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Cellulose beads emerge as carriers for poorly water-soluble drugs due to their eco-friendly raw materials and favorable porous structure. However, drug dissolution may be limited by their poor swelling ability and the presence of closed pores caused by shrinkage of the pristine cellulose beads. In this study, novel cellulose beads that can swell in acidic environment were prepared by introducing ethylenediamine (EDA) on dialdehyde cellulose (DAC), thereby addressing the shrinkage and closed pore problem of cellulose beads. The effect of the ratio of EDA on the swelling behavior and amine content of beads was studied. Three model drugs with different physicochemical properties were selected to study the physical state of loaded drugs and their release behavior. According to the results of XRPD and DSC, indomethacin and itraconazole loaded in the beads were amorphous at a drug loading of 20%, but fenofibrate was partially crystalline. Both bead size and the ratio of amine groups influenced the release behavior of the model drugs. The in vitro dissolution results showed that the cationic beads greatly improved the solubility and dissolution rate of the drug compared with the crystalline drug. Beads with a small size and high ratio of EDA tend to achieve a better drug dissolution rate and cumulative release percentage. Physical stability studies of the itraconazole-loaded beads were also implemented under four different temperature/humidity conditions for up to two months. The results showed that crystallization only appeared after two months of storage at 40°/75% RH, and the drug maintained a non-crystalline state in the other three storage conditions (0 °C/0 %RH, 0 °C/32 %RH, 25 °C/32 %RH). In conclusion, the novel pH-responsive cationic cellulose beads show great potential as a carrier for improving the rate and extent of dissolution of poorly soluble drugs and maintaining supersaturation.
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Background: There is need to investigate whether phytochemicals along with surgical detorsion could serve as better managements options in TT patients rather than surgical detorsion (SD) alone. Methods: The descriptive cross-sectional part of this study is questionnaire-based addressing sociodemographic characteristics of participants and their experience in management of TT. In the experimental part, male rats (n = 32) were grouped into: sham, Ischemia-reperfusion injury (IRI), dichloromethane (DCM) and ethanol fraction (100 mg/kg) of CO. Evaluation of tissue GPx, total thiol, SOD, MDA and H2O2 was done. Serum estimations of nitrite, TNF-α and IL-6, MPO, sperm motility, count and viability was also carried out. Tissue expression of bax and caspase 3 was assessed. Results: 68.9 % respondents agreed that SD alone is non-effective in the management of TT while 83.6 % reported a need to augment surgery with medications. Oxidative stress markers like H2O2, MDA and nitrite increased by IRI were decreased in post-treatment groups, along with a significant increase in the tissue level of GSH, GST, SOD, GPx, and total thiol. Inflammatory mediators were elevated in IRI while post-treatment rats showed significant decrease. IRI decreased sperm count significantly this was reversed by post-treatment. Bax and caspase 3 was increased in IRI rats and post-treatment with CO fractions reduced them. Conclusions: Quantitative cross-sectional study has revealed through experience of clinicians that surgical detorsion alone is not effective in managing TT. Augmented treatment with CO leaf fractions suppressed testicular IRI through inhibition of pro-apoptotic proteins expression, oxidative stress and inflammation.
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Scaffold hopping refers to computer-aided screening for active compounds with different structures against the same receptor to enrich privileged scaffolds, which is a topic of high interest in organic and medicinal chemistry. However, most approaches cannot efficiently predict the potency level of candidates after scaffold hopping. Herein, we identified potent PDE5 inhibitors with a novel scaffold via a free energy perturbation (FEP)-guided scaffold-hopping strategy, and FEP shows great advantages to precisely predict the theoretical binding potencies ΔG FEP between ligands and their target, which were more consistent with the experimental binding potencies ΔG EXP (the mean absolute deviations | Δ G FEP - Δ G EXP | < 2 kcal/mol) than those ΔG MM-PBSA or ΔG MM-GBSA predicted by the MM-PBSA or MM-GBSA method. Lead L12 had an IC50 of 8.7 nmol/L and exhibited a different binding pattern in its crystal structure with PDE5 from the famous starting drug tadalafil. Our work provides the first report via the FEP-guided scaffold hopping strategy for potent inhibitor discovery with a novel scaffold, implying that it will have a variety of future applications in rational molecular design and drug discovery.
