α-Mangostin Promotes In Vitro and In Vivo Degradation of Androgen Receptor and AR-V7 Splice Variant in Prostate Cancer Cells.

A major limitation of current prostate cancer pharmacotherapy approaches is the inability of these compounds to target androgen receptor variants or mutants that develop during prostate cancer progression. The demand for novel therapeutics to prevent, slow, and treat prostate cancer is significant because FDA approved anti-androgens are associated with adverse events and can eventually drive drug-resistant prostate cancer. This study evaluated α-mangostin for its novel ability to degrade the androgen receptor and androgen receptor variants. α-Mangostin is one of more than 70 isoprenylated xanthones isolated from Garcinia mangostana that we have been evaluating for their anticancer potential. Prostate cancer cells treated with α-mangostin exhibited decreased levels of wild-type and mutated androgen receptors. Immunoblot, immunoprecipitation, and transfection experiments demonstrated that the androgen receptor was ubiquitinated and subsequently degraded via the proteasome, which we hypothesize occurs with the assistance of BiP, an ER chaperone protein that we have shown to associate with the androgen receptor. We also evaluated α-mangostin for its antitumor activity and promotion of androgen receptor degradation in vivo. In summary, our study demonstrates that androgen receptor degradation occurs through the novel activation of BiP and suggests a new therapeutic approach for prostate cancer.

Pharmacokinetic characterization of carnosol from rosemary (Salvia Rosmarinus) in male C57BL/6 mice and inhibition profile in human cytochrome P450 enzymes.

Rosemary (Salvia Rosmarinus) is a rich source of dietary diterpenes with carnosol as one of the major polyphenols used to standardize rosemary extracts approved as a food preservative, however, at present there is not any information on the murine pharmacokinetic profile of carnosol or its potential for drug interactions. The present study utilizes cell-free, cell-based, and animal-based experiments to define the pharmacokinetic profile of the food based phytochemical carnosol. Mice were administered carnosol (100 mg/kg body weight) by oral gavage and plasma levels were analyzed by LC-MS/MS to establish a detailed pharmacokinetic profile. The maximum plasma concentration exceeded 1 µM after a single administration. The results are significant as they offer insights on the potential for food-drug interactions between carnosol from rosemary and active pharmaceutical ingredients. Carnosol was observed to inhibit selected CYP450 enzymes and modulate metabolic enzymes and transporters in in vitro assays.

Pharmacokinetic Analysis of Carnosic Acid and Carnosol in Standardized Rosemary Extract and the Effect on the Disease Activity Index of DSS-Induced Colitis.

Rosemary extract (RE) is an approved food preservative in the European Union and contains dietary phytochemicals that are beneficial for gastrointestinal health. This study investigated the effects of RE on dextran sodium sulfate (DSS)-induced colitis and also determined the pharmacokinetics of dietary phytochemicals administered to mice via oral gavage. Individual components of rosemary extract were separated and identified by LC-MS/MS. The pharmacokinetics of two major diterpenes from RE, carnosic acid (CA) and carnosol (CL), administered to mice via oral gavage were determined. Then, the effect of RE pre-treatment on the disease activity index (DAI) of DSS-induced colitis in mice was investigated. The study determined that 100 mg/kg RE significantly improved DAI in DSS-induced colitis compared to negative control. Sestrin 2 protein expression, which increased with DSS exposure, was reduced with RE treatment. Intestinal barrier integrity was also shown to improve via fluorescein isothiocyanate (FITC)-dextran administration and Western blot of zonula occludens-1 (ZO-1), a tight junction protein. Rosemary extract was able to improve the DAI of DSS-induced colitis in mice at a daily dose of 100 mg/kg and showed improvement in the intestinal barrier integrity. This study suggests that RE can be an effective preventative agent against IBD.

Inhibition of CDK2/CyclinE1 by xanthones from the mangosteen (Garcinia mangostana): a structure-activity relationship study.

