Pradeep Paudel

Characterization of the inhibitory activity of natural tanshinones from Salvia miltiorrhiza roots on protein tyrosine phosphatase 1B

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator that plays an important role in many signaling pathways, especially those associated with insulin resistance. In this study, we investigated the anti-diabetic potential of 12 natural tanshinones isolated from Salvia miltiorrhiza (S. miltiorrhiza) Bunge (Lamiaceae), deoxyneocryptotanshinone (1), grandifolia F (2), ferruginol (3), cryptotanshinone (4), tanshinone IIA (5), tanshinol B (6), tanshinone IIB (7), tanshinonal (8), methyl tanshinonate (9), 15,16-dihydrotanshinone I (10), tanshinone I (11), and dehydrodanshenol A (12) and evaluated their inhibitory activity against PTP1B. Tanshinones 4, 6 and 12 exhibited potent PTP1B inhibitory activity with IC50 values of 5.5xa0±xa00.9, 4.7xa0±xa00.4 and 8.5xa0±xa00.5xa0μM, respectively. In addition, tanshinones 1-3, 5 and 7-11 showed promising dose-dependent inhibition of PTP1B over IC50 values ranging from 18.6 to 254.8xa0μM. Enzyme kinetic analysis of PTP1B inhibition revealed that 4 and 6 were mixed -noncompetitive type inhibitors, whereas 12 was a classical-noncompetitive type inhibitor. Furthermore, 4, 6 and 12 were docked with the PTP1B enzyme using molecular docking simulations (AutoDock 4.2) and exhibited negative binding energy (-6.4 to -8.7xa0kcal/mol), indicating high binding affinity to PTP1B active site residues. Structure-activity relationships (SAR) analysis revealed that structural modifications of ring A and furan or dihydrofuran ring D on the basic structure of tanshinones influenced their activity. Overall, results indicated that tanshinones from S. miltiorrhiza are potential anti-diabetic candidates that should be explored in the development of preventive and therapeutic modalities for the treatment of diabetes as well as diabetes-associated complications.

Structure-related protein tyrosine phosphatase 1B inhibition by naringenin derivatives

Naturally occurring flavonoids co-exist as glycoside conjugates, which dominate aglycones in their content. To unveil the structure-activity relationship of a naturally occurring flavonoid, we investigated the effects of the glycosylation of naringenin on the inhibition of enzyme systems related to diabetes (protein tyrosine phosphatase 1B (PTP1B) and α-glycosidase) and on glucose uptake in the insulin-resistant state. Among the tested naringenin derivatives, prunin, a single-glucose-containing flavanone glycoside, potently inhibited PTP1B with an IC50 value of 17.5±2.6µM. Naringenin, which lacks a sugar molecule, was the weakest inhibitor compared to the reference compound, ursolic acid (IC50: 5.4±0.30µM). In addition, prunin significantly enhanced glucose uptake in a dose-dependent manner in insulin-resistant HepG2 cells. Regarding the inhibition of α-glucosidase, naringenin exhibited more potent inhibitory activity (IC50: 10.6±0.49µM) than its glycosylated forms and the reference inhibitor, acarbose (IC50: 178.0±0.27µM). Among the glycosides, only prunin (IC50: 106.5±4.1µM) was more potent than the positive control. A molecular docking study revealed that prunin had lower binding energy and higher binding affinity than glycosides with higher numbers of H-bonds, suggesting that prunin is the best fit to the PTP1B active site cavity. Therefore, in addition to the number of H-bonds present, possible factors affecting the protein binding and PTP1B inhibition of flavanones include their fit to the active site, hydrogen-bonding affinity, Van der Waals interactions, H-bond distance, and H-bond stability. Furthermore, this study clearly depicted the association of the intensity of bioactivity with the arrangement and characterization of the sugar moiety on the flavonoid skeleton.

