In vitro and in vivo antioxidant activities of the aqueous extract of Celosia argentea leaves

Objective:

The aqueous extract of Celosia argentea var. cristata L. leaves at 100, 200, and 400 mg/kg body weight (b.w.) was investigated against cadmium (Cd)-induced oxidative stress in Wistar rats. The in vitro antioxidant of the extract was evaluated using ammonium thiocyanate, reducing power, and membrane stabilizing models.

Materials and Methods:

For the in vivo study, 30 male rats (Rattus norvegicus) weighing 138.02 ± 7.02 g were completely randomized into 6 groups (A–F) of 5 animals each. Animals in groups A and B received 0.5 ml of distilled water and the same volume containing 8 mg/kg b.w. of Cd, respectively, for 7 days orally. Animals in groups C, D, E, and F were treated like those in group B except that they received 100 mg/kg b.w. of ascorbic acid, and 100, 200, and 400 mg/kg b.w. of the extract, respectively, in addition to Cd.

Results:

Phytochemical screening revealed the presence of alkaloids (0.61%), saponins (2.93%), cardiac glycosides (0.21%), cardenolides (0.20%), phenolics (3.26%), and flavonoids (2.38%). A total of 10 mg/ml of the extract inhibited linoleic acid oxidation by 67.57%. The highest reducing power was 100 mg/ml as against 10 mg/ml for ascorbic acid. In addition, 2 mg/ml of the extract produced a membrane stabilizing activity of 63.49% as against 77.46% for indomethacin. Compared with the distilled water control group, the administration of Cd alone significantly (P < 0.05) decreased the alkaline phosphatase activity of the rat liver and brain. This decrease was accompanied by a corresponding increase in the serum enzyme. The simultaneous administration of the extract and Cd produced an enzyme activity that compared favorably (P > 0.05) with the animals that received Cd and ascorbic acid. In addition, the reduction in the superoxide dismutase and catalase activity of the liver and brain of the animals, serum uric acid, albumin and bilirubin, and also the increase in the serum malondialdehyde content in animals treated with Cd alone was attenuated by the extract; the values compared well (P > 0.05) with those simultaneously administered with Cd and ascorbic acid.

Conclusion:

Overall, the results indicated that the aqueous extract of C. argentea leaves attenuated Cd-induced oxidative stress in the animals, with the best result at 400 mg/kg b.w. The antioxidant activity of the extract may be attributed to the phenolic and flavonoid components of the extract. The induction of antioxidant enzymes and scavenging of free radicals may account for the mechanism of action of the extract as an antioxidant.     

In vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid

Dietary polyphenols are thought to be beneficial for human health as antioxidants. Coffee beans contain a common polyphenol, chlorogenic acid. Chlorogenic acid is the ester of caffeic acid and quinic acid. Although these polyphenols have received much attention, there is little evidence indicating a relationship between the effect and the rate of absorption. In this study, we focused on the beneficial effects of chlorogenic acid and caffeic acid, a major metabolite of chlorogenic acid. We carried out in vitro and in vivo experiments. In the in vitro study, caffeic acid had stronger antioxidant activity than that of chlorogenic acid. The uptake of chlorogenic acid by Caco-2 cells was much less than that of caffeic acid. The physiological importance of an orally administered compound depends on its availability for intestinal absorption and subsequent interaction with target tissues. We then used an intestinal ischemia-reperfusion model to evaluate antioxidant activities in vivo. We found that both chlorogenic acid and caffeic acid had effects on intestinal ischemia-reperfusion injury. Since caffeic acid has a stronger antioxidant activity than that of chlorogenic acid and chlorogenic acid is hydrolyzed into caffeic acid in the intestine, it is possible that caffeic acid plays a major role in the protective effect of chlorogenic acid against ischemia-reperfusion injury.     

In vitro antioxidant properties of morphine.

