBD THC CBDA THCA A Fruits 3.16 x 10 − 1 1.93 x 10 − 3 6.68 x 10 − 2 1.66 x 10 − 2 Inflorescences 9.84 x 10 − 3 1.85 x 10 − 3 1.13 x 10 − 1 1.90 x 10 − 2 Leaves 4.43 x 10 − 3 1.05 x 10 − 3 5.78 x 10 − 2 9.86 x 10 − 3 Branches n.d. n.d. n.d. n.d. n.d. = not detected The results presented in Table 3 bring several answers to the original questions. Firstly, part of the main question (from the title) was answered: there are cannabinoids (THC. CBD, CBDA, and THCA A) in Trema micranthum . They were not detected in stems (branches), but similar concentrations of the target cannabinoids were observed in leaves and inflorescences, except in fruits, where CBD was observed at more than 100 times the THC concentration. For clinical applications, CBD is advantageous because it does not induce euphoric effects and is non-psychoactive. However, when combined with a balanced portion of THC, it modulates effects in hippocampal neurons distinctly from pure compounds. Moreover, CBD can reduce the psychoactive side effects of THC, enhancing synergy and providing greater therapeutic effects compared to their isolated compounds 35 , 36 . In the fruits, inflorescence and leaf parts of T. micranthum , CBDA and THCA A were detected in higher concentrations than your neutral analogues. As in Cannabis , in the fresh material of T. micranthum , the cannabinoids produced by the plant's metabolism are carboxylic acids, such as THCA A and CBDA 37 . The other question also addressed in this study was whether this plant could serve as an alternative source of CBD without the concerns associated with THC. It was observed that the leaves contained 4.43 x 10 − 3 µg of CBD per gram of the plant extract, and 1.05 x 10 − 3 µg/g of THC, which is significant given that leaves constitute the bulk of the plant's biomass. The key difference between hemp and marijuana (varieties of Cannabis sativa ) lies in their chemical profiles. For a plant to be classified as hemp, it must have less than 0.3% THC by dry weight, whereas marijuana exceeds this threshold. Marijuana cultivated for recreational use typically contains low levels of cannabidiol (CBD), while hemp is rich in CBD 31 . A difference between T. micranthum and C. sativa is related to the higher content of THC in the upper parts of the plant. In Cannabis , about 10–12% of THC is found in the flowers, followed by 1–2% in the leaves, 0.1–0.3% in the stem, and less than 0.03% in the root. In T. micranthum , about 0,2% is found in the fruits, 0,02% in inflorescences and 0,01% in the leaves 38 . The presence of THC in Cannabis seeds is still debatable. Some authors suggest that the Cannabis seed is not free from THC, with its concentration being less than 0.5 ug/g 39 . According to the UNODC, there is no THC in the seeds, what actually happens is that the detection of THC is linked to its contact with the bracts and/or flowers of the plant, which have a high content of this cannabinoid 27 . C. sativa leaves have a high CBD content (473 µg/g) and approximately 36% THC (175 µg/g), per gram of dry leaf tissue, in relation to total CBD 32 . Therefore, it would be unfeasible to use T. micranthum leaves as an alternative source for obtaining cannabinoids since the quantity is about a hundred times lesser than in C. sativa . Indeed, agrological improvements and also the optimization of the extraction processes, could be performed. In C. sativa , the highest concentrations of these cannabinoids were found flowers, with a concentration of CBD 1594 µg/g and THC 510 µg/g, per gram of dry leaf tissue 40 . In contrast, for T. micrathum the THC content in flowers was higher than that of CBD. The concentrations listed in Table 3 are significantly lower when compared to the CBD content present in medicinal cannabis extracts 32 . However, the species could potentially be used as a raw material for cannabinoid isolation, optimizing solvent extraction methods that can improve the yield of THC and CBD. Conclusion Analytical methods using high-resolution mass spectrometry allowed the identification of previously undetected cannabinoids in Threma micranthum . The use of standards (the reference materials) for CBD, THC, CBDA and THCA A facilitated excellent resolution of the cannabinoid isomers, with well-resolved peaks for the neutral compounds (CBD and THC) and their acid precursors (CBDA and THCA A). Consequently, the identification of cannabinoids in the samples was achieved through accurate mass and retention time comparisons under UHPLC conditions. Quantification of cannabinoids using the PRM method allowed the detection of compounds at very low concentrations, due to its greater selectivity and sensitivity. With this approach, all investigated cannabinoids were identified in the leaves, fruits and inflorescences of Threma micranthum , except in the stems. In fruits, inflorescences and leaves, the acidic forms CBDA and THCA A were detected at higher concentrations than their neutral counterparts. These analyses may guide future research in the search for cannabinoids in other species of the Cannabaceae family, potentially providing additional sources for the isolation of these herbal compounds. Furthermore, despite the low levels of these identified cannabinoids, the data may guide the optimization of extraction methods using selective solvents to achieve better yields of THC and CBD. Declarations Funding The project received funding from FAPERJ (Grant Numbers E-26/200.512/2023, E-26/211.315/2021), and CNPq (Grant number 310782/2022-8). Author Contribution All authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by Rayssa Ribeiro, Yasmin Cunha da Silva, Ricardo Finotti, Gabriel Reis Alves Carneiro, Gustavo Ramalho Cardoso dos Santos, and Monica Costa Padilha. The first draft of the manuscript was written by Monica Costa Padilha, Valdir F. Veiga Jr, and Rayssa Ribeiro. The final version was written by Valdir F. Veiga Jr, Monica Costa Padilha, and Henrique Marcelo Gualberto Pereira. All authors commented on previous versions of the manuscript. Availability of Data and Materials statement Not applicable. All data generated or analyzed during this study are included in this published article. References Jin, D., Dai, K., Xie, Z. & Chen, J. Secondary metabolites profiled in cannabis inflorescences, leaves, stem barks, and roots for medicinal purposes. Sci. Rep. 10 , 3309 (2020). Bonini, S. A. et al. Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history. J. Ethnopharmacol. 227 , 300–315 (2018). Hartsel, J. A., Eades, J., Hickory, B. & Makriyannis, A. Cannabis sativa and Hemp. in Nutraceuticals 735–754Elsevier, (2016). Hanuš, L. O., Meyer, S. M., Muñoz, E., Taglialatela-Scafati, O. & Appendino, G. Phytocannabinoids: a unified critical inventory. Nat. Prod. Rep. 33 , 1357–1392 (2016). Cox, C. Implications of the 2018 Canadian Cannabis Act: Should regulation differ for medicinal and non-medicinal cannabis use?⋆. Health Policy (New York) . 125 , 12–16 (2021). Tettey, J. N. A. et al. 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Jardim Botânico do Rio de Janeiro. Disponível em: < (2024). https://floradobrasil.jbrj.gov.br/FB106893 . Acesso em: 04 ago. https:// powo.science.kew.org/taxon/urn:lsid:ipni.org:names :1018059–1018052 Jin, J. et al. Born migrators: Historical biogeography of the cosmopolitan family Cannabaceae. J. Syst. Evol. 58 , 461–473 (2020). Napiroon, T. et al. Cannabinoids from inflorescences fractions of Trema orientalis (L.) Blume (Cannabaceae) against human pathogenic bacteria. PeerJ . 