Rp-Hplc Method Development and Validation for the Estimation of All-Trans Retinoic Acid in Plant Extract of Broccoli and Spinach.

The present study is aimed to develop a validated HPLC method for the quantification of All-trans retinoic acid in plant extracts namely Broccoli (Brassica oleracea), Spinach (Spinacia oleracea). Analytical methods development and validation [1-4] plays a crucial role in the discovery, development, and manufacture of pharmaceuticals. Pharmaceutical products formulated with more than one drug, typically referred to as combination products, are intended to meet previously unmet patients need by combining the therapeutic effects of two or more drugs in one product. These combination products can present daunting challenges to the analytical chemist responsible for the development and validation of analytical methods. High performance liquid chromatography (HPLC) for drug products containing more than one active ingredient. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products. The drugs may be either new entities or partial structural modification of the existing one. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical methods for such drugs. Importance of analytical chemistry is to gain information about the qualitative and quantitative composition of substance and chemical species, that is, to find out what a substance is composed of and exactly how much it is present. Pharmaceutical analysis deals with the analysis of the pharmaceutical substances. It is generally known that pharmaceutical is a chemical entity of therapeutic interest. Pharmaceutical analysts in research and development (R&D) of pharma industry plays a vital role in new drug development and follow up activities to assure that, a new drug product meets the established standards, its stability, and continued to meet the purported quality throughout its shelf life. The developed RP-HPLC method allows rapid and precise determination of All-trans retinoic acid content in Broccoli ( Brassica oleracea) and Spinach (Spinacia oleracea) extract with an economical mobile phase. All-trans retinoic acid was higher in 6th week crop (0.08mg) of Broccoli ( Brassica oleracea) and 4th week crop (0.370mg) of Spinach ( Spinacia oleracea). The scope of the present work is to expand the optimization of the chromatographic conditions, to develop RP-HPLC method, a series of mobile phases were tried, among the various mobile phases ortho phosphoric acid and acetonitrile mixture (55:45) was found to be an ideal mobile phase, since it gave a good resolution and peak shapes with perfect optimization. The flow rate was optimized at 1 ml/min. The Linearity and correlation coefficient of All-trans retinoic acid was found to be 10-50 μg/ml 0.9998 respectively. The limit of detection for All-trans retinoic acid was found to be 40 μg/ml and the limit of quantification was found to be 120 μg/ml. The method was known to be accurate with %recoveries ranging from 97.52 to 98.97%. The accuracy and precision study was performed at two levels intra-day and inter-day. The developed method showed good accuracy and precision. The inter-day accuracy ranges between 99.14-100.94% with %RSD between 0.83-0.86. The intra-day accuracy range between 99.30-100.24% with %RSD between 0.81-0.87. The isocratic elution technique developed for the determination of All-trans retinoic acid in Broccoli (Brassica oleracea) and Spinach ( Spinacia oleracea) extract is ideally suited for rapid and routine analysis. This method shows good reproducibility of the results. Furthermore this method was simple, sensitive, accurate and can be applied to all kinds of plant extract

Development and validation of dissolution test for Metoprolol sustained release capsules

Dissolution test for sustained release capsules of Metoprolol 125 mg was developed and validated according to FDA and ICH guidelines. Metoprolol coated pellets were coated with microcrystalline wax and glyceryl distearate for slow release of drug. The dissolution method which uses USP apparatus I (Basket) with rotating at 100 rpm, 900 ml of different dissolution medium, ultra violet spectroscopy for quantification was demonstrated to be robust, discriminating and transferable. Dissolution tests conditions were selected after it was demonstrated that the Metoprolol rapidly dissolved in the aqueous media over the pH range of 1.2 to 7.

Natural genetic variation for root traits among diversity lines of maize (Zea Mays L.)

Maize (Z. mays L.) is the third most important food grain for humankind after rice and wheat. Maize is mostly grown under rain-fed conditions and among the cereals, it is the second most susceptible to drought next to rice. Constitutive variation for root traits is an important adaptation under drought prone conditions. The objective of this study is to screen the twenty five diverse parental lines used in the maize nested association mapping panel along with the common parental line, B73, for constitutive root traits (including rooting depth and root biomass) and shoot traits. All the lines were grown with five replications in 72 cm deep pots containing a turface:sand mixture (2:1 v/v) for 30 days under well-watered conditions in a temperature and humidity controlled green house. Significant variation existed among the diverse lines for root length, root biomass, shoot length, and leaf area. The average root length ranged from 17.5 to 106 cm. The genotypes with a deep root system also recorded greater root biomass and leaf area. The natural genetic variation exhibited by these lines could be exploited to identify potential quantitative trait loci controlling root architecture. Using the nested association mapping populations that were developed from these diverse lines, would allow for in-depth analysis and fine-mapping of prospective candidate genes for root architecture in maize

