Deacetylepoxyazadiradione ameliorates diabesity in in-vivo zebrafish larval model by influencing the level of regulatory adipokines and oxidative stress.

Obesity and diabetes constitute significant global health issues associated with one another. In contrast to diabetes, which is characterised by oxidative stress that enhances cellular damage and the following complications. Obesity dynamics involve chronic inflammation that promotes insulin resistance and metabolic disruptions. Anti-inflammatory and antioxidant agents, therefore, hold promise for synergistic effects, addressing inflammation and oxidative stress, key factors in managing obesity and diabetes. These agents can be utilized in novel drug delivery approaches. The complex interactions between deacetylepoxyazadiradione (DEA) and zebrafish larva subjected to metabolic impairment due to a high-fat diet (HFD) are examined in this study. The survival assay showed a significantly lower rate (79% survival rate) in the larvae exposed to HFD. Contrastingly, DEA treatment showed significant results with survival rates increasing dose-dependently (84%, 89%, and 94% at concentrations of 50 µM, 100 µM, and 150 µM, respectively). Further investigations revealed that DEA could reduce hyperlipidemic and hyperglycemic conditions in zebrafish larvae. Glucose levels significantly dropped in the DEA treatment, which was associated with a decline in larval weight, lipid accumulation, oxidative stress and apoptosis. Enzyme assays revealed higher antioxidant enzyme concentrations in DEA treated in-vivo larval models, which were associated with reduced expression of pro-inflammatory genes. In conclusion, the results demonstrate that DEA can alleviate oxidative stress and inflammation, effectively easing the diabesity-like state in zebrafish larvae. This offers potential avenues for developing DEA as a valuable drug candidate to manage the intricate diabesity condition.

Analysis of Inhibition Potential of Nimbin and its Analogs against NF-κB Subunits P50 and P65: A Molecular Docking and Molecular Dynamics Study.

BACKGROUND: Cancer remains the major cause of morbidity and mortality. The nuclear factor kappa-B (NF-κB) plays an indispensable role in cancer cell proliferation and drug resistance. The role of NF-κB is not only limited to tumor cell proliferation and suppression of apoptotic genes but it also induces EMT transition responsible for metastasis. Inhibition of the NF-κB pathway in cancer cells by herbal derivatives makes it a favorable yet promising target for cancer therapeutics. AIM: The purpose of the study is to explore the inhibition potential of Nimbin and its analogs against NF-κB subunits p50 and p65. METHODS: In the present study, an herbal compound Nimbin and its derivative analogs were investigated to examine their impact on the p50 and p65 subunits of the NF-κB signaling pathway using in-silico tools, namely molecular docking and simulation. RESULTS: The molecular docking analysis revealed that Nimbin and its analogs may bind to p50 and p65 subunits with dG bind values ranging from -33.23 to -50.49Kcal/mol. Interestingly, molecular dynamic simulation for the NO5-p65 complex displayed a stable conformation and convergence when compared to the NO4-p50 complex. CONCLUSION: These results indicate that NO5 may have a potential inhibitory effect against NF-κB subunit p65, which needs to be further validated in in-vitro and in-vivo systems. Also, the results obtained emphasize and pave the way for exploring the Nimbin scaffold against NF-κB inhibition for cancer therapeutics.

Nimbin analogs stimulate glucose uptake and glycogen storage in the insulin signalling cascade by enhancing the IRTK, PI3K and Glut-4 mechanism in myotubes.

