Cytotoxicity of extract of Malaysian Mitragyna Speciosa Korth and its dominant Alkaloid Mitragynine
Creators: name: Saidin, Nor Aini
Publisher: name: Imperial College London
Year: 2008

description: Mitragyna speciosa Korth (Kratom), a herb of the Rubiaceae family is indigenous in southeast Asia mainly in Malaysia and Thailand. It is used as an opium substitute and has been increasingly abused by drug addicts in Malaysia. Recently, the potent analgesic effect of plant extract and its dominant alkaloid mitragynine (MIT) were confirmed in vivo and in vitro. MIT acted primarily on μ- and δ-opioid receptors, suggesting that MIT or similar compounds could be promising alternatives for future pain management treatments. However the potential cytotoxicity of this plant is unknown. Therefore, the cytotoxicity of methanol-chloroform extract (MSE) and MIT on human cell lines (HepG2, HEK 293, MCL-5, cHol and SH-SY5Y cells) has been examined. MSE appeared to exhibit dose-dependant inhibition of cell proliferation in all cell lines examined, at concentration &gt; 100 μg/ml with substantial cell death at 1000 μg/ml. SH-SY5Y was the most sensitive cell line examined. MIT showed a similar response. Clonogenicity assay was performed to assess the longer- term effects of MSE and MIT. The colony forming ability of HEK 293 and SH-SY5Y cells was inhibited in a dose-dependant manner. Involvement of metabolism in cytotoxicity was further assessed by clonogenicity assay using rat liver S9 (induced by Arochlor 1254); toxicity increased 10-fold in both cell lines. To determine if cytotoxicity was accompanied by DNA damage, the Mouse lymphoma tk gene mutation assay was used. The results were negative for both MSE and MIT. Studies on the involvement of metabolism in cytotoxicity of MSE and MIT were performed using MCL-5 and it appeared that CYP 2E1 is involved in activation of cytotoxicity. Studies with opioid antagonists were performed using SH-SY5Y cells treated with MSE and MIT. Naloxone (μ and δ receptor antagonists), naltrindole (δ receptor antagonist) and cyprodime hydrobromide (μ receptor antagonist) confirmed that MSE cytotoxicity was associated with μ and δ receptor while MIT mainly acted on μ receptor. Studies on mechanism of MSE and MIT cytotoxicity showed that cell death observed at high dose was preceded by cell cycle arrest, however MSE cell arrest was independent of p53 and p21 while MIT showed opposite result. Studies have been undertaken to examine the nature of this cell death. Morphological examinations showed that cell death induced by MSE was cell type dependant, in which SH-SY5Y cells appeared to die via apoptosis-like cell death while HEK 293 and MCL-5 cells predominantly via necrosis. Biochemical assessments confirmed that MSE induced cell death independent of p53 or caspases pathway while MIT cell death appeared to be associated with p53 and caspases pathway. The involvement of reactive oxygen species (ROS) generation in MSE and MIT mediating cell death was performed using SH-SY5Y cells. The results appeared negative for both MSE and MIT treated cells. Collectively, the findings of these studies suggest that MSE and its dominant alkaloid MIT produced cytotoxicity effects at high dose. Thus, the consumption of Mitragyna speciosa Korth leaves may pose harmful effects to users if taken at high dose and the evidence for involvement of CYP 2E1 in increasing the MSE cytotoxicity suggests that caution may be required if the leaves are to be taken with CYP 2E1 inducers.