Development of novel polymer haemoglobin based particles as an antioxidant, antibacterial and an oxygen carrier agents.

This innovative work aims to develop highly biocompatible and degradable nanoparticles by encapsulating haemoglobin (Hb) within poly-ε-caprolactone for novel biomedical applications. We used a modified double emulsion solvent evaporation method to fabricate the particles. A Scanning electron microscope (SEM) characterized them for surface morphology. Fourier Transform Infrared Spectroscopy (FTIR) and Ultraviolet-visible spectroscopies (UV-visible) elucidated preserved chemical and biological structure of encapsulated haemoglobin. The airproof equilibrium apparatus obtained the oxygen-carrying capacity and P50 values. The DPPH assay assessed free radical scavenging potential. The antibacterial properties were observed using four different bacterial strains by disk diffusion method. The MTT assay investigates the cytotoxic effects on mouse fibroblast cultured cell lines (L-929). The MTT assay showed that nanoparticles have no toxicity over large concentrations. The well-preserved structure of Hb within particles, no toxicity, high oxygen affinity, P50 value, and IC50 values open the area of new research, which may be used as artificial oxygen carriers, antioxidant, and antibacterial agents, potential therapeutic agents as well as drug carrier particles to treat the cancerous cells. The novelty of this work is the antioxidant and antibacterial properties of developed nanoparticles are not been reported yet. Results showed that the prepared particles have strong antioxidant and antibacterial potential.

Artemisia brevifolia Wall. Ex DC Enhances Cefixime Susceptibility by Reforming Antimicrobial Resistance.

(1) Background: A possible solution to antimicrobial resistance (AMR) is synergism with plants like Artemisia brevifolia Wall. ex DC. (2) Methods: Phytochemical quantification of extracts (n-hexane (NH), ethyl acetate (EA), methanol (M), and aqueous (Aq)) was performed using RP-HPLC and chromogenic assays. Extracts were screened against resistant clinical isolates via disc diffusion, broth dilution, the checkerboard method, time-kill, and protein quantification assays. (3) Results: M extract had the maximum phenolic (15.98 ± 0.1 µg GAE/mgE) and flavonoid contents (9.93 ± 0.5 µg QE/mgE). RP-HPLC displayed the maximum polyphenols in the M extract. Secondary metabolite determination showed M extract to have the highest glycosides, alkaloids, and tannins. Preliminary resistance profiling indicated that selected isolates were resistant to cefixime (MIC 20-40 µg/mL). Extracts showed moderate antibacterial activity (MIC 60-100 µg/mL). The checkerboard method revealed a total synergy between EA extract and cefixime with 10-fold reductions in cefixime dose against resistant P. aeruginosa and MRSA. Moreover, A. brevifolia extracts potentiated the antibacterial effect of cefixime after 6 and 9 h. The synergistic combination was non- to slightly hemolytic and could inhibit bacterial protein in addition to cefixime disrupting the cell wall, thus making it difficult for bacteria to survive. (4) Conclusion: A. brevifolia in combination with cefixime has the potential to inhibit AMR.

Cardioprotective effect of 2-methoxy phenol derivatives against oxidative stress-induced vascular complications: An integrated in vitro, in silico, and in vivo investigation.

BACKGROUND: Oxidative stress and inflammation play crucial roles in macro/microvascular complications. Phenolic compounds and their derivatives show promise as therapeutic agents for diseases like cancer, metabolic disorders, and cardiovascular diseases. With their antioxidant and anti-inflammatory properties, these compounds hold potential for mitigating vascular complications and improving overall health. METHODOLOGY: This study aimed to assess the therapeutic potential of five 2-methoxy phenol derivatives (T2, T5, T6, T7, and T8) as antioxidants, anti-inflammatory agents, and vasorelaxants using in vitro, in silico, and in vivo approaches. RESULTS: Among all, T2 exhibited substantial antioxidant potential against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals with IC50 (27.97 µg/mL), nitric oxide (NO) radicals (IC50 = 34.36 µg/mL), hydroxyl (OH) radicals (IC50 = 34.83 µg/mL) and Iron chelation (IC50 = 24.32 µg/mL). Molecular docking analysis confirms that all derivatives, particularly T2, exhibit favorable binding energies with the target proteins, ACE (-7.7 Kcal/mol), ECE-1 (-7.9 Kcal/mol), and COX-1 (-7.8 Kcal/mol). All of the compounds demonstrated satisfactory physicochemical and pharmacokinetic characteristics, and showed minimal to no toxicity during in silico, in vitro, and in vivo assessments. In isolated aortic rings from Sprague Dawley rats, pre-contracted with high K+ (80 mM), T2 induced vasorelaxation in dose dependent manner and shifted calcium response curves to the right as compared to verapamil. T2 also showed substantial platelet aggregation inhibition in a dose dependent manner with IC50 21.29 µM. All derivatives except T7 exhibited significant conservation of endogenous antioxidants i.e. catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and reduced glutathione (GSH) and significantly suppressed serum levels of inflammatory markers i.e. nitric oxide (NO), peroxides (TBARS), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2). CONCLUSION: The study concludes that T2 has significant antioxidant potential and vasorelaxant effects with adequate pharmacokinetics, making it a promising lead compound for further molecular investigation in cardiovascular disorders.