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In the microscale, bacteria with helical body shapes have been reported to yield advantages in many bio-processes. In the human society, there are also wisdoms in knowing how to recognize and make use of helical shapes with multi-functionality. Herein, we designed atypical chiral mesoporous silica nano-screws (CMSWs) with ideal topological structures (e.g., small section area, relative rough surface, screw-like body with three-dimension chirality) and demonstrated that CMSWs displayed enhanced bio-adhesion, mucus-penetration and cellular uptake (contributed by the macropinocytosis and caveolae-mediated endocytosis pathways) abilities compared to the chiral mesoporous silica nanospheres (CMSSs) and chiral mesoporous silica nanorods (CMSRs), achieving extended retention duration in the gastrointestinal (GI) tract and superior adsorption in the blood circulation (up to 2.61- and 5.65-times in AUC). After doxorubicin (DOX) loading into CMSs, DOX@CMSWs exhibited controlled drug release manners with pH responsiveness in vitro. Orally administered DOX@CMSWs could efficiently overcome the intestinal epithelium barrier (IEB), and resulted in satisfactory oral bioavailability of DOX (up to 348%). CMSWs were also proved to exhibit good biocompatibility and unique biodegradability. These findings displayed superior ability of CMSWs in crossing IEB through multiple topological mechanisms and would provide useful information on the rational design of nano-drug delivery systems.
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Scaffold hopping refers to computer-aided screening for active compounds with different structures against the same receptor to enrich privileged scaffolds, which is a topic of high interest in organic and medicinal chemistry. However, most approaches cannot efficiently predict the potency level of candidates after scaffold hopping. Herein, we identified potent PDE5 inhibitors with a novel scaffold via a free energy perturbation (FEP)-guided scaffold-hopping strategy, and FEP shows great advantages to precisely predict the theoretical binding potencies ΔG FEP between ligands and their target, which were more consistent with the experimental binding potencies ΔG EXP (the mean absolute deviations | Δ G FEP - Δ G EXP | < 2 kcal/mol) than those ΔG MM-PBSA or ΔG MM-GBSA predicted by the MM-PBSA or MM-GBSA method. Lead L12 had an IC50 of 8.7 nmol/L and exhibited a different binding pattern in its crystal structure with PDE5 from the famous starting drug tadalafil. Our work provides the first report via the FEP-guided scaffold hopping strategy for potent inhibitor discovery with a novel scaffold, implying that it will have a variety of future applications in rational molecular design and drug discovery.
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Insulin derivatives such as insulin detemir and insulin degludec are U.S. Food and Drug Administration (FDA)-approved long-acting insulin currently used by millions of people with diabetes. These derivatives are modified in C-terminal B29 lysine to retain insulin bioactivity. New and efficient methods for facile synthesis of insulin derivatives may lead to new discovery of therapeutic insulin. Herein, we report a new method using sortase A (SrtA)-mediated ligation for the synthesis of insulin derivatives with high efficiency and functional group tolerance in the C-terminal B chain. This new insulin molecule (Ins-SA) with an SrtA-recognizing motif can be conjugated to diverse groups with N-terminal oligoglycines to generate new insulin derivatives. We further demonstrated that a new insulin derivative synthesized by this SrtA-mediated ligation shows strong cellular and in vivo bioactivity. This enzymatic method can therefore be used for future insulin design and development.
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Ferroptosis is a type of cell death accompanied by iron-dependent lipid peroxidation, thus stimulating ferroptosis may be a potential strategy for treating gastric cancer, therapeutic agents against which are urgently required. Jiyuan oridonin A (JDA) is a natural compound isolated from Jiyuan Rabdosia rubescens with anti-tumor activity, unclear anti-tumor mechanisms and limited water solubility hamper its clinical application. Here, we showed a2, a new JDA derivative, inhibited the growth of gastric cancer cells. Subsequently, we discovered for the first time that a2 induced ferroptosis. Importantly, compound a2 decreased GPX4 expression and overexpressing GPX4 antagonized the anti-proliferative activity of a2. Furthermore, we demonstrated that a2 caused ferrous iron accumulation through the autophagy pathway, prevention of which rescued a2 induced ferrous iron elevation and cell growth inhibition. Moreover, a2 exhibited more potent anti-cancer activity than 5-fluorouracil in gastric cancer cell line-derived xenograft mice models. Patient-derived tumor xenograft models from different patients displayed varied sensitivity to a2, and GPX4 downregulation indicated the sensitivity of tumors to a2. Finally, a2 exhibited well pharmacokinetic characteristics. Overall, our data suggest that inducing ferroptosis is the major mechanism mediating anti-tumor activity of a2, and a2 will hopefully serve as a promising compound for gastric cancer treatment.
