Acute and subchronic oral toxicity assessments of Combretum micranthum (Combretaceae) in Wistar rats.

BACKGROUND: Combretum micranthum (CM) (Combretaceae) is widely used in traditional medicine throughout West Africa for the treatment of diabetes, hypertension, inflammation, malaria and liver ailments. In our recent research we demonstrated that CM has nephroprotective potentials in diabetes mellitus, hypertension and renal disorders. However, to the best of our knowledge, no systematic study concerning its toxicity profile has been reported. AIM OF THE STUDY: The study carried out to evaluates the potential toxicity of the hydroalcoholic extract from leaves of the CM, through the method of acute and sub-chronic oral administration in rats. MATERIALS AND METHODS: During the acute toxicity study, male and female rats were orally administrated with CM extract at single doses of 5000 mg/kg (n = 5/group/sex). Abnormal behaviour, toxic symptoms, weight, and death were observed for 14 consecutive days to assess the acute toxicity. For sub-chronic toxicity study, the extract was administered orally at doses of 500 and 1000 mg/kg (n = 5/group/sex) daily to Wistar rats for 28 days. The general behaviour and body weight of the rats was observed daily. A biochemical, haematological, macroscopical and histopathological examinations of several organs were conducted at the end of the treatment period. The CM extract was subjected to Fourier transform infrared spectrophotometric examination in order to detect the presence or absence of cyanide toxic compounds. RESULTS: The absence of absorbance peaks between the 2220-2260 cm-1 region of FT-IR spectrum of CM, indicating the absence of cyanide groups. This suggested that the CM extract may not contain toxic substances. During the acute toxicity test, no mortality or adverse effects were noted at the dose of 5000 mg/kg. In the subchronic study, the CM extract induced no mortality or treatment-related adverse effects with regard to body weight, general behaviour, relative organ weights, hematological, and biochemical parameters. Histopathological examination of vital organs showed normal architecture suggesting no morphological alterations. CONCLUSION: The present study revealed that oral administration of CM extract for 28 days, at dosage up to 1000 mg/kg did not induce toxicological damage in rats. From acute toxicity study, the median lethal dose (LD50) of the extract was estimated to be more than 5000 mg/kg.

Nephroprotective activity of Combretum micranthum G. Don in cisplatin induced nephrotoxicity in rats: In-vitro, in-vivo and in-silico experiments.

Nephrotoxicity is known to be a major complication during cisplatin chemotherapy in cancer patients. In the present study, the protective effect of a hydroalcoholic extract of Combretum micranthum (CM) against cisplatin (CP)-induced renal damage was evaluated using in-vitro human embryonic kidney (HEK)-293 cells and in-vivo experiments. Further, in-silico molecular docking and dynamic experiments were carried out with bioactive compounds of the title plant against nuclear factor kappa B (NF-κB) and soluble epoxide hydrolase (sEH). Incubation of HEK-293 cells with cisplatin resulted in a significant increase in cell death with changes in normal cellular morphology. Co-treatment of HEK-293 cells with CP and CM extract at varying concentrations resulted in significant enhancement of cell growth compared to CP treatment indicating the cytoprotective activity of CM with an EC50 8.136 µg/mL. In vivo nephroprotective activity was evaluated by administering CM (200 and 400 mg/kg, p.o) to rats for 10 days followed by single intraperitonial injection of CP (7.5 mg/kg) on the 5th day of the experiment. Nephrotoxicity induced by CP was apparent by elevated levels of serum and urine kidney function markers, transaminases, oxidative stress markers and histopathological alterations in kidney. Pre-treatment with CM normalized the renal function at both the doses by ameliorating the CP-induced renal damage markers, oxidative stress and histopathological variations. In-silico studies showed that, out of the thirty bioactive compounds, isovitexin and gallic acid exhibited a higher docking score of -22.467, -21.167 kcal/mol against NF-κB. Cianidanol and epicatechin exhibited a higher docking score of -14.234, -14.209 kcal/mol against sEH. The protective effect of CM extract in CP-induced nephrotoxicity might be attributed to its antioxidant, anti-inflammatory activity by inhibiting NF-κB and sEH upregulation.

Antioxidant and nephroprotection activities of Combretum micranthum: A phytochemical, in-vitro and ex-vivo studies.