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.

Appraisal of selected ethnomedicinal plants as alternative therapies against onychomycosis: Evaluation of synergy and time-kill kinetics.

Introduction: This study aims at the biological profiling of Allium sativum, Zingiber officinale, Nigella sativa, Curcuma longa, Mentha piperita, Withania somnifera, Azadirachta indica, and Lawsonia inermis as alternatives against onychomycosis to combat the treatment challenges. Methods: An extract library of aqueous (DW), ethyl acetate (EA), and methanol (M) extracts was subjected to phytochemical and antioxidant colorimetric assays to gauge the ameliorating role of extracts against oxidative stress. RP-HPLC quantified therapeutically significant polyphenols. Antifungal potential (disc diffusion and broth dilution) against filamentous (dermatophytes and non-dermatophytes) and non-filamentous fungi (yeasts; Candida albicans), synergistic interactions (checkerboard method) with terbinafine and amphotericin-B against resistant clinical isolates of dermatophytes (Trichophyton rubrum and Trichophyton tonsurans) and non-dermatophytes (Aspergillus spp., Fusarium dimerum, and Rhizopus arrhizus), time-kill kinetics, and protein estimation (Bradford method) were performed to evaluate the potential of extracts against onychomycosis. Results: The highest total phenolic and flavonoid content along with noteworthy antioxidant capacity, reducing power, and a substantial radical scavenging activity was recorded for the extracts of Z. officinale. Significant polyphenolics quantified by RP-HPLC included rutin (35.71 ± 0.23 µg/mgE), gallic acid (50.17 ± 0.22 µg/mgE), catechin (93.04 ± 0.43 µg/mgE), syringic acid (55.63 ± 0.35 µg/mgE), emodin (246.32 ± 0.44 µg/mgE), luteolin (78.43 ± 0.18 µg/mgE), myricetin (29.44 ± 0.13 µg/mgE), and quercetin (97.45 ± 0.22 µg/mgE). Extracts presented prominent antifungal activity against dermatophytes and non-dermatophytes (MIC-31.25 µg/ml). The checkerboard method showed synergism with 4- and 8-fold reductions in the MICs of A. sativum, Z. officinale, M. piperita, L. inermis, and C. longa extracts and doses of amphotericin-B (Amp-B) and terbinafine (against non-dermatophytes and dermatophytes, respectively). Furthermore, the synergistic therapy showed a time-dependent decrease in fungal growth even after 9 and 12 h of treatment. The inhibition of fungal proteins was also observed to be higher with the treatment of synergistic combinations than with the extracts alone, along with the cell membrane damage caused by terbinafine and amp-B, thus making the resistant fungi incapable of subsisting. Conclusion: The extracts of A. sativum, Z. officinale, M. piperita, L. inermis, and C. longa have proven to be promising alternatives to combat oxidative stress, resistance, and other treatment challenges of onychomycosis.