BACKGROUND: Diabetes Mellitus is a metabolic disorder characterized by insulin dysfunction or failure of the pancreatic ß-cells to produce insulin resulting in hyperglycemia. Adverse effects of hyperglycemic conditions continue to be common, reducing treatment adherence. Intensified therapies are required for the constant loss of endogenous islet reserve. AIM: This study aimed to evaluate the effect of Nimbin semi-natural analogs (N2, N5, N7, and N8) from A. indica on high glucose-induced ROS and apoptosis with insulin resistance in L6 myotubes evaluated along with Wortmannin and Genistein inhibitors and the expression of key genes in the insulin signalling pathway. MATERIALS AND METHODS: The analogs were screened for anti-oxidant and anti-diabetic activity using cell-free assays; The ability of analogs to suppress ROS and prevent apoptosis induced by High glucose and uptake glucose and glycogen storage in L6 myotubes was evaluated using DCFH-DA, AO-PI and 2NBDG staining. Further, the glucose uptake was performed in the presence of Insulin Receptor Tyrosine Kinase (IRTK) inhibitors, and the expression of key genes PI3K, Glut-4, GS and IRTK in the insulin signalling pathway were evaluated. KEY FINDINGS: The Nimbin analogs were not toxic to the L6 cells, and the analogs could scavenge ROS and suppress cellular damage induced due to high glucose. Enhanced glucose uptake was observed in N2, N5 and N7 compared to N8. The maximum activity of optimum concentration was found to be 100 µM. The N2, N5 and N7 showed an increase in IRTK, which is equivalent to insulin at a concentration of 100 µM. The IRTK inhibitor with Genistein (50 µM) confirmed the presence of IRTK-dependent glucose transport activation; it also supports the expression of key genes PI3K, Glut-4, GS and IRTK. As a result of PI3K activation, N2, N5, and N7 exhibited the insulin-mimetic effect by enhancing glucose uptake and glycogen conversion regulating glucose metabolism. SIGNIFICANCE: N2, N5 and N7 could therapeutically benefit against insulin resistance by glucose metabolism modulation, insulin secretion, ß-cell stimulation, inhibition of gluconeogenic enzymes and ROS protection.

Trihydroxy piperlongumine protects aluminium induced neurotoxicity in zebrafish: Behavioral and biochemical approach.

Aluminium (Al) is proven to be a potent environmental neurotoxin involved in progressive neurodegeneration. Al primarily induces oxidative stress by free radical generation in the brain, followed by neuronal apoptosis. Antioxidants are promising therapeutic options for Al toxicity. Piperlongumine is traditionally long known for its medicinal properties. Therefore, the present study has been designed to explore the antioxidant role of trihydroxy piperlongumine (THPL) against Al-induced neurotoxicity in the zebrafish model. Zebrafish exposed to AlCl3 exhibited higher oxidative stress and altered locomotion. Adult fish displayed anxiety comorbid with depression phenotype. THPL increases antioxidant enzyme activity by quenching Al-induced free radicals and lipid peroxidation, thus minimizing oxidative damage in the brain. THPL rescues behavior deficits and improves anxiety-like phenotype in adult fish. Histological alterations caused by Al were also attenuated on administration with THPL. Results of the study demonstrate the neuroprotective role of THPL against Al-induced oxidative damage and anxiety, which could be exploited as a psychopharmacological drug.

Functionalized Sulfur-Containing Heterocyclic Analogs Induce Sub-G1 Arrest and Apoptotic Cell Death of Laryngeal Carcinoma In Vitro.

In this study, we speculate that the hydroxyl-containing benzo[b]thiophene analogs, 1-(3-hydroxybenzo[b]thiophen-2-yl) ethanone (BP) and 1-(3-hydroxybenzo[b]thiophen-2-yl) propan-1-one hydrate (EP), might possess antiproliferative activity against cancer cells. Hydroxyl-containing BP and EP show selectivity towards laryngeal cancer cells (HEp2), with IC50 values of 27.02 ± 1.23 and 35.26 ± 2.15 µM, respectively. The hydroxyl group present in the third position is responsible for the anticancer activity and is completely abrogated when the hydroxyl group is masked. BP and EP enhance the antioxidant enzyme activity and reduce the ROS production, which are correlated with the antiproliferative effect in HEp-2 cells. An increase in the BAX/BCL-2 ratio occurs during the BP and EP treatment and activates the caspase cascade, resulting in apoptosis stimulation. It also arrests the cells in the Sub-G1 phase, indicating the induction of apoptosis. The molecular docking and simulation studies predicted a strong interaction between BP and the CYP1A2 protein, which could aid in combinational therapy by enhancing the bioavailability of the drugs. BP and EP possess an antioxidant property with low antiproliferative effects (~5.18 µg/mL and ~7.8 µg/mL) as a standalone drug, therefore, they can be combined with other drugs for effective chemotherapy that might trigger the effect of pro-oxidant drug on healthy cells.

