AIM OF STUDY: To investigate the effect of mitragynine after chronic morphine treatment on cyclic AMP (cAMP) level and mRNA expression of mu-opioid receptor (MOR) in human neuroblastoma SK-N-SH cell.
METHOD AND MATERIALS: Mitragynine was isolated from the Mitragyna speciosa plant using the acid-base extraction method. The cAMP level upon forskolin stimulation in the cells was determined using the Calbiochem(®) Direct Immunoassay Kit. The mRNA expression of the MOR was carried out using quantitative RT-PCR.
RESULT: Cotreatment and pretreatment of morphine and mitragynine significantly reduced the production of cAMP level at a lower concentration of mitragynine while the higher concentration of this compound could lead to the development of tolerance and dependence as shown by the increase of the cAMP level production in foskolin stimulation. In MOR mRNA expression study, cotreatment of morphine with mitragynine significantly reduced the down-regulation of MOR mRNA expression as compared to morphine treatment only.
CONCLUSION: These finding suggest that mitragynine could possibly avoid the tolerance and dependence on chronic morphine treatment by reducing the up-regulation of cAMP level as well as reducing the down-regulation of MOR at a lower concentration of mitragynine.
AIMS: In the present study, we investigated the effects of mitragynine on spatial learning and synaptic transmission in the CA1 region of the hippocampus.
METHODS: Male Sprague Dawley rats received daily (for 12 days) training sessions in the Morris water maze, with each session followed by treatment either with mitragynine (1, 5, or 10 mg/kg; intraperitoneally), morphine (5 mg/kg; intraperitoneally) or a vehicle. In the second experiment, we recorded field excitatory postsynaptic potentials in the hippocampal CA1 area in anesthetized rats and assessed the effects of mitragynine on baseline synaptic transmission, paired-pulse facilitation, and long-term potentiation. Gene expression of major memory- and addiction-related genes was investigated and the effects of mitragynine on Ca2+ influx was also examined in cultured primary neurons from E16-E18 rats.
RESULTS/OUTCOMES: Escape latency results indicate that animals treated with mitragynine displayed a slower rate of acquisition as compared to their control counterparts. Further, mitragynine treatment significantly reduced the amplitude of baseline (i.e. non-potentiated) field excitatory postsynaptic potentials and resulted in a minor suppression of long-term potentiation in CA1. Bdnf and αCaMKII mRNA expressions in the brain were not affected and Ca2+ influx elicited by glutamate application was inhibited in neurons pre-treated with mitragynine.
CONCLUSIONS/INTERPRETATION: These data suggest that high doses of mitragynine (5 and 10 mg/kg) cause memory deficits, possibly via inhibition of Ca2+ influx and disruption of hippocampal synaptic transmission and long-term potentiation induction.
AIM OF THE STUDY: To assess the potential applicability of M. speciosa alkaloids (mitragynine, speciociliatine or paynantheine) as chemosensitizers for cisplatin in Nasopharyngeal carcinoma (NPC) cell lines.
MATERIALS AND METHODS: The cytotoxic effects of the extracts, fractions and compounds were determined by conducting in vitro cytotoxicity assays. Based on the cytotoxic screening, the alkaloid extract of M. speciosa exhibited potent inhibitory effect on the NPC cell line NPC/HK1, and therefore, was chosen for further fractionation and purification. NPC cell lines NPC/HK1 and C666-1 were treated with combinations of cisplatin and M. speciosa alkaloids combinations in 2D monolayer culture. The effect of cisplatin and mitragynine as a combination on cell migration was tested using in vitro wound healing and spheroid invasion assays.
RESULTS: In our bioassay guided isolation, both methanolic and alkaloid extracts showed mild to moderate cytotoxic effect against the NPC/HK1 cell line. Both NPC cell lines (NPC/HK1 and C666-1) were insensitive to single agent and combination treatments of the M. speciosa alkaloids. However, mitragynine and speciociliatine sensitized the NPC/HK1 and C666-1 cells to cisplatin at ~4- and >5-fold, respectively in 2D monolayer culture. The combination of mitragynine and cisplatin also significantly inhibited cell migration of the NPC cell lines. Similarly, the combination also of mitragynine and cisplatin inhibited growth and invasion of NPC/HK1 spheroids in a dose-dependent manner. In addition, the spheroids did not rapidly develop resistance to the drug combinations at higher concentrations over 10 days.
CONCLUSION: Our data indicate that both mitragynine and speciociliatine could be potential chemosensitizers for cisplatin. Further elucidation focusing on the drug mechanistic studies and in vivo studies are necessary to support delineate the therapeutic applicability of M. speciosa alkaloids for NPC treatment.