Structure Related Inhibition of Enzyme Systems in Cholinesterases and BACE1 In Vitro by Naturally Occurring Naphthopyrone and Its Glycosides Isolated from Cassia obtusifolia

Cassia obtusifolia Linn. have been used to improve vision, inflammatory diseases, and as hepatoprotective agents and to promote urination from ancient times. In the present study, we investigated the influence of glycosylation of components of C. obtusifolia and structure-activity relationships (SARs) with respect to the inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are related to Alzheimer’s disease (AD). All six C. obtusifolia-derived compounds, rubrofusarin (1), rubrofusarin 6-O-β-d-glucopyranoside (2), rubrofusarin 6-O-β-d-gentiobioside (3), nor-rubrofusarin 6-O-β-d-glucoside (4), isorubrofusarin 10-O-β-d-gentiobioside (5), and rubrofusarin 6-O-β-d-triglucoside (6) showed promising inhibitory activity against AChE/BACE1. Compounds 3 and 4 showed most significant inhibition against AChE and BACE1, respectively. The SARs results emphasized the importance of gentiobiosyl moiety in the rubrofusarin for AChE inhibition, whereas the presence of hydroxyl group at C-8 and the glucosyl moiety at the C-6 position in the nor-rubrofusarin appeared to largely determine BACE1 inhibition. Kinetics and docking studies showed the lowest binding energy and highest affinity for mixed-type inhibitors, 3 and 4. Hydrophobic bonds interactions and the number of hydrogen bonds determined the strength of the protein-inhibitor interaction. These results suggest that C. obtusifolia and its constituents have therapeutic potential, and that the SARs of its active components are further explored with a view towards developing a treatment for AD.

Protein Tyrosine Phosphatase 1B Inhibition and Glucose Uptake Potentials of Mulberrofuran G, Albanol B, and Kuwanon G from Root Bark of Morus alba L. in Insulin-Resistant HepG2 Cells: An In Vitro and In Silico Study

Type II diabetes mellitus (T2DM) is the most common form of diabetes and has become a major health problem across the world. The root bark of Morus alba L. is widely used in Traditional Chinese Medicine for treatment and management of diabetes. The aim of the present study was to evaluate the enzyme inhibitory potentials of three principle components, mulberrofuran G (1), albanol B (2), and kuwanon G (3) in M. alba root bark against diabetes, establish their enzyme kinetics, carry out a molecular docking simulation, and demonstrate the glucose uptake activity in insulin-resistant HepG2 cells. Compounds 1–3 showed potent mixed-type enzyme inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase. In particular, molecular docking simulations of 1–3 demonstrated negative binding energies in both enzymes. Moreover, 1–3 were non-toxic up to 5 µM concentration in HepG2 cells and enhanced glucose uptake significantly and decreased PTP1B expression in a dose-dependent manner in insulin-resistant HepG2 cells. Our overall results depict 1–3 from M. alba root bark as dual inhibitors of PTP1B and α-glucosidase enzymes, as well as insulin sensitizers. These active constituents in M. alba may potentially be utilized as an effective treatment for T2DM.

Computational insights into β-site amyloid precursor protein enzyme 1 (BACE1) inhibition by tanshinones and salvianolic acids from Salvia miltiorrhiza via molecular docking simulations

The rhizome of Salvia miltiorrhiza has emerged as a rich source of natural therapeutic agents, and its several compounds are supposed to exhibit favorable effects on Alzheimers disease (AD). The present work investigate the anti-AD potentials of 12 tanshinones, three salvianolic acids and three caffeic acid derivatives from S. miltiorrhiza via the inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Among the tested compounds, deoxyneocryptotanshinone (1), salvianolic acid A (13) and salvianolic acid C (15) displayed good inhibitory effect on BACE1 with IC50 values of 11.53u202f±u202f1.13, 13.01u202f±u202f0.32 and 9.18u202f±u202f0.03u202fμM, respectively. Besides this, enzyme kinetic analysis on BACE1 revealed 13, a competitive type inhibitor while 1 and 15 showed mixed-type inhibition. Furthermore, molecular docking simulation displayed negative binding energies (AutoDock 4.2.6u202f=u202f-10.0 to -7.1u202fkcal/mol) of 1, 13, and 15 for BACE1, indicating these compounds bound tightly to the active site of the enzyme with low energy and high affinity. The results of the present study clearly demonstrate that S. miltiorrhiza and its constituents have potential anti-AD activity and can be used as a therapeutic agent for the treatment of AD.