Management of chronic renal failure is exceedingly expensive. Despite of encouraging experimental outcomes, there is a lack of potent nephroprotective drugable molecules in a clinics or market. To develop a nephroprotective phytomedicine, the present study was designed to do a literature survey on reported phytochemical and biological analysis of Combretum micranthum and to carry out chemoprofiling, in-vitro antioxidant and ex-vivo nephroprotective capacity of the title plant. The phytochemical and biological activity survey of C. micranthum has reveals the presence of many bioactive compounds such as flavonoids, terpenoids, steroids and alkaloids with many biological activities. Phytochemical investigation re-confirmed the presence of these compounds. Hydroalcoholic extract of C. micranthum (CM extract) showed a strong antioxidant activity by scavenging AAPH, DPPH, nitric oxide, hydrogen peroxide and chelating metal ions. CM extract exhibited significant (P < 0.001) dose dependent inhibition of ferric chloride-ascorbic acid induced lipid peroxidation. Diabetic nephropathy is a serious and common complication leading to end stage renal disease. Therefore, in the present study, glucose-induced toxicity was also studied in human embryonic kidney cells (HEK-293) as an in vitro model for diabetic nephropathy. The results showed that exposure of cells to high glucose (100 mM) for 72 h significantly reduced the cell viability resulting in morphological changes such as cell shrinkage, rounded cell shape and cytoplasmic vacuolation. Treatment with CM extract at 10 and 25 µg/mL resulted in significant improvement in cell viability from 10 to 23% compared to the high glucose control. This study demonstrated the potential antioxidant and nephroprotective properties of C. micranthum, justifying its traditional use in the treatment of various diseases.

Antipsychotic activity of embelin isolated from Embelia ribes: A preliminary study.

BACKGROUND: Embelia ribes is claimed in Indian traditional medical practice to be useful in the treatment of nervous diseases. Embelin, an alkyl substituted hydroxy benzoquinone, is a major active constituent of E. ribes. The present preliminary study was intended to evaluate antipsychotic activity of embelin against apomorphine-induced climbing behaviour in mice and stereotyped behaviour in rats. METHODS: Two doses of embelin (5 and 10mg/kg) were administered once daily for 15days before exposure to apomorphine. On the concluding day of pre-treatment, after apomorphine-injection, the rodents were assessed for climbing and stereotyped behaviours according to the published scoring system. Thereafter, neurotransmitters (dopamine, noradrenaline and serotonin) levels were estimated in rodent brains. RESULTS: Embelin pre-treatment significantly inhibited apomorphine-induced climbing and stereotyped behaviours in mice and rats, respectively. Further, embelin also statistically reversed elevated levels of dopamine, noradrenaline and serotonin neurotransmitters in the brain of mice and rats. Embelin showed more significant results at high dose (10mg/kg) than low dose (5mg/kg) in both the tested models. CONCLUSION: Considering the present pharmacological profile of embelin, it is suggested that embelin possesses antipsychotic activity in the treatment of psychotic disorders. However, further research is warranted for evaluating its exact mechanism of action.

Ameliorative effect of chromium-d-phenylalanine complex on indomethacin-induced inflammatory bowel disease in rats.

Present study was designed to evaluate the effect of chromium-d-phenylalanine complex (Cr (d-phe)3) on indomethacin-induced inflammatory bowel disease (IBD) in rats. Adult Wistar rats were pretreated with vehicle/Cr (d-phe)3 (30, 60 and 90µg/kg, p.o.) for 11days. On day 8 and 9, after one h of the above mentioned treatment, indomethacin (7.5mg/kg/day,s.c.) was administered to induce IBD. On day 12, blood samples were collected from animals for lactate dehydrogenase (LDH) estimation and ileum was isolated for macroscopic scoring, biochemical estimation (lipid peroxidation, reduced glutathione and myeloperoxidase activity) and histopathological study. Administration of indomethacin significantly altered the serum LDH, macroscopic and microscopic appearance and biochemical parameters in ileum tissue. Cr (d-phe)3, at all the tested doses, caused a significant reversal of changes induced by indomethacin. Present study demonstrates the protective effect of Cr (d-phe)3 against indomethacin-induced IBD in rats. The observed protective effect might be attributed to the antioxidant and anti-inflammatory properties of Cr (d-phe)3.

Antidiabetic activity of Embelia ribes, embelin and its derivatives: A systematic review and meta-analysis.