Hepatoprotective effect of dihydroxy piperlongumine in high cholesterol-induced non-alcoholic fatty liver disease zebrafish via antioxidant activity.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are a growing epidemic and the most common liver diseases. Consumption of a western diet with high fats alters redox status, induces inflammation, and impairs the physiological function of hepatocytes. However, the pharmacological market lacks anti-NAFLD/NASH drugs. Long pepper (Piper longum L) is used in traditional Mongolian medicine for treating hyperlipidemia. Piperlongumine (PL) is a bioactive compound of Piper longum L, which usually possesses anticancer activities due to its ROS elevation property. However, when PL was demethylated they behave as an antioxidant. Previously, we found dihydroxy piperlongumine (DHPL) possesses high antioxidant activity among the hydroxy piperlongumines, which makes us curious to reveal the anti-NAFLD effect. A high-cholesterol diet (HCD) was chosen to induce NAFLD zebrafish model, and the antioxidant and lipid-lowering effects of DHPL were evaluated. Histological alterations of NAFLD were also scored along with gene expression to explore the molecular mechanism. DHPL reduced lipid accumulation in both short-term and long-term feeding trials. DHPL increases antioxidant activity and lipid-lowering gene expression and decreases hepatic triglyceride, oxidative stress, and lipogenic genes. In conclusion, DHPL halted the progression of HCD-induced NAFLD in the zebrafish model.

6-Gingerdione Reduces Apoptotic Conditions in HepG2 Cells and Inhibits Inflammatory Cytokine Gene Expression in Alcoholic Liver Injured Zebrafish Larvae.

Antioxidant natural products and their analogs especially phenolic compounds, exhibit diverse biological properties, including anti-inflammatory, antioxidant, and anticancer activities. Ginger which is widely used worldwide for various beneficial effects also contains several phenolic antioxidants, and 6-gingerol is one of the natural products studied extensively. However, the molecular mechanism of synthetically synthesized 6-gingerdione (compound 1) from 6-gingerol was not known. In this study, compound 1 and methylated 6-gingerdione (compound 2) were obtained semi synthetically from 6-gingerol. Compound 1 and 2 are subjected to SwissADME prediction. Then the protective effect of compound 1 was analyzed in 2 % EtOH induced HepG2 cells and zebrafish larvae. Hydroxyl and nitric oxide scavenging assays reveal that compound 1 showed more antioxidant activity than compound 2 at 50 µM. Moreover, compound 1 exhibited good anti-inflammatory activity via lipoxygenase inhibition and proteinase inhibition. Apoptosis and oxidative stress in HepG2 cells were induced by 2 % EtOH and treated with compound 1. Compound 1 significantly inhibited the EtOH induced nitric oxide production, apoptosis, and ROS generation in HepG2 cells. Encouraged by the in-vitro antioxidant and anti-inflammatory activities, compound 1 was then investigated for its protective effect in 2 % EtOH induced ALD zebrafish larva. Compound 1 protected the zebrafish larvae from liver injury by suppressing inflammatory (COX-2, TNF-α, and IL-1ß) and lipogenic genes (C/EBP-α, SREBP1, and IL-1ß) while upregulating the antioxidant gene. Our findings indicate that compound 1 synthesized from 6-gingerol ameliorated liver injury that likely, contributes to its potential antioxidant and anti-inflammatory properties.

Nimbin analog N2 alleviates high testosterone induced oxidative stress in CHO cells and alters the expression of Tox3 and Dennd1a signal transduction pathway involved in the PCOS zebrafish.