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Recently, we reported high antibacterial efficiency of Loranthus acaciae (LA) against different standard strains of bacteria including Methicillin-Resistant Staphylococcus aureus (MRSA). Therefore, this study aimed to confirm the effectiveness of LA against clinically isolated Staphylococcus aureus (SA) including ß-lactamase producer (Blac) and MRSA. Forty-eight SA isolates collected from various clinical samples were used in this study. Antibiotics susceptibility profile was determined for twenty different antibiotics using automated Microscan Walkaway 96 Plus system as recommended by Clinical and Laboratory Standards Institute (CLSI) guidelines. This system also identified ß-lactamase producers and MRSA. In the meantime, LA ethanolic extract was fractionated using liquid-liquid fraction method to hexane, dichloromethane DCM and methanol 80% fractions. Antimicrobial activities of LA extract and fraction were performed with agar well diffusion method for all SA isolates, MIC and MBC were also recorded. Phytochemical screening for various phyto-constituent classes of LA ethanolic extract was determined. Out of 48 SA isolates, Cefoxitin-positive MRSA represent 31 (64.6%), Blac 17 (35.4%), and 41 (85.4%) were multidrug-resistant SA, which was resistant at least to one antibiotic from three different categories. All isolates were resistant to ampicillin and penicillin. Antimicrobial activities of LA extract and fractions revealed that ethanol extract was active against all isolated SA with inhibition zone ranged from 33 ± 2.00 to 25 ± 3.05 mm. While DCM exhibited the largest inhibition zone range from 37 ± 3.00 to 33 ± 2.00 mm. This study is first of its kind conforming the high antibacterial activity of LA against SA isolated from a different source of infection. The study concluded that LA extract and fractions are active and give positive result for all isolated SA. Therefore, suitable pharmacological formulation of LA extract as a promising antibacterial agent for the treatment of SA infection should be given extreme priority.
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Due to traffic accidents, injuries, burns, congenital malformations and other reasons, a large number of patients with tissue or organ defects need urgent treatment every year. The shortage of donors, graft rejection and other problems cause a deficient supply for organ and tissue replacement, repair and regeneration of patients, so regenerative medicine came into being. Stem cell therapy plays an important role in the field of regenerative medicine, but it is difficult to fill large tissue defects by injection alone. The scientists combine three-dimensional (3D) printed bone tissue engineering scaffolds with stem cells to achieve the desired effect. These scaffolds can mimic the extracellular matrix (ECM), bone and cartilage, and eventually form functional tissues or organs by providing structural support and promoting attachment, proliferation and differentiation. This paper mainly discussed the applications of 3D printed bone tissue engineering scaffolds in stem cell regenerative medicine. The application examples of different 3D printing technologies and different raw materials are introduced and compared. Then we discuss the superiority of 3D printing technology over traditional methods, put forward some problems and limitations, and look forward to the future.
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Ferroptosis is a type of cell death accompanied by iron-dependent lipid peroxidation, thus stimulating ferroptosis may be a potential strategy for treating gastric cancer, therapeutic agents against which are urgently required. Jiyuan oridonin A (JDA) is a natural compound isolated from Jiyuan
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Insulin derivatives such as insulin detemir and insulin degludec are U.S. Food and Drug Administration (FDA)-approved long-acting insulin currently used by millions of people with diabetes. These derivatives are modified in C-terminal B29 lysine to retain insulin bioactivity. New and efficient methods for facile synthesis of insulin derivatives may lead to new discovery of therapeutic insulin. Herein, we report a new method using sortase A (SrtA)-mediated ligation for the synthesis of insulin derivatives with high efficiency and functional group tolerance in the C-terminal B chain. This new insulin molecule (Ins-SA) with an SrtA-recognizing motif can be conjugated to diverse groups with N-terminal oligoglycines to generate new insulin derivatives. We further demonstrated that a new insulin derivative synthesized by this SrtA-mediated ligation shows strong cellular and
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Human dihydroorotate dehydrogenase (DHODH) is a viable target for the development of therapeutics to treat cancer and immunological diseases, such as rheumatoid arthritis (RA), psoriasis and multiple sclerosis (MS). Herein, a series of acrylamide-based novel DHODH inhibitors as potential RA treatment agents were designed and synthesized. 2-Acrylamidobenzoic acid analog 11 was identified as the lead compound for structure-activity relationship (SAR) studies. The replacement of the phenyl group with naphthyl moieties improved inhibitory activity significantly to double-digit nanomolar range. Further structure optimization revealed that an acrylamide with small hydrophobic groups (Me, Cl or Br) at the 2-position was preferred. Moreover, adding a fluoro atom at the 5-position of the benzoic acid enhanced the potency. The optimization efforts led to potent compounds 42 and 53â55 with IC50 values of 41, 44, 32, and 42 nmol/L, respectively. The most potent compound 54 also displayed favorable pharmacokinetic (PK) profiles and encouraging in vivo anti-arthritic effects in a dose-dependent manner.