Indigofera linifolia ameliorated CCl4 induced endoplasmic reticulum stress in liver of rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigofera linifolia (L.f.) Retz. is used in subcontinent for liver disorders, in wounds, febrile eruption and as diuretic. AIM OF STUDY: The current study evaluates the protective effects of the methanol extract of Indigofera linifolia (ILM) on CCl4-induced endoplasmic reticulum (ER) stress in liver of rat. METHODS: ILM was analyzed for phytochemical classes, total phenolic (TPC) and flavonoid content (TFC) as well as multidimensional in vitro antioxidant assays. Male (Sprague Dawley) rats were dispersed into seven groups (6 rats/group) receiving 0.9% saline (1 ml/kg bw), CCl4 (1 ml/kg bw) diluted in olive oil (3:7 v/v), silymarin (200 mg/kg bw) + CCl4 (30% v/v), ILM (150 mg/kg bw) + CCl4 (30% v/v), ILM (300 mg/kg bw) + CCl4 and ILM alone (either 150 mg/kg bw or 300 mg/kg bw). RESULTS: ILM extract was constituted of different phytochemical classes. Co-administration of ILM along with CCl4 to rat revert the level of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total bilirubin in blood serum and antioxidant parameters in liver. Further, CCl4 increased the level of ER stress markers and inflammatory mediators while decreased level of GCLC and Nrf-2 in liver tissues of rat. CCl4-induced histopathological variations were reduced with ILM co-administration in liver tissues. CONCLUSION: The results suggest that active phyto-constituents of I. linifolia might be responsible for its antioxidant, anti-inflammatory and gene-regulating activities.

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.

Gonadoprotective ability of Vincetoxicum arnottianum extract against bisphenol A-induced testicular toxicity and hormonal imbalance in male Sprague Dawley rats.

Vincetoxicum arnottianum (Wight) of family Apocynaceae is a rich source of therapeutic alkaloids, phenolics and flavonoids. Study aims to evaluate the protective potential of methanol extract of Vincetoxicum arnottianum (VAM) on bisphenol A (BPA)-induced testicular toxicity in male Sprague Dawley rat. Quantitative analysis of VAM for total phenolic (TPC), total flavonoid (TFC) and total alkaloid content (TAC) along with HPLC analysis for polyphenolics was carried out. BPA-induced testicular toxicity was determined through analysis of antioxidant enzymes, DNA damages and testicular histopathology along with reproductive hormones in serum of rat. VAM was constituted of TFC (382.50 ± 1.67 µg GAE/mg), TPC (291.17 ± 0.82 µg RE/mg), TAC (16.5 ± 0.5%), ferulic acid (2.2433 µg/mg) and vanillic acid (2.1249 µg/mg). VAM co-administration to BPA-treated rats attenuated the toxic effects of BPA and restored the body and testis weights. Altered level of luteinizing hormone (LH), testosterone and follicle-stimulating hormone (FSH) in serum, and level of antioxidants (GSH, POD, CAT and SOD) and nitric oxide in testis tissues of BPA-induced toxicity were significantly restored by VAM. Histological and comet assay studies also sanctioned the protective potential of VAM in BPA-intoxicated rats. The presence of polyphenols and alkaloids might contribute towards the scavenging and ameliorative potential of VAM in testicular toxicity induced by BPA.

Vincetoxicum arnottianum ameliorate inflammation by suppressing oxidative stress and pro-inflammatory mediators in rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Vincetoxicum arnottianum (Wight) Wight (Family Apocynaceae) are used by local communities for inflammation, healing of wound and injuries and also for urticaria. AIM OF STUDY: Extract/fractions of V. arnottianum were evaluated for potential anti-inflammatory activity in rat. METHODS: Methanol extract of aerial parts of V. arnottianum (VAM) was partitioned on polarity for n-hexane (VAH), ethyl acetate (VAE), butanol (VAB) and aqueous (VAA) fractions. The extract/fractions were evaluated during in vitro assay for protection against heat induced protein denaturation and Carrageenan induced paw inflammation in rat. VAM and VAE were evaluated for anti-inflammatory potential against formalin and Freund's complete adjuvant (FCA) induced inflammation in paw of rat while croton oil induced inflammation in ear of rat, respectively. The level of inflammatory mediators; IL-17, IL-1ß, IL-6, TNF-α and nitric oxide (NO) was estimated in serum of rat. RESULTS: All the extract/fractions used in this study exhibited anti-inflammatory activity. However, VAE (300 mg/kg) exhibited potential anti-inflammatory activity in carrageenan (78.06 ± 4.6%), formalin (54.71 ± 0.34%) and croton oil (73.12 ± 1.9%) induced edema in rat. In FCA induced inflammation model VAM and VAE showed admiring proficiencies against alteration of body weight and organ weight indices, paw edema and histological studies. In serum increased level of pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-17) and NO during adjuvant-induced inflammation were more efficiently restored with VAE treatment to rat. Presence of polyphenolics; rutin, gallic acid, caffeic acid, apigenin, myricetin and quercetin was indicated in VAE. CONCLUSION: The results suggest the presence of anti-inflammatory constituents in V. arnottianum.