Morphine is implicated in diverse functions, from development to immune modulation in the central and peripheral nervous systems. It has also been used extensively in the clinical management of pain due to its potent analgesic effect. This study was designed to evaluate the in vitro antioxidant capacity of morphine using different antioxidant tests, including total antioxidant activity, reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities. Morphine exhibited strong total antioxidant activity. The concentrations of 25, 50 and 75 microgml(-1) of morphine showed 79.1, 84.3 and 92.3% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, at 75 microgml(-1) concentration of standard antioxidant, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and alpha-tocopherol, exhibited 88.7, 94.5 and 70.4% inhibition on peroxidation of linoleic acid emulsion, respectively. In addition, morphine had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities at the same concentrations (25, 50 and 75 microgml(-1)). These various antioxidant activities were compared to standard antioxidants such as BHA, BHT and alpha-tocopherol.     

In vitro and in vivo antitumor activity of a methanol extract of Dregea volubilis leaves with its antioxidant effect

Context: In India, Dregea volubilis (L.f.) Benth. ex Hook.f. (Asclepediaceae), a large twining shrub with a woody vine, is used to treat tumors traditionally.

Objective: This study evaluated the in vitro and in vivo antitumor activity of the methanol extract of Dregea volubilis leaves (MEDV) and elucidated its possible mechanism of action.

Materials and methods: In vitro antitumor activity of MEDV was evaluated against Ehrlich ascites carcinoma (EAC) cell-line. In vivo antitumor and antioxidant activity of MEDV at three dose levels (50, 100, and 200?mg/kg) were determined against EAC tumor-bearing mice. After 24?h of EAC inoculation, the extract was administered for 9 consecutive days. After the administration of the last dose on the 9th day followed by 18?h fasting, mice from all groups were sacrificed to determine antitumor activity and hematological profiles along with liver related biochemical parameters like lipid peroxidation, antioxidant enzymatic activity, etc.

Results: For in vitro antitumor activity, IC₅₀ value of MEDV for EAC tumor cells was 85.51?±?4.07 µg/ml. The MEDV showed a decrease in tumor volume, packed cell volume and viable cell count and an increase in the non-viable cell count of the EAC tumor-bearing mice (p?<?0.001). Hematological profile reverted near to normal level in extract treated mice. MEDV decreased the hepatic lipid peroxidation level and enhanced superoxide dismutase and catalase level in tumor-bearing mice (p?<?0.001).

Discussion and conclusion: MEDV exhibited in vitro and in vivo antitumor activity in EAC tumor-bearing mice mediated through augmenting antioxidant defense system.     

Appraisal of in vitro and in vivo antioxidant activity potential of cornelian cherry leaves

The antioxidant activity of the 80% methanolic extract of Cornus mas L. leaves (CMM) was evaluated by various methods both in vitro and in vivo. In vitro screening tests indicated that CMM had high antioxidant activity in terms of free radical scavenging and metal reducing activity. In vivo antioxidant activity studies in normal healthy rats demonstrated that the total antioxidant capacity of liver homogenates were increased, although no changes were observed in the activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase or in the level of lipid peroxidation. Studies on CCl4-treated rats also showed that CMM restored the activities of antioxidant enzymes, lowered the level of lipid peroxidation and elevated the total antioxidant capacities of both the total blood and liver homogenates of the animals. Further activity-guided fractionation studies led to the isolation of gallic acid, a well-known antioxidant, as one of the active components.     

连翘叶中熊果酸的分离纯化

建立从连翘叶中分离纯化熊果酸的方法.采用8倍量95％乙醇,80℃提取3次,每次1h.提取液经活性炭脱色和AB-8大孔吸附树脂富集纯化得到纯度80.2％的熊果酸,再经乙醇重结晶纯度可达96％,得率约0.6％(以干燥叶计).     

In vitro aging of red blood cells and lipid peroxidation

Incubating isolated erythrocytes in phosphate buffered saline supplied with sufficient glucose (20 mM) for several days resulted in methemoglobin formation and decrease in glycolytic and antioxidant enzyme activities. Volatile hydrocarbon gas release (ethane, ethylene, propane, butane, isobutane, pentane) and loss of the polyunsaturated fatty acids, arachidonic acid (204) and docosahexaenoic acid (226) in the erythrocyte membrane indicated possible involvement of peroxidative reactions in cellular aging processes.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

In vivo effects of indomethacin--I. Activity of antioxidant enzymes and lipid peroxidation.