Embelia ribes (ER) has been documented in Ayurveda for treating various diseases, including diabetes mellitus (DM). The present systematic review and meta-analysis evaluated the efficacy and safety of ER and its active bio-marker, embelin and its derivatives in the treatment of DM. Literature search was performed in PubMed/MEDLINE, EMBASE, Scopus, ScienceDirect, Scifinder, and Google Scholar. Using Review Manager, meta-analysis of ER/embelin/derivatives of embelin versus diabetic control was performed with inverse-variance model, providing mean differences (MDs) and 95% confidence intervals (CIs). Heterogeneity was determined by I2 statistic. A total of 13 studies were included in the systematic review and meta-analysis, and were conducted in experimental rats. ER and embelin significantly (P≤0.01) resorted blood glucose (MD, -231.30; CI, -256.79, -205.82; and MD, -154.70; CI, -168.65, -140.74) and glycosylated haemoglobin (MD, -6.36; CI, -8.33, -4.39; and MD,-4.68; CI, -7.76, -1.60), respectively. Meta-analysis findings also reported considerable restoration of insulin, lipid profile, haemodynamic parameters, serum and oxidative stress markers. The derivatives of embelin, 6-bromoembelin and vilangin, also improved diabetic condition. In addition, treatments also ameliorated body weight changes due to diabetes. The present systematic review and meta-analysis supports scientific evidence for the antidiabetic activity of ER/embelin/derivatives of embelin. However, further research is warranted in clinical trials to validate the present findings.

Effect of embelin against 3-nitropropionic acid-induced Huntington's disease in rats.

3-Nitropropionic acid (3-NP) causes severe neurotoxicity in animals, which depicts Huntington's disease (HD) in humans. Embelin, the main active constituent of Embelia ribes, has been reported to possess various pharmacological actions, mainly anti-inflammatory, antioxidant, anticonvulsant and neuroprotective. The aim of the present study was to evaluate the neuroprotective effect of embelin against 3-NP induced experimental HD in rats. Adult Wistar rats were pretreated with vehicle/embelin (10 and 20mg/kg p.o.) for 7 days. From 8th day onwards, embelin was co-treated with 3-NP (15mg/kg, i.p.) for 7 days. At the end of the treatment schedule, animals were evaluated for behavioral alterations and brain homogenates were used for estimation of oxidative stress parameters (lipid peroxidation, reduced glutathione, catalase and glutathione-S-transferase). 2,3,5-Triphenyl tetrazolium chloride (TTC) stained brain slices were used for lesion size measurement. Administration of 3-NP significantly altered the behavioral and neuronal antioxidant status and caused significant neuronal damage in striatal region. Embelin, at both the tested doses, caused a significant reversal of behavioral and antioxidant status alterations and reversed the striatal neuronal damage induced by 3-NP. These findings suggest the neuroprotective effect of embelin against HD. The observed protective effect might be attributed to the antioxidant properties of embelin.

Antiepileptic and antipsychotic activities of standardized Silajatu (Shilajit) in experimental animals.

BACKGROUND: Silajatu (Shilajit; SJ) is claimed in traditional Indian medical practice to be useful in the treatment of nervous disorders, epilepsy and as antistress. AIM: To investigate whether SJ possesses antiepileptic and antipsychotic activities in rodents. MATERIALS AND METHODS: Isonicotinyl hydrazine (INH), pentylenetetrazole (PTZ), apomorphine, phenytoin, diazepam, haloperidol and other chemicals of analytical grade were procured from standard companies. The antiepileptic activity of SJ was assessed using maximal electro shock (MES)-induced seizures in rats, INH and PTZ-induced seizures in mice. The antipsychotic effect of SJ was evaluated using apomorphine-induced climbing and stereotyped behaviours respectively, in mice and rats. SETTINGS AND DESIGNS: SJ (25 and 50 mg/kg, p.o.) was given orally once daily for 15 days in all the rodent models. On the test day, SJ was administered 1 h prior to electric shock or chemical inducers (INH/PTZ/apomorphine) in experimental animals; the animals were then observed for different phases of seizures and psychotic behaviours. In addition, gamma-aminobutyric acid (GABA) content in the brain of rats and mice was estimated in seizure models. STATISTICAL ANALYSIS: The data were expressed as mean ± standard error of mean. Statistical comparisons were performed by one-way ANOVA followed by Tukey's post-test using Graph Pad Prism version 5.0, USA. A P < 0.05 was considered significant. RESULTS AND CONCLUSIONS: SJ pretreatment significantly inhibited the seizures induced by MES, INH and PTZ in a dose dependent manner. Further, SJ augmented brain GABA levels to normal, decreased by INH and PTZ in mice brain. SJ pretreatment also significantly inhibited the climbing and stereotyped behaviours induced by apomorphine. The present data seems to confirm the antiepileptic activity of SJ which may be because of enhancing the GABAergic system. The antipsychotic activity observed may be due to anti-dopaminergic and/or GABA-mimetic actions.

