Anticancer activity of Cocculus hirsutus against Dalton’s lymphoma ascites (DLA) cells in mice

Context: Cocculus hirsutus (L.) Diels (Menispermaceae) is used in Indian folk system of alternative medicine for rheumatism, eczema, diabetics, inflammation, and neuralgia.

Objective: To evaluate antitumor activities of C. hirsutus in vitro and in vivo.

Materials and methods: C. hirsutus was successively extracted using hexane, petroleum ether, chloroform, ethyl acetate, methanol, and water. In vitro cytotoxicity was assessed by the MTT assay. Phytochemical analyses were conducted with methanol extract of C. hirsutus (MECH) and in vivo antitumor activity was carried out with MECH using Dalton’s lymphoma ascites (DLA) mouse model. Antioxidant properties were assessed by estimating superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation.

Results and discussion: Phytochemical studies indicated a high content of total alkaloid (165.6?mg/100?g), total phenolic (43.5 GAE mg/g), and total flavanoid (4.97 RE mg/g) in MECH. Anti-proliferative activity against the breast cancer cell line MCF-7 showed IC₅₀ values of 221.5?±?16.68, 255?±?17.88, 213?±?8.4, 147?±?7.9, and 229?±?8.02?µg/ml with hexane, petroleum ether, chloroform, ethyl acetate, methanol, and aqueous extracts, respectively. A significant (p?p?Conclusion: C. hirsutus exhibited significant in vitro and in vivo antitumor activities that are reasonably attributed to endogenous antioxidant mechanisms.     

Antitumor activity and antioxidant property of Curcuma caesia against Ehrlich’s ascites carcinoma bearing mice

Abstract

Context: Curcuma caesia Roxb. (Zingiberaceae), commonly known as “Kala Haldi” in Bengali, has been traditionally used for the treatment of cancer, bruises, inflammation and as an aphrodisiac.

Objective: To evaluate the antitumor activity and antioxidant status of the methanol extract of Curcuma caesia (MECC) rhizomes on Ehrlich’s ascites carcinoma (EAC)-treated mice.

Materials and methods: In vitro cytotoxicity assay of MECC was evaluated by using Trypan blue method. Determination of in vivo antitumor activity was performed after 24?h of EAC cells (2?×?10⁶?cells/mouse) inoculation; MECC (50 and 100?mg/kg i.p.) was administered daily for nine consecutive days. On day 10, half of the mice were sacrificed and the rest were kept alive for assessment of increase in lifespan. Antitumor effect of MECC was assessed by the study of tumor volume, tumor weight, viable and non-viable cell count, hematological parameters and biochemical estimations. Furthermore, antioxidant parameters were assayed by estimating liver and kidney tissue enzymes.

Results: MECC showed direct cytotoxicity (IC₅₀ 90.70?±?8.37?μg/mL) on EAC cell line. MECC exhibited significant (p?<?0.01) decrease in tumor volume, tumor weight, viable cell count and percentage increased the lifespan (57.14 and 88.09%) of EAC-treated mice. Hematological profile, biochemical estimation, tissue antioxidant assay significantly (p?<?0.01) reverted to normal level in MECC-treated mice.

Conclusion: MECC possesses potent antitumor activity that may be due to its direct cytotoxic effect or antioxidant properties. Further research is in progress to find out the active principle(s) of MECC for its antitumor activity.     

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.     

Terpenoids of methanol extract of Clerodendrum infortunatum exhibit anticancer activity against Ehrlich’s ascites carcinoma (EAC) in mice

Context: Clerodendrum infortunatum Linn. is a widely used plant in the Indian indigenous system of medicine for the treatment of tumors.

Objective: The present study evaluated the anticancer activity of methanol extract of C. infortunatum (MECI) against Ehrlich’s ascites carcinoma (EAC) bearing Swiss albino mice and isolation of bioactive terpenoids from it.

Methods: HPLC analysis of the methanol extract showed the presence of three major components. Out of those, two compounds were isolated and characterized as oleanolic acid and clerodinin A. The anticancer activity of MECI was assessed by measuring the tumor growth response, percentage increase of life span, study of hematological parameters, lipid peroxidation, antioxidant enzyme activity like glutathion and CAT. In vitro cytotoxicity assay was also performed using EAC cell lines.