Uncontrolled regulation of cyclin dependent kinases (CDKs) has negative implications in many cancers and malignancies and has recently led to the approval of select CDK inhibitors. Herein we present data reporting that xanthones, a class of compounds isolated from the purple mangosteen (Garcinia mangostana) fruit, can inhibit CDK2/CyclinE1. We evaluated nine different xanthones, including α-mangostin, ß-mangostin, γ-mangostin, gartanin, 8-desoxygartanin, garcinone C, garcinone D, 9-hydroxycalabaxanthone, and 3-isomangostin for toxicity in 22Rν1 (prostate cancer cells) and MDA-MB-231 (breast cancer cells). All compounds dose-dependently inhibited the viability of both cell lines. A cell free biochemical assay was performed to determine if selected phytochemicals inhibited CDK2/CyclinE1. γ-Mangostin and α-mangostin were the strongest inhibitors, respectively. The results suggest that the position of key functional groups including hydroxyl and isoprenyl groups contribute to the CDK2 inhibitory effect. Taken together, the evidence suggests that xanthones can directly target CDK2 providing a possible explanation for their therapeutic potential.

Development of a method for clinical pharmacokinetic testing to allow for targeted Melphalan dosing in multiple myeloma patients undergoing autologous transplant.

AIMS: High dose melphalan (HDM) and autologous stem cell transplant (ASCT) is standard of care for multiple myeloma (MM), but there is significant variability in melphalan exposure (area under the plasma drug concentration-time curve, AUC) when using body surface area-based dosing. Our aim was to establish a method of pharmacokinetic (PK) testing for real-time melphalan dose adjustments. METHODS: We performed a prospective PK study of melphalan 140 or 200 mg/m2 in MM patients undergoing ASCT. Twenty MM patients were administered HDM on days -2 and - 1, with PK sampling at 8-10 time points. PK testing was performed on day -2 in all patients, and on day -1 in 5 patients. RESULTS: Less than 20% interpatient variation in the day -2 and - 1 AUC was observed. The day -2 range in AUC (4.95-11.28 mg h/L) confirmed significant interpatient variability. The hypothetical total dose ranged from 133-302 mg/m2 to achieve the total median AUC. A 4-time point AUC (0, 30, 150 and 240 min) highly correlated with the AUC from the 8-time point schedule. A higher AUC correlated with increased risk of febrile neutropenia (P = .05). CONCLUSION: Here we outline the methods to establish novel melphalan dosing using PK testing in MM patients undergoing ASCT to target a desired melphalan AUC.

Structure activity relationship of xanthones for inhibition of Cyclin Dependent Kinase 4 from mangosteen (Garcinia mangostana L.).

The mangosteen fruit is a popular Southeast Asian fruit consumed for centuries. There have been a variety of xanthones isolated from the fruit, bark, roots and leaves with each having unique chemical and physical properties. Previously, the most abundant xanthone α-mangostin has been shown to inhibit CDK4. Herein we describe the role of selected xanthones from the mangosteen inhibiting CDK4. The evidence we provide here is that key functional groups are required to inhibit the CDK4 protein to prevent the phosphorylation of downstream targets critical to inhibiting uncontrolled cell cycle progression. To define the properties of xanthones for inhibiting CDK4 we utilized a cell free biochemical assay to identify inhibitors of CDK4. The following xanthones were used for the analysis: α-mangostin, ß-mangostin, γ-mangostin, gartanin, 8-desoxygartanin, garcinone C and garcinone D, 9-hydroxycalabaxanthone, and 3-isomangostin These results further substantiate the unique pharmacological properties of individual xanthones and how a mixture of xanthones may be responsible for a multi-targeted effect in cell based pharmacology systems.

Carnosol, a dietary diterpene from rosemary (Rosmarinus officinalis) activates Nrf2 leading to sestrin 2 induction in colon cells.

BACKGROUND: Rosemary is abundant with phytochemicals and has recently been approved as an antioxidant food preservative in the European Union. The safety of rosemary is well established, however, the benefits on gastrointestinal health are less known. Our overall hypothesis is that the phytochemicals in rosemary including carnosol have the potential to promote gastrointestinal health by activation of the antioxidant sestrin-2 when consumed in our diet. METHODS: Colon cells HCT116 and SW480 were treated with carnosol and evaluated by MTT, immunofluorescence, ELISA, and Western blot analysis to understand the modulation of the PERK/Nrf2/Sestrin-2 pathway. RESULTS: Carnosol was found to modulate PERK and increase the concentration of nuclear Nrf2. Furthermore, a downstream marker of Nrf2 expression, Sestrin-2 was shown to be upregulated. CONCLUSION: Based on these observations carnosol modulates the PERK and Nrf2 pathways along with increased expression of sestrin-2, a known stress inducible antioxidant.