9 , e11446 (2021). Teixeira, S. P., Nascimento, I. C., Grejo, M. P., Leite, V. G. & Machado, S. R. The putative cannabinoid-secreting trichome of Trema micrantha (L.) Blume (Cannabaceae). Protoplasma . 261 , 463–475 (2024). Márquez, U. C., Binnqüist, C. L. & Castillo, P. N. Una especie multiusos del trópico mexicano Trema micrantha (L.) Blume. Ciencias . Wiraswati, H. L. et al. Biological potential of eight medicinal plants collected in the restored landscape after mining in South Kalimantan. Discover Appl. Sci. 6 , 1–15 (2024). Munoz, V. et al. The search for natural bioactive compounds through a multidisciplinary approach in Bolivia. Part II. Antimalarial activity of some plants used by Mosetene indians. J. Ethnopharmacol. 69 , 139–155 (2000). Barbera, R., Trovato, A., Rapisarda, A. & Ragusa, S. Analgesic and antiinflammatory activity in acute and chronic conditions of Trema guineense (Schum. et Thonn.) Ficalho and Trema micrantha Blume extracts in rodents. Phytother. Res. 6 , 146–148 (1992). Schoenfelder, T., Cirimbelli, T. M. & Citadini-Zanette, V. Acute effect of Trema micrantha (Ulmaceae) on serum glucose levels in normal and diabetic rats. J. Ethnopharmacol. 107 , 456–459 (2006). de Oliveira, G. Does Trema micranthum (L.) Blume Produce Cannabinoids? Plants . 13 , 1951 (2024). Quevedo, L. S. et al. Toxic pneumopathy by Trema micrantha in sheep in the State of Santa Catarina, Brazil. Pesquisa Veterinária Brasileira . 42 , e07128 (2022). Frimmel, A. E., Peixoto, J. L. B., Sarragiotto, M. H. & Vidotti, G. J. Vitexin, paprazine and terpenoids from Trema micrantha. Biochem. Syst. Ecol. 28 , 495–496 (2000). Viana dos Santos, M. B. et al. In Vitro and in Vivo Antimalarial Activity, Cytotoxicity and Phytochemical HRMS2 Profile of Plants from the Western Pará State, Brazilian Amazonia. Chem. Biodivers. 21 , e202301082 (2024). ElSohly, M. A., Stanford, D. F. & Murphy, T. P. Chemical fingerprinting of cannabis as a means of source identification. Marijuana Cannabinoids 51–66 (2007). Carvalho, V. M., Aguiar, A. F. L., Baratto, L. C., Souza, F. L. C. & Rocha, E. D. Quantificação de canabinoides em extratos medicinais de cannabis por cromatografia líquida de alta eficiência. Quim. Nova . 43 , 90–97 (2020). United Nations Office On Drugs And Crime & –, U. N. O. D. C. – Vienna. Recommended Methods for the Identification and Analysis of Cannabis and Cannabis Products. (2022). https://www.unodc.org/unodc/en/scientists/recommended-methods-for-the-identification-and-analysis-of-cannabis-and-cannabis-products.htm . Accessed August 25, 2024. Bordin, D. C., Messias, M., Lanaro, R., Cazenave, S. O. S. & Costa, J. L. Análise forense: pesquisa de drogas vegetais interferentes de testes colorimétricos para identificação dos canabinóides da maconha ( Cannabis sativa L ). Quím Nova . 35 , 2040–2043 (2012). World Anti-Doping Agency. Minimum criteria for chromatography-mass spectrometric confirmation of the identity of analytes for doping control purposes. Wada Technical Document – TD2023IDCR. http://www.wada-ama.org . Accessed on August 27, 2024. De Backer, B. et al. Innovative development and validation of an HPLC/DAD method for the qualitative and quantitative determination of major cannabinoids in cannabis plant material. J. Chromatogr. B . 877 (32), 4115–4124 (2009). Citti, C., Braghiroli, D., Vandelli, M. A. & Cannazza, G. Pharmaceutical and biomedical analysis of cannabinoids: A critical review. J. Pharm. Biomed. Anal. 147 , 565–579 (2018). Anvisa aprova mais três produtos. de Cannabis para uso medicinal — Agência Nacional de Vigilância Sanitária - Anvisa (www.gov.br). SANTOS, Nayara, A. et al. Analysis of isomeric cannabinoid standards and Cannabis products by UPLC-ESI-TWIM-MS: a comparison with GC-MS and GC× GC-QMS. J. Braz. Chem. Soc. 30 , 60–70 (2019). Citti, C. et al. Cannabinoid profiling of hemp seed oil by liquid chromatography coupled to high-resolution mass spectrometry. Front. Plant. Sci. 10 , 120 (2019). Russo, E. B. & Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. Br. J. Pharmacol. 163 , 1344–1364 (2011). Anand, U., Pacchetti, B., Anand, P. & Sodergren, M. H. Cannabis-Based Medicines and Pain: A Review of Potential Synergistic and Entourage Effects. Pain Manag . 11 , 395–403 (2021). Vanhove, W., Van Damme, P. & Meert, N. Factors determining yield and quality of illicit indoor Cannabis ( Cannabis spp .) production. Forensic Sci. Int. 212 , 158–163 (2011). Elsohly, M. A. Chemical constituents of Cannabis. In: Grotenhermen, Franjo. Cannabis and Cannabinoids. Pharmacology, Toxicology and Therapeutic Potential. Harworth, Binghamton, New York, 27–38, (2002). Handbook of Cannabis Therapeutics From Bench to Beside. Editors Ethan B. Russo and Franjo Grotenhermen (Routledge Taylor & Francis Group –, 2006). Esposito, L. G. A. et al. Immune responses are differentially regulated by root, stem, leaf, and flower extracts of female and male CBD hemp (cannabis sativa L.) plants. Immuno . 1 , 369–379 (2021). Additional Declarations No competing interests reported. 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Veiga-Junior","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYFAC5gaJBDANxB+AmI2doBZGhBbGGSAtzMRogVvIA7MOHzA4frDxxsM9DIn97OwXP9v82ibPx8zA+OFjDh4tZxKbLRKeMSTObOYpls7tu23YxszALDlzG24tkg2JbRIJBxiMDQ7zJEjn9txmBGphY+bFp6X/IUSL/WGe5N+WPbftCWrhl4DYImfAzH5MmuHH7UQitDwE+uWAhJzEYR42y96G28ltzIzNeP3Cxp988OaPAzY8/P3HH9/48ee27fz25oMfPuLRAgWgqOExYGBsA3EYGwiqhwL2BwwMf4hVPApGwSgYBSMJAACzSEy9RSnOwwAAAABJRU5ErkJggg==","orcid":"","institution":"Military Institute of Engineering - Rio de Janeiro/RJ","correspondingAuthor":true,"prefix":"","firstName":"Valdir","middleName":"F.","lastName":"Veiga-Junior","suffix":""}],"badges":[],"createdAt":"2024-09-20 20:26:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5125664/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5125664/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-80857-6","type":"published","date":"2024-11-28T15:58:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69013342,"identity":"0dd0c868-53fb-4969-91e3-6ebb0303b271","added_by":"auto","created_at":"2024-11-14 14:21:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1371116,"visible":true,"origin":"","legend":"\u003cp\u003eParts of the plant \u003cem\u003eTrema micranthum\u003c/em\u003e (L.) Blume: leaves, inflorescences, fruits and branches.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5125664/v1/3149b0a4c7201cbc50ef9331.png"},{"id":69013343,"identity":"99c477ba-567c-48e2-9d0d-5b7be9b53dc5","added_by":"auto","created_at":"2024-11-14 14:21:20","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130175,"visible":true,"origin":"","legend":"\u003cp\u003eExtracted-ion chromatograms for each analyte monitored in this work and their retention time. (\u003cstrong\u003ea\u003c/strong\u003e) Chromatogram of the standard mixture of CBD (Rt = 12.31 min) and THC (Rt = 13.14 min); (\u003cstrong\u003eb, c and d\u003c/strong\u003e) Chromatogram of \u003cem\u003eTrema micranthum \u003c/em\u003efruits, inflorescences and leaves extracts´, highlighting the cannabinoids present.