Result and conclusion: Treatment with MECI causes significant decrease in the tumor cell volume and increase in the life span. The median survival time (MST) of EAC control group was found as 19.42?±?0.91 d, whereas the MST was increased to 23.44?±?2.69 d and 27.57?±?2.57 d for the groups treated with MECI at 100 and 200?mg/kg, respectively. All the hematological parameters, malonaldehyde content and antioxidant enzymes’ activity were restored towards the normal level. IC₅₀ value of MECI was found as 498.33 µg/mL in cytotoxicity study. The experimental results suggested that MECI has significant anticancer activity, which can be attributed to the presence of oleanolic acid and clerodinin A.     

Molecular changes in the esophageal epithelium after a subchronic exposure to cigarette smoke in the presence of bile-acid reflux

Background: Gastroesophageal reflux of bile acids plays an important role in the development of Barrett’s esophagus (BE)–associated esophageal adenocarcinoma (EAC). Cigarette smoke has been demonstrated to exacerbate the effects of reflux and thus the initial stages of EAC carcinogenesis. To date, no in vivo studies have been conducted to look at the concomitant effects of cigarette smoke and bile acids on EAC incidence.

Methods: In this pilot study, rats that underwent esophagoduodenal anastomosis (EDA) surgery to induce reflux were exposed to whole-body cigarette smoke 3 weeks after surgery. Smoke exposure (135?mg/m³/day) was done for 4?h/day for 5 consecutive days and animals were euthanized after a 48-h recovery period.

Results: Exposure to EDA-smoke accelerated the development of BE when compared to EDA-air. The presence of reflux caused a significant 3.5-fold increase in nuclear factor-κB-inducing kinase (NIK) staining (1.47?±?0.6; p?=?0.01). Animals with both reflux and smoking had the highest (10-fold; 4?±?0.9) induction of cyclooxygenase-2 (COX-2) expression (p?<?0.05). Similarly, there was a 10-fold increase in 4-aminobiphenyl (4-ABP) protein adducts identified in all smoke-exposed animals (p?<?0.01).

Conclusion: Cigarette smoke aggravates reflux-induced BE and potentially accelerates the progression of BE to EAC through the loss of manganese superoxide dismutase (MnSOD), and overexpression of NF-κB- and COX-2-mediated factors.     

Nephroprotective and curative effects of Ficus religiosa latex extract against cisplatin-induced acute renal failure

Abstract

Context. Ficus religiosa L. (Moraceae) is widely planted in the tropics. Its chemical constituents include tannin, saponin gluanol acetate, β-sitosterol, leucoanthocyanidin and leucoanthocyanin which are used for the treatment of pain, inflammation, impotence, menstrual disturbances, uterine tonic and urine related problems.

Objective: To determine the possible nephroprotective and curative effects of F. religiosa latex methanol extract against cisplatin induced acute renal failure.

Materials and methods: Methanol extract was obtained by maceration process. Rats were divided in five groups. Group 1 was administered acacia (2% w/v) of 5?ml/kg throughout the experiment; group 2 was treated with single dose of cisplatin (5?mg/kg i.p.) on the 1st day; group 3 (200?mg/kg p.o.) of extract control for the 1st to 10th day, group 4 (200?mg/kg p.o.) of extract from the 1st to 10th day and a single dose of cisplatin (5?mg/kg, i.p.) on 11th day while group 5 received the same dose of cisplatin on day 1 and extract (200?mg/kg p.o.) from the 7th to 16th day.

Results: Phytochemical screening of the extract revealed the presence of glycoside, alkaloids, tannins (phenolic compounds), flavonoids and amino acids. The half maximal inhibitory concentration (IC₅₀) values of the extract were 31.75?±?0.12 and 18.35?±?0.48?µg/ml, respectively. The cisplatin-treated group 2 showed significant changes; renal functions, biochemical parameters and histopathology were significantly (**p?<?0.01) recovered by 200?mg/kg curative and protective groups.

Discussion and conclusion: These findings demonstrated that F. religiosa latex and constituents have excellent nephroprotective and curative activities and thus have great potential as a source for natural health products.     

Antitumor efficacy and amelioration of oxidative stress by Trichosanthes dioica root against Ehrlich ascites carcinoma in mice

Context: Trichosanthes dioica Roxb. (Cucurbitaceae) is a dioecious climber, traditionally used in India for several medicinal purposes.

Objective: The present study assessed the hydroalcoholic extract of T. dioica root (TDA) for antitumor effect and antioxidant influence against Ehrlich ascites carcinoma (EAC) in Swiss albino mice.