Histopathological and morphological studies on natural Cysticercus fasciolaris infection in liver of Wistar rats.

Descriptive morphology of multifocal hepatic cysts found in eight of forty five (17.78 %) Wistar rats sacrificed during pharmacological studies related to herbal formulations was studied. The creamish to white cysts were of varying sizes, ranging from 3-8 mm in diameter. Morphological studies of these cysts depicted the presence of metacestodes of Taenia taeniaeformis i.e. Cysticercus fasciolaris inside them. The scolex of metacestode revealed four suckers and rostellum armed with two distinct rows of characteristic pen knife shaped hooks (characteristics of taeniid cestodes). The average size of large hooks was 392.92 ± 10.12 µ and that of small hooks was 240.64 ± 14.26 µ. The average size of suckers was 304.36 ± 12.33 µ. Histopathology of hepatic tissue surrounding the cysts revealed zones of fatty change, inflammation, granulation tissue and metaplasia. However, the histopathology of stomach and small intestines didn't show any significant lesions.

Acaricidal activity of leaf extracts of Dalbergia sissoo Roxb. (Fabaceae) against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus.

Resistance status of Rhipicephalus (Boophilus) microplus against synthetic pyrethroids was assessed by larval packet test which revealed level I and II resistance against cypermethrin and deltamethrin, respectively. Adult immersion test was employed to study the acaricidal activity of leaf extracts of Dalbergia sissoo (sheesham) against these ticks. Mortality and fecundity of ticks exposed to sheesham leaf aqueous (SLA) and ethanolic (SLE) extracts were evaluated at concentrations of 0.625, 1.25, 2.5, 5.0 and 10.0% and controls (distilled water and 10% ethanol). Higher acaricidal activity was recorded in SLA with a lower LC50 (95% CL) value of 1.58% (0.92-2.71%) than SLE [5.25% (4.91-5.63%)]. A significant decrease in egg mass weight and reproductive index was recorded in treated ticks along with an increase in percent inhibition of oviposition. A complete inhibition of hatching was recorded in eggs laid by ticks treated with higher concentrations of SLA, whereas, SLE exhibited no effect on hatching percentage.

Emu oil offers protection in Crohn's disease model in rats.

BACKGROUND: Emu oil is a product of animal origin used for the treatment of inflammation, burns etc. as a part of aboriginal medicine in Australia. Crohn's disease is a common inflammatory manifestation in humans and other animal species relating to the ulceration and digestive disturbances in upper gastro-intestinal tract. Aloe vera is commonly used substance from plant sources for inflammation, wound healing and various other properties. Given the difference in the source of the substances all the while playing a similar therapeutic role in different parts of the world, the present investigation was undertaken to evaluate the protective effect of aloe vera and emu oil alone and in combination; in comparison to sulfasalazine (Allopathic drug) as an alternative for the treatment of Crohn's disease. METHODS: Wistar albino rats were divided into six groups with two sub-groups of six animals each. After pre-treating the animals with sulfasalazine, aloe vera, emu oil and their combination for five consecutive days, the animals were sub-cutaneously administered indomethacin on 4(th) and 5(th) day and each sub-group was sacrificed on day 6 and 9. After sacrifice, serum and intestine of these animals was collected. Intestine length from duodenum till caecum was measured for estimating relative organ weight and disease activity index. Part of intestine was preserved in formalin for histopathology while the rest was used for analysis of oxidative parameters and myeloperoxidase. Serum collected was used for measuring alkaline phosphatase and cholesterol. RESULTS: Assessment of the parameters in treatment groups indicated that the combination of aloe vera and emu oil resulted in better protection by suppressing the oxidative (P < 0.05) and histomorphological changes indicating a enhanced effect of these two agents which was found to be better than sulfasalazine. CONCLUSION: The combination of emu oil and aloe vera exhibited enhanced effect resulting in significant protection from indomethacin induced ulceration. This might be due to the different mechanism of anti-inflammatory effects (Salicylic acid in aloe vera and n3, n6 fatty acids acting as pseudosubstrates to cyclooxygenase enzyme) of components of the animal and plant products tested.