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5125664/v1/82739f713008d73e2aa7e305.jpeg"},{"id":69013341,"identity":"fd5a3161-c05b-40b3-a09a-1c208f98e3ac","added_by":"auto","created_at":"2024-11-14 14:21:20","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117801,"visible":true,"origin":"","legend":"\u003cp\u003eExtracted-ion chromatograms for each analyte monitored in this work and their retention time. (\u003cstrong\u003ea\u003c/strong\u003e) Chromatogram of the standard mixture of CBDA (Rt = 12.67 min) and THCA A (Rt = 14.08 min); (\u003cstrong\u003eb, c and d\u003c/strong\u003e) Chromatogram of \u003cem\u003eTrema micranthum \u003c/em\u003efruits, inflorescences and leaves extracts´, respectively, highlighting the cannabinoids present.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5125664/v1/404de2836e44b2a4685bbc19.jpeg"},{"id":70391133,"identity":"e6e55ff6-e6f5-453e-b811-1591c20fff47","added_by":"auto","created_at":"2024-12-02 17:30:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2696255,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5125664/v1/3acefc0b-37a0-41e1-9220-0370e3a4fe56.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Can Trema micranthum (L.) Blume (Cannabaceae) become a new source of cannabinoids? A UHPLC- HRMS/MS analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCannabidiol (CBD) is a non-psychoactive cannabinoid with important therapeutic benefits. The recent FDA approval for formulation containing CBD, such as Epidiolex\u0026reg; used to treat seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, or tuberous sclerosis complex in patients, gave new impetus for research around the theme\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the huge health importance and wellness and drug industries interest, the use of CBD formulation still faces several commercialization challenges. In many countries, CBD is regulated differently depending on its source, hemp or marijuana buds (varieties of \u003cem\u003eCannabis sativa\u003c/em\u003e L. \u0026ndash; Cannabaceae Martinov). In addition, the content of Δ\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e-tetrahydrocannabinol (THC), the main psychoactive cannabinoid and responsible for several important pharmacological properties but also for recreational use, brings a lot of controverses to the discussion\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCannabis sativa\u003c/em\u003e buds are commonly extracted with ethanol and, after the solvent evaporates, but before adding coconut oil (or similar) to produce the commercially material known as Cannabis oil, the extract needs to be activated. The carboxylic acid precursors of THC and CBD present in the extracts are mainly Δ\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e-tetrahydrocannabinoic acid (THCA) and cannabidiolic acid (CBDA), respectively. The extract needs to be heated to allows the decarboxylation process to produce the bioactives CBD and THC, when it is finally considered activated. However, this process can sometimes lead to the degradation or evaporation of other bioactive constituents, such as the volatile sesquiterpene caryophyllene. Sources of cannabinoids with natural high content of CBD is highly desired, without the need to activation and avoiding the presence of THC\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBesides the production issues and the social preconception against the use of \u003cem\u003eCannabis\u003c/em\u003e L., the main difficulties of CBD-based products commerce lie on the complex and often lack of a standardized regulatory background. As consequence, there is a clear concern about the product quality, safety, and labelling, leading to a framework of laws that can confuse consumers and complicate commercial operations, challenging international trade\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe legal issues related to the presence of THC are also a consistent justification for the search for new CBD-producing species. The main alternative for obtaining cannabinoids is the use of other species of Cannabaceae botanical family that can be easy to obtain and produce. Diversifying cannabinoid sources can address sustainability and environmental concerns associated with large-scale cultivation more efficiently or under less resource-intensive conditions. This approach can mitigate the overreliance on a single species, thereby reducing the risks associated with crop failure due to pests, diseases, or changing climatic conditions. A reasonable outcome is the stimulus to innovation in agriculture and biotechnology. Such advancements would not only benefit the pharmaceutical industry, but also have implications for other sectors, including food, cosmetics, and wellness products\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eTrema\u003c/em\u003e Lour (Cannabaceae) is pantropical with nearly 20 species, six of them being cited for Americas\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eTrema micranthum\u003c/em\u003e L. (Blume), synonym \u003cem\u003eTrema micrantha\u003c/em\u003e, is the one traditionally registered for Brazil\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It is a shrub present as ruderal species, native from tropical and subtropical America, occurring naturally at all the Brazilian biomes and in diverse vegetational forest types (ever-green, semi-decidual and gallery forests)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Phylogenetically, \u003cem\u003eTrema-Parasponia\u003c/em\u003e group is a sister group of \u003cem\u003eHumulus-Cannabis\u003c/em\u003e group, been diverged nearly from 58.5 to 46.3 Ma, between the late Paleogene and the early Neogene\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Other species of the previous cited groups are known cannabinoid producers as \u003cem\u003eCannabis sativa\u003c/em\u003e L. and \u003cem\u003eTrema orientale\u003c/em\u003e (L) Blume\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, glandular trichomes of \u003cem\u003eT. micranthum\u003c/em\u003e flowers and leaves were described as cannabinoid secreting structures\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Its popular uses are described in Bolivia, to cough and wounds, and in Mexico, where traditional use includes timber products for house construction (wood for wall and roof support) and Mexican popular medicine with leaves used in baths for skin injuries\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Previous studies with \u003cem\u003eT. micranthum\u003c/em\u003e extracts showed antioxidant properties at 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase assays\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and some antimicrobial activities \u003cem\u003ein vitro\u003c/em\u003e (62%) and \u003cem\u003ein vivo\u003c/em\u003e (81% at 100 mg/kg)\u003csup\u003e18\u003c/sup\u003e. Ether and ethanol extracts of \u003cem\u003eT. micranthum\u003c/em\u003e also presented \u003cem\u003ein vivo\u003c/em\u003e (mice and rats) anti-inflammatory and antiarthritic activities, with inhibition of carrageenin-induced oedema and adjuvant arthritis, and this effect was comparable to that of indomethacin\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The ethanolic extract also showed hypoglycaemic activity in acute treatment in rats (250 and 1000 mg/kg doses)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The consumption of \u003cem\u003eT. micranthum\u003c/em\u003e by animals has been reported to cause several toxic effects, including hepatocellular necrosis in cattle, sheep, and goats and oedema, and cerebral haemorrhage in horses\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The exact mechanism of action and the substance responsible for the toxicity remain unidentified.