Methods: Twenty four hours after intraperitoneal inoculation of tumor (EAC) cells in mice, TDA was administered at 5 and 10?mg/kg body weight daily for 9 consecutive days. On the 10th day, half of the mice were sacrificed for estimation of tumor proliferation, hematological, and liver antioxidant parameters viz. lipid peroxidation, reduced glutathione (GSH), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT); and the rest were kept alive for assessment of increase in life span. The antitumor effect of TDA was assessed by evaluating tumor weight, tumor volume, packed cell volume, viable and non-viable tumor cell counts, median survival time and percentage increase in life span of EAC bearing mice.

Results and discussion: TDA exhibited dose dependent and significant (p?<?0.001) decrease in tumor weight, tumor volume, packed cell volume and viable cell count and extended the life span of EAC bearing hosts. Hematological profiles were significantly (p?<?0.001) restored near to normal in TDA treated mice as compared to EAC control. TDA treatment significantly (p?<?0.001) modulated the aforesaid liver antioxidant parameters as compared to EAC control.

Conclusion: The present study demonstrated that TDA possessed promising antitumor efficacy in mice, plausibly mediated by amelioration of oxidative stress by multiple mechanisms.     

Antihyperglycemic,antilipid peroxidation,and insulin secretory activities of Otostegia persica shoot extract in streptozotocin-induced diabetic rats and in vitro C187 pancreatic β-cells

Context: Otostegia persica Boiss (Lamiaceae) contains antioxidant agents and is used in traditional medicine for treatment of diabetes mellitus complications.

Objectives: The acute antihyperglycemic, antilipid peroxidation, and insulin secretory activities of methanol extract of O. persica aerial parts were investigated.

Materials and methods: The extract [200, 300, 400?mg/kg body weight (b.w.)] was given orally to rats and glucose (2?g/kg b.w. orally) was administered 30?min later. Glucose and insulin serum levels were measured before and 30, 60, 120, and 240?min after administration of the test samples in normal and diabetic rats. The in vitro insulin secretory activity of extract was evaluated in C187 pancreatic β-cells and its antilipid peroxidation effect was determined by measuring malondialdehyde (MDA) and glutathione (GSH) levels in rat livers after 240?min. The identification of the major phytoconstituents of the extract was carried out using gas chromatography-mass spectrometry.

Results: The extract (300?mg/kg b.w.) significantly decreased the serum glucose level in diabetic rats at 1 h (494?±?13.4 vs. 426?±?12.9), 2 h (472.8?±?17.8 vs. 396?±?22), and 4?h (438.8?±?25 vs. 346?±?19) after treatment. Accordingly, the serum insulin level increased at the same times. The extract significantly increased glucose-induced insulin secretion in C187 β-cells. Moreover, the extract significantly decreased MDA and increased GSH levels in the liver of diabetic rats. Phytochemical analysis revealed thymol as the major phytoconstituent in the extract.

Discussion and conclusion: O. persica shoot extract has antihyperglycemic, antilipid peroxidation, and insulin secretory properties.     

High drug payload curcumin nanosuspensions stabilized by mPEG-DSPE and SPC: in vitro and in vivo evaluation

Context: Curcumin (CUR) is a promising drug candidate based on its broad bioactivities and good antitumor effect, but the application of CUR is potentially restricted because of its poor solubility and bioavailability.

Objective: This study aims at developing a simple and effective drug delivery system for CUR to enhance its solubility and bioavailability thus to improve its antitumor efficacy.

Materials and methods: Curcumin nanosuspensions (CUR-NSps) were prepared by precipitation-ultrasonication method using mPEG2000-DSPE and soybean lecithin as a combined stabilizer.

Results: CUR-NSps with a high drug payload of 67.07% were successfully prepared. The resultant CUR-NSps had a mean particle size of 186.33?±?2.73?nm with a zeta potential of ?19.00?±?1.31?mV. In vitro cytotoxicity assay showed that CUR-NSps exhibited enhanced cytotoxicity compared to CUR solution. The pharmacokinetics results demonstrated that CUR-NSps exhibited a significantly greater AUC_0–24 and prolonged MRT compared to CUR injections after intravenous administration. In the biodistribution study, CUR-NSps demonstrated enhanced biodistribution compared with CUR injections in liver, spleen, kidney, brain, and tumor. The CUR-NSps also showed improved antitumor therapeutic efficacy over the injections (70.34% versus 40.03%, p?Conclusions: These results suggest that CUR-NSps might represent a promising drug formulation for intravenous administration of CUR for the treatment of cancer.     

