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As with the p53 effects noted previously MIT had little effect on p21 levels (Fig. Smoking Kratom Joint p21 levels of MSE treated SH-SY5Y cells at different time points (6 12 24 and 48 hr). The blots were representatives of duplicate experiments. P21 levels of MIT treated SH-SY5Y cells at different time points (6 12 24 and 48 hr).

The control and low dose groups however did express kratom with opiates p21 protein consistent with the p53 expression. In the parallel Smoking Kratom Joint experiment with MIT again p21 was expressed in a time-dependant manner that correlated with p53 expression. MIT exerts weaker toxicity effects compared to MSE. Collectively the current findings suggest that MSE induces a cycle arrest that appears to be independent of p53 Smoking Kratom Joint pathway. In contrast MIT appears to induce cell cycle arrest that is p53 dependant.

S9 did not influence the MIT metabolism as the cells number were within the similar range as cells in negative control kratom therapy premium bali groups or positive control group and the RSG values were high and not much different with other groups. Interestingly in the absence of S9 MIT showed dose-dependant cytotoxicity (low RSG) on its own. The preliminary data shown here are the results taken after 2 days expression period prior to plating. There was no significant difference in cell numbers compared to negative control or positive control groups; however based on the formula which takes into account the suspension growth for two days culturing period low dose-dependant RSG was calculated. The low suspension growth was noted even after 24 hr post treatment (data not shown).

Ten thousand cells were analysed by CellQuest Pro software and the subG1 population representing apoptotic cells were gated manually. Reactive oxygen species (ROS) analysis in SH-SY5Y cells treated with MSE and MIT ROS generation assay was carried out using SH-SY5Y cells by using a fluorescent dye 27-dichlorofluorescein diacetate (DCFH-DA). Principally this dye diffuses through the cell membrane and is hydrolysed enzymatically by intracellular esterases to form monofluorescent dichlorofluorescein (DCFH) in the presence of ROS. The intensity of the fluorescence is therefore proportional to the levels of intracellular ROS (Galvano et al 2002). A fluorescence-based ethod to measure ROS generation in live cells was a modification of the procedure described by Esposti and McLennan (1998).

In the present study it is suggested that the toxicity effects seen for MSE were predominantly due to MIT as shown by similar IC50 values for MIT and MSE treated SH-SY5Y cells. The role of metabolism was also assessed in which the toxicity of MSE treated SH-SY5Y cells was found to increase 10-fold when the metabolic activation system post mitochondrial rat liver S9 induced by Arochlor 1254 was added to the treatment. However contradictory results were noted when metabolically competent MCL-5 cells appeared to detoxify MSE rather than activate it.

Other alkaloids present include other indoles and oxindoles such as ajmalicine corynanthedine mitraversine rhychophylline and stipulatine. The kratom leaf capsules dominant alkaloid in this species is mitrajavine which has not yet been pharmacologically tested. Kratom has a very unique aroma

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that is wonderful for the fine art of incense creation. It is used for its relaxing mood-lifting effects. Herbal-x is located in the USA.

P53 levels of MSE treated SH-SY5Y cells after 24 hr treatment. Bars are the mean of three experiments with SEM. P53 levels of MSE treated SH-SY5Y cells at different time points (6 12 24 and 48 hr). P53 levels of MIT treated SH-SY5Y cells after 24 hr treatment. P53 kratom powder illegal levels of MIT treated SH-SY5Y cells at different time points (6 12 24 and 48 hr).

Values of each phase of the cell cycle were the mean of the three experiments with SEM. Human lymphoblastoid – MCL-5 cells For this cell line the cell cycle

analysis was carried out using Cellquest Pro software and the aggregated cells (doublet cells) were gated out. The DNA profiles were determined using Modfit LT cell cycle analysis software (Verity Software Topsham ME).

M MIT where cells accumulated at G1 phase and the population shifted to the right side of the scale. This phenomenon implies that the treated cells have taken up more PI dye thus leading to a shift to the right. Due to the amount of MIT compound available repetition of this experiment was not possible. Effects of MSE on the cell cycle distribution of SH-SY5Ycells after 48 hr of treatment.