\u003c/p\u003e \u003cp\u003eA recent publication reported the analyses of leaves, branches, fruits, and inflorescences of the methanolic extracts obtained from \u003cem\u003eT. micranthum\u003c/em\u003e using HPLC-DAD and GC-MS. Despite these traditional tools employed in natural products research, no cannabinoids were detected\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, anecdotic information from different sources creates the hypotheses of the production of CBD without THC formation.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, only two studies have accurately described the chemical composition of \u003cem\u003eTrema micranthum\u003c/em\u003e leaves with the identification of the ubiquitous sterols β-sitosterol and 3β-\u003cem\u003eO\u003c/em\u003e-β-D-glucopyranosyl sitosterol, the triterpenes ursolic acid and 2α,3β-dihydroxyurs-12-en-28-oic acid, the amide paprazine ((2E)-3-(4-hydroxyphenyl)-\u003cem\u003eN\u003c/em\u003e-[2-(4-hydroxyphenyl)ethyl]prop-2-enamide) and the flavonoids vitexin (8-glucopyranosyl-4,5,7-trihydroxyflavone)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and also vitexin (or isovitexin) and orientin (or isoorientin) arabinosides (or xylosides) derivatives\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe lack of a deep investigation relative to the molecular composition of \u003cem\u003eTrema micranthum\u003c/em\u003e opens the perspective of contributions using the \u0026ldquo;State of the Art\u0026rdquo; in organic analytes identification, using high quality approaches already well tested in different fields such as analytical toxicology and food residue analysis. The present study intends to perform a UHPLC-HRMS/MS (Orbitrap) search for the cannabinoids CBD and THC and their main precursors, THCA and CBDA A, at leaves, inflorescences, fruits, and branches of \u003cem\u003eTrema micranthum\u003c/em\u003e extracts to verify their possible use as an alternative to \u003cem\u003eCannabis\u003c/em\u003e and source of CBD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eFor sample preparation, methanol and n-hexane, both GC grade, were acquired from Tedia (Fairfield, OH, USA). CBD and THC standards were provided by AB Cient\u0026iacute;fica Ltda (Esp\u0026iacute;rito Santo, Brazil). CBDA standards were supplied by LGC group (North Charleston, United States). THCA A was provided Sigma-Aldrich (Darmstadt, Germany). For LC-HRMS/MS analysis, methanol HPLC grade, formic acid 98%-100%, and ammonium formiate were provided by Tedia (Fairfield, OH, USA), Merck (Darmstadt, Germany) and Spectrum Chemical (Gardena, CA, USA), respectively. Ultrapure water (18.2 MΩ.cm) was obtained from a Millipore Milli-Q purification system (Billerica, MA, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExtraction process\u003c/h3\u003e\n\u003cp\u003eTwigs, leaves, inflorescences, and fruits were collected from an individual of \u003cem\u003eTrema micranthum\u003c/em\u003e found at Parque Nacional da Tijuca, close to the Pedra da G\u0026aacute;vea, Rio de Janeiro-RJ, Brazil, on July 10th, 2024, with a voucher deposited at RB 648300 Herbarium. his work was registered in the National Management System of Genetic Heritage and Associated Traditional Knowledge (SISGEN) under number xx. Ten grams of plant parts fresh, including branches, leaves, and inflorescences, were extracted using a methanol/hexane solvent mixture in a 9:1 (v/v) ratio. One gram of macerated fruits fresh was weighed, and the same solvent mixture was used for extraction. The plant materials immersed in the solvents were subjected to extraction under agitation in a shaker at 150 rpm for 12 hours. The resulting organic extract was centrifuged at 4000 rpm for 20 minutes and evaporated under nitrogen flow at 40\u0026deg;C. The dry residue was reconstituted with methanol to obtain a final concentration of 10 \u0026micro;g/mL. The extracts were vortexed for 30 seconds, 1.5 mL was transferred to an Eppendorf tube, and centrifuged at 13000 rpm for 15 minutes at 15\u0026deg;C. The supernatant was transferred to a vial and injected into LC-HRMS.\u003c/p\u003e\n\u003ch3\u003eUHPLC-HRMS Instrumentation and analysis\u003c/h3\u003e\n\u003cp\u003eA Dionex Ultimate 3000 ultra-high performance liquid chromatography (UHPLC) system coupled to a QExactive Plus hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ionization (ESI) source was used. Separation was performed in a reversed-phase column (kinetex 2.6 \u0026micro;m PS C18, 100 \u0026Aring;, 100 mm x 2,1 mm), at 40\u0026deg;C, constant flow rate of 300 \u0026micro;L/min and injection volume of 5 \u0026micro;L. A gradient chromatographic run started at 5% of mobile phase B (methanol with 0.1% formic acid) and 95% of mobile phase A (water with 5 mM ammonium formiate and 0.1% formic acid). Mobile phase B was increased to 10% at 1.0 minute, then to 25% at 2 minutes, and then 90% at 10 minutes. After reaching 100% of B at 14 minutes and maintaining this ratio until 16 minutes, the initial chromatographic condition was restored from 16.1 to 20.0 minutes.\u003c/p\u003e \u003cp\u003eThe LC effluent was pumped to the mass spectrometer operating in a positive and negative ESI mode, calibrated daily with a manufacturer's calibration solution (Thermo Fisher Scientific, Bremen, Germany). ESI parameters were further optimized, with the final setup: spray voltage of 2.9 kV, S-lens voltage of 80 V, the capillary temperature of 380\u0026deg;C, auxiliar gas heater temperature of 350\u0026deg;C, nitrogen sheath, auxiliary, and sweep gas were set at 30, 10, and 1 arbitrary unit, respectively. Full-scan data were acquired in a range of \u003cem\u003em/z\u003c/em\u003e 70\u0026ndash;1050 at a resolution of 70,000 full width at half maximum (FWHM), automatic gain control (AGC) of 1 x 10\u003csup\u003e6\u003c/sup\u003e and maximum injection time (IT) of 100 ms. Targeted mass spectrometry-based approaches were performed using the parallel reaction monitoring technique (PRM), the precursor ions were fixed at a resolution of 17,500 full width at half maximum (FWHM), automatic gain control (AGC) of 1 x 10\u003csup\u003e6\u003c/sup\u003e, maximum injection time (IT) of 100 ms and quadrupole isolation window of \u003cem\u003em/z\u003c/em\u003e 2.