Oligonol promotes glucose uptake by modulating the insulin signaling pathway in insulin-resistant HepG2 cells via inhibiting protein tyrosine phosphatase 1B

Insulin resistance and protein tyrosine phosphatase 1B (PTP1B) overexpression are strongly associated with type 2 diabetes mellitus (T2DM), which is characterized by defects in insulin signaling and glucose intolerance. In a previous study, we demonstrated oligonol inhibits PTP1B and α-glucosidase related to T2DM. In this study, we examined the molecular mechanisms underlying the anti-diabetic effects of oligonol in insulin-resistant HepG2 cells. Glucose uptake was assessed using a fluorescent glucose tracer, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose, and the signaling pathway was investigated by western blotting. Oligonol significantly increased insulin-provoked glucose uptake and decreased PTP1B expression, followed by modulation of ERK phosphorylation. In addition, oligonol activated insulin receptor substrate 1 by reducing phosphorylation at serine 307 and increasing that at tyrosine 895, and enhanced the phosphorylations of Akt and phosphatidylinositol 3-kinase. Interestingly, it also reduced the expression of two key enzymes of gluconeogenesis (glucose 6-phosphatase and phosphoenolpyruvate carboxykinase), attenuated oxidative stress by scavenging/inhibiting peroxynitrite, and reactive oxygen species (ROS) generation, and augmented the expression of nuclear factor kappa B. These findings suggest oligonol improved the insulin sensitivity of insulin-resistant HepG2 cells by attenuating the insulin signaling blockade and modulating glucose uptake and production. Furthermore, oligonol attenuated ROS-related inflammation and prevented oxidative damage in our in vitro model of type 2 diabetes. These result indicate oligonol has promising potential as a treatment for T2DM.

Two new naphthalenic lactone glycosides from Cassia obtusifolia L. seeds

Two new naphthalenic lactone glycosides, (3S)-9,10-dihydroxy-7-methoxy-3-methyl-1-oxo-3,4-dihydro-1H-benzo[g]isochromene-3-carboxylic acid 9-O-β-d-glucopyranoside (1) and (3R)-cassialactone 9-O-β-d-glucopyranoside (2) were isolated from seeds of Cassia obtusifolia Linn., along with five known compounds: (3R)-cassialactone 9-O-β-d-gentiobioside (3), emodin 1-O-β-gentiobioside (4), 1-hydroxyl-2-acetyl-3,8-dimethoxy-naphthalene 6-O-β-d-apiofuranosyl-(1u2009→u20092)-β-d-glucopyranoside (5), rubrofusarin 6-O-β-d-gentiobioside (6), rubrofusarin 6-O-β-d-triglucoside (7). Structures of 1 and 2 were elucidated by NMR and HR-ESI-MS spectroscopic analysis. Their stereochemistry was determined by CD experiment. All compounds were tested for their ability to inhibit the formation of advanced glycation end-products in vitro. Compounds 1, 2, 3, 5, and 6 showed significant in vitro inhibitory activities (IC50 values of 11.63, 23.40, 7.32, 89.03, and 38.89xa0µM, respectively).

Anthraquinone and naphthopyrone glycosides from Cassia obtusifolia seeds mediate hepatoprotection via Nrf2-mediated HO-1 activation and MAPK modulation

