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.

Interplay of N acetyl cysteine and melatonin in regulating oxidative stress-induced cardiac hypertrophic factors and microRNAs.

Early and specific diagnosis of oxidative stress linked diseases as cardiac heart diseases remains a major dilemma for researchers and clinicians. MicroRNAs may serve as a better tool for specific early diagnostics and propose their utilization in future molecular medicines. We aimed to measure the microRNAs expressions in oxidative stress linked cardiac hypertrophic condition induced through stimulants as Endothelin and Isoproterenol. Cardiac hypertrophic animal models were confirmed by BNP, GATA4 expression, histological assays, and increased cell surface area. High oxidative stress (ROS level) and decreased antioxidant activities were assessed in hypertrophied groups. Enhanced expression of miR-152, miR-212/132 while decreased miR-142-3p expression was observed in hypertrophic condition. Similar pattern of these microRNAs was detected in HL-1 cells treated with H2O2. Upon administration of antioxidants, the miRNAs expression pattern altered from that of the cardiac hypertrophied model. Present investigation suggests that oxidative stress generated during the cardiac pathology may directly or indirectly regulate anti-hypertrophy pathway elements through microRNAs including antioxidant enzymes, which need further investigation. The down-regulation of free radical scavengers make it easier for the oxidative stress to play a key role in disease progression.

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.

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.

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.

Evaluation of anti-inflammatory potential of the leaves of Wendlandia heynei (Schult.) Santapau & Merchant in Sprague Dawley rat.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of W. heynei (family: Rubiaceae) are used by the folklore in swelling, wounds and body aches. In this study anti-inflammatory potential of W. heynei leaves has been evaluated during in vitro studies and in rat. METHODS: Methanol extract of the leaves of W. heynei (WHLM) and its fractions; n-hexane (WHLH), chloroform (WHLC), ethyl acetate (WHLE), n-butanol (WHLB) and residual soluble aqueous (WHLA) were screened for phytochemical analysis and several active constituents (alkaloids, flavonoids, saponins, tannins, terpenoids, ß-carotene and lycopene) were also quantified. Heat induced albumin denaturation assay and in vitro cell cultures was carried out for in vitro anti-inflammatory activity, while various in vivo assays like TPA induced ear edema, croton oil induced anus edema, formalin and carrageenan-induced hind paw edema was investigated in Sprague-Dawley rats. Alterations on levels of tumor necrosis factor (TNF-α), Interleukin-1ß (IL-1ß), IL-6 and prostaglandins (PGE2) induced with WHLE was studied in serum after carrageenan induced paw edema in rat. Meanwhile, the dose dependent WHLE inhibition of NFκB pathway via regulation of the phosphorylation of IKKs, IκBα, and p65 subunit was studied in LPS-induced rat peritoneal macrophages. On account of marked anti-inflammatory activity of WHLE its bioactive components were analyzed by HPLC-DAD analysis. RESULTS: The phytochemical analysis yielded alkaloids, saponins, tannins, coumarins, glycosides, quinones and vitamin C in WHLM and in all fractions. Fraction (WHLE) was enriched with alkaloids (20.20 ±â€¯2.5%), flavonoids (25.26 ±â€¯2.11%) and tannins (307.2 ±â€¯2.03 mg of GAE/g of extract), while terpenoids (21.60 ±â€¯1.65%) were the major constituents of WHLH. Ethyl acetate fraction convincingly protected heat induced albumin denaturation. WHLE exhibited highest edema inhibition in models of TPA-induced ear edema (74.51 ±â€¯2.05) and croton oil-induced anal edema (75.38 ±â€¯2.83). The pretreatment with WHLE significantly (p < 0.05) reduced the paw edema with formalin (78.99 ±â€¯2.26%) assessed after 6 h and in carrageenan (75.71 ±â€¯4.46%) was detected after 4 h. Level of anti-inflammatory markers; IL-1ß, IL-6, TNF-α and PGE2 in carrageenan induced paw edema in serum of rat was significantly (p < 0.001) decreased with WHLE pretreatment to rat. WHLE significantly inhibited the NFκB by reducing the phosphorylation of IKKs, IκBα, and p65 subunit in LPS-induced inflammation in rat peritoneal macrophages. HPLC-DAD analysis of WHLE indicated the presence of rutin, gallic acid, catechin, caffeic acid and myricetin. CONCLUSIONS: It is concluded that WHLM fractions have marked anti-inflammatory activity and this study endorsed the folklore use of W. heynei leaves for swelling related disorders.