MATERIALS AND METHODS: Male and female Sprague-Dawley rats received three doses of mitragynine (1, 10, 100mg/kg, p.o) for 28 days respectively. Food intake and relative body weight were measured during the experiment. After completion of drug treatment biochemical, hematological, and histological analyses were performed.
RESULTS: No mortality was observed in any of the treatment groups. The groups of rats treated with the lower and intermediate doses showed no toxic effects during the study. However, the relative body weight of the group of female rats treated with the 100mg/kg dose was decreased significantly. Food intake also tended to decrease in the same group. Only relative liver weight increased after treatment with the high dose of mitragynine (100mg/ kg) in both the male and female treatment groups of rats. Biochemical and hematological parameters were also altered especially in high dose treatment group which corresponds to the histopathological changes.
CONCLUSIONS: The study demonstrated that mitragynine is relatively safe at lower sub-chronic doses (1-10mg/kg) but exhibited toxicity at a highest dose (sub-chronic 28 days: 100mg/kg). This was confirmed by liver, kidney, and brain histopathological changes, as well as hematological and biochemical changes.
AIM OF THE STUDY: To evaluate kratom's effects towards hematological and clinical-chemistry parameters among regular kratom users in Malaysia.
METHODS: A total of 77 subjects (n=58 regular kratom users, and n=19 healthy controls) participated in this cross-sectional study. All the surveys were conducted through face-to-face interview to elicit subject's socio-demographic characteristics and kratom use history. A full-blood test was also administered. Laboratory analysis was conducted using GC-MS to determine mitragynine content in the acquired kratom samples in order to relate mitragynine consumption with possible alterations in the blood parameters of kratom users.
RESULTS: Findings showed that there were no significant differences in the hematological and clinical-chemistry parameters of traditional kratom users and healthy controls, except for HDL and LDL cholesterol values; these were found to be above the normal reference range for the former. Similarly, long-term kratom consumption (>5 years), and quantity of daily kratom use (≥3 ½ glasses; mitragynine content 76.3-114.8mg) did not appear to alter the hematological and biochemical parameters of kratom users.
CONCLUSION: These data suggest that even long-term and heavy kratom consumption did not significantly alter the hematological and clinical-chemistry parameters of kratom users in a traditional setting.
PURPOSE: We adopted a combinatorial approach with the joint application of γ-tocotrienol and jerantinine A at lower concentrations in order to minimize toxicity towards non-cancerous cells while improving the potency on brain cancer cells.
METHODS: The antiproliferative potency of individual γ-tocotrienol and jerantinine A as well as combined in low-concentration was firstly evaluated on U87MG cancer and MRC5 normal cells. Morphological changes, DNA damage patterns, cell cycle arrests and the effects of individual and combined low-concentration compounds on microtubules were then investigated. Finally, the potential roles of caspase enzymes and apoptosis-related proteins in mediating the apoptotic mechanisms were investigated using apoptosis antibody array, ELISA and Western blotting analysis.
RESULTS: Combinatorial study between γ-tocotrienol at a concentration range (0-24µg/ml) and fixed IC20 concentration of jerantinine A (0.16µg/ml) induced a potent antiproliferative effect on U87MG cells and led to a reduction on the new half maximal inhibitory concentration of γ-tocotrienol (i.e.tIC50=1.29µg/ml) as compared to that of individual γ-tocotrienol (i.e. IC50=3.17µg/ml). A reduction on undesirable toxicity to MRC5 normal cells was also observed. G0/G1 cell cycle arrest was evident on U87MG cells receiving IC50 of individual γ-tocotrienol and combined low-concentration compounds (1.29µg/ml γ-tocotrienol + 0.16µg/ml jerantinine A), whereas, a profound G2/M arrest was evident on cells treated with IC50 of individual jerantinine A. Additionally, individual jerantinine A and combined compounds (except individual γ-tocotrienol) caused a disruption of microtubule networks triggering Fas- and p53-induced apoptosis mediated via the death receptor and mitochondrial pathways.
CONCLUSIONS: These findings demonstrated that the combined use of lower concentrations of γ-tocotrienol and jerantinine A induced potent cytotoxic effects on U87MG cancer cells resulting in a reduction on the required individual concentrations and thereby minimizing toxicity of jerantinine A towards non-cancerous MRC5 cells as well as probably overcoming the high-dose limiting application of γ-tocotrienol. The multi-targeted mechanisms of action of the combination approach have shown a therapeutic potential against brain cancer in vitro and therefore, further in vivo investigations using a suitable animal model should be the way forward.