Thymus linearis Extracts Ameliorate Indices of Metabolic Syndrome in Sprague Dawley Rats.

Materials and Methods: The extract library (n-hexane (NH), ethyl acetate (EA), methanol (M), distilled water (DW), and combined extract (CE)) was standardized using in vitro phytochemical, antioxidant, and α-amylase inhibition assays, after which the protective effect of selected "hit," i.e., CE against metabolic syndrome, was determined in vivo, using rats fed a high-fat diet supplemented with additional cholesterol administration. CE was administered to Sprague Dawley rats in high dose as 100 mg/kg in carboxymethyl cellulose (CMC) (1 ml; 0.75% in DW) and low-dose group as 50 mg/kg in CMC (0.5 ml; 0.75% in DW). After 10 weeks, the effects of CE on insulin resistance, lipid metabolism, nonalcoholic fatty liver disease (NAFLD), oxidative stress, and genotoxicity were assessed through histological, biochemical, and hematological investigations. Results: Phytochemical analysis including RP-HPLC analysis of the extracts showed that flavonoids and phenolics (myricetin, kaempferol, and apigenin), previously known to be effective against obesity and diabetes, are present in the extracts. Antioxidant studies revealed that the plant possesses a highly significant (p < 0.05) concentration of antioxidants. Satisfactory α-amylase inhibitory activity was also observed in in vitro experiments. In vivo studies showed that CE-administered animals had significantly (p < 0.05) lower weight gain and smaller adipocytes than the control group. Moreover, CE resisted any significant (p < 0.05) change in the organ weights. Analogous to findings from its traditional use, the plant extract had a positive modulatory effect on insulin resistance and hyperglycemia. The study also indicated that CE resisted high-fat diet-induced disturbance in lipid profile and countered any pathological changes in liver enzymes caused by fat-infused diet. Furthermore, a study on endogenous antioxidant levels indicated that CE was effective in maintaining catalase and peroxidase levels within the normal range and resisted the effects of lipid peroxidation of thiobarbituric acid reactive substances. Conclusion: In principle, the current study's findings scientifically validate the implication of T. linearis in metabolic syndrome and recommend further studies on molecular insights of the observed therapeutic activity.

Toxicity evaluation induced by single and 28-days repeated exposure of withametelin and daturaolone in Sprague Dawley rats.

Safe preclinical dose determination is predictive of human toxicity and can have a profound impact on the overall progress of the compound in early drug discovery process. In this respect, current study sought to investigate for the first time the acute and subacute oral toxicity of two pharmacologically active natural compounds i.e., withametelin and daturaolone in Sprague Dawley rats following OECD guideline 420 and 407, respectively. As per acute toxicity studies, withametelin and daturaolone were characterized as Globally Harmonized System (GHS) category 4 and 5 compounds, respectively. Sub-acute daily dose of withametelin was 5, 2.5, and 1.25 mg/kg but, for daturaolone, it was 10, 5, and 2.5 mg/kg. High dose (5 and 2.5 mg/kg) withametelin groups showed dose dependent changes in the general, hematological, biochemical and histopathological parameters in both sexes, the most prominent being hyperthyroidism while no toxicity was observed at lower doses (1.25 and 0.75 mg/kg), No Observable Adverse Effect Level (NOAEL) being 1.25 mg/kg. Daturaolone was comparatively safer and showed dose dependent significant changes in hepatic enzyme (Alanine Transaminase), bilirubin, creatinine, and glucose levels while histological changes in testes were also observed. Lower doses (5, 2.5, and 1.25 mg/kg) of daturaolone showed no significant toxic effects and 5 mg/kg was declared as its NOAEL. Depending upon our findings, starting effective oral dose levels of 1.25 mg/kg/day for withametelin and 5 mg/kg/day for daturaolone are proposed for repeated dose (up to 28 days) preclinical pharmacological evaluation models. Long term studies with more behavioral, biochemical, histopathological and hormonal parameters are proposed to strengthen the findings.