Polycystic ovarian syndrome (PCOS) is a hormonal disorder that causes enlargement of ovaries and follicular maturation arrest, which lacks efficient treatment. N2, a semi-natural triterpenoid from the neem family, was already reported to have antioxidant and antiinflammatory properties in our previous report. This study investigated the anti-androgenic property of N2 on testosterone-induced oxidative stress in Chinese Hamster Ovarian cells (CHO) and PCOS zebrafish model. The testosterone exposure disrupted the antioxidant enzymes and ROS level and enhanced the apoptosis in both CHO cells and PCOS zebrafish. However, N2 significantly protected the CHO cells from ROS and apoptosis. N2 improved the Gonado somatic index (GSI) and upregulated the expression of the SOD enzyme in zebrafish ovaries. Moreover, the testosterone-induced follicular maturation arrest was normalized by N2 treatment in histopathology studies. In addition, the gene expression studies of Tox3 and Denndla in zebrafish demonstrated that N2 could impair PCOS condition. Furthermore, to confirm the N2 activity, the in-silico studies were performed against PCOS susceptible genes Tox3 and Dennd1a using molecular docking and molecular dynamic simulations. The results suggested that N2 alleviated the oxidative stress and apoptosis in-vitro and in-vivo and altered the expression of PCOS key genes.

Deacetyl epoxyazadiradione protects aminoglycoside antibiotic-induced renal cell apoptosis, in vitro.

Aminoglycoside antibiotics such as gentamicin are used frequently to treat bacterial infections in humans. Excessive consumption of these antibiotics lead to renal dysfunction. One of the factors contributing to renal dysfunction is oxidative damage, which causes apoptosis. Hence, this study investigates the effect of the antioxidant compound deacetyl epoxyazadiradione (DEA) in reducing cell death induced by gentamicin treatment in kidney cells (Madin-Darby canine kidney cells). The antioxidant experiments showed that reactive oxygen species level is decreased up to 27.06 ± 0.18% in 150 µM of DEA treatment. At this concentration, the activity of antioxidant enzymes such as superoxide dismutase increased from 0.4 ± 0.04 to 1.46 ± 0.05 µmol/min/L and catalase increased from 7.48 ± 0.39 to 17.6 ± 0.74 U/mg. The relative folds of gene expression of mitochondrial enzymes such as GST, GPx and GR restored from 0.596 ± 0.019, 0.521 ± 0.013 and 0.775 ± 0.014 to 0.866 ± 0.013, 0.669 ± 0.015 and 0.8615 ± 0.028, respectively. Consequently, the percentage of cell viability increases upto 91.8 ± 2.01 from 61.93 ± 1.63 with much less fragmentation in genomic DNA. Additionally, molecular docking results showed that DEA could bind to Bax, Bcl- 2, Caspase- 3 and Caspase- 9 proteins. These results indicate that DEA could reduce cell apoptosis by reducing oxidative stress due to antibiotics and interrupting the apoptotic signal pathway in kidney cells.

Withaferin A targets the membrane of Pseudomonas aeruginosa and mitigates the inflammation in zebrafish larvae; an in vitro and in vivo approach.

Infections due to multidrug-resistant Pseudomonas aeruginosa are prevalent among patients with cystic fibrosis. The emergence of antibiotic-resistant pathogens necessitated the development of novel low-risk natural antibacterial compounds. Herbal medicines are used from dates of the origin of mankind and still serve their purpose as therapeutic agents. We demonstrated the antibacterial activity of Withaferin A extracted from the traditional herb, ashwagandha or winter cherry (Withania somnifera). Withaferin A exhibits strong antibacterial activity against P. aeruginosa with a minimum inhibitory concentration of 60 µM and minimum bactericidal concentration of 80 µM. Results obtained from membrane stabilization assay and electron microscopic analysis showed that Withaferin A acts by damaging the cell membrane of P. aeruginosa. Additionally, we investigated oxidative stress and inflammatory response after Withaferin A treatment in P. aeruginosa infected zebrafish larvae model. The results indicate that the level of ROS, and its related lipid peroxidation and apoptosis were significantly reduced after treated with Withaferin A. Consequently, an increment in antioxidant enzymes level such as superoxide dismutase (SOD) and catalase (CAT) was observed. Macrophage localization experiment showed a smaller number of localized macrophages in zebrafish, which indicates the reduction in inflammatory response. In conclusion, Withaferin A could serve as an alternative natural product in the treatment of infections caused by P. aeruginosa.

