Characterizing the Stability of Angiotensin II in 0.9% Sodium Chloride Using High Performance Liquid Chromatography and Liquid Chromatography Tandem Mass Spectrometry.

Purpose: The purpose of this study was to evaluate the stability of angiotensin II in 0.9% sodium chloride for up to 5 days. Methods: We prepared angiotensin II dilutions, by aseptically diluting 2.5 mg (1 mL) in 249 mL 0.9% sodium chloride creating a solution of 10 000 ng/mL. Admixtures were stored under refrigeration (5 ± 3°C). Stability of the dilution was assessed by: preservation of clarity, consistency of pH, and retention of concentration. Solutions were sampled at times 0, 24, 48, 72, 96, 120 hours. Solutions were analyzed via High-Performance Liquid Chromatography (HPLC-UV) and Liquid Chromatography Mass Spectrometry (LC-MS/MS). Retention of concentration was set a priori at > 90% of initial concentration. Results: Clarity, color, and pH at all sample time points remained constant. Both methods of analysis confirmed similar results. When stored under refrigeration, the concentration of angiotensin II solution remained above 90% of initial concentration throughout the entire sampling period. Conclusions: Angiotensin II in 0.9% sodium chloride stored in infusion bags under refrigeration (5 ± 3°C) maintained at least 90% of their original concentrations for up to 5 days. Stability was also demonstrated based on turbidity, color, and pH assessment.

Terpenes: Modulating anti-inflammatory signaling in inflammatory bowel disease.

Inflammatory Bowel Disease (IBD) are autoimmune diseases characterized by chronic intestinal inflammation. Considered a western disease, IBD incidence in newly developed countries is skyrocketing. Accordingly, global prevalence is steadily increasing. There are two major IBD phenotypes, ulcerative colitis (UC) and Crohn's disease (CD). UC manifests as uninterrupted inflammation localized in the colon and rectum. Meanwhile, CD presents as interrupted inflammation that can occur throughout the digestive tract. As a result, therapeutics have focused on anti-inflammatory approaches for its treatment. Unfortunately, only 50% of patients benefit from current Food and Drug Administration approved treatments, and all are associated with serious adverse effects. Thus, there is a need for safer and novel therapeutics to increase the efficacy in this population. One aspect that is critical in understanding IBD is how food and phytochemicals therein may be associated with modifying the pathogenesis of IBD. A variety of retrospective and prospective studies, and clinical trials have shown benefits of plant-rich diets on the prevention and symptomatic improvement of IBD. The Mediterranean diet is rich in vegetables, fruits, legumes, and herbs; and characterized by the abundance of anti-inflammatory phytochemicals. An understudied phytochemical class enriched in this diet is terpenes; isoprene-based molecules are widely available in Mediterranean herbs and citrus fruits. Various terpenes have been evaluated in different IBD models. However, some present contradictory or inconclusive results. Therefore, in this review we evaluated preclinical studies of terpenes modulating basic inflammatory signaling related to IBD.

Covalent Modification of Proteins by Plant-Derived Natural Products: Proteomic Approaches and Biological Impacts.

Plant-derived natural products (NPs) with electrophilic functional groups engage various subsets of the proteome via covalent modification of nucleophilic cysteine residues. This electrophile-nucleophile interaction can change protein conformation, alter protein function, and modulate their biological action. The biological significance of these covalent protein modifications in health and disease is increasingly recognized. One way to understand covalent NP-protein interactions is to utilize traditional proteomics and modern mass spectrometry (MS)-based proteomic strategies. These strategies have proven effective in uncovering specific NP protein targets and are critical first steps that allow for a much deeper understanding of the ability of NPs to modulate cellular processes. Here, plant-derived NPs that covalently modify proteins are reviewed, the biological significance of these covalent modifications, and the different proteomic strategies that have been employed to study these NP-protein interactions.

Prenylated xanthones from mangosteen (Garcinia mangostana) activate the AhR and Nrf2 pathways and protect intestinal barrier integrity in HT-29 cells.

Xanthones from the tropical fruit mangosteen (Garcinia mangostana) display anti-inflammatory and anti-oxidative activities. Here, we isolate and identify potential inducers of the aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways from mangosteen using a bioassay-guided strategy. Mangosteen fruit pericarp extracts were subjected to sequential solvent extractions, followed by chromatography coupled with NMR spectroscopy and mass spectrometric analyses for identification and isolation of pure compounds. Isolation of active fractions led to seven prenylated xanthones that were identified and subsequently evaluated for bioactivity. In vitro luciferase reporter cellular assays using H1L6.1c3 (AhR induction) and HepG2-ARE (Nrf2 induction) were used to identify AhR and Nrf2 activators. All seven prenylated xanthones displayed AhR inducing activity, whereas only five xanthones activated Nrf2. Garcinone D (GarD) significantly upregulated AhR/Cyp1a1 and Nrf2/HO-1 protein expression and enhanced zonula occludens-1 and occludin protein levels in HT-29 cells. In addition, GarD inhibited oxidative stress-induced intestinal epithelial barrier dysfunction by enhancing tight junction (TJ) proteins and inhibition of reactive oxygen species production. Inhibition of AhR by pretreating cells with an AhR antagonist revealed that the AhR pathway is required for the improved epithelial barrier functions of GarD. These results highlight a dual mechanism by GarD that confers protection against intestinal epithelial barrier dysfunction.