Lipid-lowering property of Clausena anisum-olens in hypercholesterolemic rats

Context: Clausena anisum-olens (Blanco) Merr. (Rutaceae) is a medicinal shrub which has been reported to have various pharmacological uses. No study regarding the effects of C. anisum-olens on cholesterol-lowering has been reported.

Objective: The effects of the ethanol extract of C. anisum-olens leaves on the cholesterol level of hypercholesterolemic rats were evaluated.

Materials and methods: Acute oral toxicity of the extract (175, 550 and 2000?mg/kg) was determined using female Sprague-Dawley rats, as described in OECD 425 Main test guidelines. The lipid-lowering assay utilized 30 male Sprague-Dawley rats divided into five groups (A–E). Triton X-100 was administered to induce hypercholesterolemia. After hypercholesterolemia induction, oral treatment of Atorvastatin and crude ethanol extract was given daily to the treatment groups for 14 days. The total cholesterol, triglycerides, HDL and LDL were determined before induction, after induction, after first week of treatment and after second week of treatment.

Results: Acute oral toxicity showed the crude extract is nontoxic up to 2000?mg/kg. The lipid-lowering assay indicated reduction of serum cholesterol (87.21?±?5.10?mg/dL), triglycerides (58.09?±?4.10?mg/dL) and LDL (27.82?±?4.11?mg/dL) for 200?mg/kw extract. Reduction in serum cholesterol (74.72?±?3.64?mg/dL), triglycerides (52.79?±?2.98?mg/dL) and LDL (12.06?±?5.51?mg/dL) were observed for 400?mg/kg group. The result is comparable to Atorvastatin, which showed serum cholesterol (80.90?±?9.72?mg/dL), triglycerides (55.94?±?7.19?mg/dL) and LDL (22.09?±?7.60?mg/dL) reduction.

Discussion and conclusion: The crude extract of C. anisum-olens proved to be useful in lowering of cholesterol.     

Influence of mefenamic acid on the intestinal absorption and metabolism of three bioactive flavones in Radix Scutellariae and potential pharmacological impact

Context: Mefenamic acid (MEF) and the dried root of Scutellaria baicalensis Georgi (Radix Scutellariae, RS) share a high possibility of combined medication to treat inflammation.

Objective: The present study investigates the impact of MEF on absorption/disposition of three major components in RS (baicalein, B; wogonin, W; oroxylin A, OA) and further pharmacological changes.

Materials and methods: The apparent permeability (P_app) and percentage of metabolism of B, W and OA at 10?μΜ were measured at the absence/presence of MEF (100?μΜ) in the Caco-2 cell monolayer model. A modified whole blood assay was employed to quantify prostaglandin E₂ (PGE₂) 4, 6 and 8?h post-oral administration with water suspension of MEF at 40?mg/kg and RS at 200?mg/kg.

Results: In the presence of MEF, P_app of B, W and OA were increased from 1.69?±?0.89?×?10^?6, 1.57?±?0.10?×?10^?6 and 3.09?±?0.70?×?10^?6?cm/sec to 5.24?±?0.27?×?10^?6, 6.08?±?0.19?×?10^?6 and 4.13?±?0.38?×?10^?6, whereas their percentage of metabolism was decreased from 72.75?±?2.44%, 73.27?±?3.25% and 89.84?±?2.99% to 21.11?±?0.69%, 17.90?±?5.55% and 45.44?±?3.38%. PGE₂ level was much lower in the co-administration group (49.04?±?2.03?pg/ml) than in the MEF group (73.13?±?3.03?pg/ml) or RS group (494.37?±?11.75?pg/ml) 4?h post MEF dosing, suggesting a synergic effect.

Discussion and conclusion: Co-administration of MEF and RS could induce potential alterations in their pharmacokinetic profiles and anti-inflammatory effects.     

Growth inhibition and apoptosis of Ehrlich ascites carcinoma cells by the methanol extract of Eucalyptus camaldulensis

Context: Eucalyptus camaldulensis Dehnh. (Myrtaceae) is a tall evergreen tree found commonly in Bangladesh. Its use in traditional folk medicine for the treatment of various health complications are well known.

Objective: To explore the in vivo antitumor effect of Eucalyptus camaldulensis stem bark methanol extract (ME) against Ehrlich’s ascites carcinoma (EAC) in Swiss albino mice.