Datura stramonium Leaf Extract Exhibits Anti-inflammatory Activity in CCL4-Induced Hepatic Injury Model by Modulating Oxidative Stress Markers and iNOS/Nrf2 Expression.

Background: Inflammation is a frequent phenomenon in the pathogenesis of hepatic disorders leading to fibrosis and cirrhosis. Phytopharmaceuticals developed from traditional medicine can provide effective therapeutic alternatives to conventional medications. Datura stramonium (DS) has reported traditional uses in inflammatory diseases. In this study, we have tried to validate its potential as a source of anti-inflammatory agents. Methods: Powdered leaf part of DS was extracted using ethyl acetate (EA) to provide the extract (DSL-EA). Lymphocyte and macrophage viability and acute toxicity assays established the safety profile, while nitric oxide (NO) scavenging assay estimated the in vitro anti-inflammatory potential. Noninvasive anti-inflammatory, antidepressant, and antinociceptive activities were monitored using BALB/c mice using low and high doses (150 and 250 mg/kg). Major inflammatory studies were performed on Sprague-Dawley male rats using CCl4-induced liver injury model. Disease induction was initiated by intraperitoneal injections of CCl4 (1 mL/kg of 30% CCl4 in olive oil). The rats were divided into six groups. The anti-inflammatory potential of DSL-EA in low and high doses (150 and 300 mg/kg, respectively) was assessed through hematological, biochemical, liver antioxidant defense, oxidative stress markers, and histological studies as well as the expression of Nrf2 and iNOS. Results: DSL-EA exhibited prominent in vitro NO scavenging (IC50: 7.625 ± 0.51 µg/mL) and in vivo anti-inflammatory activity in paw and anal edema models. In CCl4 model, hematological investigations revealed vasotonic effects. Liver functionality was significantly (P < 0.001 - 0.05) improved in DSL-EA-treated rats. The activity level of endogenous antioxidant enzymes in liver tissues was improved in a manner identical to silymarin. The extract reduced the percent concentration of oxidative stress markers in liver tissues. Furthermore, DSL-EA displayed restorative effects on histological parameters (H and E and Masson's trichrome staining). Immunohistochemistry studies showed marked decline in Nrf2 expression, while overexpression of iNOS was also observed in disease control rats. The damage was distinctly reversed by the extract.

Caralluma tuberculata N.E.Br Manifests Extraction Medium Reliant Disparity in Phytochemical and Pharmacological Analysis.

Solubility of phytoconstituents depends on the polarity of the extraction medium used, which might result in the different pharmacological responses of extracts. In line with this, ethnomedicinally important food plant (i.e., Caralluma tuberculata extracts) have been made in fourteen distinct solvent systems that were then analyzed phytochemically via total phenolic amount estimation, total flavonoid amount estimation, and HPLC detection and quantification of the selected polyphenols. Test extracts were then subjected to a battery of in vitro assays i.e., antioxidants (DDPH scavenging, antioxidant capacity, and reducing power estimation), antimicrobial (antibacterial, antifungal, and antileishmanial), cytotoxic (brine shrimps, THP-1 human leukemia cell lines and normal lymphocytes), and protein kinase inhibition assays. Maximum phenolic and flavonoid contents were computed in distilled water-acetone and acetone extracts (i.e., 16 ± 1 µg/mg extract and 8 ± 0.4/mg extract, respectively). HPLC-DAD quantified rutin (0.58 µg/mg extract) and gallic acid (0.4 µg/mg extract) in methanol-ethyl acetate and methanol extracts, respectively. Water-acetone extract exhibited the highest DPPH scavenging of 36 ± 1%. Total reducing potential of 76.0 ± 1 µg/mg extract was shown by ethanol chloroform while maximum total antioxidant capacity was depicted by the acetone extract (92.21 ± 0.70 µg/mg extract). Maximal antifungal effect against Mucor sp., antileishmanial, brine shrimp cytotoxicity, THP-1 cell line cytotoxicity, and protein kinase inhibitory activities were shown by ethyl acetate-methanol (MIC: 50 µg/disc), n-hexane (IC50: 120.8 ± 3.7 µg/mL), ethyl acetate (LD50: 29.94 ± 1.6 µg/mL), distilled water-acetone (IC50: 118 ± 3.4 µg/mL) and methanol-chloroform (ZOI: 19 ± 1 mm) extracts, respectively. Our findings show the dependency of phytochemicals and bioactivities on the polarity of the extraction solvent and our preliminary screening suggests the C. tuberculata extract formulations to be tested and used in different ailments, however, detailed studies remain necessary for corroboration with our results.

