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. 2024 Nov 28;14:29620. doi: 10.1038/s41598-024-80857-6
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 Δ9-tetrahydrocannabinol (THC), this study sought to explore the presence of CBD, THC, and their precursors, Δ9-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 × 10−3 µg/g of CBD and 1.05 × 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.
Introduction
Cannabidiol (CBD) is a non-psychoactive cannabinoid with important therapeutic benefits. The recent FDA approval for formulations 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 theme1.
Despite the huge health importance and wellness and drug industries’ interest, 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 Δ9-tetrahydrocannabinol (THC), the main psychoactive cannabinoid and responsible for several important pharmacological properties but also for recreational use, brings a lot of controversies to the discussion2.
Cannabis sativa buds are commonly extracted with ethanol and, after the solvent evaporates, but before adding coconut oil (or similar) to produce the commercial 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 Δ9-tetrahydrocannabinoic acid (THCA) and cannabidiolic acid (CBDA), respectively. The extract must be heated to allow the decarboxylation process to produce the bioactive CBD and THC when they are finally 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 naturally high content of CBD are highly desired, without the need to activate and avoid the presence of THC3,4.
Besides the production issues and the social preconception against the use of Cannabis L., the main difficulties of CBD-based products commerce lie in the complex and often lack of a standardized regulatory background. Consequently, there is a clear concern about product quality, safety, and labeling, leading to a framework of laws that can confuse consumers and complicate commercial operations, challenging international trade5,6.
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 the 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 products7,8.
The genus Trema Lour (Cannabaceae) is pantropical with nearly 20 species, six of them being cited for Americas9. Trema micranthum L. (Blume), synonym Trema micrantha, is the one traditionally registered for Brazil10. It is a shrub present as a ruderal species, native to tropical and subtropical America, occurring naturally in all the Brazilian biomes and diverse vegetational forest types (ever-green, semi-decidual, and gallery forests)11,12. Phylogenetically, the Trema-Parasponia group is a sister group of the Humulus-Cannabis group, been diverged nearly from 58.5 to 46.3 Ma, between the late Paleogene and the early Neogene13. Other species of the previously cited groups are known cannabinoid producers as Cannabis sativa L. and Trema orientale (L) Blume14.
Recently, glandular trichomes of T. micranthum flowers and leaves were described as cannabinoid secreting structures15. Its popular uses are described in Bolivia, for 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 injuries16. Previous studies with T. micranthum extracts showed antioxidant properties at 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase assays17 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 indomethacin19. 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 hemorrhage in horses22. 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 detected21. 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) derivatives24.
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 analyte 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 648,300 Herbarium. This work was registered in the National Management System of Genetic Heritage and Associated Traditional Knowledge (SISGEN) under number AE058C8. 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 h. The resulting organic extract was centrifuged at 4000 rpm for 20 min 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 s, 1.5 mL was transferred to an Eppendorf tube, and centrifuged at 13,000 rpm for 15 min 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 min, then to 25% at 2 min, and then 90% at 10 min. After reaching 100% of B at 14 min and maintaining this ratio until 16 min, the initial chromatographic condition was restored from 16.1 to 20.0 min.
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, auxiliary 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 × 106, 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 × 106, 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 × 10− 4 µg/g for CBD and 3.7 × 10− 4 µg/g for THC, 1.4 × 10− 3 µg/g for CBDA and 1.0 × 10− 3 µg/g for THCA A per gram of extract. The estimated limit of quantification established by PRM was 1.7 × 10− 3 µg/g for CBD and 1.2 × 10− 3 µg/g for THC, 4.7 × 10− 3 µg/g for CBDA, and 3.5 × 10− 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 × 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 Δ9-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 formed3,4. 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.
Fig. 1.
The extracts were obtained following a solvent system suggested based on a huge literature search and analysis (results not presented) as the most efficient to extract cannabinoids: maceration on methanol/hexane 9:126. 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 (%) | 3.0 | 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 (UNODC), a combination of colorimetric tests, thin-layer chromatography, and physical (macroscopic and microscopic) examination is considered an acceptable minimum analytical approach for the identification of cannabinoids in plant material27. 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 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 test28.