Synthesis, pharmacological screening and in silico studies of new class of Diclofenac analogues as a promising anti-inflammatory agents.

A novel series of 5-[2-(2,6-dichlorophenylamino)benzyl]-3-(substituted)-1,3,4-oxadiazol-2(3H)-thione (4a-k) derivatives have been synthesized by the Mannich reaction of 5-[2-(2,6-dichlorophenylamino)benzyl]-1,3,4-oxadiazol-2(3H)-thione (3) with an appropriately substituted primary/secondary amines, in the presence of formaldehyde and absolute ethanol. Structures of these novel compounds were characterized on the basis of physicochemical, spectral and elemental analysis. The title compounds (4a-k) were screened for in vivo acute anti-inflammatory and analgesic activities at a dose of 10mg/kg b.w. Compound 4k exhibited the most promising and significant anti-inflammatory profile while compounds 4a, 4d, 4e, 4i, and 4j showed moderate to good inhibitory activity at 2nd and 4thh, respectively. These compounds were also found to have considerable analgesic activity (acetic acid induced writhing model) and antipyretic activity (yeast induced pyrexia model). In addition, the tested compounds were also found to possess less degree of ulcerogenic potential as compared to the standard NSAIDs. Compounds that displayed promising anti-inflammatory profile were further evaluated for their inhibitory activity against cyclooxygenase enzyme (COX-1/COX-2), by colorimetric COX (ovine) inhibitor screening assay method. The results revealed that the compounds 4a, 4e, 4g and 4k exhibited effective inhibition against COX-2. In an attempt to understand the ligand-protein interactions in terms of the binding affinity, docking studies were performed using Molegro Virtual Docker (MVD-2013, 6.0) for those compounds, which showed good anti-inflammatory activity. It was observed that the binding affinities calculated were in agreement with the IC50 values.

Effect of tramadol on behavioral alterations and lipid peroxidation after transient forebrain ischemia in rats.

N-methyl-D-aspartate (NMDA) antagonists and γ-aminobutyric acid (GABA) agonists are proven protective in various animal models of ischemic brain damage. Tramadol, a centrally acting opioid analgesic reportedly possesses NMDA antagonistic and GABA agonistic properties, with additional ion channel blocking activity. The aim of the present study was to evaluate the possible neuroprotective effect of tramadol hydrochloride in a rat model of transient forebrain ischemia. Male Wistar rats were pretreated with tramadol hydrochloride at doses of 10 and 20 mg/kg b.w. intraperitoneally for 4 days and were subjected to 30 min occlusion of bilateral common carotid arteries followed by reperfusion for 24 h. Impairment in sensorimotor functions was evaluated by beam walking task, spontaneous locomotor activity and hanging wire test. Animals were sacrificed and the brain homogenates were used for estimating the levels of lipid peroxidation, a marker for extent of oxidative stress. Ischemic rats exhibited a significant decrease in locomotion, grip strength and increase in beam walking latency. Tramadol attenuated the post ischemic motor impairment evidenced by improvement in the performance in sensorimotor tests. The extent of lipid peroxidation was significantly (p < 0.001) reduced by tramadol pretreatment which was higher in ischemic control. This study demonstrates the neuroprotective effect of tramadol against transient forebrain ischemia in rats.

Antidiabetic, antihyperlipidemic and antioxidant activities of methanolic extract of Amaranthus viridis Linn in alloxan induced diabetic rats.

The aim of this study was to investigate the antidiabetic, antihyperlipidemic and antioxidant activities of methanolic extract of whole plant of Amaranthus viridis (MEAV) in alloxan (ALX) induced diabetic rats. Diabetes was confirmed after 5 days of single intraperitoneal injection of ALX (140 mg/kg) in albino Wister rats. MEAV (200 and 400 mg/kg) and glibenclamide (10 mg/kg, p.o.) orally administered daily for 15 days, blood was withdrawn for glucose determination on 0, 1, 10 and 15 days respectively. On the 15th day, overnight fasted rats were sacrificed and blood was collected for the determination of high density lipoproteins cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), total cholesterol (TC), total glycerides (TG) and total proteins (TP). For in vivo antioxidant activity of MEAV, liver tissues were homogenized and the assay of lipid peroxidation and was measured as Malondialdehyde (MDA), glutathione (GSH), catalase (CAT) and total thiols (TT) were performed in control, ALX and MEAV treated rats. MEAV at doses of 200 and 400 mg/kg showed significant reduction is blood glucose, lipid profiles and significant improvement in MDA, GSH, CAT and TT when compared to diabetic control group. In vitro α-amylase inhibition activity of MEAV was also studied. We concluded that MEAV possess antidiabetic, antihyperlipidemic and antioxidant activities.