Deacetylated nimbin analog N2 fortifies alloxan-induced pancreatic ß-cell damage in insulin-resistant zebrafish larvae by upregulating phosphoenolpyruvate carboxykinase (PEPCK) and insulin levels.

This study aims to evaluate the protective behaviour of N2, a semi-natural analog of nimbin, for its anti-diabetic efficacy against alloxan-induced oxidative damage and ß-cell dysfunction in in-vivo zebrafish larvae. A 500 µM of alloxan was exposed to zebrafish larvae for 24 h to induce oxidative stress in the pancreatic ß-cells and co-exposed with N2 to study the protection of N2 by inhibiting ROS by DCFH-DA, DHE and NDA staining along with Cellular damage, apoptosis and lipid peroxidation. The zebrafish was further exposed to 500 µM alloxan for 72 h to induce ß-cell destruction along with depleted glucose uptake and co-exposed to N2 to study the protective mechanism. Glucose levels were estimated, and PCR was used to verify the mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK) and insulin. Alloxan induced (24 h) oxidative stress in the pancreatic ß-cells in which N2's co-exposure inhibited ROS by eliminating O-2 radicals and restoring the glutathione levels, thus preventing cellular damage and lipid peroxidation. The zebrafish exposed to 500 µM alloxan for 72 h was observed with ß-cell destruction along with depleted glucose uptake when stained with 2NBDG, wherein N2 was able to protect the pancreatic ß-cells from oxidative damage, promoted high glucose uptake and reduced glucose levels. N2 stimulated insulin production and downregulated PEPCK by inhibiting gluconeogenesis, attenuating post-prandial hyperglycemia. N2 may contribute to anti-oxidant protection against alloxan-induced ß-cell damage and anti-hyperglycemic activity, restoring insulin function and suppressing PEPCK expression.

Deacetylepoxyazadiradione Derived from Epoxyazadiradione of Neem (Azadirachta indica A. Juss) Fruits Mitigates LPS-Induced Oxidative Stress and Inflammation in Zebrafish Larvae.

Reactive oxygen species (ROS) produced by cell metabolism have a duplex role in oxidation and inflammation reactions which involve cell damage or repair responses. Excess ROS production has detrimental effects on the survival of cells. We examined the protective effect of a semi-natural compound NF2 (deacetylepoxyazadiradione), for its protective activity against free radical-mediated stress and inflammatory response to lipopolysaccharide (LPS) using zebrafish larvae. Preliminary antioxidant assays indicated an increase in scavenging of free radicals from NF2 than NF1 (Epoxyazadiradione) in a concentration-dependent manner. Cell cytotoxicity was determined using rat myoblast cell lines (L6), and more than 95 % of cell viability was obtained. Zebrafish developmental toxicity test indicated that NF2 is not toxic even at 150 µM. The percentage of ROS, lipid peroxidation, nitric oxide and apoptosis were reduced significantly in NF2 treated LPS-stressed zebrafish larvae. The reduced number of employed macrophages on NF2 treatment was observed in neutral red dye-marked macrophage localization images. Relative expression of antioxidant genes in zebrafish larvae after treatment with NF2 is significantly increased. The RT-PCR quantification of antioxidant and anti-inflammatory gene expression indicated decreased relative folds of pro-inflammatory cytokines, iNOS and increased relative folds of mitochondrial antioxidant genes (GR, GST and GPx) in LPS stressed zebrafish larvae after treatment with NF2. From the overall obtained results, it can be concluded that NF2 reduced the oxidative stress and inflammatory response by scavenging free radicals caused by LPS.

Nanoencapsulation of Ru(p-cymene) Complex Bearing Ginger-based Natural Product into Liposomal Nanoformulation to Improve Its Cellular Uptake and Antiproliferative Activity.