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.

Wound healing potential of oil extracted from Parrotiopsis jacquemontiana (Decne) Rehder.

ETHNOPHARMACOLOGICAL RELEVANCE: Oil extracted from Parrotiopsis jacquemontiana stem traditionally used for wound healing, body aches and dermatitis. In this study we have evaluated oil for its phytoconstituents, antioxidant, antimicrobial and wound healing activities. METHODS: Phytochemical characterization of oil was determined by standard qualitative procedures, gas chromatography mass spectrometry technique (GC-MS) and Fourier transform infra-red spectroscopy (FT-IR). The in vitro antioxidant aptitude was determined by scavenging of DPPH radical, hydroxyl ion, nitric oxide, inhibition of ß-carotene bleaching assay and iron chelation power assay. The antimicrobial potential of oil was investigated by disc diffusion method against multidrug resistant (MDR) bacterial isolates and fungal strains. Wound healing was performed in vivo with determination of wound contraction rates, histopathology, hemostatic potential and hydroxyproline estimation. RESULTS: GC-MS analysis indicated that oil was constituted mainly of 2, 6-dimethyl-8-oxoocta-2, 6-dienoic acid, methyl ester (18.2%), syringol (17.8%), catechol (12.4%), guaiacol (5.2%), p-cresol (5.4%) and phenol, 2-propyl- (3.7%). FT-IR analysis revealed several important functional groups in its chemical composition especially phenolic O-H compound stretching. Scavenging of DPPH radical, hydroxyl ion, nitric oxide, inhibition of ß-carotene oxidation and iron chelation power assays indicated strong antioxidant activities of oil. Further it efficiently inhibited growth of multidrug resistant isolates of Staphylococcus aureus, S. lugdenesis, Klebsiella pneumoniae, Escherichia coli, Coagulase -ve staphylococci and Pseudomonas aeruginosa. The minimum inhibitory concentrations ranged between (32-256) (µg/mL) of oil. The oil also strongly inhibited the growth of various fungal isolates with low level of minimum inhibitory concentrations (64-256) µg/mL. Remarkable rate for wound closure and epithelization, hemostatic potential and marked increase (p < 0.05) in hydroxyproline content was observed for oil during wound healing in rat. CONCLUSION: The results suggested that oil can be used as a potential source of wound healing therapeutics.

Protective aptitude of Periploca hydaspidis Falc against CCl4 induced hepatotoxicity in experimental rats.

In the present study the antioxidant capacity of Periploca hydaspidis was assessed through various in vitro assays and by the hepatoprotective potential on CCl4 induced toxicity in rat. Phytochemical analysis of different extracts of P. hydaspidis indicated existence of various phytochemical classes. HPLC-DAD analysis of methanol extract indicated the existence of rutin, gallic acid and caffeic acid. Total phenolic (TPC) and total flavonoid content (TFC) exhibited significant (p < 0.05) correlation with 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide, hydroxyl ion, inhibition of ß-carotene oxidation, iron chelation, reducing power and total antioxidant capacity. In hepatic sample of rat, CCl4 administration increased (p < 0.05) the level of nitrite, hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) whereas a decline was recorded in antioxidant enzymes; superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and in reduced glutathione (GSH). Concentration of alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST) and globulin increased (p < 0.05) whereas level of total protein and albumin decreased in serum of CCl4 treated rats. Level of pro-inflammatory cytokines; tumor necrosis factor-α (TNF-α), tumor growth factor-ß1 (TGF-ß1) and resistin was increased (p < 0.05) in serum whereby anti-inflammatory markers; interleukin-10 (IL-10), adiponectin and nuclear factor erythroid 2- related factor 2 (Nrf-2) decreased (p < 0.05) in hepatic tissues of CCl4 treated rats. DNA damages and histopathological alterations were induced with administration of CCl4 to rat. The altered levels of various parameters provoked by CCl4 toxicity restored towards the control level by the methanol extract of P. hydaspidis in a dose dependent manner. These results suggested the presence of antioxidant and anti-inflammatory phyto-constituents in methanol extract of P. hydaspidis.

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.