Methanol extract of Iphiona aucheri ameliorates CCl4 induced hepatic injuries by regulation of genes in rats.

We have investigated the protective potential of methanol extract of Iphiona aucheri (IAM) on the expression of endoplasmic reticulum (ER) stress associated genes and inflammatory genes on carbon tetrachloride (CCl4) induced hepatic toxicity in rats. Hepatic damage markers: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin were elevated while the content of antioxidants: catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and reduced glutathione (GSH) were decreased significantly (p < 0.05) in CCl4 treated rats as compared to the control group. The CCl4 intoxication induced a higher expression of glucose-regulated protein 78 kDa (GRP78), X-box-binding protein 1 total (XBP1t), spliced X-box-binding protein 1 (XBP1s), unspliced X-box-binding protein 1 (XBP1u), C/EBP homologous protein (CHOP) and genes involved in inflammation and fibrosis: tumor necrosis factor alpha (TNF-α), transforming growth factor-beta (TGF-ß), mothers against DPP homolog 3 (SMAD3), alpha skeletal muscle actin (αSMA) and collagen type I alpha 1 chain (COL1A1). The intoxicated rats showed a low expression of the glutamate-cysteine ligase catalytic subunit (GCLC), protein disulfide isomerase (PDI) and nuclear factor (erythroid-derived 2) like-2 (Nrf2). The administration of IAM to intoxicated rats restored the expression of ER stress, inflammatory, fibrosis and antioxidant genes in a dose dependent manner. Our results indicated that IAM can impede the ER stress and inflammatory genes and it could be a complementary and alternative therapeutic agent for oxidative stress associated disorders.

In vitro antioxidant efficacy and the therapeutic potential of Wendlandia heynei (Schult.) Santapau & Merchant against bisphenol A-induced hepatotoxicity in rats.

The aim of present study was to access the antioxidant and ameliorative efficacy of Wendlandia heynei stem bark's crude methanol extract (WHBM) against bisphenol A (BPA)-induced hepatotoxicity in the rat moel. WHBM and its derived fractions exhibited promising activity for the scavenging of DPPH, hydroxyl and nitrite radicals, iron chelation, and for the inhibition of ß-carotene oxidation. The administration of BPA to Sprague Dawley rats (25 mg kg-1) for 28 days resulted in an elevated (p < 0.01) level of aspartate transaminase, alanine transaminase, alkaline phosphatase, and globulin, and at the same time a decrease (p < 0.01) in the level of total protein and albumin in the serum of the rats. In hepatic samples, the levels of catalase, peroxidase, superoxide dismutase, glutathione-S-transferase, and reduced glutathione were decreased (p < 0.05), whereas thiobarbituric acid reactive substances, hydrogen peroxide, and the nitrite content were increased (p < 0.05) with BPA treatment to the rats. The administration of WHBM to BPA-intoxicated rats restored the altered levels of these parameters toward the control animals. Histopathological alterations of the hepatic tissues induced with BPA were restored with WHBM co-treatment to the rats. HPLC-DAD analysis ensured the occurrence of rutin, catechin, and caffeic acid in WHBM and WHBE. The results of this study suggested that the presence of phenolics and flavonoids in W. heynei bark might be responsible for it exhibiting antioxidant potential during the in vitro and in vivo studies and hence it has potential to be used as a therapeutic agent against oxidative stress associated diseases.

Brachychiton populneus (Schott & Endl.) R.Br. ameliorate carbon tetrachloride induced oxidative stress through regulation of endoplasmic reticulum stress markers and inflammatory mediators in Sprague-Dawley male rats.