Comparative toxicity of green and chemically synthesized CuO NPs during pregnancy and lactation in rats and offspring: Part I -hepatotoxicity.

Widespread applications of nanomaterials in food and health sciences have inevitable toxicological outcomes. Among nanomaterials, copper oxide nanoparticles (CuO NPs) are commonly used in all fields due to its distinctive characteristics. The study was designed to investigate the comparative hepatotoxic effect of green (GNPs) and chemically synthesized (CNPs) CuO NPs on spargue-dawely rats and their offspring. NPs (50 and 100 mg/kg) were orally administered to rats twice a week starting before mating. After birth the parents were continued dosing while pups were only on mother feed. Antioxidant, lipid peroxidation, genotoxicity, and histology were performed on liver tissue. In addition serum biochemistry of parents and offspring was also performed. The levels of catalase, peroxidase, and glutathione were significantly lowered only in CNPs treated parents while lipid peroxidation level was increased in these groups. Maximum genotoxicity (2.3%) in terms of percent tail DNA was observed in parent rats administered with high CNPs dose while other groups did not exhibit significant variation in genetoxic parameters. Gender and dose dependent effects were observed on liver function tests especially ALP and ALT in parents however no obvious differences were observed in offspring. Furthermore, dose dependent dilation and congestion of sinusoids was observed on CNPs administration. In GNPs treated rats and offspring histological alterations were observed. The study concludes that chemically synthesized CuO NPs exhibit dose dependent toxic effects on liver as compared to green synthesized CuO NPs. Furthermore lactation does not play significant role in the hepatotoxicity of offspring though minor oxidative stress was observed only on CNPs administration. The study also shows that pharmacological application of green synthesized NPs can be accomplished due to their biocompatible nature.

Preclinical anticancer studies on the ethyl acetate leaf extracts of Datura stramonium and Datura inoxia.