\u003c/p\u003e \u003cp\u003eIn the Full MS approach, the target exact mass was \u003cem\u003em/z\u003c/em\u003e 315.23184 ([M - H]\u003csup\u003e+\u003c/sup\u003e) to CBD and THC, and \u003cem\u003em/z\u003c/em\u003e 357.20713 ([M - H]\u003csup\u003e\u0026minus;\u003c/sup\u003e) to CBDA and THCA A. The detection limit established by PRM was 5.2 x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e \u0026micro;g/g for CBD and 3.7 x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e \u0026micro;g/g for THC, 1.4 x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g/g for CBDA and 1.0 x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g/g for THCA A per gram of extract. The estimated limit of quantification established by PRM was 1.7 x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g/g for CBD and 1.2 x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g/g for THC, 4.7 x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g/g for CBDA and 3.5 x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g/g for THCA A per gram of extract. In the PRM approach, precursor ions of \u003cem\u003em/z\u003c/em\u003e 315.23184 [M - H]\u003csup\u003e+\u003c/sup\u003e (CBD and THC), and \u003cem\u003em/z\u003c/em\u003e 357.20713 [M - H]\u003csup\u003e\u0026minus;\u003c/sup\u003e (CBDA and THCA A) were fragmented in a higher energy collisional dissociation (HCD) cell with (N)CE of 40%.\u003c/p\u003e \u003cp\u003eTo detect and quantify CBD, CBDA, THC and THCA A in the plant parts, the standard mixtures were prepared in methanol at a concentration of 3.16 x 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026micro;g/g.\u003c/p\u003e \u003cp\u003eData was acquired and processed using Thermo ScientificTM TraceFinderTM 4.1 software (Thermo Fisher Scientific, Austin, TX, USA), with a\u0026thinsp;\u0026plusmn;\u0026thinsp;6 ppm mass tolerance.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003ePhytocannabinoids are usually biosynthesized as acids. Neutral forms are produced through non-enzymatic decarboxylation. Cannabigerolic acid (CBGA) is the bioprecursor of other acidic cannabinoids in the plant, such as Δ\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These substances give rise to their respective neutral forms, THC and CBD. Sequentially, isomerization and oxidation products are formed\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In the present study, to confirm the presence and estimate the concentration of the cannabinoids CBD, THC, CBDA and THCA A in \u003cem\u003eTrema micranthum\u003c/em\u003e, its fruits, inflorescences, branches and leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were analysed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe extracts were obtained following a solvent system that was suggested based on a huge literature search and analysis (results not presented) as the most efficient to extract cannabinoids: maceration on methanol/hexane 9:1\u003csup\u003e26\u003c/sup\u003e. The yield obtained from the extracts is present in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, resulting in some very interesting amounts, such as 6.6% at the leaves, that could allow sustainable production.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eYield of \u003cem\u003eTrema micranthum\u003c/em\u003e extracts.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eExtracts of fresh \u003cem\u003eTrema micranthum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFruits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInflorescences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBranches\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant material (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtracts (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtract yield (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGiven that recently published papers have unsuccessfully searched for cannabinoids in this species, a brief discussion of the methods typically used is relevant. From the analytical point of view of the United Nations Office on Drugs and Crime, a combination of colorimetric tests, thin-layer chromatography, and physical (macroscopic and microscopic) examination is considered an acceptable minimum analytical approach for identification of cannabinoids in plant material\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. One of the colorimetric tests used in Brazil, specifically for marijuana samples, is the Fast Blue BB or B Salt. This assay is based on a colorimetric reaction attributed to the phenolic nature of the chemical structures of cannabinoids. Regarding to colorimetric tests, in addition to sensitivity, there is a lack of specificity. In the case described, phenolic compounds present in plant extracts would potentially yield a false positive result in the test\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGas chromatography coupled with flame ionization detector (GC-FID) or coupled with mass spectrometry, as well as liquid chromatography coupled with mass spectrometry with single analyzer or in tandem and any other combinations could be used. The selection of the appropriate method depends on the purpose of the analysis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Gas chromatography is considered one of the most used techniques in studying the chemical profile of \u003cem\u003eCannabis\u003c/em\u003e and its products. However, the principle of this technique assumes the sample aliquot is heated in the vaporization chamber, leading a high potential of degradation of polar groups. Acid carboxylic groups seem to be particular sensitive to this phenomenon, resulting in the loss of the acid moiety. Consequently, acidic compounds are converted to neutral ones, often incompletely\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To prevent decarboxylation, derivatization reactions are used beforehand. Derivatization reactions often do not achieve complete conversion and can introduce an additional analytical step, potentially leading to systematic errors.\u003c/p\u003e \u003cp\u003eThe introduction of electrospray ionization changes the paradigm of polar analytes. At liquid chromatography, DAD detector depends of the presence of a chromophore group, presenting low specificity to target analysis, especially in the presence of complex matrix. Based on that, LC-MS allows direct determination of neutral and acidic cannabinoid species is possible\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Indeed, the used of ultra-high performance liquid chromatography coupled with high resolution mass spectrometry analysis allow a completely different perspective of specificity and sensitivity, when compared with colorimetric assays, TLC, or even to detector as diode array.