The organometallic compounds are prospective candidates in the row of developing metallochemotherapeutics with the aim of overcoming the limitations of platinum drugs. In order to explore the anticancer properties of organometallic compounds with the natural medicines, two Ru(II)-p-cymene complexes containing the natural products, viz., 6-gingerol (6G) and benzylated-6-gingerdione (B-6GD) have been synthesized and characterized well. The phenolic group of the Ru(6G) complex facilitates its higher cell-free antioxidant activity than its analogue complex. Also, the same complex shows higher cytotoxicity toward A549 lung and HeLa-S3 cervical cancer cells than the Ru(B-6GD) complex but lower cytotoxicity toward A2058 metastatic melanoma cancer cells. Both complexes are shown to easily accumulate in melanoma cancer cells, and their degree of cytotoxicity in the same cells is found to be positively correlated with cell uptake. The cytotoxicity of complexes arises from their intracellular activity, mainly due to the induction of singlet oxygen production in cancer cells. The subcellular fractionation study shows that mitochondria and ER-Golgi membranes might be their predominant targets. Also, the mechanistic investigation revealed that Ru(B-6GD) induces caspase-dependent non-apoptotic cell death whereas Ru(6G) can induce caspase-independent non-apoptotic cell death. Furthermore, both complexes are found to moderately alter the adhesion properties of cancer cells, which is beneficial for antimetastatic treatment. Despite the potential pharmacological activity, Ru(6G) is encapsulated into polymer-supported liposomes to reduce its toxicity and further improve its anticancer potency. The π-conjugated yne-ene chain of polydiacetylene aids in the development of a stable nanoformulation, which achieved a slow release of the complex. Most importantly, the cancer cell uptake of the liposome-encapsulated Ru(6G) complex is 20 times enhanced and the total ROS formation in cancer cells is significantly increased compared to the non-encapsulated complex. However, the nanoformulation does not alter the antimetastatic potency of the encapsulated complex.

Amelioration of acrylamide induced neurotoxicity by benzo[b]thiophene analogs via glutathione redox dynamics in zebrafish larvae.

Acrylamide is a thermal process contaminant, which gets global attention due to its neurotoxic nature and its omnipresence in carbohydrate-rich foods. Chronic exposure to acrylamide leads to neuronal deterioration and motor dysfunction. Acrylamide could severely affect the antioxidant defense system, especially in the developing brain leading to premature neurological disorders. Acrylamide forms adduct in presynaptic neurons leading to neuroinflammation which is also a factor to consider. In this present study, we have explored whether our benzo[b]thiophene analogs, 1-(3-hydroxybenzo[b]thiophen-2-yl) ethanone (BP) and 1-(3-hydroxybenzo[b]thiophen-2-yl) propan-1-one hydrate (EP) with antioxidant activity, could inhibit the acrylamide-induced neurotoxicity-like behavior in zebrafish larvae. The experiment was set up to expose 3 days post fertilized (dpf) larvae to acrylamide (0.75 mM) for 3 days with or without compounds (80 µM). Locomotion behavioral analysis, antioxidants, glutathione, and acetylcholineesterase activity in the head region were analyzed after one day of the experimental procedure. We witnessed a restoration effect on glutathione redox dynamics. Since glutathione plays a crucial role in the detoxification of acrylamide, it is necessary to maintain the glutathione redox cycle to eliminate acrylamide from the body. BP and EP reduced the pro-inflammatory transcript in the head, which correlates with the reduction in oxidative stress. Finally, BP and EP showed a positive effect on synaptic vesicle cycling transcript and partially restores the motor neuron response to stimuli. Findings in this study showed the ability of compound BP and EP possess therapeutic value in oxidative stress-associated neurological disorders.

Reverse pharmacology of Nimbin-N2 attenuates alcoholic liver injury and promotes the hepatoprotective dual role of improving lipid metabolism and downregulating the levels of inflammatory cytokines in zebrafish larval model.