Materials and methods: The antitumor activity of ME was studied by determining viable tumor cell count, recording tumor weight and survival time, observing morphological changes and nuclear damage of EAC cells, and estimating hematological as well as biochemical parameters of experimental mice (25, 50 and 100?mg/kg/day for 5?d, i.p.).

Results: ME showed 96% (p?p?p?50 value (1120?mg/kg) of ME indicated its low host toxic effects. ME-treated EAC cells showed membrane blebbing, chromatin condensation, nuclear fragmentation (apoptotic features) in Hoechst 33342 staining under fluorescence microscope. The DNA profile in agarose gel (1.5%) electrophoresis also confirmed that ME caused EAC cell death by apoptosis.

Discussion and conclusion: Results showed that ME exhibits strong anticancer activity through apoptosis and stimulation of host immunity. Thus, E. camaldulensis may be considered as a promising resource in cancer chemotherapy.     

The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen–glucose deprivation-induced injury

Context: The effects of icariin, a chief constituent of ?avonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown.

Objective: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability.

Materials and methods: PC12 cells were treated with icariin (10^?7, 10^?6 or 10^?5?mol/L) for 3?h (1?h before oxygen–glucose deprivation (OGD) plus 2?h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: After 2?h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3?±?1.9?ng/L, HSP-60: 199?±?16?ng/L, HSP-70: 195?±?17?ng/L, NSE: 1487?±?125?ng/L), and cell viability was significantly decreased (0.26?±?0.03), while icariin (10^?7, 10^?6, or 10^?5?mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1?±?1.4, 22.6?±?1.8, 15.7?±?2.1, HSP-60: 100?±?12, 89?±?6, 113?±?11, HSP-70: 139?±?9, 118?±?7, 95?±?9 and NSE: 1121?±?80, 1019?±?52, 731?±?88), and improved cell viability (0.36?±?0.03, 0.38?±?0.04, 0.37?±?0.03) in OGD-treated PC12 cells.

Discussion and conclusion: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.     

Leonurine hydrochloride induces apoptosis of H292 lung cancer cell by a mitochondria-dependent pathway

Abstract

Context: Leonurine hydrochloride (LH), a major alkaloid compound extracted from Leonurus japonicas Houtt. (Labiatae), is considered to have antitumor roles.

Objective: This study investigated its effects on human non-small cell lung cancer (NSCLC) H292 cells and illustrated the possible mechanism involved.

Materials and methods: After treatment with different concentrations of LH (0, 10, 25, and 50?μmol/L) for 6, 12, 24, 48, and 72?h, the cell viability was assessed by the MTT assay. After exposed to different doses of LH for 24?h, cell-cycle distribution, cell apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were monitored by flow cytometry. RT-PCR and western blot were used to detect the expression of apoptosis-related genes.

Results: LH significantly inhibited the proliferation of H292 cells in a time- and dose-dependent manner, and induced G0/G1 cell-cycle arrest. Coincidentally, LH treatment at a dose of 10, 25, and 50?μmol/L for 24?h increased apoptotic ratio from 4.9?±?0.43% to 11.5?±?1.12%, 19.3?±?1.16%, and 61.3?±?6.69%, respectively. The inhibition effect of LH on H292 cells was associated with the loss of MMP and the generation of ROS. The phosphorylation level of p38 was increased and Akt phosphorylation was reduced by LH treatment. Furthermore, LH treatment increased the expression levels of caspase-3, caspase-9 and Bax/Bcl-2.

Conclusions: LH inhibits the proliferation and induces the apoptosis of H292 cells in a mitochondria-dependent pathway, and the specific mechanism need to be further explored.     

In vitro antioxidant and anti-cholinesterase activities of Rhizophora mucronata

Context: Rhizophora mucronata Lam. (Rhizophoraceae), commonly known as Asiatic mangrove, has been used traditionally among Asian countries as folk medicine.

Objective: This study investigates the cholinesterase inhibitory potential and antioxidant activities of R. mucronata.

Materials and method: Rhizophora mucronata leaves were successively extracted using solvents of varying polarity and a dosage of 100–500?µg/ml were used for each assay. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were assessed according to the method of Ellman. In vitro antioxidant activity was assessed using free radical scavenging, reducing power, and metal-chelating activity (duration – 3 months). Total phenolic and flavonoid content were quantified spectrophotometrically. Compound characterization was done using column chromatography, NMR, FTIR, and LC-MS analysis.