\u003c/p\u003e \u003cp\u003eThe ability to identify an analyte depends on the analytical method, and in this research, we refined the approach to enable cannabinoid detection in compliance with the high analytical standards set by the World Anti-Doping Agency (WADA). The identification of an analyte using chromatography coupled with mass spectrometry is based upon a comparison of the Retention Time (RT) and Relative Abundances of the Diagnostic Ions (RADI) of the analyte detected in the sample with those in a reference specimen analysed in the same analytical sequence. The principles applied at the present study were the use of the retention time of the analyte\u0026rsquo;s chromatographic peak in the sample that shall not differ by more than one percent (%) or \u0026plusmn;\u0026thinsp;0.1 minutes, from that of the same analyte in a standard. A maximum ΔRT is set at the FWHM of the reference peak in the standard. Similarly, the MS criteria for identification applied were at least two diagnostic ions, the signal-to-noise ratio of all diagnostic ions shall be greater than three to one. The greater one was used in the diagnostic. The abundance of Diagnostic Ions shall be determined from the peak area or height in the integrated selected ion chromatograms. The relative abundance of the Diagnostic Ions did not differ by more than (\u0026le;) the amount from the corresponding relative abundances of the same ions acquired from the sample\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this project, a chromatographic system based on a column with stationary phase particles with a diameter of 2.5 \u0026micro;m or smaller achieved high separation efficiency and resolution power achieved. The coupling with a high-resolution mass spectrometer working at 70,000 full widths at half maximum (FWHM) deliver an analytical capability unmatched when compared with another works dedicated to identification of the component in \u003cem\u003eThrema micranthum\u003c/em\u003e. This approach allowed us to identify previously undetected cannabinoids in this species.\u003c/p\u003e \u003cp\u003eThe compounds CBD and THC are constitutional isomers, meaning the same exact mass. The same can be said to their respective acid precursors, CBDA and THCA. Considering these structural similarities, the observation of similar mass spectrometry fragmentation profiles was already expected. The main MS outcomes must be recognized as a critical key to eliminating the impact of potential interferents from the matrix, while complementary and refined analytical strategy could be used to guarantee the trustiness of the conclusions. The comparison of chromatography and MS data with the ones obtained using reference material gives the analytical capability of the confident identification of the analytes.\u003c/p\u003e \u003cp\u003eAs observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the conditions of the developed UHPLC method for the reference material allowed excellent separation of the analytes, with the well-resolved peaks of CBD and THC eluting in less than 14 minutes of chromatographic run time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe same quality in separation can be observed for CBDA and THCA A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). After confirming the retention times with reference material under UHPLC conditions, the identification of CBD, THC, CBDA and THCA in the samples was performed by exact mass and retention time comparison (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c, d and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c, d).\u003c/p\u003e \u003cp\u003eIn Brazil, the commercialization of CBD and THC products is regulated by ANVISA, an official agency similar to the US FDA. CBD-based products, with concentrations ranging from 2500 \u0026micro;g/g to 200 x 103 \u0026micro;g/g of the extract, and without the presence of THC, are the most recommended for therapeutic purposes. Generally, the absence of THC is considered to be very low concentrations, or below 0.2%. At these THC concentrations, CBD-based products are permitted with a specific medical prescription for patients in terminal states or where there is no therapeutic alternative\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. These products can be found in various pharmaceutical forms, such as oils, sprays, creams and ointments. Oils are the most commonly used and are obtained by extraction, presenting greater efficacy and a wider therapeutic window than synthetic products. This is because all the substances present in the plant act synergistically (entourage effect). Clinically, they are used to treat conditions such as epilepsy, chronic pain, anxiety and other neurological conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor quantitative analysis of the cannabinoids at the extracts, Parallel Reaction Monitoring (PRM) was applied (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PRM is a targeted method of high-resolution mass spectrometry quantification in which a full scan of each precursor ion transition is monitored, and all fragments derived from the precursor ion are monitored. Collision-induced dissociation (CID) experiments were performed to identify specific fragments for the analytes and corroborate previously published data in the literature\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs commented above, CBD and THC present similar fragmentation profiles. A neutral loss of 56 Da (\u003cem\u003em/z\u003c/em\u003e 315 \u0026rarr; \u003cem\u003em/z\u003c/em\u003e 259) represents the cleavage of the cannabinoid molecule's side chain (butyl group, C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003e), resulting in the fragment [C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e. A neutral loss of 122 Da (\u003cem\u003em/z\u003c/em\u003e 315 \u0026rarr; \u003cem\u003em/z\u003c/em\u003e 193) represents an ether function and a terpene ring cleavage. A neutral loss of 180 Da (\u003cem\u003em/z\u003c/em\u003e 315 \u0026rarr; \u003cem\u003em/z\u003c/em\u003e 135) is also observed.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMonitored compounds, their molecular formulas, mass transitions and (N)CE applied.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular formula [M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRetention\u003c/p\u003e \u003cp\u003etime\u003c/p\u003e \u003cp\u003e(min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrecursor ion (\u003cem\u003em/z\u003c/em\u003e) [M-H]\u003csup\u003e+\u003c/sup\u003e or [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(N)CE (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProduct ion (\u003cem\u003em/z\u003c/em\u003e) [M-H]\u003csup\u003e+\u003c/sup\u003e or [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e12.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e315.2318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e193.1223\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e259.1691\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e135.1168\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTHC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e13.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e315.2318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e193.1223\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e259.1691\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e135.1168\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eCBDA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eC\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e12.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e357.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e339.1970\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e311.2022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e245.15494\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e271.1345\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTHCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eC\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e14.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e357.