1. The in vivo effects of indomethacin on the activity of antioxidant enzymes and on lipid peroxidation in erythrocytes, liver and small intestines of rats were examined. 2. The activity of the enzymes studied increased or remained unchanged depending on the preparation and model used: treatment with "therapeutic" or "ulcerogenic" dose of indomethacin. 3. Indomethacin inhibited lipid peroxidation in the liver but not in the erythrocytes. 4. The results suggest that the stimulation of antioxidant enzymes, probably through in vivo formed metal complexes, is an alternative mechanism of the antiinflammatory action of indomethacin.     

In vivo and in vitro toxicological evaluations of aqueous extract from Cecropia pachystachya leaves

ABSTRACT

Cecropia pachystachya

leaves are popularly used to treat asthma and diabetes. Despite the widespread consumption of this plant, there are few scientific studies regarding its toxicological potential. In order to conduct a thorough study concerning the potential adverse effects, the aim of this study was to assess acute and subacute toxicity tests of crude aqueous extract from C. pachystachya leaves (CAE-Cp) using in vivomodel, as well as in vitro cytotoxicity, genotoxicity and antioxidant activity. In addition, genotoxicity, and cytotoxicity of chlorogenic acid (CGA) and cytotoxicity of isoorientin (ISOO) were also evaluated. The antioxidant activity was verified by DPPH, cytotoxicity using sulforhodamine B (SRB) assay and genotoxicity by comet assay on V79 cells. The phytochemical analysis of CAE-Cp detected flavonoids and tannins, CGA and ISOO as the major compounds utilizing HPLC. The total flavonoid content (6.52 mg/g EQ) and antioxidant activity (EC₅₀ = 62.15 µg/ml) of CAE-Cp were determined. In vitro evaluations with CAE-Cp showed genotoxic effects at 0.31 to 2.5 mg/ml and an expressive cytotoxicity on HT-29 (IC₅₀ = 4.43 µg/ml) cells. CGA was genotoxic against V79 cells at 0.07 mg/ml and cytotoxic against to HT-29 (IC₅₀ = 71.70 µg/ml), OVCAR-3 (IC₅₀ = 80.07 µg/ml), MCF-7 (IC₅₀ = 45.58 µg/ml) and, NCI-H460 (IC₅₀ = 71.89 µg/ml) cancer cell lines. Wistar rats treated with a single dose (2,000 mg/kg) CAE-Cp decreased hemoglobin levels after 14 days, although no significant toxicity was observed in animals after 28 days. In view of the in vitro cytotoxicity and genotoxicity detected, further studies are necessary to establish the safe use of CAE-Cp.     

In vivo and in vitro properties of anti-delta 9-tetrahydrocannabinol antibody

An immune serum to cannabinoids was produced in rabbits. It was tested in vitro by immunoprecipitation, hemagglutination and hemagglutination-inhibition tests. In vivo, when passively transferred into mice, it prevented the effect of delta 9-tetrahydrocannabinol on the barbiturate sleeping time.     

In vitro and ex vivo toxicological testing of sildenafil-loaded solid lipid nanoparticles