Valorization of pomelo (Citrus grandis Osbeck) peel: A review of current utilization, phytochemistry, bioactivities, and mechanisms of action.

Citrus grandis Osbeck, commonly known as "pomelo" or "shaddock," is the largest citrus fruit, the peel of which is a well-known agricultural residual waste. Pomelo peel offers a wide range of components such as essential oils, polysaccharides, and phytochemicals with potential food applications. Utilization of pomelo peel to recover these components is an important step toward agricultural sustainability. This review covers pomelo peel utilization opportunities beyond conventional composting and animal feed production, and critically examines value-added uses via the recovery of potentially bioactive components. The peel of pomelo accounts for approximately 30% of the total fruit weight and contains phytochemicals, including aroma-active volatiles, pectin, flavonoids, phenolic acids, carotenoids, coumarins, and polysaccharides. Recovery of these phytochemicals offers an opportunity for value-added utilization such as the development of enriched or functional foods and nutraceuticals. The health-promoting and therapeutic potential of pomelo peel extracts and isolated pure compounds have been evaluated through numerous in vitro and in vivo studies that revealed a wide range of bioactivities, including hypolipidemic, hypoglycemic, antioxidant, antimicrobial, anti-inflammatory, and anticancer effects. Preclinical evidence highlights multifaceted molecular and signaling events that possibly underlie the said bioactive potential. Overall, the pomelo processing industry offers a great opportunity to recover or produce valuable products from the large amounts of residual wastes it generates. It is envisaged that a thorough understanding of the bioactive components of pomelo peel, their functional and nutraceutical applications, and mode of actions will benefit the food industry.

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.

Lupeol inhibits proliferation of human prostate cancer cells by targeting beta-catenin signaling.

Lupeol, a dietary triterpene, was shown to decrease serum prostate-specific antigen levels and inhibit the tumorigenicity of prostate cancer (CaP) cells in vivo. Here, we show that Lupeol inhibits the proliferative potential of CaP cells and delineated its mechanism of action. Employing a focused microarray of human CaP-associated genes, we found that Lupeol significantly modulates the expression level of genes such as ERBB2, tissue inhibitor of metalloproteinases-3, cyclin D1 and matrix metalloproteinase (MMP)-2 that are known to be associated with proliferation and survival. A common feature of these genes is that all of them are known to either regulate or act as downstream target of beta-catenin signaling that is highly aberrant in CaP patients. Lupeol treatment significantly (1) reduced levels of beta-catenin in the cytoplasmic and nuclear fractions, (2) modulated expression levels of glycogen synthase kinase 3 beta (GSK3beta)-axin complex (regulator of beta-catenin stability), (3) decreased the expression level and enzymatic activity of MMP-2 (downstream target of beta-catenin), (4) reduced the transcriptional activation of T Cell Factor (TCF) responsive element (marker for beta-catenin signaling) in pTK-TCF-Luc-transfected cells and (5) decreased the transcriptional activation of MMP-2 gene in pGL2-MMP-2-Luc-transfected cells. Effects of Lupeol treatment on beta-catenin degradation were significantly reduced in CaP cells where axin is knocked down through small interfering RNA transfection and GSK3beta activity is blocked. Collectively, these data suggest the multitarget efficacy of Lupeol on beta-catenin-signaling network thus resulting in the inhibition CaP cell proliferation. We suggest that Lupeol could be developed as an agent for chemoprevention as well as chemotherapy of human CaP.

Curcumin for chemoprevention of colon cancer.

The most practical approach to reduce the morbidity and mortality of cancer is to delay the process of carcinogenesis through the use of chemopreventive agents. This necessitates that safer compounds, especially those derived from natural sources must be critically examined for chemoprevention. A spice common to India and the surrounding regions, is turmeric, derived from the rhizome of Curcuma longa. Pre-clinical studies in a variety of cancer cell lines including breast, cervical, colon, gastric, hepatic, leukemia, oral epithelial, ovarian, pancreatic, and prostate have consistently shown that curcumin possesses anti-cancer activity in vitro and in pre-clinical animal models. The robust activity of curcumin in colorectal cancer has led to five phase I clinical trials being completed showing the safety and tolerability of curcumin in colorectal cancer patients. To date clinical trials have not identified a maximum tolerated dose of curcumin in humans with clinical trials using doses up to 8000mg per day. The success of these trials has led to the development of phase II trials that are currently enrolling patients. Overwhelming in vitro evidence and completed clinical trials suggests that curcumin may prove to be useful for the chemoprevention of colon cancer in humans. This review will focus on describing the pre-clinical and clinical evidence of curcumin as a chemopreventive compound in colorectal cancer.

S100A4 accelerates tumorigenesis and invasion of human prostate cancer through the transcriptional regulation of matrix metalloproteinase 9.

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.