BACKGROUND: Cancer is a horrific disease relentlessly affecting human population round the globe. Genus Datura encompasses numerous species with reported medicinal uses. However, its potential as a source of natural anticancer agents is yet to be determined. Datura stramonium (DS) and Datura inoxia (DI) are the two species chosen for this study. METHODS: Total phenolic and flavonoid content (TPC and TFC) as well as antioxidant activity were assessed through colorimetric method. Polyphenolic quantification was done by RP-HPLC. Following extract standardization ethyl acetate leaf extracts of both species (DSL-EA and DIL-EA) were chosen for anticancer studies. In vitro cytotoxicity using various models including cancer cell lines was monitored. Following toxicity studies, benzene (0.2 ml) was used to induce leukemia in Sprague-Dawley rats. Extracts were orally administered to preventive (100 and 200 mg/kg) and treatment (200 mg/kg only) groups. The antileukemic potential of extracts was assessed through haematological, biochemical, endogenous antioxidants and histological parameters. RESULTS: Significant TPC and TFC were estimated in DSL-EA and DIL-EA. RP-HPLC quantified (µg/mg extract) rutin (0.89 ± 0.03), gallic acid (0.35 ± 0.07), catechin (0.24 ± 0.02) and apigenin (0.29 ± 0.09) in DSL-EA while rutin (0.036 ± 0.004) and caffeic acid (0.27 ± 0.03) in DIL-EA. Both extracts exhibited significant brine shrimp cytotoxicity (LC50 < 12.5 µg/ml). DIL-EA exhibited greater cytotoxicity against PC-3, MDA-MB 231 and MCF-7 cell lines (IC50 < 3 µg/ml in each case) as well as higher protein kinase inhibitory action (MIC: 25 µg/disc) compared to DSL-EA. Leukemia induced in rats was affirmed by elevated serum levels of WBCs (7.78 ± 0.012 (× 103) /µl), bilirubin (7.56 ± 0.97 mg/dl), Thiobarbituric acid reactive substances (TBARs) (133.75 ± 2.61 nM/min/mg protein), decreased RBCs (4.33 ± 0.065 (× 106)/µl), platelets (344 ± 3.19 (× 103)/µl), total proteins (2.14 ± 0.11 g/dl), Glutathione S-transferases (GST) (81.01 ± 0.44 nM/min/ml), endogenous antioxidant enzymes levels and abnormal liver and kidney functionality in disease control rats. Both species revealed almost identical and significant (p < 0.05) alleviative effects in benzene induced leukemia. CONCLUSION: Comprehensive screening divulged the tremendous potential of selected species as potent source of natural anticancer agents in a variety of cancers particularly leukemia. Present study might provide useful finger prints in cancer research and mechanistic studies are prerequisite in logical hunt of this goal.

Matrine alleviates neurobehavioral alterations via modulation of JNK-mediated caspase-3 and BDNF/VEGF signaling in a mouse model of burn injury.

RATIONALE: The c-Jun N-terminal kinase (JNK) pathway and neurotrophic factor dysregulation play a critical role in the pathogenesis of neurobehavioral disorders (anxiety and depression). Targeting the JNK pathway and BDNF/VEGF signaling may signify a new avenue for the treatment of neurobehavioral disorders. OBJECTIVES: The present study investigated the effect of matrine (Mat) against anxiety- and depressive-like emotional status in an acute mouse model of burn injury and explores its underlying mechanism. METHODS: In the mouse model of thermal injury, anxiety- and depression-related behaviors were evaluated using the elevated plus-maze test, the light-dark box test, the open-field test, the forced swimming test, and the tail suspension test. The JNK/caspase-3 and BDNF/VEGF proteins were determined by immunohistochemistry. Additionally, proinflammatory cytokine, antioxidant, nitric oxide, and corticosterone levels were also measured. RESULTS: The results showed that treatment with Mat significantly improves anxiety- and depressive-like behaviors. It remarkably reduced the levels of proinflammatory cytokines, malondialdehyde, and nitric oxide in the hippocampus and prefrontal cortex of a mouse brain. It considerably improved burn-induced alteration in the antioxidant status, corticosterone, and BDNF/VEGF. It also inhibited burn-induced apoptotic signaling by downregulating the expression of JNK/caspase-3. Similarly, it prevented DNA damage and histopathological changes in the dentate gyrus of the hippocampus. Furthermore, molecular docking results showed that Mat possess better binding affinity for JNK/caspase-3 and BDNF/VEGF proteins. CONCLUSIONS: These findings provide convincing evidence that Mat improves anxiety- and depressive-like emotional status through modulation of JNK-mediated inflammatory, oxidative stress, apoptotic, and BDNF/VEGF signaling in an acute mouse model of burn injury.

Withametelin: a biologically active withanolide in cancer, inflammation, pain and depression.