Abstract

The aim of this study was to investigate the potential cytotoxicity of solid lipid nanoparticles (SLN) loaded with sildenafil. The SLNs were tested as a new drug delivery system (DDS) for the inhalable treatment of pulmonary hypertension in human lungs. Solubility of sildenafil in SLN lipid matrix (30:70 phospholipid:triglyceride) was determined to 1% sildenafil base and 0.1% sildenafil citrate, respectively. Sildenafil-loaded SLN with particle size of approximately 180?nm and monomodal particle size distribution were successfully manufactured using a novel microchannel homogenization method and were stable up to three months. Sildenafil-loaded SLN were then used in in vitro and ex vivo models representing lung and heart tissue. For in vitro models, human alveolar epithelial cell line (A459) and mouse heart endothelium cell line (MHEC5-T) were used. For ex vivo models, rat precision cut lung slices (PCLS) and rat heart slices (PCHS) were used. All the models were treated with plain SLN and sildenafil-loaded SLN in a concentration range of 0–5000?µg/ml of lipid matrix. The toxicity was evaluated in vitro and ex vivo by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Median lethal dose 50% (LD₅₀) values for A549 cells and PCLS were found to be in the range of 1200–1900?µg/ml while for MHEC5-T cells and precision cut heart slices values were found between 1500 and 2800?µg/ml. PCHS showed slightly higher LD₅₀ values in comparison to PCLS. Considering the toxicological aspects, sildenafil-loaded SLN could have potential in the treatment of pulmonary hypertension via inhalation route.     

Influence of lipid lowering fibrates on P-glycoprotein activity in vitro

Statin/fibrate combinations are frequently used to treat mixed dyslipidemia. However, these combinations may cause life-threatening drug interactions (e.g. rhabdomyolysis) possibly induced by modifications of cytochrome P450 isozyme activities. Some statins are also transported by P-glycoprotein (Pgp) and may act as inhibitors of this drug efflux pump. So far, nothing is known about possible Pgp modulating effects of fibrates. We tested whether gemfibrozil, fenofibrate, fenofibric acid, and bezafibrate inhibit Pgp in vitro using a calcein acetoxymethylester (calcein-AM) uptake assay and confocal laser scanning microscopy with bodipy-verapamil as substrate in L-MDR1 cells, which overexpress human Pgp. In uptake assays in cells with (L-MDR1) and without (LLC-PK1) human Pgp we also investigated whether these compounds are transported by Pgp. Intracellular concentrations were measured by liquid chromatography tandem mass spectrometry. Of the tested fibrates, only fenofibrate increased calcein-AM uptake into cells indicating an inhibition of Pgp mediated transport by this compound. The potency of fenofibrate (mean+/-SD: 7.1+/-3.2 microM), evaluated by calculating the concentration needed to double baseline fluorescence (f2), was similar to that of simvastatin (5.8+/-1.5 microM), lovastatin (10.1+/-1.0), and verapamil (4.7+/-0.8 microM). For simvastatin and fenofibrate Pgp inhibition was confirmed with confocal laser scanning microscopy. Fenofibrate, fenofibric acid, gemfibrozil, and bezafibrate showed no difference in the cellular uptake between LLC-PK1 and L-MDR1, indicating that the tested fibrates are not Pgp substrates. In conclusion, this study demonstrates that fenofibrate inhibits Pgp in vitro with a potency similar to simvastatin.     

In vitro release--in vivo microbiological and toxicological studies on ketoconazole lipid granules

In some multidrug therapy programs, ketoconazole (KTZ) may be administered with some antacids that could modify its dissolution rate and reduce its absorption, thus leading to therapeutic failures. The primary aim of this study was to evaluate the influence of Compritol HD5 ATO and Compritol 888 ATO on this interaction in comparison with commercial KTZ tablets. The second aim was to prepare lipid granules of KTZ that could be an alternative to the commercial formulation. Therefore, six KTZ sustained-release granules were prepared with different lipid concentrations, because they were found to be more suitable than tablets that are dissolved only in gastric medium. The results confirmed that the dissolution rate of KTZ granules was significantly reduced in the presence of antacids. The ideal formulation was selected as granules including 5% of Compritol lipids in relation to the suitability of the target profile. Therapeutic effects of orally administered, ideal KTZ granule formulations, and commercial tablets were evaluated in vivo by the experimental model of murine vulvo-vaginal candidiasis (VVC) with and without antacids. It was found that formulations were very effective on VVC, and the therapeutic effect decreased significantly in the presence of antacids. Histopathological studies were carried out for vagina, stomach, and liver tissues and hepatoxicity was also examined. The levels of reduced glutathione (GSH) were measured to assess the oxidative stress induced by KTZ and function of the liver. It was observed that orally administered formulations of KTZ were successful in treating candidiasis in mice without irritancy in stomach. However, liver tissues were damaged. The decreased GSH levels indicated toxicity in our study. This study suggested that in vitro release and in vivo microbiological-toxicological properties of KTZ were affected by antacids and drug-excipient interactions. Lipid granules of KTZ prepared with Compritol 888 ATO could be proposed as a new KTZ solid dosage form with optimum dissolution and therapeutic characteristics.     