Nummularic acid, a triterpenoid, from the medicinal plant Fraxinus xanthoxyloides, induces energy crisis to suppress growth of prostate cancer cells.

We recently identified and characterized nummularic acid (NA) as a major chemical constituent of Fraxinus xanthoxyloides, a medicinal plant used for over hundred years in traditional medicine. In this study, we describe its potential anti-cancer activity using prostate cancer (PCa) cells as a model. We found that NA treatment (5-60 µM) significantly reduced the proliferation and colony formation capabilities of PCa DU145 and C4-2 cells in a time and dose dependent manner, reduced the migratory and invasive properties and increased apoptotic cell population. Mechanistically, we found that NA treatment to PCa cells resulted in a sustained activation of adenosine monophosphate-activated protein kinase (AMPK). NA simultaneously increased acetyl CoA carboxylase phosphorylation and decreased pS6 phosphorylation, the two major substrates of AMPK. Further, NA treatment significantly elevated the cellular ADP/ATP ratio and altered glycolytic rate. We further observed a reversible decrease in oxygen consumption rate in NA treated cells when compared to the control. Finally, we performed global untargeted metabolomics which showed that NA treatment alters PCa cell metabolism at multiple sites including glycolysis, tricarboxylic acid, and glutamine metabolism which supported our observation of a possible AMPK activation. In summary, we report NA as a novel small molecule activator of AMPK that alters cellular metabolism to induce energy crisis and ultimately cancer cell death. Because of its unique mechanism NA could be potentially applicable against other cancer types.

Euphorbia dracunculoides L. abrogates carbon tetrachloride induced liver and DNA damage in rats.

BACKGROUND: Evaluation of Euphorbia dracunculoides of family Euphorbiaceae during previous studies had established the in vitro antioxidant and in vivo anti-inflammatory activities. The plant is used by the local communities of Pakistan for various disorders including rheumatism and edema. In this investigation we have evaluated the hepatoprotective effects against CCl4 induced toxicity in rat. METHODS: Dry powder of the aerial parts of E. dracunculoides was extracted with 95% methanol to get the extract (EDME). To investigate the hepatoprotective effects of EDME the Sprague-Dawley male rats were divided in to 8 groups with 6 rats in each. Group I and II were the normal and vehicle treated while the Groups III-VI were injected intraperitoneally with 1 ml of CCl4 (30% in olive oil). Rats of Group IV were orally administered with silymarin (50 mg/kg) while the Group V and VI with 200 mg/kg and 400 mg/kg of EDME, respectively. Animals of Group VII (200 mg/kg) and VIII (400 mg/kg) were treated with EDME alone. The treatments were given thrice a week for 4 weeks. Effects of EDME were evaluated for the protective effects against oxidative stress and genotoxicity induced with CCl4 in liver of rat. RESULTS: Analysis of serum indicated significant (p < 0.05) rise in the level of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and globulin whereas decrease was recorded for the total protein and albumin in CCl4 treated rats. In liver tissues the activity level of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione (GSH) was decreased while the level of lipid peroxides; thiobarbituric acid reactant substances (TBARS), nitrite and hydrogen peroxide increased in CCl4 treated rats as compared to the control group. Histopathological injuries and DNA damages were recorded in liver of rat with CCl4 treatment. However, co-administration of EDME, dose dependently, ameliorated the CCl4-induced hepatic toxicity in these parameters. CONCLUSIONS: These results suggested that the phyto-constituents of EDME were able to ameliorate the oxidative stress induced with CCl4 and can be a useful therapeutic agent for oxidative stress related disorders.