Sedative and antiepileptic effects of Anthocephalus cadamba Roxb. in mice and rats.

OBJECTIVE: The aim of the present study was to evaluate the sedative and antiepileptic activities of ethanolic extract of Anthocephalus cadamba (ACE) bark in various experimental animal models. MATERIALS AND METHODS: ACE was tested at three doses viz. 100, 200 and 400 mg/kg p.o. We used ketamine-induced sleeping time model to test the sedative property of the extract where, onset and duration of sleep were observed. A paradigm of anticonvulsant models (pentylenetetrazole, isoniazid and maximal electroshock-induced seizures) were used to evaluate its protective effect against absence and generalized types of seizures. Onset of clonic convulsions, tonic extension and time of death were observed in PTZ and INH-induced seizure models. In MES model, duration of tonic hind leg extension and onset of stupor were observed. RESULTS: ACE showed significant increase in ketamine induced sleeping time. It also exhibited significant increase (P<0.05, 0.01 and 0.001) in latency to clonic convulsion, tonic extension and time of death in PTZ and INH models at all tested doses, whereas in the MES model, the lower dose was found to be effective when compared with the higher doses (200 and 400 mg/kg, p.o.). CONCLUSION: The results of the present investigation demonstrated that ACE possesses sedative and antiepileptic activities.

Effect of curcumin and curcumin copper complex (1:1) on radiation-induced changes of anti-oxidant enzymes levels in the livers of Swiss albino mice.

The effect of mononuclear copper (II) complex of curcumin in 1:1 stoichiometry (hereafter referred to as complex) administered 30 min before gamma-irradiation (4.5 Gy) on alterations in antioxidant and Thiobarbituric acid reactive substances (TBARS) levels in livers was studied in comparison to curcumin at a dose of 50 mg/kg. The different antioxidants like GSH, GST, catalase, SOD, TBARS and total thiols were estimated in the liver homogenates excised at different time intervals (1, 2 and 4 h) post irradiation using colorimetric methods. There was a radiation-induced decrease in the levels of all the studied enzymes at 1 h post irradiation, while an increase was observed at later time points. Both curcumin and complex treatment in sham-irradiated mice decreased the levels of GSH and total thiols, whereas there was an increase in the levels of catalase, GST and SOD compared to normal control. Under the influence of irradiation, both curcumin and complex treatment protected the decline in the levels of GSH, GST, SOD, catalase and total thiols, and inhibited radiation-induced lipid peroxidation. Further, the complex was found to be more effective in protecting the enzymes at 1 h post irradiation compared to curcumin treated group. This may be due to the higher rate constants of the complex compared to curcumin for their reactions with various free radicals.

Effect of sesamol on radiation-induced cytotoxicity in Swiss albino mice.

The radio-protective ability of sesamol (SM) at various doses viz., 0, 10, 25, 40, 50, 70 and 100mg/kg bw, administered intraperitoneally 30min prior to 9.5Gy whole-body gamma-irradiation was studied in Swiss albino mice. Radiation toxicity and mortality were observed during a period of 30 days and the percentage mortality was calculated. SM pretreatment with 50mg/kg bw was found to be the most effective dose in maintaining body weight and in reducing the percentage mortality, while 100mg/kg bw was found to be more effective in maintaining the spleen index and in stimulation of endogenous spleen colony-forming units. Pretreatment with SM (50mg/kg bw) in mice irradiated with 15Gy significantly reduced dead, inflammatory, mitotic and goblet cells in irradiated jejunum. SM at 50mg/kg bw also increased crypt cells, maintained villus height, and prevented mucosal erosion. Nuclear enlargement in epithelial cells was found less in SM-treated mice compared with the irradiated control. The radiation-induced decrease in endogenous antioxidant enzymes (GSH, GST, catalase) and the increase in lipid peroxidation were also reduced by pretreatment with SM [50 and 100mg/kg bw] at all monitored post-irradiation intervals. There was no protection at a dose less than 25mg/kg bw.