In vivo antioxidant activity of total flavonoids from indocalamus leaves in aging mice caused by D-galactose

Objective: The aim of this thesis is to explore antioxidant activity of total flavonoids extracted from indocalamus leaves. Method: Aging mice model was established by D-galactose induction. Three groups of mice were treated with total flavonoids extracted from indocalamus leaves at doses of 20, 40 and 80 mg/kg d bw respectively. The antioxidant status in the aging mice was measured by determining the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and total anti-oxidant capability (T-AOC) in the serum and liver and malondialdehyde (MDA) content in the serum, liver and brain. Results: Compared with control group, extracts of indocalamus leaves significantly enhanced activities of SOD, GSH-Px, CAT in the serum and liver, and decreased MDA content in the serum, liver and brain at the tested doses. Conclusion: Total flavonoids extracted from indocalamus leaves demonstrated the potent antioxidant activity.     

In vitro and in vivo properties of usnic acid encapsulated into PLGA-microspheres

Microparticles will probably play a promising role in the future of chemotherapy. These polymeric delivery systems are capable of maximizing the therapeutic activity while reducing side effects of anti-cancer agents. Usnic acid (UA) is a secondary metabolite produced by lichens, which exhibits an anti-tumour activity. In this study, PLGA-microspheres containing usnic acid from Cladonia substellata were prepared by the double emulsion method, with or without PEG as stabilizer. The morphology of the microspheres was examined by optical and scanning electron microscopy. The in vitro kinetic profile of usnic acid loaded-microspheres was carried out by dissolution testing. The usnic acid content was analysed by HPLC. The cytotoxicity of free and encapsulated usnic acid was evaluated against HEp-2 cells using the MTT method. The anti-tumour assay was performed in mice against Sarcoma-180 tumour (UA 15?mg?kg^?1 weight body/day) during 7 days. Animals were then sacrificed and tumour and organs were excised for histopathological analysis. Microspheres presented a smooth spherical surface with a mean diameter of 7.02?±?2.72?µm. The usnic acid encapsulation efficiency was ～100% (UA 10?mg 460?mg^?1 microspheres). A maximum release of 92% was achieved at the fifth day. The IC₅₀ values for free and encapsulated usnic acid were 12 and 14?µg?ml^?1, respectively. The encapsulation of usnic acid into microspheres promoted an increase of 21% in the tumour inhibition as compared with the free usnic acid treatment. In summary, usnic acid was efficiently encapsulated into PLGA-microspheres and the microencapsulation improved its anti-tumour activity.     

In vitro and in vivo properties of usnic acid encapsulated into PLGA-microspheres

Microparticles will probably play a promising role in the future of chemotherapy. These polymeric delivery systems are capable of maximizing the therapeutic activity while reducing side effects of anti-cancer agents. Usnic acid (UA) is a secondary metabolite produced by lichens, which exhibits an anti-tumour activity. In this study, PLGA-microspheres containing usnic acid from Cladonia substellata were prepared by the double emulsion method, with or without PEG as stabilizer. The morphology of the microspheres was examined by optical and scanning electron microscopy. The in vitro kinetic profile of usnic acid loaded-microspheres was carried out by dissolution testing. The usnic acid content was analysed by HPLC. The cytotoxicity of free and encapsulated usnic acid was evaluated against HEp-2 cells using the MTT method. The anti-tumour assay was performed in mice against Sarcoma-180 tumour (UA 15 mg kg(-1) weight body/day) during 7 days. Animals were then sacrificed and tumour and organs were excised for histopathological analysis. Microspheres presented a smooth spherical surface with a mean diameter of 7.02 +/- 2.72 microm. The usnic acid encapsulation efficiency was approximately 100% (UA 10 mg 460 mg(-1) microspheres). A maximum release of 92% was achieved at the fifth day. The IC50 values for free and encapsulated usnic acid were 12 and 14 microg ml(-1), respectively. The encapsulation of usnic acid into microspheres promoted an increase of 21% in the tumour inhibition as compared with the free usnic acid treatment. In summary, usnic acid was efficiently encapsulated into PLGA-microspheres and the microencapsulation improved its anti-tumour activity.     