Alcoholic liver disease is one of the most prominent liver diseases in the world. Lipid accumulation accompanied by oxidative stress and inflammation in the liver is the most important pathogenesis of ALD. This study was designed to investigate the anti-oxidative, fat metabolism-regulating, and anti-inflammatory potential of N2, a seminatural analog of Nimbin. The ethanol exposure was found to induce liver injury on zebrafish larvae, such as liver inflammation, lipid accumulation, oxidative stress, and hepatocytes apoptosis. N2 was subjected to ADMET screening in-silico, and it was observed N2's co-exposure decreased the ROS, apoptosis, lipid peroxidation, and macrophage accumulation in the liver of larval zebrafish. To further study the mechanism behind ethanol hepatotoxicity and the hepatoprotective behavior of N2, gene expression changes were determined in zebrafish. The results of this study revealed that ethanol exposure upregulated mRNA expressions of SREBP1, C/EBP-α, FAS and provoked more severe oxidative stress and hepatitis via upregulation of inflammatory cytokines TNF-α, IL-10, IL-1ß, iNOS, COX-2. However, the N2 co-exposure protected the hepatocyte damage and almost reversed the condition by downregulating the mRNA levels. The study suggested that N2 could be an effective therapeutic agent for the treatment of ALD and other inflammatory conditions.

Hydroxyl containing benzo[b]thiophene analogs mitigates the acrylamide induced oxidative stress in the zebrafish larvae by stabilizing the glutathione redox cycle.

AIMS: This study aims to elucidate a systematic free-radical quenching ability of synthesized benzo[b]thiophene derivatives using in vitro assays and acrylamide induced oxidatively stressed model in zebrafish larvae. MATERIALS AND METHODS: Antioxidant activity of the compounds was evaluated using in vitro methods. The toxicity of the compounds was evaluated in Madin-Darby Canine Kidney (MDCK) cell line and zebrafish embryos. Oxidative stress was generated by acrylamide (1 mM) in zebrafish larvae and treated with compounds to evaluate the in vivo antioxidant ability. Specific fluorescence dyes were used to detect ROS generation, lipid peroxidation, and cell death followed by gene expression using RT PCR. Density functional theory (DFT) and in silico pharmacokinetics were also studied. KEY FINDINGS: Compound BP and EP have a greater in vitro free radical scavenging ability. The maximum tolerated concentration (MTC) of the compounds in zebrafish larvae is 80 µM. The antioxidant system in zebrafish larvae was dysregulated due to acrylamide exposure and improvement was found while treating acrylamide exposed larvae with compounds 1-(3-hydroxybenzo[b]thiophen-2-yl) ethanone (BP) and 1-(3-hydroxybenzo[b]thiophen-2-yl) propan-1-one hydrate (EP). Compound BP and EP enhanced the SOD and CAT activity, reduced the ROS and lipid peroxidation level, thus decreasing cell death in zebrafish larvae. Compound BP and EP also improved the glutathione redox cycle by stabilizing glutathione-related gene expressions. SIGNIFICANCE: Hydroxyl-containing compounds BP and EP are promising lead molecules for pathological conditions related to oxidative stress, which showed an attenuated effect on acrylamide-induced oxidative stress in zebrafish larvae by enhancing the glutathione redox cycle and enzymatic antioxidants.

Anti-inflammatory role demonstrated both in vitro and in vivo models using nonsteroidal tetranortriterpenoid, Nimbin (N1) and its analogs (N2 and N3) that alleviate the domestication of alternative medicine.

Human health may benefit from the study of natural compounds and phytoconstituents that can protect from inflammation. We investigated Nimbin (N1), a member of the ring C Seco-tetranortriterpenoids family, and its semi-natural analog deacetyl Nimbin namely N2 and N3 for their anti-inflammatory properties. As key findings, N1, N2, and N3 were able to improve wound healing by cell proliferation in a period of 24 h and were able to reduce the reactive oxygen species (ROS) production in Madin-Darby Canine Kidney cells which were screened using dichloro-dihydro fluorescein diacetate (DCF-DA) staining. When the zebrafish larvae were subjected to DCF-DA assay N1, N2, and N3 were able to substantially reduce the ROS levels in a dose-dependent manner. In zebrafish larvae, the cell death indicates the fluorescent intensity due to acridine orange staining that was found to be dramatically decreasing upon the treatment of N1, N2, and N3. The cell membrane lipid peroxidation levels were also reduced in a dose-dependent manner upon the treatment of Nimbin and its analogs indicating lesser blue fluorescent levels. Among the Nimbin and its analogs, N2 was subjected to have better activity. To confirm the activity of N1, N2, and N3, in silico characterization was performed using Density functional theory and molecular docking. As a result, N2 exhibited the lowest electronegative value and highest binding energy when docked with anti-inflammatory and antioxidant proteins CAT, COX, GP, IL-1, and MPO. Furthermore, the therapeutic potential of N2 must be explored at the molecular level as well as in clinical studies for the treatment of inflammation-associated diseases.