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Proteolysis targeting chimeras (PROTACs) are dual-functional hybrid molecules that can selectively recruit an E3 ubiquitin ligase to a target protein to direct the protein into the ubiquitin-proteasome system (UPS), thereby selectively reducing the target protein level by the ubiquitin-proteasome pathway. Nowadays, small-molecule PROTACs are gaining popularity as tools to degrade pathogenic protein. Herein, we present the first small-molecule PROTACs that can induce the α 1A-adrenergic receptor (α 1A-AR) degradation, which is also the first small-molecule PROTACs for G protein-coupled receptors (GPCRs) to our knowledge. These degradation inducers were developed through conjugation of known α 1-adrenergic receptors (α 1-ARs) inhibitor prazosin and cereblon (CRBN) ligand pomalidomide through the different linkers. The representative compound 9c is proved to inhibit the proliferation of PC-3 cells and result in tumor growth regression, which highlighted the potential of our study as a new therapeutic strategy for prostate cancer.
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OBJECTIVE: To prepare a novel Chitosan (CS)-coated-PLGA-NPs of catechin hydrate (CTH) and to improve lungs bioavailability via direct nose to lungs-delivery for the comparative assessment of a pulmokinetics study by the first-time UHPLC-MS/MS developed method in the treatment of lungs cancer via anticancer activities on H1299 lung cancer cells. MATERIAL AND METHODS: PLGA-NPs was prepared by solvent evaporation (double emulsion) method followed by coated with chitosan (CS) and evaluated based on release and permeation of drug, a comparative pulmokinetics study with their anticancer activities on H1299 lung cancer cells. RESULTS: The particle size, PDI and ZP of the optimized CAT-PLGA-NPs and CS-CAT-PLGA-NPs were determined 124.64⯱â¯12.09â¯nm and 150.81⯱â¯15.91â¯nm, 0.163⯱â¯0.03 and 0.306⯱â¯0.03, -3.94⯱â¯0.19â¯mV and 26.01⯱â¯1.19â¯mV respectively. Furthermore, higher entrapment efficiency was observed for CS-CAT PLGA NPs. The release pattern of the CS-CAT-PLGA NPs was found to favor the release of entrapped CAT within the cancer microenvironment. CS-CAT-PLGA-NPs exposed on H1299 cancer cells upto 24.0â¯h was found to be higher cytotoxic as compared to CAT-solution (CAT-S). CS-CAT-PLGA-NPs showed higher apoptosis of cancer cells after their exposure as compared to CAT-S. CS-CTH-PLGA-NPs showed tremendous mucoadhesive-nature as compared to CTH-S and CS-CTH-PLGA NPs by retention time (RT) of 0.589â¯min, and m/z of 289.21/109.21 for CTH alongwith RT of 0.613â¯min and m/z of 301.21/151.21 was found out for IS (internal standard), i.e. Quercetin). Likewise, for 1-1000â¯ngâ¯mL-1 (linear range) of % accuracy (92.01-99.31%) and %CV (inter & intra-day, i.e. 2.14-3.33%) was determined. The improved Cmax with AUC0-24 was observed extremely significant (pâ¯<â¯0.001) via i.n. as compared oral and i.v. in the wistar rat's lungs. The CS-approach was successfully designed and safely delivered CAT to the lungs without causing any risk. CONCLUSION: CS-CTH-PLGA-NPs were showed a significant role (pâ¯<â¯0.001) for the enhancement of lungs-bioavailability and potentially promising approach to treat lung cancers. CS-CTH-PLGA-NPs did not cause any toxicity, it showed safety and have no obvious toxic-effects on the rat's lungs and does not produce any mortality followed by no abnormal findings in the treated-rats.