In vivo and in vitro assessment of the role of glutathione antioxidant system in anthracycline-induced cardiotoxicity

The clinical usefulness of anthracycline antineoplastic drugs is limited by their cardiotoxicity. Its mechanisms have not been fully understood, although the induction of oxidative stress is widely believed to play the principal role. Glutathione is the dominant cellular antioxidant, while glutathione peroxidase (GPx) together with glutathione reductase (GR) constitutes the major enzymatic system protecting the cardiac cells from oxidative damage. Therefore, this study aimed to assess their roles in anthracycline cardiotoxicity. Ten-week intravenous administration of daunorubicin (DAU, 3 mg/kg weekly) to rabbits induced heart failure, which was evident from decreased left ventricular ejection fraction and release of cardiac troponins to circulation. However, no significant changes in either total or oxidized glutathione contents or GR activity were detected in left ventricular tissue of DAU-treated rabbits when compared with control animals. GPx activity in the cardiac tissue significantly increased. In H9c2 rat cardiac cells, 24-h DAU exposure (0.1–10 μM) induced significant dose-dependent toxicity. Cellular content of reduced glutathione was insignificantly decreased, oxidized glutathione and GR activity were unaffected, and GPx activity was significantly increased. Neither buthionine sulfoximine (BSO, glutathione biosynthesis inhibitor) nor 2-oxo-4-thiazolidine-carboxylic acid (OTC, glutathione biosynthetic precursor) had significant effects on DAU cytotoxicity. This contrasted with model oxidative injury induced by hydrogen peroxide, which cytotoxicity was increased by BSO and decreased by OTC. In conclusion, our results suggest that the dysfunction of glutathione antioxidant system does not play a causative role in anthracycline cardiotoxicity.     

In vitro and in vivo evaluation of antioxidant and antigenotoxic potential of Punica granatum leaf extract

Context: Several studies have reported the antioxidant activity and potential therapeutic properties of Punica granatum L. (Lythraceae) fruit. Medicinal properties have also been attributed to other parts of P. granatum tree, which are rich in bioactive phytochemicals.

Objective: To explore the phytochemical characteristics, in vitro and in vivo antioxidant and in vivo antigenotoxic potential of P. granatum leaf extract (PLE).

Materials and methods: The in vitro antioxidant potential of PLE was assessed by DPPH (1,1-diphenyl-2-picrylhydrazyl), ferric reducing antioxidant power (FRAP). Inhibition of lipid peroxidation (LPO) and the total phenolic content of the samples were also determined. Thirty-six male Swiss albino mice were divided into six groups (six animals each). Group 1 (control) and group 2 mice received vehicle and genotoxin alone, respectively. Groups 3, 4 and 5 were pretreated with PLE (400, 600 and 800 mg/kg body weight, respectively) prior to the administration of genotoxin. Group 6 received highest test dose of PLE. DNA damage in the bone marrow cells, hepatic LPO and antioxidants were recorded.

Results: Phytochemical analysis of PLE showed the presence of flavonoids, phenols, phytosterols, tannins and carbohydrates. Aqueous PLE demonstrated free radical scavenging activity, reducing power and inhibition of LPO with the EC₅₀ values of 10.25, 59.88 and 20.05, respectively. A significant protective effect was observed against cyclophosphamide induced DNA damage and inhibition of hepatic LPO with concomitant increase in reduced glutathione (GSH) glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) in mice pretreated with PLE.

Discussion and conclusion: PLE demonstrated a significant antioxidant and antigenotoxic potential and hence can be a potential natural source in health and medicine.