A second incubation time point at 18 hr also showed negative results. The next step was investigating the possibility of involvement of executioner caspases such as caspase 3 and 7. The executioner caspases are also known as downstream caspases as they depend on active initiator caspases for their activation by proteolytic cleavage (Srinivasula et al 2001). Red Vein Kratom Extract as anticipated there was no activation of caspases 3 and 7 activities in cells treated bulk mitragyna speciosa wenham with high dose of MSE at both 4 hr and 18 hr incubation time points. Interestingly for MIT there was a clear significant difference of caspases 3 and 7 activities at both concentrations of MIT tested. This finding suggests that the mode of the cell death of MIT treated Red Vein Kratom Extract cells is dependant on caspase 3 and 7 activation pathway. There were no significant differences in the subG1 population (apoptosis population) between treated groups (caspase 3 inhibitor caspase 8 inhibitor caspase 9 inhibitor and general caspase inhibitor treated with high dose of MSE) and the control and negative control groups.
For HEK 293 cells the nature of cell death was more necrotic than apoptotic as morphologically the cell membrane integrity was compromised leaving a reduced stained intensity and indicating lysis of cell membrane and subsequent lost of cell content. Although Rapi-Diff staining is often used for cell morphology in this case the quality of staining was not as good as Wright-Giemsa staining however it still provided an indication of the different modes of cell death of MCL-5 cells. MSE with control and lower dose groups showed there was a clear necrotic appearance with swelling of cells lysis of cell membrane and lost of cell content.
Unsuccessful repair processes may lead the cells to undergo apoptosis. In mammalian cells an important protein that plays a central role in cell cycle arrest is p53. Norman et al 2005). These reports confirm the complexity of maintenance of the cell cycle. HEK 293 MCL-5 and SH-SY5Y cells were used in this analysis. The cells were cultured and maintained as described in chapter 2 section 2.
Discussion Mitragyna speciosa Korth (Kratom) leaves have been used by humans for decades. There are no reports of increased cancer associated with consumption of Kratom leaves although such associations have never been examined in a proper controlled study. Neither is there any information available concerning the genotoxic potential of Kratom leaves. As part of establishing a database on the toxicological potential of the use of this plant I have attempted to examine the possible toxicological effects this plant might have including potential for carcinogenicity via genotoxicity testing. The basic toxicology data established in the previous chapter has informed us
on the potential cytotoxicity of MSE and MIT on several human cell lines which
generally shows cytotoxicity with high dose. The lethal effect of the extract and major alkaloid (MIT) on the cells examined prompted the question whether cell death was accompanied by DNA damage.
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The bacterial tryptophan reverse mutation assay with Escherichia coli WP2. Rapid colorimteric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Immunol Methods 65: 5563.
This result again indicated no generation kratom maeng da extract of ROS upon treatment with MIT. However an interesting finding was noted upon microscopic observation of the cells pre-treated with NAC as the majority of them were floating and very few cells appeared attached to the bottom of wells. This observation is in contrast of what was seen for MSE pre-treated NAC groups.
Some people report
that after using the plant they experience headaches and nausea which usually ceases after a short while. There are some known possible negative effects to kratom use especially after a longer period of regular Red Vein Kratom Extract consumption. In East Asia it is also often used as a substitute for opium when opium is unavailable or to moderate opium addiction. Mitragynine is used to gradually wean the user off narcotics. Within a few days the addict would stop use of the narcotic they are addicted to and the cravings and withdrawal will be moderated by the binding of mitragynine to the delta receptors.