In this study hepatoprotective aptitude of Brachychiton populneus against carbon tetrachloride (CCl4) instigated liver injuries in rats was investigated. High-performance liquid chromatography (HPLC) with a diode array detector (DAD) analysis of methanol extract of B. populneus (BPM) indicated existence of rutin, catechin and myricetin. Administration of CCl4 to rat decreased (p < 0.01) the level of catalase (CAT), total superoxide dismutase (SOD), peroxidase (POD), soluble protein and reduced glutathione (GSH) whereas elevated the concentration of H2O2, thiobarbituric acid reactive substances and nitrite in hepatic samples. In serum the level of hepatic markers; aspartate transaminase, alanine transaminase, alkaline phosphatase and total bilirubin increased with CCl4 treatment against control animals. In hepatic samples the expression level of endoplasmic reticulum stress associated genes like glucose regulated protein (GRP78), x-box binding protein- 1 total (XBP-1 t), x-box binding protein- 1 spliced (XBP-1 s), x-box binding protein- 1 unspliced (XBP-1 u), glutamate-cysteine ligase catalytic subunit (GCLC) and pro-inflammatory cytokines; tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) was elevated many fold with CCl4 administration to rat. Co-administration of BPM along with CCl4 to rats decreased (p < 0.05) the expression of above genes except GCLC where expression level was enhanced as compared to CCl4 treatment. Histopathology of liver showed injuries of hepatocytes, infiltration of leukocytes and damaged central lobule in CCl4 treated rats. However, BPM administration to CCl4 intoxicated rats restored the altered parameters towards the control rats. These results suggested the presence of antioxidant and anti-inflammatory constituents in methanol extract of B. populneus.

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.

Quercus dilatata Lindl. ex Royle ameliorates BPA induced hepatotoxicity in Sprague Dawley rats.

Quercus dilatata Lindl. ex Royle was evaluated for in vitro polyphenol content and antioxidant potential as well as in vivo protective role against bisphenol A (BPA) induced hepatotoxicity. The distilled water-acetone (QDDAE) and methanol-ethyl acetate (QDMEtE) extracts were standardized and administered in high (300 mg/kg body weight (BW) and low (150 mg/kg BW) doses to Sprague Dawley rats, injected with BPA (25 mg/kg BW). Silymarin (50 mg/kg BW) was used as positive control. Subsequently, blood and liver homogenates were collected after four weeks of treatment, and the defensive effects of both extracts against oxidative damage and genotoxicity were assessed via hematological and biochemical investigations, determination of endogenous expression of enzymes as well as levels of free radicals and comet assay. Between the two extracts, maximum phenolics (213 ±â€¯0.15 µg gallic acid equivalent/mg dry extract (DE) and flavonoids (55.6 ±â€¯0.16 µg quercetin equivalent/mg DE) content, DPPH scavenging activity (IC50: 8.1 ±â€¯0.5 µg/ml), antioxidant capacity (53.7 ±â€¯0.98 µg ascorbic acid equivalent (AAE)/mg DE) and reducing potential (228.4 ±â€¯2.4 µg AAE/mg DE) were observed in QDMEtE. In in vivo analysis, a dose dependent hepatoprotective activity was exhibited by both the extracts. QDDAE demonstrated maximum reduction in levels of alanine transaminase (49.77 ±â€¯3.83 U/l), thiobarbituric acid reactant substances (33.46 ±â€¯0.70 nM/min/mg protein), hydrogen peroxide (18.08 ±â€¯0.01 ng/mg tissue) and nitrite (55.64 ±â€¯1.79 µM/ml), along with decline in erythrocyte sedimentation rate (4.13 ±â€¯0.072 mm/h), histopathological injuries and DNA damage in BPA intoxicated rats as compared with QDMEtE. Likewise, QDDAE also significantly restored activity levels of endogenous antioxidants, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (POD) and GSH with values of 6.46 ±â€¯0.15 U/mg protein, 6.87 ±â€¯0.1 U/min, 11.94 ±â€¯0.17 U/min and 16.86 ±â€¯1.56 nM/min/mg protein, respectively. Comparative results were obtained for QDMEtE. In conclusion, the present study endorses the significant hepatoprotective potential of standardized extracts of Q. dilatata with known polyphenolics content and validates the traditional use of this plant in natural medicine to manage disorders like hepatotoxicity.

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.