Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography–tandem mass spectrometry

Purpose Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography–triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QqTOFMS). Methods 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS. Results The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems. Conclusions The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

COMPARISON OF ANTIMICROBIAL, LARVICIDAL AND ANTHELMINTIC ACTIVITY OF CURCUMA AROMATICA SALISB. COW URINE EXTRACT

The present study was undertaken to evaluate and compare antimicrobial, larvicidal and anthelmintic activity of Curcuma aromatica Salisb. cow urine extracts. The antimicrobial activity was performed using five microbial strains, v.i.z, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Aspergillus niger and Aspergillus flavus; larvicidal activity was evaluated using 3rd and 4th instar stage larvaes, and anthelmintic activity using red earthworms (Lumbricus rubellus) with raw and photoactivated cow urine. Antimicrobial activity of photoactivated cow urine extract of C. aromatica (CAPUE) showed significant results when compared with raw cow urine extract of C. aromatic (CAUE). Larvicidal activity of CAPUE at a concentration of 25% V/V i.e. 40.2% V/V was found to show better mortality rate of larvae when compared with CAUE at a concentration of 25% v/v i.e. 45.2% V/V. Anthelmintic activity of CAPUE was found to show better paralysis and death time at a concentration of 10 μg/mL i.e 36.3 min for paralysis and 58.9 min for death when compared to CAUE i.e. 38.3 min paralysis rate at a concentration of 10 μg/mL and death rate is found 69.9 min at 10 μg/mL concentration.

Antitumor Activity of Human Urine Extract Cda-2 against Multiple Myeloma Cell Lines Via PI3K/Akt Signaling Pathway in a Caspase-3-Dependent Manner

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment although a number of new agents have recently been shown to be effective. There were still some patients who did not respond to GCs or develop resistance. The aim of this study was to investigate the antitumor activity of human urine extract against MM and Dexamethasone-resistant and sensitive MM cell lines. These cell lines were used to examine the effects of a human urine preparation CDA-2 on the induction of growth arrest and apoptosis. Apoptotic proteins including caspase family, Bcl-2 family were studied. Phosphoinositide 3-kinase (PI3K)/Akt survival signaling pathway was also examined. The caspase-3 inhibitor Z-DEVD-FMK was used to examine the involvement of caspase-3 and PARP. In current study we found that CDA-2 could induce growth arrest and apoptosis of MM cells in vitro. Using MTT assay, we determined the effects of CDA-2 on RPMI8226, U266, MM.1R and MM.1S cells as well as peripheral blood monouclear cells(PBMCs) from three normal volunteers. Fifty percents of growth inhibition (IC50) was measured in these cells treated with CDA-2 at concentrations ranging from 2 to 16 mg/ml for 24 to 72h. CDA-2 exerted substantial growth inhibition in RPMI8226, U266, MM.1R and MM.1S cell lines, while it did not induce cytotoxicity in normal PBMCs. The 24h and 48h IC50 mean values of CDA-2 showed 4.61 and 3.47 mg/ml in RPMI8226 cells(P=0.043<0.05),6.09 and 5.71 mg/ml in U266 cells(P>0.05),5.07and 4.11 mg/ml in MM.1R cells(P=0.002<0.01), 3.26 and 2.13mg/ml in MM.1S cells(P=0.007<0.01). The results indicated that cell viability in the presence of CDA-2 decreased almostly in a dosedependent manner. The inhibitory rates of cell growth were positively correlated with CDA-2 concentrations except U266 cell lines. While in contrasts, CDA-2 did not induce cytotoxicity in PBMCs from three normal volunteers (IC50=103.92 mg/ml, p=0.006<0.01). The mechanism of CDA-2 in MM and Dexmethasone-resistant and sensitive MM cell lines was related to the inhibition of PI3Kp110α expression in protein level, which inactivated the phosphorylation of Akt involving dephosphorylation of Bad protein, downregulation of Bcl-xl protein, and triggered the activation of caspase cascades. We contrasted the grey scale of the PI3Kp110αprotein level after treated with the different concentrations of CDA-2. The grey scale values of 8226 cells and MM.1R cells after treated with 4mg/ml CDA-2 were 3.71 A2.11 respectively, while the grey scale values of the untreated control cells of 8226 and MM.1R were 6.92 A7.46 respectively (P=0.001<0.01). This phenomenon could be inhibited by the caspase-3 inhibitor Z-DEVD-FMK. In addition, CDA-2 could also downregulate the level of DNA maintenance methylation enzyme, DNMT-1 in MM and Dexamethasone-sensitive and resistant MM cell lines. Therefore, our results demonstrate the presence of active components in the human urine extract that can induce growth arrest and apoptosis of Multiple Myeloma and Dexamethasone-resistant and sensitive Multiple Myeloma cell lines and may involve the PI3K/Akt signaling pathway in a caspase-3 dependent manner, associated with downregulation the level of the DNA maintenance methylation enzyme. This may provide new insights for the treatment of MM, especially the drug-resistant Multiple Myeloma.

Antimicrobial Property of a Herbal Preparation Containing Dalbergia Sissoo and Datura Stramonium with Cow Urine against Pathogenic Bacteria

In this study, a herbal preparation containing Dalbergia sissoo and Datura stramoium with cow urine (DSDS), was evaluated for its antibacterial potential against pathogenic strains of gram-positive ( Staphylococcus aureus and Streptococcus pneumoniae) and gram-negative ( Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) bacteria. Antibacterial activity was compared to standard antibiotic drugs i.e. Chloramphenicol (30 mcg), Ampicillin (10 mcg), Nalidixic acid (10 mcg) and Rifampicin (30 mcg). Cow urine extract was found to be most active against both gram-positive as well as gram-negative bacteria. Clinical isolate of S. aureus showed higher sensitivity towards cow urine extract of DSDS than standard strains, and inhibited growth on most regulatory levels such as inhibition of protein, DNA, RNA and peptidoglycan synthesis. The results of the present study shows that the cow urine extract of DSDS may be used as a potent antiseptic preparation for prevention and treatment of chronic bacterial infections.