Withanolides are natural medicinal agents whose safety and therapeutic profiles make them valuable to mankind. Among multiple withanolides, withametelin is underexplored. The present study was aimed to create a general biological profile of isolated withametelin from Datura innoxia Mill. targeting different biological models. In-silico studies include drug-likeliness, pharmacokinetics, toxicity, molecular targets and cytotoxicity to cancer cell lines predictions. In silico directed preliminary in-vitro evaluation comprised of cancer/normal cell cytotoxicity, DPPH and protein kinase inhibition assays while in-vivo bioactivities include antiinflammatory, analgesic, antidepressant and anticoagulant assays. Pharmacological findings were strengthened by molecular docking studies to check interactions with various proteins and to propose the future path of studies. Results indicated compliance with Lipinski drug-likeliness rule (score -0.55). ADMET prediction showed strong plasma protein binding, GI absorption (Caco-2 cells permeability = 46.74 nm/s), blood brain barrier penetration (Cbrain/Cblood = 0.31), efflux by P-glycoprotein, metabolism by CYP1A2, CYP2C19 and CYP3A4, medium hERG inhibition and non-carcinogenicity in rodents. Predicted molecular targets included mainly receptors (glucocorticoid, kappa opioid, delta opioid, adrenergic and dopamine), oxidoreductase (arachidonate 5-lipoxygenase and cyclooxygenase-2), enzymes (HMG-CoA reductase) and kinase (NFκb). Withametelin was more cytotoxic to cancer cells (DU145 IC50 7.67 ± 0.54 µM) than normal lymphocytes (IC50 33.55 ± 1.31 µM). It also showed good antioxidant and protein kinase inhibition potentials. Furthermore, withametelin (20 mg/kg) significantly reduced inflammatory paw edema (68.94 ± 5.55%), heat-induced pain (78.94 ± 6.87%) and immobility time (50%) in animals. Molecular docking showed hydrogen bonding interactions (binding energies: -11.3 to -7.8 kcal/mol) with arachidonate 5 lipoxygenase, NFκb and glucocorticoid receptor. Withametelin has potential for advance investigations for its cytotoxic, anti-inflammatory, analgesic and antidepressant activities.

Scientific Validation of Ethnomedicinal Use of Ipomoea batatas L. Lam. as Aphrodisiac and Gonadoprotective Agent against Bisphenol A Induced Testicular Toxicity in Male Sprague Dawley Rats.

Sweet potato (Ipomoea batatas L. Lam.), known as "Shakarqandi" in Pakistan, is an imperative root vegetable with large size, traditionally used as aphrodisiac, antiprostatic, anti-inflammatory, antidiabetic, cardiotonic, and anticancer agent. Present study was conducted to gauge aphrodisiac potential of Ipomoea batatas ethyl acetate (IPT-EA, IPA-EA) and methanol (IPT-M, IPA-M) extracts from tuber and aerial part, respectively, via behavioral and biochemical tests and their possible protective role in BPA-induced gonadotoxicity at the dose 300 mg/kg in male Sprague Dawley rats. Phytochemical analysis was done qualitatively and quantitatively through total phenolic and flavonoid content (TPC and TFC) and high performance liquid chromatographic (HPLC-DAD) fingerprinting while antioxidant profiling used multimode in vitro assays. To calculate sexual excitement mount latency, intromission latency, mount frequency, intromission frequency, ejaculatory latency, and postejaculatory interval were examined while for biochemical ratification semen characteristics, levels of testosterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol were measured. Gonadoprotective ability was assessed through comet assay and histomorphological examination of testes. Qualitative analysis ensured the presence of phenols, flavonoids, tannins, anthocyanin, saponins, coumarins, terpenoids, and betacyanin. Quantitatively maximal TPC (304.32±7.20 µg GAE/mg dry extract) and TFC (214.77±4.09 µg QE/mg DE) were estimated in IPA-EA extract. IPT-EA yielded maximum rutin (7.3±0.12) and myricetin (2.7±0.14 µg/mg DE) while IPA-EA and IPA-M yielded maximum caffeic acid (4.05±0.22 and 1.92±0.17 µg/mg DE, respectively) in HPLC-DAD analysis. Extracts enhanced sexual excitement, improved semen quality, levels of testosterone, FSH, LH, and estradiol, and successfully attenuated toxic effects of BPA. Levels of endogenous antioxidant enzymes (CAT, SOD, POD, and GSH) were restored and NO abundance was minimized. Significant stimulation in sexual behavior, amelioration of toxicity symptoms, elevated spermatic production, raised viability, vitalized levels of gonadal hormones, maintained endogenous enzymes, genoprotection, and reformed testicular histology endorsed I. batatas as a better aphrodisiac alternative and gonadoprotective agent.

