TLC-BIOAUTOGRAPHY GUIDED IDENTIFICATION AND GC-MS CHARACTERIZATION OF ANTIBACTERIAL COMPOUNDS FROM CANNABIS SATIVA LEAVES
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Date
2026
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busitema university
Abstract
The emergence of antibiotic-resistant bacteria has created an urgent need for novel antibacterial agents from natural sources. This study aimed to localize, recover and tentatively characterize antibacterial constituents from Cannabis sativa leaves using TLC-bioautography-guided isolation followed by GC-MS analysis. Fresh Cannabis sativa leaves were collected, dried and extracted by maceration using three solvents of increasing polarity, namely hexane, ethyl acetate and ethanol. The crude extracts were screened for antibacterial activity against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) using direct TLC-bioautography with resazurin dye as a microbial-growth indicator.
The extracts produced distinct inhibition zones (blue or colourless regions against a pink background) against both bacterial strains, with a visually more pronounced response observed for S. aureus, consistent with the absence of an outer membrane in Gram-positive bacteria; this observation is descriptive rather than quantitative, as inhibition-zone measurements and replicate testing were not performed. Ethyl acetate produced four cleanly separated spots with no co-elution under the hexane:ethyl acetate (7:3) TLC mobile-phase system used, indicating that the ethyl acetate extract yielded the cleanest chromatographic profile among the three extracts. Ethanol produced six spots with co-elution at a retardation factor (Rf) of 0.4, while hexane produced five spots with co-elution at Rf 0.5.
GC-MS analysis of the recovered active bands from the ethanol extract generated tentative, library-based annotations for several constituents, including a compound provisionally annotated as a Cannflavin-related flavonoid (consistent with 2’,4’-dimethoxy-3-hydroxy-6-methylflavone) from Spot 2 (Rf 0.5), a compound provisionally annotated as oleanolic acid from Spot 3 (Rf 0.4), and fatty-acid esters including methyl palmitate, methyl stearate and methyl oleate. The hexane and ethyl acetate extracts were associated primarily with putative alkanes (eicosane, heneicosane), fatty-acid esters and sterol-related compounds. Because these assignments rest on GC-MS spectral-library matching without confirmation by authentic standards, retention-index comparison, or complementary spectroscopy, and because no minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) or dose-response data were generated for isolated compounds, all compound identities and potency comparisons reported here should be regarded as tentative rather than confirmed.
Within this limitation, the study indicates that Cannabis sativa leaf extracts contain multiple chromatographically separable constituents associated with antibacterial activity, with the ethanol extract yielding the constituents of greatest apparent interest despite co-elution, and the ethyl acetate extract offering the cleanest analytical separation. These findings provide preliminary, exploratory support for the traditional use of Cannabis sativa in managing bacterial infections and identify candidate bands — most notably the putative Cannflavin-related flavonoid — that warrant purification, structural confirmation and quantitative antibacterial testing in future work.