Investigations on anti-inflammatory and analgesic activities of Alnus nitida Spach (Endl). stem bark in Sprague Dawley rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Stem bark of Alnus nitida (Spach) Endl. (family Betulaceae) is used by local communities in northern areas of Pakistan as a remedy for swelling, injuries and pain. However no pharmacological study of this plant has been reported to confirm these activities. In this study anti-inflammatory and analgesic effect of A. nitida stem bark have been evaluated. METHODS: Powder of the stem bark of A. nitida was extracted with methanol (ANBM) and partitioned in escalating polarity to get the n-hexane (ANBH), chloroform (ANBC), ethyl acetate (ANBE) and the residual soluble aqueous (ANBA) fractions. The methanol extract and derived fractions were evaluated for anti-inflammatory activity by using in vitro heat induced albumin denaturation assay and various in vivo assays; carrageenan-induced hind paw edema method, Freunds' complete adjuvant induced arthritis, histamine induced paw edema and xylene induced ear edema in Sprague Dawley rat. The extracts/fractions were also evaluated for analgesic effects by using hot plate analgesic test and acetic acid induced writhing test in rat. The ANBM composition was analyzed by HPLC-DAD and GC-MS analysis. RESULTS: Results of heat induced albumin denaturation activity indicated that among the extract/fractions ANBC at concentration range of 100-500µg/ml remarkably protected the heat induced albumin denaturation. The pretreatment with ANBC significantly reduced the carrageenan induced edema with 90.81±1.6% after 4h, comparing with 86.63±3.42% reduction produced by the reference drug diclofenac potassium. Histopathological alterations of the gastric and hind paw were decreased with the extract/fractions. Furthermore, anti-inflammatory effects of ANBC were evident in Freunds' complete adjuvant induced arthritis, histamine induced paw edema and xylene induced ear edema. The latency time in hot plate analgesic assay with ANBC (61.59±0.38%) after 90min was comparable to standard drug morphine (69.31±2.67%) and aspirin (67.24±2.08%). Similarly ANBC significantly (p<0.01) inhibited the pain sensation in acetic acid induced writhing test in rat. HPLC-DAD analysis of ANBM indicated the presence of gallic acid, catechin and rutin while the GC-MS analysis of ANBM indicated the presence of 30 compounds predominantly of neophytadiene, 3,7,11,15 tetramethyl-2-hexadecen-1-ol, phytol, vitamin E and linalool. CONCLUSION: The results of this study suggested that the presence of polyphenols, sterols, terpenoids and other constituents might contributed towards the anti-inflammatory and analgesic activities of the crude methanol extract of A. nitida bark and its derived fractions. This study endorsed the folklore use of A. nitida bark for inflammation related disorders.

In vitro and in vivo evaluation of anti-arthritic, antioxidant efficacy of fucoidan from Undaria pinnatifida (Harvey) Suringar.

Seaweed and their constituents have been traditionally employed for the management of various human pathologic conditions such as edema, urinary disorders and inflammatory anomalies. The current study was performed to investigate the antioxidant and anti-arthritic effects of fucoidan from Undaria pinnatifida. A noteworthy in vitro antioxidant potential at 500µg/ml in 2, 2-diphenyl-1-picrylhydrazyl scavenging assay (80% inhibition), nitrogen oxide inhibition assay (71.83%), hydroxyl scavenging assay (71.92%), iron chelating assay (73.55%) and a substantial ascorbic acid equivalent reducing power (399.35µg/mg ascorbic acid equivalent) and total antioxidant capacity (402.29µg/mg AAE) suggested fucoidan a good antioxidant agent. Down regulation of COX-2 expression in rabbit articular chondrocytes in a dose (0-100µg) and time (0-48h) dependent manner, unveiled its in vitro anti-inflammatory significance. In vivo carrageenan induced inflammatory rat model demonstrated a 68.19% inhibition of inflammation whereas an inflammation inhibition potential of 79.38% was recorded in anti-arthritic complete Freund's adjuvant-induced arthritic rat model. A substantial ameliorating effect on altered hematological and biochemical parameters in arthritic rats was also observed. Therefore, findings of the present study prospects fucoidan as a potential antioxidant that can effectively abrogate oxidative stress, edema and arthritis-mediated inflammation and mechanistic studies are recommended for observed activities.

Fraxinus xanthoxyloides leaves reduced the level of inflammatory mediators during in vitro and in vivo studies.