Results: Methanolic leaf extract (500?µg/ml) exhibited the highest inhibitory activity against AChE (92.73?±?0.54%) and BuChE (98.98?±?0.17%), with an IC₅₀ value of 59.31?±?0.35 and 51.72?±?0.33?µg/ml, respectively. Among the different solvent extracts, methanolic extract exhibited the highest antioxidant activity with an IC₅₀ value of 47.39?±?0.43, 401.45?±?18.52, 80.23?±?0.70, and 316.47?±?3.56?µg/ml for DPPH, hydroxyl, nitric oxide radical, and hydrogen peroxide, respectively. Total polyphenolic and flavonoid contents in methanolic extract were observed to be 598.13?±?1.85?µg of gallic acid equivalent and 48.85?±?0.70?μg of rutin equivalent/mg of extract. Compound characterization illustrated (+)-catechin as the bioactive compound responsible for cholinesterase inhibitory and antioxidant activities.

Conclusion: The presence of rich source of flavonoids, in particular catechin, might be responsible for its cholinesterase inhibitory and antioxidant activities.     

Effect of macronutrient deficiency on withanolides content in the roots of Withania somnifera and its correlationship with molybdenum content

Abstract

Context: The content of withanolides in the roots of Withania somnifera (L.) Dunal (Solanaceae) is important for therapeutic application. Earlier studies have shown that the deficiency of macro- and micronutrients affects the growth of W. somnifera. Therefore, we examined the effect of these deficiencies on the withanolides content of the roots.

Objective: To examine the effect of molybdenum accretion in nitrogen-, phosphorus-, calcium- and potassium-deficient soils on the accumulation of withanolides in the roots of W. somnifera. Different withanolides have different therapeutic applications hence major bioactive withanolides assume importance.

Materials and methods: Methanol extracts of the roots were subjected to HPTLC and individual withanolides were identified by comparing their R_f values with those of the authentic samples. Molybdenum was quantified by atomic absorption spectroscopy. Free radical scavenging activity was monitored by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay.

Results: Molybdenum content in roots of nitrogen-, phosphorus-, calcium-, potassium-deficient, and control plants were 7.02?±?2.1, 13.1?±?1.6, 17.1?±?0.9, 33.5?±?3.3, and 33.9?±?1.6?ppm, respectively. Levels of withaferine A increased with the increase in the Mo content in roots from 7.79?±?2.2?mg/g to 12.57?±?3.4?mg/g. Antioxidant activity of nitrogen-deficient plants was the lowest (24.7?±?2.2%) compared to other groups.

Discussion and conclusion: It was observed that nitrogen metabolism-dependent molybdenum uptake influences the withanolides accumulation in the roots.     

The role of Act1, a NF-κB-activating protein,in IL-6 and IL-8 levels induced by IL-17 stimulation in SW982 cells

Abstract

Context: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation in the synovial membrane of affected joints. It has been shown that several kinds of cytokine were increased in synovial fluid, while the underlying mechanism remains poorly understood.

Objectives: NF-κB activator 1 (Act1) is a recently identified protein binding to the IκB kinase complex. Our study aimed to investigate the expression of Act1 induced by cytokine IL-17 stimulation in SW982 cells.

Materials and methods: The human synovial sarcoma cell line SW982 and primary cultured RA fibroblast-like synovial cells were used. RT-PCR and Western blot assays were selected to investigate the genetic and protein expression of Act1. Additionally, four independent Act1 small interfering RNA (siRNA) oligonucleotides were designed and obtained according to the GenBank cDNA, the sequence of Act1 (Traf3ip2). Finally, enzyme-linked immunosorbent assay (ELISA) double antibody sandwich was used to assay supernatant IL-6 and IL-8 concentrations.

Results: The Act1 mRNA expression level increased significantly after stimulation with IL-17 (5–100?ng/ml) in SW982 cells. Additionally, the level of Act1 mRNA expression correlated positively with the concentration of IL-17 (p?<?0.01). IL-17 induced IL-6 and IL-8 in SW982 cells was in a concentration- and time-dependent way. Furthermore, ELISA assay revealed that IL-17 (20?ng/ml) significantly increased IL-6 (1927.4?±?288.77 versus 786.5?±?172.42?ng/ml, p?<?0.01) and IL-8 levels (984.8?±?95.09?ng/ml versus 307.1?±?90.83?ng/ml, p?<?0.01) compared with control group after stimulation for 24?h. However, transfection of Traf3ip2 siRNA markedly decreased IL-6 (995.9?±?115.30?ng/ml versus 1816.1?±?273.27?ng/ml, p?<?0.01) and IL-8 levels (575.6?±?65.96?ng/ml versus 929.4?±?124.39?ng/ml, p?<?0.01) compared to transfection negative control. These findings suggested that IL-6 and IL-8 level induced by IL-17 in SW982 cells could be reversed by down-regulation of Act1 expression level with Traf3ip2 siRNA.