Rapid colorimetric discrimination of cyanide ions - mechanistic insights and applications.

In this work, we have employed an intramolecular charge transfer-based DMN colorimetric probe for the rapid naked-eye detection of cyanide ions in solution as well as real water samples. The intermolecular interaction between the DMN probe and cyanide ions in solution was investigated using a combination of spectroscopic and computational methods in this study. The DMN probe exhibited a selective colorimetric response for cyanide ions over the other anions exposed. The cyanide sensing mechanism of the probe has been investigated by 1H NMR titration and density functional theory calculations. The results reveal that the colorimetric response of the DMN probe is due to the Michael adduct formation in the ß-conjugated position of the dicyanovinyl group with cyanide, which blocks intramolecular charge transfer transition. Under optimized experimental conditions, the DMN probe showed a linear plot in the concentration range of 0.01-0.25 µM, with a detection limit of 23 nM. Further, a 3D printed portable accessory for the smartphone and an open-source android application is developed to suit the DMN probe for on-site work. In addition, we have developed the microfluidic paper-based analytical device that could selectively detect cyanide ions at very low concentration using a colorimetric DMN probe. In addition, the DMN probe was effectively used to determine the cyanide ion in a variety of water samples.

6-Gingerol and Semisynthetic 6-Gingerdione Counteract Oxidative Stress Induced by ROS in Zebrafish.

6-Gingerol (1) is one of the major components in ginger and developing new synthetic methodologies could bring semisynthetic analogs with improved therapeutic properties. Towards this, multigram scale isolation of 6-gingerol with excellent purity was optimized using a simple and robust extraction, followed by column purification. Synthesis of 6-gingerdione, 7 from 6-gingerol was then achieved through selective -OTBDMS protection, DMP oxidation and deprotection reaction sequence for the first time. Compounds 1, 7 and 8 (dehydrozingerone) exhibited excellent cell-free antioxidant properties in DPPH, ABTS, superoxide radical scavenging assay and H2 O2 assay at 10-50 µM concentrations. The hemolytic study suggests that up to 50 µM, all three compounds did not exhibit toxicity to human erythrocytes. When H2 O2 treated zebrafish larvae groups (96hpf) were exposed to compounds 1, 7 and 8, it increases the SOD (19, 19.1 and 18.7 U/mg protein), CAT (18.1, 16.5, and 15.8 µmol/mg levels and decreases the lipid peroxidation level (13, 15 and 18 nmol/mg protein), respectively. In vivo ROS levels and degree of cell death were studied using DCFDA and Acridine orange assays. Compounds 1, 7 and 8 decreases the ROS and cell death level significantly. Taken together, compounds 1, 7 and 8 exhibit excellent antioxidant properties, counteract H2 O2 induced oxidative stress, reduces cell death in zebrafish larvae.

A New Chemoenzymatic Semisynthetic Approach Provides Insight into the Role of Phosphorylation beyond Exon1 of Huntingtin and Reveals N-Terminal Fragment Length-Dependent Distinct Mechanisms of Aggregation.

Huntington's disease is a neurodegenerative disorder caused by the expansion of a polyglutamine repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Httexon1 (Httex1). Herein, we investigated the aggregation properties of mutant N-terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1 (mHttex1). We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded insights into how post-translational modifications (PTMs) and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slows the aggregation of mHtt171, whereas phosphorylation at T107 and S116 accelerates the aggregation, underscoring the importance of crosstalk between different PTMs. The mHtt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mHttex1. These observations suggest that different N-terminal fragments could have distinct aggregation mechanisms and that a single polyQ-targeting antiaggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the need for further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity and disease progression.