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ABSTRACT
Mitragyna speciosa
Korth (
Kratom
), a herb of the
Rubiaceae
family is indigenousin southeast Asia mainly in Malaysia and Thailand. It is used as an opiumsubstitute and has been increasingly abused by drug addicts in Malaysia.Recently, the potent analgesic effect of plant extract and its dominant alkaloidmitragynine (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 toexhibit dose-dependant inhibition of cell proliferation in all cell lines examined, atconcentration > 100 µg/ml with substantial cell death at 1000 µg/ml. SH-SY5Ywas the most sensitive cell line examined. MIT showed a similar response.Clonogenicity assay was performed to assess the longer- term effects of MSE andMIT. The colony forming ability of HEK 293 and SH-SY5Y cells was inhibited ina 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 wasaccompanied by DNA damage, the Mouse lymphoma
tk
gene mutation assay wasused. The results were negative for both MSE and MIT. Studies on theinvolvement of metabolism in cytotoxicity of MSE and MIT were performedusing MCL-5 and it appeared that CYP 2E1 is involved in activation of cytotoxicity. Studies with opioid antagonists were performed using SH-SY5Ycells 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 MITcytotoxicity showed that cell death observed at high dose was preceded by cellcycle arrest, however MSE cell arrest was independent of p53 and p21 while MITshowed opposite result. Studies have been undertaken to examine the nature of this cell death. Morphological examinations showed that cell death induced byMSE 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 deathindependent of p53 or caspases pathway while MIT cell death appeared to beassociated with p53 and caspases pathway. The involvement of reactive oxygenspecies (ROS) generation in MSE and MIT mediating cell death was performedusing SH-SY5Y cells. The results appeared negative for both MSE and MITtreated cells. Collectively, the findings of these studies suggest that MSE and itsdominant alkaloid MIT produced cytotoxicity effects at high dose. Thus, theconsumption of
Mitragyna speciosa
Korth leaves may pose harmful effects tousers if taken at high dose and the evidence for involvement of CYP 2E1 inincreasing the MSE cytotoxicity suggests that caution may be required if theleaves are to be taken with CYP 2E1 inducers.