Ipomoea batatas L. Lam. ameliorates acute and chronic inflammations by suppressing inflammatory mediators, a comprehensive exploration using in vitro and in vivo models.

BACKGROUND: Ipomoea batatas L. Lam. is a functional food and belongs to family Convolvulaceae. It is used as an antiinflammatory, aphrodisiac, antiasthmatic, anticonvalescent, antitumor, antanemic and antidiabetic agent by local communities. This study has been planned to evaluate its antiinflammatory and antiarthritic potentials. METHODS: Dry powder of I. batatas tuber and roots were extracted with ethyl acetate (IPT-EA, IPR-EA) and methanol (IPT-M, IPR-M), respectively. These extracts were tested for total phenolic and flavonoid contents (TPC and TFC), HPLC finger printing, multidimensional in vitro and in vivo antioxidant potential and albumin denaturation inhibition. Carrageenan-induced paw edema, croton oil-induced ear and anal edema inhibition and Complete Freund's Adjuvant (CFA)-induced antiarthritic assays were executed at a dose of 300 mg/kg body weight on Sprague-Dawley rats. Serum levels of interleukins IL-1ß and IL-6 and nitric oxide (NO) were assessed to measure the inhibition of inflammation. RESULTS: Maximal TPC (319.81 ± 14.20 µg GAE/mg dry extract) and TFC (208.77 ± 9.09 µg QE/mg DE) were estimated in IPR-EA extract. IPT-EA and IPR-EA yielded the maximum amounts of rutin (7.3 ± 1.12 and 4.5 ± 0.55), caffeic acid (1.60 ± 0.25 and 2.17 ± 0.26) and myricetin (2.7 ± 0.14 and 1.01 ± 0.08 µg/mg DE), respectively in HPLC-DAD analysis. All extracts showed dose dependent response in in vitro antioxidant assays. Best inhibition (76.92 ± 3.07%) of albumin denaturation was shown by IPT-EA in comparison to ibuprofen (79.48 ± 4.71%). IPR-EA exhibited highest edema inhibition in models of carrageenan-induced paw edema (79.11 ± 5.47%) and croton oil-induced ear and anal edema (72.01 ± 7.80% and 70.80 ± 4.94%, respectively). Significant inhibition of CFA-induced arthritic edema and arthritic score were observed by IPR-EA as compared to ibuprofen. Suppression of pro-inflammatory cytokines (IL-1ß, IL-6) and NO levels was shown by IPR-EA and IPT-EA, respectively. CONCLUSION: These results depict that richness of polyphenols and phytoconstituents in I. batatas ameliorates oxidative stress and inflammation of acute and chronic nature. Dose dependent antioxidant potential and inhibition of inflammatory edema, pro-inflammatory cytokines and hematological, biochemical and histological changes prove I. batatas therapeutic potential as an antiinflammatory and antiarthritic agent.

Oxidative stress, consequences and ROS mediated cellular signaling in rheumatoid arthritis.

There are numerous extra- and intra-cellular processes involved in the production of reactive oxygen species (ROS). Augmented ROS generation can cause the damage of biomolecules such as proteins, nucleic acid and lipids. ROS act as an intracellular signaling component and is associated with various inflammatory responses, chronic arthropathies, including rheumatoid arthritis (RA). It is well documented that ROS can activate different signaling pathways having a vital importance in the patho-physiology of RA. Hence, understanding of the molecular pathways and their interaction might be advantageous in the development of novel therapeutic approaches for RA.