BACKGROUND: Different parts of Fraxinus xanthoxyloides Wall. (Oleaceae) are used traditionally in the treatment of internal wounds, bone fracture, pain, jaundice, malaria and in pneumonia. These ailments involve protective and essential mechanisms of the organism in response to infection, injury and trauma. However, prolonged inflammation may lead to inflammatory disorders. The present investigation was carried to evaluate the crude methanol extract of F. xanthoxyloides leaves and its fractions for their anti-inflammatory and analgesic effects. METHODS: Methanol extract of F. xanthoxyloides leaves was fractionated through liquid-liquid partition on escalating polarity of solvents. Acetic acid and thermal responses were used to evaluate the analgesic effects of extract/fractions in rat. Anti-inflammatory effects were monitored through in vitro; TNF-α activated NFkB in 293/NFkB-Luc HEK cells and LPS-activated nitric oxide (NO) assay in RAW 264.7 cells. For in vivo studies carrageenan induced paw edema model was used in rat. Both in vitro and in vivo studies have indicated that chloroform fraction exhibited superior anti-inflammatory effects to other extract/fractions and therefore, was used in air pouch model in rat to estimate the inhibition in leukocyte migration and synthesis of inflammatory mediators. In addition, phytochemical investigation of crude extract was carried out by GC-MS analysis. RESULTS: GC-MS studies of crude extract revealed the presence of various classes of which terpenoids (26.61 %), lactam (16.47 %), esters (15.81 %), phenols (8.37 %), and steroid (6.91 %) constituted the major categories. Among the extracts chloroform fraction (200 mg/kg bw) significantly (P <0.001) increased the percent latency time (76.13 ± 4.49 %) in hot plate test after 120 min and decreased (P <0.001) the count of writhes (77.23 ± 5.64 %) as compared to other extracts. The in vitro studies indicated that chloroform fraction at 15 µg/ml more effectively inhibited the TNF-α induced synthesis of NFkB (85.0 ± 8.12 %, IC50 = 5.98 µg/ml) and LPS-instigated nitric oxide (78.23 ± 2.39 %, IC50 = 6.59 µg/ml) synthesis. Although all the extract/fractions showed a dose dependent increase in inhibition of edema formation however, chloroform fraction (4(th) h = 77.64 ± 3.04 %) at 200 mg/kg bw exhibited relatively higher (P <0.001) anti-inflammatory activity in carrageenan-induced paw edema in rat. Moreover, chloroform fraction had the ability to decrease (P <0.001) the influx of leukocytes and the concentration of inflammatory mediators; TNF-α, NO, IL-6 and PGE2 in air pouch exudate. CONCLUSION: The study demonstrates the therapeutic potential of F. xanthoxyloides leaves against the inflammatory disorders suggesting the presence of active constituents in chloroform fraction.

Proficiencies of Artemisia scoparia against CCl4 induced DNA damages and renal toxicity in rat.

BACKGROUND: Artemisia scoparia is traditionally used in the local system of medicine in kidney disorders. This study aimed at scrutinizing the nephroprotective prospective of A. scoparia methanol extract against carbon tetrachloride (CCl4) provoked DNA damages and oxidative stress in kidneys of rat. METHODS: Dried aerial parts of A. scoparia were powdered and extracted with methanol to obtain the viscous material (ASM). Sprague Dawley male rats (42) were grouped (7) having 6 rats in each. Group I remained untreated and Group II treated intraperitoneally (i.p) with DMSO + olive oil (1 ml/kg body weight (bw). Rats of Group III - VI were treated with CCl4 (1 ml/kg bw; i.p 30 % v/v in olive oil). Animals of Group IV were co-administered with 100 mg/kg bw of silymarin whereas rats of Group V and VI with 150 mg/kg bw and 300 mg/kg bw of ASM at an interval of 48 h for four weeks. Animals of Group VII were administered with ASM (300 mg/kg bw) alone. DNA damages were investigated with comet assay in renal tissues while the oxidative injuries were estimated in serum and renal tissues. RESULTS: Co-administration of ASM to rats significantly reduced the DNA damages at 300 mg/kg dose as indicated in comet length (40.80 ± 2.60 µm), head length (34.70 ± 2.21 µm), tail length (7.43 ± 1.24 µm) and DNA content in head (88.03 ± 2.27 %) to that of CCl4 for comet length (63.16 ± 2.11 µm), head length (23.29 ± 1.50 µm), tail length (39.21 ± 2.81 µm) and DNA content of head (74.81 ± 2.18 %) in renal cell's nuclei. Increased level of urea, creatinine, bilirubin, blood urea nitrogen whereas decreased concentration of proteins in serum of CCl4 treated animals were restored towards the normal level with co-administration of ASM. CCl4 injection in rats decreased the activity level of CAT, POD, SOD, GST and γ-GT and GSH contents while elevated levels of TBARS, H2O2 and nitrite contents were observed in renal tissues. A noteworthy retrieval of all these parameters and the altered histopathological observations was notified near to the normal values after treatment with both the doses of ASM. CONCLUSION: Results obtained suggested the therapeutic role of ASM in oxidative stress related disorder of kidneys.