In vitro acaricidal activity of Murraya koenigii (L.) Spreng (Rutaceae) extracts against synthetic pyrethroid-resistant Rhipicephalus (Boophilus) microplus.

Larval packet test was used for detection of resistance status against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids in Rhipicephalus (Boophilus) microplus collected from Faridkot district, Punjab (India). The slope of mortality, lethal concentration for 50 % (LC50) and resistance levels were determined from the regression graphs of probit mortality of ticks plotted against log values of increasing concentrations of cypermethrin and deltamethrin. Results indicated presence of resistance of levels I and II against cypermethrin (resistance factor (RF) = 2.82) and deltamethrin (RF = 8.44), respectively. Adult immersion test was used to assess the acaricidal activity of aqueous (MLAq), ethanol (MLE), chloroform (MLC), acetone (MLA) and hexane (MLH) extracts of leaves of Murraya koenigii against these synthetic pyrethroid (SP)-resistant engorged adult females of R. (B.) microplus by determination of per cent adult mortality, reproductive index (RI), per cent inhibition of oviposition (%IO) and hatching rate. The per cent mortality caused by various extracts at concentrations ranging from 0.625 to 10.0% varied from 0.0 to 100.0% with maximum per cent mortality of 10.0, 100.0, 70.0, 40.0 and 10.0 recorded against MLAq, MLE, MLC, MLA and MLH, respectively. Among all extracts, the highest acaricidal property against SP-resistant R. (B.) microplus was exhibited by the MLE as it showed the minimum LC50 [95% confidence limit (CL)] values of 2.97% (2.82-3.12%), followed by MLC as 10.26% (8.84-11.91 %) and MLA as 18.22% (16.18-20.52%). The average egg mass weight recorded in live ticks treated with various concentrations of different extracts was lower than the respective control group ticks and was significantly (p < 0.01) lower in ticks treated with MLH extract. However, no significant effect on hatchability of eggs of treated groups when compared to control was recorded. A significant (p < 0.05) decrease in the RI was recorded in MLH extract-treated ticks, and the %IO varied from 0.07 to 34.73% with various extracts and was recorded maximum with highest concentration of MLH. The results of the current study indicate that the extracts of M. koenigii can be used for control of SP-resistant ticks.

Pharmacokinetic-pharmacodynamic integration of enrofloxacin and its metabolite ciprofloxacin in buffalo calves.

The present study was planned with an objective to test the pharmacokinetics of a new formulation of enrofloxacin (Flobac® SA) in buffalo calves. The drug was administered at the dose rate of 7.5 mg kg-1 body weight through the intravenous (i.v.) and intramuscular (i.m.) route followed by plasma collection and analysis at different time intervals. After analysis, using High Performance Liquid Chromatography - Ultraviolet, various pharmacokinetic parameters were calculated using visual fit for compartmental analysis, followed by integration with pharmacodynamic parameters against Escherichia coli and Pasteurella multocida. Although total area under plasma drug concentration time curve was higher through the i.v. route, mean residence time and metabolic conversion ratio was higher following administration by the i.m. route indicating longer persistence of the drug in body. Overall i.m. bioavailability of the parent compound with its metabolite was found to be 91%. Upon, Pharmacokinetic-Pharmacodynamic integration, all the parameters indicated significant antibacterial activity. It can be concluded that the dose of enrofloxacin used in the present study can be administered to contain infections caused by P. multocida and E. coli in buffalo calves.

Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum.