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Propolis is rich in diverse bioactive compounds. Propolis samples were collected from three localities of Cameroon and used in the study. Column chromatography separation of propolis MeOH:DCM (50:50) extracts yielded a new isoflavonol, 2-hydroxy-8-prenylbiochanin A (1) alongside 2',3'-dihydroxypropyltetraeicosanoate (2) and triacontyl p-coumarate (3) isolated from propolis for first time together with seven compounds: ß-amyrine (4), oleanolic acid (5), ß-amyrine acetate (6), lupeol (7), betulinic acid (8), lupeol acetate (9) and lupenone (10). These compounds were tested for their inhibitory effect on oxidative burst where intracellular reactive oxygen species (ROS) were produced from zymosan stimulated human whole blood phagocytes and on production of nitric oxide (NO) from lipopolysaccharide (LPS) stimulated J774.2 mouse macrophages. The cytotoxicity of these compounds was evaluated on NIH-3 T3 normal mouse fibroblast cells, antiradical potential on 2,2-diphenyl-1-picrylhydrazylhydrazyl (DPPH·) as well as their anti-yeast potential on four selected candida species. Compound 1 showed higher NO inhibition (IC50 = 23.3 ± 0.3 µg/mL) than standard compound L-NMMA (IC50 = 24.2 ± 0.8 µg/mL). Higher ROS inhibition was shown by compounds 6 (IC50 = 4.3 ± 0.3 µg/mL) and 9 (IC50 = 1.1 ± 0.1 µg/mL) than Ibuprofen (IC50 = 11.2 ± 1.9 µg/mL). Furthermore, compound 1 displayed moderate level of cytotoxicity on NIH-3 T3 cells, with IC50 = 5.8 ± 0.3 µg/mL compared to the cyclohexamide IC50 = 0.13 ± 0.02 µg/mL. Compound 3 showed lower antifungal activity on Candida krusei and Candida glabrata, MIC of 125 µg/mL on each strain compared to 50 µg/mL for fuconazole. The extracts showed low antifungal activities ranging from 250 to 500 µg/mL on C. albicans, C. krusei and C. glabrata and the values of MIC on Candida parapsilosis were 500 µg/mL and above. DPPH* scavenging activity was exhibited by compounds 1 (IC50 = 15.653 ± 0.335 µg/mL) and 3 (IC50 = 89.077 ± 24.875 µg/mL) compared to Vitamin C (IC50 = 3.343 ± 0.271 µg/mL) while extracts showed moderate antiradical activities with IC50 values ranging from 309.31 ± 2.465 to 635.52 ± 11.05 µg/mL. These results indicate that compounds 1, 6 and 9 are potent anti-inflammatory drug candidates while 1 and 3 could be potent antioxidant drugs.
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Diabetes or diabetes mellitus is a complex or polygenic disorder, which is characterized by increased levels of glucose (hyperglycemia) and deficiency in insulin secretion or resistance to insulin over an elongated period in the liver and peripheral tissues. Thiazolidine-2,4-dione (TZD) is a privileged scaffold and an outstanding heterocyclic moiety in the field of drug discovery, which provides various opportunities in exploring this moiety as an antidiabetic agent. In the past few years, various novel synthetic approaches had been undertaken to synthesize different derivatives to explore them as more potent antidiabetic agents with devoid of side effects (i.e., edema, weight gain, and bladder cancer) of clinically used TZD (pioglitazone and rosiglitazone). In this review, an effort has been made to summarize the up to date research work of various synthetic strategies for TZD derivatives as well as their biological significance and clinical studies of TZDs in combination with other category as antidiabetic agents. This review also highlights the structure-activity relationships and the molecular docking studies to convey the interaction of various synthesized novel derivatives with its receptor site.
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Our recent studies demonstrated that the natural product nobiletin (NOB) served as a promising multidrug resistance (MDR) reversal agent and improved the effectiveness of cancer chemotherapy in vitro. However, low aqueous solubility and difficulty in total synthesis limited its application as a therapeutic agent. To tackle these challenges, NOB was synthesized in a high yield by a concise route of six steps and fourteen derivatives were synthesized with remarkable solubility and efficacy. All the compounds showed improved sensitivity to paclitaxel (PTX) in P-glycoprotein (P-gp) overexpressing MDR cancer cells. Among them, compound 29d exhibited water solubility 280-fold higher than NOB. A drug-resistance A549/T xenograft model showed that 29d, at a dose of 50 mg/kg co-administered with PTX (15 mg/kg), inhibited tumor growth more effective than NOB and remarkably increased PTX concentration in the tumors via P-gp inhibition. Moreover, Western blot experiments revealed that 29d inhibited expression of NRF2, phosphorylated ERK and AKT in MDR cancer cells, thus implying 29d of multiple mechanisms to reverse MDR in lung cancer.