Molecular Modeling-Based Evaluation of hTLR10 and Identification of Potential Ligands in Toll-Like Receptor Signaling

Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogens based on distinct molecular signatures. The human (h)TLR1, 2, 6 and 10 belong to the hTLR1 subfamilies, which are localized in the extracellular regions and activated in response to diverse ligand molecules. Due to the unavailability of the hTLR10 crystal structure, the understanding of its homo and heterodimerization with hTLR2 and hTLR1 and the ligand responsible for its activation is limited. To improve our understanding of the TLR10 receptor-ligand interaction, we used homology modeling to construct a three dimensional (3D) structure of hTLR10 and refined the model through molecular dynamics (MD) simulations. We utilized the optimized structures for the molecular docking in order to identify the potential site of interactions between the homo and heterodimer (hTLR10/2 and hTLR10/1). The docked complexes were then used for interaction with ligands (Pam3CSK4 and PamCysPamSK4) using MOE-Dock and ASEDock. Our docking studies have shown the binding orientations of hTLR10 heterodimer to be similar with other TLR2 family members. However, the binding orientation of hTLR10 homodimer is different from the heterodimer due to the presence of negative charged surfaces at the LRR11-14, thereby providing a specific cavity for ligand binding. Moreover, the multiple protein-ligand docking approach revealed that Pam3CSK4 might be the ligand for the hTLR10/2 complex and PamCysPamSK4, a di-acylated peptide, might activate hTLR10/1 hetero and hTLR10 homodimer. Therefore, the current modeled complexes can be a useful tool for further experimental studies on TLR biology

A continuous infusion of a minor histocompatibility antigen-immunodominant peptide induces a delay of male skin graft rejection.

We previously reported that an inhibition of antigen-specific Interferon-gamma release and cytotoxicity occurs after a continuous infusion of an HY immunodominant peptide although this treatment is not able to cause a significant delay of male skin grafts rejection. In vivo administration of high doses of an HY peptide, through mini-osmotic pumps, in naïve female mice was used to study the effects on the male skin grafts rejection. A continuous infusion of 1mg of an HY peptide induces a significant delay of male skin graft rejection. In vitro HY-specific Interferon-gamma release was inhibited adding peptide-specific suppressor cells: the ability to inhibit Interferon-gamma release was evident when two HY peptides were present on the same dendritic cells indicating that the suppressor cells exert "linked-suppression". The phenotype of the suppressor cells is CD8(+)CD28(-) and these cells express more CD62 ligand and FOXP3 than controls. Suppressor cells were able to cause a significant delay of rejection of male skin grafts when injected in naive female mice. The inhibitory effects of these suppressor cells seem to be due to the impairment of antigen presentation; down-regulation of B7 molecules on dendritic cells occurred. Taken all together, our data demonstrate that a continuous infusion of an immunodominant HY peptide induces a T CD8 suppressor subset able to inhibit immune responses to male tissues and cells

Structure-Function Relationship of Cytoplasmic and Nuclear IκB Proteins: An In Silico Analysis

Cytoplasmic IκB proteins are primary regulators that interact with NF-κB subunits in the cytoplasm of unstimulated cells. Upon stimulation, these IκB proteins are rapidly degraded, thus allowing NF-κB to translocate into the nucleus and activate the transcription of genes encoding various immune mediators. Subsequent to translocation, nuclear IκB proteins play an important role in the regulation of NF-κB transcriptional activity by acting either as activators or inhibitors. To date, molecular basis for the binding of IκBα, IκBβ and IκBζ along with their partners is known; however, the activation and inhibition mechanism of the remaining IκB (IκBNS, IκBε and Bcl-3) proteins remains elusive. Moreover, even though IκB proteins are structurally similar, it is difficult to determine the exact specificities of IκB proteins towards their respective binding partners. The three-dimensional structures of IκBNS, IκBζ and IκBε were modeled. Subsequently, we used an explicit solvent method to perform detailed molecular dynamic simulations of these proteins along with their known crystal structures (IκBα, IκBβ and Bcl-3) in order to investigate the flexibility of the ankyrin repeat domains (ARDs). Furthermore, the refined models of IκBNS, IκBε and Bcl-3 were used for multiple protein-protein docking studies for the identification of IκBNS-p50/p50, IκBε-p50/p65 and Bcl-3-p50/p50 complexes in order to study the structural basis of their activation and inhibition. The docking experiments revealed that IκBε masked the nuclear localization signal (NLS) of the p50/p65 subunits, thereby preventing its translocation into the nucleus. For the Bcl-3- and IκBNS-p50/p50 complexes, the results show that Bcl-3 mediated transcription through its transactivation domain (TAD) while IκBNS inhibited transcription due to its lack of a TAD, which is consistent with biochemical studies. Additionally, the numbers of identified flexible residues were equal in number among all IκB proteins, although they were not conserved. This could be the primary reason for their binding partner specificities