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e313.2177\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e245.1545\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor the CBDA, the suggested fragmentations begin with the transition from \u003cem\u003em/z\u003c/em\u003e 357 to \u003cem\u003em/z\u003c/em\u003e 339, represented by the neutral loss of 18 Da (H\u003csub\u003e2\u003c/sub\u003eO), resulting in the fragment \u003cem\u003em/z\u003c/em\u003e 339. Next, the transition from \u003cem\u003em/z\u003c/em\u003e 339 to \u003cem\u003em/z\u003c/em\u003e 311 involves the neutral loss of 28 Da (CO), forming the fragment \u003cem\u003em/z\u003c/em\u003e 311, which then loses 40 Da to form \u003cem\u003em/z\u003c/em\u003e 271.\u003c/p\u003e \u003cp\u003eFor THCA, the initial transition from \u003cem\u003em/z\u003c/em\u003e 357 to \u003cem\u003em/z\u003c/em\u003e 313 was observed, which can be related to the decarboxylation process, involving the neutral loss of 44 Da (CO\u003csub\u003e2\u003c/sub\u003e), resulting in the formation of the deprotonated THC molecule (\u003cem\u003em/z\u003c/em\u003e 313). Next, the transition from \u003cem\u003em/z\u003c/em\u003e 313 to \u003cem\u003em/z\u003c/em\u003e 245 involves the neutral loss of 68 Da (C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003e). Estimates of cannabinoid concentrations mapped in the present study in \u003cem\u003eT. micranthum\u003c/em\u003e are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimated concentration of CBD, THC, CBDA and THCA in \u003cem\u003eTrema micranthum\u003c/em\u003e extracts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eCannabinoids (\u0026micro;g.g \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCBD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCBDA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTHCA A\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFruits\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.16 x 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.93 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.68 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.66 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInflorescences\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.84 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.85 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.13 x 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.90 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.43 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.05 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.78 x 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.86 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBranches\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.d.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003en.d. = not detected\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e bring several answers to the original questions. Firstly, part of the main question (from the title) was answered: there are cannabinoids (THC. CBD, CBDA, and THCA A) in \u003cem\u003eTrema micranthum\u003c/em\u003e. They were not detected in stems (branches), but similar concentrations of the target cannabinoids were observed in leaves and inflorescences, except in fruits, where CBD was observed at more than 100 times the THC concentration. For clinical applications, CBD is advantageous because it does not induce euphoric effects and is non-psychoactive. However, when combined with a balanced portion of THC, it modulates effects in hippocampal neurons distinctly from pure compounds. Moreover, CBD can reduce the psychoactive side effects of THC, enhancing synergy and providing greater therapeutic effects compared to their isolated compounds\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In the fruits, inflorescence and leaf parts of \u003cem\u003eT. micranthum\u003c/em\u003e, CBDA and THCA A were detected in higher concentrations than your neutral analogues. As in \u003cem\u003eCannabis\u003c/em\u003e, in the fresh material of \u003cem\u003eT. micranthum\u003c/em\u003e, the cannabinoids produced by the plant's metabolism are carboxylic acids, such as THCA A and CBDA\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe other question also addressed in this study was whether this plant could serve as an alternative source of CBD without the concerns associated with THC. It was observed that the leaves contained 4.43 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g of CBD per gram of the plant extract, and 1.05 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e \u0026micro;g/g of THC, which is significant given that leaves constitute the bulk of the plant's biomass. The key difference between hemp and marijuana (varieties of \u003cem\u003eCannabis sativa\u003c/em\u003e) lies in their chemical profiles. For a plant to be classified as hemp, it must have less than 0.3% THC by dry weight, whereas marijuana exceeds this threshold. Marijuana cultivated for recreational use typically contains low levels of cannabidiol (CBD), while hemp is rich in CBD\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A difference between \u003cem\u003eT. micranthum\u003c/em\u003e and \u003cem\u003eC. sativa\u003c/em\u003e is related to the higher content of THC in the upper parts of the plant. In \u003cem\u003eCannabis\u003c/em\u003e, about 10\u0026ndash;12% of THC is found in the flowers, followed by 1\u0026ndash;2% in the leaves, 0.1\u0026ndash;0.3% in the stem, and less than 0.03% in the root. In \u003cem\u003eT. micranthum\u003c/em\u003e, about 0,2% is found in the fruits, 0,02% in inflorescences and 0,01% in the leaves\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe presence of THC in \u003cem\u003eCannabis\u003c/em\u003e seeds is still debatable. Some authors suggest that the \u003cem\u003eCannabis\u003c/em\u003e seed is not free from THC, with its concentration being less than 0.5 ug/g\u003csup\u003e39\u003c/sup\u003e. According to the UNODC, there is no THC in the seeds, what actually happens is that the detection of THC is linked to its contact with the bracts and/or flowers of the plant, which have a high content of this cannabinoid\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. sativa\u003c/em\u003e leaves have a high CBD content (473 \u0026micro;g/g) and approximately 36% THC (175 \u0026micro;g/g), per gram of dry leaf tissue, in relation to total CBD\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, it would be unfeasible to use \u003cem\u003eT. micranthum\u003c/em\u003e leaves as an alternative source for obtaining cannabinoids since the quantity is about a hundred times lesser than in \u003cem\u003eC. sativa\u003c/em\u003e. Indeed, agrological improvements and also the optimization of the extraction processes, could be performed.