Cassia obtusifolia L. seed is one of the most popular traditional Chinese medicine for mutagenicity, genotoxicity, hepatotoxicity, and acute inflammatory diseases. We evaluated the hepatoprotective activity of anthraquinone and naphthopyrone glycosides isolated from the butanol fraction of C. obtusifolia seeds and explored their effects on cell signaling pathways. Continuous chromatographic separation led to the isolation of 1-desmethylaurantio-obtusin 2-O-β-D-glucopyranoside (1), rubrofusarin 6-O-β-D-apiofuranosyl-(1u2009→u20096)-O-β-D-glucopyranoside (2) and rubrofusarin 6-O-β-gentiobioside (3). All glycosides were non-toxic at concentrations up to 80xa0µM. The increased intracellular reactive oxygen species (ROS) and decreased glutathione levels observed after tert-butylhydroperoxide (t-BHP) intoxication were ameliorated by all three glycosides, with compound 3 being the most active. Pretreatment with the three glycosides increased nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated heme oxidase-1 (HO-1) expression. All the glycosides enhanced the phosphorylation of c-Junxa0N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and the dephosphorylation of p38. The protective effects of the anthraquinone and naphthopyrone glycosides against t-BHP-induced oxidative damage in human liver-derived HepG2 cells were due to the prevention of ROS generation and up-regulated activity of HO-1 via Nrf2 activation and modulation of the JNK/ERK/MAPK signaling pathway. The data indicate the potential of these compounds as hepatoprotective agents in pharmaceuticals and/or nutraceuticals.

Comparative study of formulations of ondansetron hydrochloride orodispersible tablets by effervescent and sublimation methods

