In vitro and in vivo anti-colorectal cancer properties and potential mechanisms of Lemmaphyllum drymoglossoides essential oil.

Lemmaphyllum drymoglossoides (Baker) Ching is a widely used folk herbal remedy that has been employed in cancer treatment.

However, studies on the anti-colorectal properties and phytochemicals of L. drymoglossoides essential oil (EO) are currently lacking. The purpose of this study was to examine the in vivo and in vitro anti-colorectal cancer properties of L. drymoglossoides EO and to examine its chemical composition. Using a Clevenger-type apparatus, L. drymoglossoides was extracted via hydrodistillation to obtain L. drymoglossoides EO. The cytotoxicity of L. drymoglossoides EO was evaluated using the MTT assay against the colorectal cancer HCT-116 cell line and the non-cancer NCM460 and L929 cell lines.

Clone formation assays were employed to detect the effects of L. drymoglossoides EO on HCT-116 cell proliferation, and its effect on the cell cycle was assessed using flow cytometry.

Moreover, EO's ability to induce apoptosis was evaluated using Hoechst 33258 staining, Annexin V-APC/PI staining, AO/EB staining, and morphological analysis. We evaluated the inhibitory activity of L. drymoglossoides EO against HCT-116 cell metastasis using Transwell migration and invasion detections and wound healing assays.

Furthermore, western blotting was utilized to evaluate its impact on the expression of proteins related to cell proliferation, apoptosis, and metastasis. To further verify its in vivo anticancer effect, a nude mouse model of HCT-116 cell xenograft tumors was established. We analyzed the in vivo anticancer effect of L. drymoglossoides EO (100 mg/kg, intraperitoneal injection) by measuring tumor volume and weight, calculating tumor inhibition rate, and performing H&E and TUNEL staining. We employed an Agilent GC-MS/FID to perform chemical composition detection of L. drymoglossoides EO.

L. drymoglossoides EO displayed selective cytotoxic activity with stronger cytotoxicity toward the HCT-116 cancer cell line (IC50: 34.44 ± 1.15 μg/mL) and lower cytotoxicity against the non-cancer cell lines NCM460 (IC50: 70.37 ± 1.39 μg/mL) and L929 (IC50: 85.32 ± 3.70 μg/mL). L. drymoglossoides EO induced G1 phase arrest by increasing p21 expression and decreasing Cyclin E1, CDK2, Cyclin D3, and CDK4 expression, thereby suppressing cell proliferation.

Additionally, L. drymoglossoides EO elevated the Bax/Bcl-2 ratio, decreased ΔΨm, facilitated the release of Cyt c, and stimulated Caspase 9 and Caspase 3 activation, resulting in PARP cleavage, and triggering apoptosis by regulating mitochondrial apoptosis-related markers. L. drymoglossoides EO downregulated N-cadherin and upregulated E-cadherin, thereby restricting cell metastasis. L. drymoglossoides EO effectively inhibited the growth of HCT-116 xenograft tumors by inducing apoptosis in nude mice.

Furthermore, GC-MS/FID analysis identified the major components of L. drymoglossoides EO as 1-hexanol, hexanal, linalool, hexanal dihexyl acetal, pimaradiene, trans-2-hexenal, trans-β-ionone, α-terpineol, and 3-hexenol. L. drymoglossoides EO possesses promising anti-colorectal properties and holds development potential in the pharmaceutical field.