Gas chromatography coupled with flame ionization detector (GC-FID) or coupled with mass spectrometry, as well as liquid chromatography coupled with mass spectrometry with a single analyzer or in tandem, and any other combinations could be used. The selection of the appropriate method depends on the purpose of the analysis29. 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 to a high potential for degradation of polar groups. Acid carboxylic groups seem to be particularly sensitive to this phenomenon, resulting in the loss of acid moiety. Consequently, acidic compounds are converted to neutral ones, often incompletely30. 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, the diode-array detector (DAD) depends on the existence of a chromophore group, presenting low specificity to target analysis, especially in a complex matrix. Based on that, LC–MS allows direct determination of neutral and acidic cannabinoid species is possible31. Indeed, the use of ultra-high performance liquid chromatography coupled with high resolution mass spectrometry analysis allows a completely different perspective of specificity and sensitivity, when compared with colorimetric assays, TLC, or even to LC-DAD.
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 in 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 1% (%) or ± 0.1 min, 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 sample27.
In this project, a chromatographic system based on a column with stationary phase particles with a diameter of 2.6 μm or smaller achieved high separation efficiency and resolution power. The coupling with a high-resolution mass spectrometer working at 70,000 full widths at half maximum (FWHM) delivers an unmatched analytical capability compared with other works dedicated to identifying the component in Trema micranthum21. 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 for 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 min of chromatographic run time (Fig. 2a).
Fig. 2.
The same quality in separation can be observed for CBDA and THCA A (Fig. 3a). 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 (Figs. 2b–d and 3b–d). The additional peak after THCA A is a constitutional isomer of CBDA and THCA A, with an exact mass of m/z 357.2071. There are several possibilities for cannabinoid isomers with the molecular formula C22H30O4, among which the following stand out: CBCA, CBLA, Δ9-THCA A, and Δ8-THCA.
Fig. 3.
In Brazil, the commercialization of CBD and THC products is regulated by ANVISA, an official agency like the US FDA. CBD-based products, with concentrations ranging from 2500 µg/g to 200 × 103 µg/g of the extract, and without the presence of THC, are the most recommended for therapeutic purposes. Generally, it is suggested the absence of THC or in very low concentrations, 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 alternative32. These products can be found in various pharmaceutical forms, such as oils, sprays, creams, and ointments. Oils are the most used and are obtained by solvent 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 in 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 literature33,34.
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 | C21H30O2 | 12.52 | 315.2318 | 40 | 193.1223 |
| 259.1691 | |||||
| 135.1168 | |||||
| THC | C21H30O2 | 13.26 | 315.2318 | 40 | 193.1223 |
| 259.1691 | |||||
| 135.1168 | |||||
| CBDA | C22H30O4 | 12.68 | 357.2 | 40 | 339.1970 |
| 311.2022 | |||||
| 245.15494 | |||||
| 271.1345 | |||||
| THCA | C22H30O4 | 14.12 | 357.2 | 40 | 313.2177 |
| 245.1545 |
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, C4H8), resulting in the fragment [C17H22O2 + 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.
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 (H2O), 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 (CO2), 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 (C5H8). 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 × 10− 1 | 1.93 × 10− 3 | 6.68 × 10− 2 | 1.66 × 10− 2 |
| Inflorescences | 9.84 × 10− 3 | 1.85 × 10− 3 | 1.13 × 10− 1 | 1.90 × 10− 2 |
| Leaves | 4.43 × 10− 3 | 1.05 × 10− 3 | 5.78 × 10− 2 | 9.86 × 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 compounds35,36. In the fruits, inflorescence, and leaf parts of T. micranthum, CBDA, and THCA A were detected in higher concentrations than their neutral analogs. 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 CBDA37.
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 × 10− 3 µg of CBD per gram of the plant extract, and 1.05 × 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 CBD31. 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 leaves38.
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/g39. According to the UNODC, there is no THC in the seeds, what 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 cannabinoid27.
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 about total CBD32. 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 less than in C. sativa. Indeed, agrological improvements and the optimization of the extraction processes could be performed.
In C. sativa, the highest concentrations of these cannabinoids were found in flowers, with a concentration of CBD 1594 µg/g and THC 510 µg/g, per gram of dry leaf tissue40. 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 extracts32. 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 Trema 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 Trema 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.
Author contributions
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.
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).
Data availability
The data are provided in the manuscript and not as supplementary information.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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