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Histone lysine specific demethylase 1 (LSD1) has been recognized as an important modulator in post-translational process in epigenetics. Dysregulation of LSD1 has been implicated in the development of various cancers. Herein, we report the discovery of the hit compound 8a (IC50 = 3.93⯵mol/L) and further medicinal chemistry efforts, leading to the generation of compound 15u (IC50 = 49â¯nmol/L, and K i = 16â¯nmol/L), which inhibited LSD1 reversibly and competitively with H3K4me2, and was selective to LSD1 over MAO-A/B. Docking studies were performed to rationalize the potency of compound 15u. Compound 15u also showed strong antiproliferative activity against four leukemia cell lines (OCL-AML3, K562, THP-1 and U937) as well as the lymphoma cell line Raji with the IC50 values of 1.79, 1.30, 0.45, 1.22 and 1.40⯵mol/L, respectively. In THP-1 cell line, 15u significantly inhibited colony formation and caused remarkable morphological changes. Compound 15u induced expression of CD86 and CD11b in THP-1 cells, confirming its cellular activity and ability of inducing differentiation. The findings further indicate that targeting LSD1 is a promising strategy for AML treatment, the triazole-fused pyrimidine derivatives are new scaffolds for the development of LSD1/KDM1A inhibitors.
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Hyaluronic acid (HA) is a natural ligand of tumor-targeted drug delivery systems (DDS) due to the relevant CD44 receptor overexpressed on tumor cell membranes. However, other HA receptors (HARE and LYVE-1) are also overexpressing in the reticuloendothelial system (RES). Therefore, polyethylene glycol (PEG) modification of HA-based DDS is necessary to reduce RES capture. Unfortunately, pegylation remarkably inhibits tumor cellular uptake and endosomal escapement, significantly compromising the in vivo antitumor efficacy. Herein, we developed a Dox-loaded HA-based transformable supramolecular nanoplatform (Dox/HCVBP) to overcome this dilemma. Dox/HCVBP contains a tumor extracellular acidity-sensitive detachable PEG shell achieved by a benzoic imine linkage. The in vitro and in vivo investigations further demonstrated that Dox/HCVBP could be in a "stealth" state at blood stream for a long circulation time due to the buried HA ligands and the minimized nonspecific interaction by PEG shell. However, it could transform into a "recognition" state under the tumor acidic microenvironment for efficient tumor cellular uptake due to the direct exposure of active targeting ligand HA following PEG shell detachment. Such a transformative concept provides a promising strategy to resolve the dilemma of natural ligand-based DDS with conflicting two processes of tumor cellular uptake and in vivo nonspecific biodistribution.
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The aim of the study is to levels of nicotine and cotinine were elevated the oxidative stress malondialdehyde (MDA) and inflammation as nitric oxide (NO2 and NO3) may possibly be associated with decreased antioxidant enzyme activities and can sensitively indicate the production of reactive oxygen species (ROS). To evaluate the quantitative analysis of nicotine and cotinine levels and the alterations in the selected parameters of antioxidant metabolisms during nitroxidative stress in the saliva of smokeless tobacco consumers. Saliva nicotine and cotinine was measured by HPLC method and nitric oxide, lipid peroxidation and activities of antioxidant enzymes were estimated by spectrophotometric methods. Significant increase in concentrations of nicotine and cotinine levels of saliva in smokeless tobacco users in comparison to controls. Saliva lipid peroxidation was increased in experimental subjects (gutkha group 39.28% and khaini group 25.00%) as compared to controls and nitric oxide in the form of nitrites and nitrates was significantly increased in the saliva of smokeless tobacco users compared to controls. The activity levels of antioxidant enzymes were decreased in the saliva of the smokeless tobacco users in comparison with normal controls. A strong positive correlation of nicotine and cotinine with nitroxidative stress markers in gutkha and khaini users. Increased expression of inducible nitric oxide synthase (iNOS) enzyme leads to intoxication in saliva and indirectly induces inflammation process. Increased production of reactive oxygen species (ROS) and decrease in the activity levels of antioxidant enzymes in the saliva of smokeless tobacco users indicate conspicuous cell and tissue damage.