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eC. sativa\u003c/em\u003e, the highest concentrations of these cannabinoids were found flowers, with a concentration of CBD 1594 \u0026micro;g/g and THC 510 \u0026micro;g/g, per gram of dry leaf tissue\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In contrast, for \u003cem\u003eT. micrathum\u003c/em\u003e the THC content in flowers was higher than that of CBD. The concentrations listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e are significantly lower when compared to the CBD content present in medicinal cannabis extracts\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, the species could potentially be used as a raw material for cannabinoid isolation, optimizing solvent extraction methods that can improve the yield of THC and CBD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAnalytical methods using high-resolution mass spectrometry allowed the identification of previously undetected cannabinoids in \u003cem\u003eThrema micranthum\u003c/em\u003e. The use of standards (the reference materials) for CBD, THC, CBDA and THCA A facilitated excellent resolution of the cannabinoid isomers, with well-resolved peaks for the neutral compounds (CBD and THC) and their acid precursors (CBDA and THCA A). Consequently, the identification of cannabinoids in the samples was achieved through accurate mass and retention time comparisons under UHPLC conditions. Quantification of cannabinoids using the PRM method allowed the detection of compounds at very low concentrations, due to its greater selectivity and sensitivity. With this approach, all investigated cannabinoids were identified in the leaves, fruits and inflorescences of \u003cem\u003eThrema micranthum\u003c/em\u003e, except in the stems. In fruits, inflorescences and leaves, the acidic forms CBDA and THCA A were detected at higher concentrations than their neutral counterparts. These analyses may guide future research in the search for cannabinoids in other species of the Cannabaceae family, potentially providing additional sources for the isolation of these herbal compounds. Furthermore, despite the low levels of these identified cannabinoids, the data may guide the optimization of extraction methods using selective solvents to achieve better yields of THC and CBD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe project received funding from FAPERJ (Grant Numbers E-26/200.512/2023, E-26/211.315/2021), and CNPq (Grant number 310782/2022-8).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by Rayssa Ribeiro, Yasmin Cunha da Silva, Ricardo Finotti, Gabriel Reis Alves Carneiro, Gustavo Ramalho Cardoso dos Santos, and Monica Costa Padilha. The first draft of the manuscript was written by Monica Costa Padilha, Valdir F. Veiga Jr, and Rayssa Ribeiro. The final version was written by Valdir F. Veiga Jr, Monica Costa Padilha, and Henrique Marcelo Gualberto Pereira. All authors commented on previous versions of the manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials statement\u003c/h2\u003e \u003cp\u003e Not applicable.\u003cbr\u003e All data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJin, D., Dai, K., Xie, Z. \u0026amp; Chen, J. Secondary metabolites profiled in cannabis inflorescences, leaves, stem barks, and roots for medicinal purposes. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 3309 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonini, S. A. et al. Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history. \u003cem\u003eJ. 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Immune responses are differentially regulated by root, stem, leaf, and flower extracts of female and male CBD hemp (cannabis sativa L.) plants. \u003cem\u003eImmuno\u003c/em\u003e. \u003cb\u003e1\u003c/b\u003e, 369\u0026ndash;379 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cannabaceae, Trema micranthum, Cannabinoids, CBD/THC, UHPLC-HRMS/MS","lastPublishedDoi":"10.21203/rs.3.rs-5125664/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5125664/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eTrema micranthum\u003c/em\u003e (Cannabaceae) has emerged as a promising new source of cannabinoids, including cannabidiol (CBD). Given the substantial medicinal demand for cannabinoids and the regulatory challenges associated with \u003cem\u003eCannabis sativa\u003c/em\u003e due to the presence of Δ\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e-tetrahydrocannabinol (THC), this study sought to explore the presence of CBD, THC, and their precursors, Δ\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e-tetrahydrocannabinolic acid A (THCA A) and cannabidiolic acid (CBDA), in various parts of \u003cem\u003eTrema micranthum\u003c/em\u003e using UHPLC-HRMS/MS (Orbitrap). Extracts from fruits, leaves, inflorescences, and stems were obtained using a methanol/hexane (9:1, v/v) solvent mixture. UHPLC coupled with an Orbitrap mass spectrometer was employed for cannabinoid identification and quantification, with standard mixtures prepared in methanol. The extracts yielded significant quantities, such as 6.6%/g from leaves and 3%/g from fruits. Cannabinoids were detected in fruits, leaves, and inflorescences, with acidic forms (CBDA and THCA A) present in higher concentrations than their neutral counterparts. Notably, leaves contained 4.43 x 10⁻\u0026sup3; \u0026micro;g/g of CBD and 1.05 x 10⁻\u0026sup3; \u0026micro;g/g of THC. These findings, facilitated by high-resolution analytical methods, underscore the potential of \u003cem\u003eTrema micranthum\u003c/em\u003e as an alternative source for cannabinoids, guiding future research in this area.\u003c/p\u003e","manuscriptTitle":"Can Trema micranthum (L.) Blume (Cannabaceae) become a new source of cannabinoids? A UHPLC- HRMS/MS analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 14:21:14","doi":"10.21203/rs.3.rs-5125664/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-25T08:01:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-18T12:26:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-17T14:11:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139145241360937722079810747414188286896","date":"2024-10-08T11:42:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272795952844962409450087118135586761327","date":"2024-10-08T10:12:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-08T09:57:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-08T09:54:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-08T03:27:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-07T06:03:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-20T20:24:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5930ef9-8bd3-40ad-8d07-68a48d412299","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39409488,"name":"Biological sciences/Chemical biology/Natural products"},{"id":39409489,"name":"Biological sciences/Biochemistry"},{"id":39409490,"name":"Biological sciences/Biotechnology"},{"id":39409491,"name":"Biological sciences/Chemical biology"},{"id":39409492,"name":"Biological sciences/Drug discovery"},{"id":39409493,"name":"Biological sciences/Plant sciences"},{"id":39409494,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2024-12-02T17:26:22+00:00","versionOfRecord":{"articleIdentity":"rs-5125664","link":"https://doi.org/10.1038/s41598-024-80857-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-11-28 15:58:29","publishedOnDateReadable":"November 28th, 2024"},"versionCreatedAt":"2024-11-14 14:21:14","video":"","vorDoi":"10.1038/s41598-024-80857-6","vorDoiUrl":"https://doi.org/10.1038/s41598-024-80857-6","workflowStages":[]},"version":"v1","identity":"rs-5125664","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5125664","identity":"rs-5125664","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}