Does Trema micranthum (L.) Blume Produce Cannabinoids?

Abstract

There are inconclusive claims in the scientific literature that the species Trema micranthum, widely distributed throughout the Brazilian territory, may produce phytocannabinoids, potentially serving as an alternative to Cannabis sativa. In this study, we conducted a comprehensive investigation to assess the presence of phytocannabinoids in two Trema micranthum samples collected in the Midwest region of Brazil. In trying to detect cannabinoids in T. micranthum, a recommended cannabis screening test was employed, the Fast Blue BB Salt (FBBBS) colorimetric assay, followed by thin-layer chromatography (TLC) and instrumental techniques: high-performance liquid chromatography coupled to diode array detector (HPLC-DAD) and gas chromatography coupled to mass spectrometry (GC-MS). When employed without chloroform extraction, the FBBBS reagent yielded positive results for extracts from all parts of T. micranthum (leaves, branches, fruits, and inflorescences). However, these initial positive results from the FBBBS test, suggesting the presence of cannabinoids, were not corroborated by FBBBS followed by chloroform extraction, TLC, or the instrumental techniques used in this study. These additional outcomes suggest that the positive FBBBS test results were likely due to the presence of other phenolic compounds rather than phytocannabinoids. For example, the presence of vitexin-like compounds in T. micranthum extracts might explain the positive FBBBS test results. Therefore, new assertions that T. micranthum produces cannabinoids will require the support of more selective experiments to avoid false-positive claims based on less selective screening tests.

Keywords: Cannabaceae; FBBBS; cannabidiol; phytocannabinoids.

Figure 1

Leaves, inflorescences, unripe and ripe fruits, and branches from one of the Trema micranthum (L.) Blume specimens included in this work.

Figure 2

Colorimetric assay results using Fast Blue BB Salt (FBBBS). Reaction blank (A), and methanol extracts from Trema micranthum branches (B), leaves (C), ripe fruits (D), unripe fruits (E), and inflorescences (F), in comparison with the analytical standards of delta-9-tetrahydrocannabinol (THC), cannabichromene (CBC), cannabidiol (CBD), and cannabinol (CBN). Photos were taken after adding the methanol extracts to FBBBS, showing color changes indicative of phytocannabinoids.

Figure 3

FBBBS colorimetric assay in biphasic mode for Trema micranthum leaves (A) and cannabidiol (B), and results of this test applied to the analytical standard vitexin (in methanol), step by step (C–E): (C) addition of vitexin solution to FBBBS, (D) addition of 0.1 mol/L NaOH and mixing, and (E) addition of chloroform and mixing.

Figure 4

TLC analyses of methanol extracts from Trema micranthum and methanol solutions of analytical standards of cannabinoids and vitexin, using toluene (A) or ethyl acetate—acetic acid—formic acid—water (100:11:11:26, v/v) (B) as mobile phase. A mixture of the analytical standards CBN, THC, and CBD at 10 µg/mL (1), leaves (2), branches (3), inflorescences (4), unripe fruits (5), and ripe fruits (6) extracts of T. micranthum; analytical standards at 1000 µg/mL of vitexin (7), CBN (8), THC (9), and CBD (10). Detection reagent: Fast Blue BB Salt.

Figure 5

Chromatographic profile from Trema micranthum extracts and the main cannabinoids from Cannabis sativa. Pink line: analytical standards of CBD (~20 µg/mL), CBN (~10 µg/mL), and Δ⁹-THC (~30 µg/mL). Dark green line: T. micranthum methanol extracts of unripe fruits; yellow line: T. micranthum methanol extracts of inflorescences; light green line: T. micranthum methanol extracts of leaves; red line: T. micranthum methanol extracts of ripe fruits.

Figure 6

HPLC-DAD data using polyphenols method. (A,B) Chromatogram of methanol extracts of Trema micranthum leaves (at 280 nm in (A), and from 230 to 400 nm in (B)); (C) UV spectrum of the major peak in the chromatogram of T. micranthum leaves, at 25.2 min (left) and of the analytical standard of vitexin (right).

Figure 7

Total ion chromatograms (TIC) of Cannabis sativa and Trema micranthum samples, including leaves, inflorescences, unripe fruits, and ripe fruits. Some compounds detected in the Cannabis sativa samples are labeled: Delta-9-Tetrahydrocannabiorcol (Δ⁹-THCO); tetrahydrocannabivarin (THCV); cannabicyclol (CBL); cannabidiol (CBD); Delta-9-Tetrahydrocannabinol (Δ⁹-THC); cannabigerol (CBG); cannabinol (CBN). Unknown compounds from T. micranthum: Unk 1 and Unk 2.

Figure 8

Mass spectra of peaks from Cannabis sativa (A) and Trema micranthum ripe fruit (B) samples at retention time 9.548 min.

Figure 9

Total ion chromatograms (TIC) of analytical standards of CBD, CBN, and Δ⁹-THC and Trema micranthum samples, including branches, leaves, inflorescences, and fruits. T. micranthum samples did not present peaks with retention times or MS spectra of any cannabinoid.