H metal ion pollution is a serious environment problem that has attracted more and more attentions in recent years. Mercury ion (Hg2+) and lead ion (Pb2+) are two of the most toxic metallic pollutants even at an extremely low concentration. Copper ion (Cu2+), an essential micronutrient element for human life, can cause adverse health effects when present in high concentrations. Considerable efforts have been devoted to the detection of heavy metal ions due to their high toxicity towards the ecosystem and human health. Unlike the traditional detection technologies, we have developed some simple and sensitive biosensors without the requirement of sophisticated instrumentation and skilled personnel. With the combination of DNAzyme, DNA machine and lateral flow biosensor, the visual instrument-free method offers a point-of-use solution for heavy metal ion analysis and provides a basis for the future work aiming at the development of household devices for sensitive detection of various analytes.Purpose: The aim of our present work was to develop and validate a reverse phase high-performance liquid chromatography (RP-HPLC) method for the determination of Decitabine (DCB). The developed method was further applied to observe the degradation of DCB under various stress conditions. Methods: Chromatographic separation was achieved on C18, 250 × 4.6 mm, particle size 5 μm, Agilent column, using ammonium acetate (0.01M) as mobile phase with flow rate of 1mL/min and injection volume was 20 μL. Quantification was carried out with UV detector at 230 nm with a linear calibration curve in the concentration range of 10–100 μg/mL based on peak area. Thus, developed method was validated for linearity, accuracy, precision, and robustness. Results: Linearity was found to be in the range between 10–100 μg/mL with a significantly higher value of correlation coefficient r = 0.9994. The limits of detection (LOD) and the limits of quantification (LOQ) were found to be 1.92μg/mL and 5.82 μg/mL respectively. Moreover, validated method was applied to study the degradation profile of DCB under various stress degradation conditions. Examination of different stress conditions on degradation of DCB showed that its degradation was highly susceptible to oxidative condition as 31.24% of drug was degraded. In acidic and alkaline conditions, the drug was degraded by 21.03% and 12.16% respectively, while thermal and photolytic condition causes least degradation, i.e. 0.21% and 0.3% respectively. Conclusion: The proposed method was found to be sensitive, specific and was successfully applied for the estimation of DCB in bulk drug, and lipid based nanoparticles.Abstract: A validated stability-indicating HPLC method has been reported for the determination of Ilaprazole in bulk drug and tablet. The drug was subjected to the various stress conditions as per the ICH guidelines. The degradation behavior of Ilaprazole was studied under hydrolytic, oxidative, photolytic and thermal conditions and was found to be unstable in almost all conditions except under alkaline and photolytic conditions. The separation of drug and its degraded products was carried out on Kinetex C-18 100A (5µ, 250×4.6 mm) column. The initial mobile phase composition used was Acetonitrile and water in the ratio 50:70v/v for 1 min then changed to 70:30v/v in next 6 min and finally equilibrated back to initial composition in 14min. The method was applied for the determination of Ilaprazole in marketed tablet formulation. The detection was carried at 305 nm using PDA detector with a flow rate of 1.0ml/min and injection volume 20µl. The validation of developed method was performed for linearity, accuracy, precision, selectivity and specificity and robustness.In the present work orodispersible tablets of Ondansetron hydrochloride were formulated by effervescent and sublimation method using factorial design. In the effervescent method, crospovidone (1.31%-4.68%) was used as superdisintegrant whereas citric acid (7.95%-18.04%) and sodium bicarbonate (5.22%-28.77%) were used as effervescent agent. In the sublimation method, crospovidone (1.58%-4.41%) was used as superdisintegrant whereas camphor (6.34%-17.35%) was used as subliming agent. The prepared batches of tablets were evaluated for hardness, friability, drug content uniformity, wetting time, disintegration time and In Vitro drug release pattern. Total 15 batches were formulated by effervescent method and 9 batches were formulated by sublimation method. Disintegration time of the formulations prepared by effervescent method was found between 13-36 secs whereas formulations prepared by sublimation method showed disintegration time between 7-39 secs. Among all the formulations, the formulation (S6) prepared by sublimation method using crospovidone 4% and camphor 8% was found to have the minimum disintegration time (7 seconds). Finally, it was concluded that Orodispersible tablet of Ondansetron Hydrochloride can be successfully formulated using effervescent and sublimation method and can be used as a novel drug dosage form for pediatric and geriatric with improved patient compliance and enhanced bioavailability.I this presentation, the interaction between cilostazol and two different cyclodextrins (β-CD and DM-β-CD) is studied by using LC. The capacity factors (k) of cilostazol were monitored in the presence of increasing concentrations of β-CD or DM-β-CD from the reduction of the retention time (tR). It was observed that cilostazol forms a 1:1 inclusion complex with β-cyclodextrin (β-CD) and dimethyl-β-cyclodextrin (DM-β-CD) at 250C, 370C and 450C. The interaction of cilostazol with DM-β-CD was more efficient and the highest the formation constant (K) was found for DM-β-CD (23.82M-1) at 250C. Moreover, the values of K decreased as the system temperature increased. To obtain the information on the mechanism of cilostazol affinity for β-CD and DM-β-CD, the thermodynamic parameters of the complexation (ΔG, ΔH, and ΔS) were studied. Finally, a comparison of the K values obtained for the two different cyclodextrins revealed that the K values of the complexation are dependent upon the structure of the host molecule. The change in the thermodynamic parameters suggested that the complexation could proceed spontaneously (ΔG<0) along with the releasing of heat (ΔH<0) and the decrease of entropy (ΔS<0).T light scattering of single nanoparticle, such as gold and silver nanoparticles, is stable and efficient, hence the applications of single nanoparticle-based dark-field microscopic imaging have attracted extensive attention. Both the changes of single nanoparticle scattering imaging color and scattering intensity can be used as effective detection signal in analytical chemistry. In the author’s group, to study the localized surface plasmon resonance (LSPR) light scattering of single nanoparticle, first the shape effect of single silver nanoparticle on the scattering light color and refractive index sensitivities was investigated. It was found that particles with a large radial ratio or a tip structure always had a higher sensitivity which could be due to the LSPR maxima at long wavelengths and the strong electric field intensity distribution. Then single nanoparticle-based RGB analytical method was established by coding the colors of the scattering light of individual nanoparticles with the RGB system, and the imaging date was manipulated with the IPP software. In addition, the scattering light intensity of single gold nanoparticle was digitized and expressed as digital information through the aforementioned software. Based on the RGB system and digitized method, it was established new scattering analytical method which had been certified in our other energy transfer experiment. Furthermore, the single nanoparticle-based dark-field microscopic imaging was also used to monitoring some chemical reactions. Real-time monitoring of the etching process of gold nanoparticles by iodine, in situ growth of single Ag@Hg nanoalloys and photochemical reaction were successfully achieved by dark-field microscopic imaging. In addition to the chemical reactions, the single nanoparticle-based dark-field microscopic imaging has a potential to real-time monitoring the intracellular biochemical reactions and infection process of bacteria and virus which may provide a new method for the diagnosis of the disease.