Larval packet test was used for detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids, in the multi-host tick Hyalomma anatolicum collected from district Moga, Punjab (India). Results indicated the presence of level I resistance against deltamethrin (RF = 2.81), whereas the tick isolate was susceptible to cypermethrin (RF = 0.2). The aqueous and ethanolic extracts of leaves of Cymbopogon winterianus, Vitex negundo and Withania somnifera along with roots of Vitex negundo were assessed for their acaricidal activity against the larvae of deltamethrin resistant H. anatolicum. The efficacy was assessed by measuring per cent larval mortality and determination of LC50 values. The various ethanolic extracts produced a concentration dependent increase in larval tick mortality, whereas the aqueous extracts exhibited a much lower mortality. The highest mortality (93.7 ± 0.66 %) was observed at the 5.0 % concentration of ethanolic extract of leaves of C. winterianus and the lowest LC50 value (0.011 %) was recorded for ethanolic extracts of leaves of V. negundo. The results indicated that these plant extracts have potential to be developed as herbal acaricides.

Hematological Alterations on Sub-acute Exposure to Flubendiamide in Sprague Dawley Rats.

BACKGROUND: Pesticide poisoning is a common occurrence around the world. Pesticides can act on various body systems resulting in toxicity. Flubendiamide is a new generation pesticide, reported to have better activity against Lepidopteran insects. The present study was carried out with an objective to analyze the effects of flubendiamide sub-acute exposure on hematology of rats. MATERIALS AND METHODS: Male and female Sprague Dawley (SD) rats (9-11 weeks) were divided into five groups with six animals in each group. First group served as control, while the rest were exposed to ascending oral doses of flubendiamide (125, 250, 500 and 1000 mg/kg) for 28 days. After the trial period, blood was collected in heparinized vials and analyzed using Siemens ADVIA 2120(®) autoanalyzer. Various erythrocytic, platelet and leukocyte parameters were measured and analyzed using statistical tests by one-way analysis of variance (ANOVA) and t-test using Statistical Package for Social Sciences (SPSS)(®) 20 software. RESULTS: After processing the data through statistical analysis, it was observed that the effect of flubendiamide exposure on female rats was negligible. The only significant change observed in the female rats was that in total erythrocytic count, while rest of the parameters showed non-significant bidirectional changes. In males, many parameters viz., total leukocyte count (TLC), total erythrocyte count (TEC), packed cell volume (PCV), mean corpuscular volume (MCV), platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), hemoglobin distribution width (HDW), large platelets (LPT) and plateletcrit (PCT) expressed significant difference when compared to control. CONCLUSION: Many of the changes were dose independent, but sex specific. This lead to the hypothesis that saturation toxicokinetics might be one of the reasons for this varied response, which can only be evaluated after further testing.

Acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus.

Detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids (SPs) against Rhipicephalus (Boophilus) microplus collected from Moga, Punjab (India) was carried out using larval packet test. Results indicated the presence of resistance of level I and III against cypermethrin (resistance factors (RF) = 4.67) and deltamethrin (RF = 34.2), respectively. Adult immersion test was used to assess the acaricidal activity of aqueous and ethanolic extracts of leaves of Cymbopogon winterianus, Vitex negundo, and Withania somnifera along with roots of V. negundo against the SP resistant engorged females of R. (B.) microplus. The efficacy of various extracts was assessed by estimation of percent adult mortality, reproductive index (RI), percent inhibition of oviposition (%IO), and hatching rate. A concentration dependent increase in tick mortality was recorded which was more marked with various ethanolic extracts, and highest mortality was recorded in ticks treated with ethanolic extract of leaves of C. winterianus. The LC50 values were determined by applying regression equation analysis to the probit transformed data of mortality for various aqueous and ethanolic extracts. Acaricidal property was recorded to be higher in ethanolic extracts, and high activity was found with the ethanolic extract of leaves of C. winterianus with LC50 (95% CL) values of 0.46% (0.35-0.59%), followed by W. somnifera as 5.21% (4.45-6.09%) and V. negundo as 7.02% (4.58-10.74%). The egg mass weight of the live ticks treated with different concentrations of the various extract was significantly (p < 0.01) lower than that of control ticks; consequently, the RI and the %IO value of the treated ticks were reduced. Further, complete inhibition of hatching was recorded in eggs laid by ticks treated with ethanolic extracts of leaves of V. negundo and aqueous extracts of leaves of W. somnifera. The results of the current study indicate that extracts of C. winterianus, V. negundo, and W. somnifera can be used for the control of SP resistant ticks.