Conclusion: Our results suggested that Act1 might play a key role in the pathophysiology and the treatment of RA.     

Prevention of arsenic-mediated reproductive toxicity in adult female rats by high protein diet

Abstract

Context: The detrimental effects of arsenic on female reproductive functions may involve overt oxidative stress. Casein and pea [Pisum sativum Linn. (Fabaceae)] proteins have antioxidant properties.

Objective: To investigate the role of casein- and pea-supplemented high-protein diet (HPD) in utero-ovarian protection from arsenic toxicity.

Materials and methods: Adult female Wistar rats were orally gavaged with vehicle (Gr-I) or arsenic at 3?ppm/rat/d (Gr-II and Gr-III) for 30 consecutive days, when they were maintained on either regular diet containing 18% protein (Gr-I and Gr-II), or HPD containing 27% protein in the form of casein (20%) and pea (7%) (Gr-III). Reproductive functions were evaluated using a battery of biochemical and histological techniques.

Results: As compared to Gr-I, the Gr-II rats suffered from loss of estrous cyclicity, reduction in weight (mg/100?g body weight) of ovary (Gr-I: 54.3?±?4.2 versus Gr-II: 35.8?±?1.6; p?<?0.001) and uterus (Gr-I: 161.7?±?24.6 versus Gr-II: 94.44?±?13.2; p?<?0.05), utero-ovarian degeneration, attenuated ovarian activities (unit/mg tissue/h) of Δ⁵, 3β-hydroxysteroid dehydrogenase (Gr-I: 3.41?±?0.12 versus Gr-II: 2.31?±?0.09; p?<?0.01) and 17β-hydroxysteroid dehydrogenase (Gr-I: 3.82?±?0.57 versus Gr-II: 1.24?±?0.19; p?<?0.001), and decreased serum estradiol level (pg/ml) (Gr-I: 61.5?±?2.06 versus 34.1?±?2.34; p?<?0.001). Ovarian DNA damage was preponderant with blatant generation of malondialdehyde (nM/mg tissue; Gr-I: 15.10?±?2.45 versus Gr-II: 29.51?±?3.44; p?<?0.01) and attenuated superoxide dismutase activity (unit/mg tissue) (Gr-I: 2.18?±?0.19 versus Gr-II: 1.33?±?0.18; p?<?0.05). The Gr-III rats were significantly protected from these ill effects of arsenic.

Discussion and conclusion: HPD, by way of antioxidant properties, may find prospective role in the protection of reproductive damage caused by arsenic.     

Antioxidant and anti-cholinesterase activity of Sargassum wightii

Abstract

Context. Sargassum has been used in traditional Chinese medicine since the eighth century AD to treat goiter. Sargassum wightii Greville (Sargassaceae) is a major source of alginic acid used widely in food and drug industries.

Objective: To evaluate the anti-Alzheimer potential of S. wightii through evaluation of antioxidant and cholinesterase inhibitory activities.

Materials and methods: Successive extraction was done using solvents of varying polarity. Solvent extracts (100–500?µg/mL) were employed for all the antioxidant assays. Free radical scavenging activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl, OH?, H₂O₂ radical scavenging assay. The reducing power of the seaweed was evaluated by ferric reducing antioxidant power and reducing power assay. Cholinesterase (ChE) inhibitory activity was evaluated and the Km, Vmax and Ki were calculated. Further, compound characterization was done by GC-MS analysis.

Results: The non-polar extracts were found to possess significant antioxidant activity. At 100?μg/mL, petroleum ether, hexane, benzene and dichloromethane extracts showed significant ChE inhibitory activity with IC₅₀ values of 19.33?±?0.56, 46.81?±?1.62, 27.24?±?0.90, 50.56?±?0.90?µg/mL, respectively, for AChE, and 17.91?±?0.65, 32.75?±?1.00, 12.98?±?0.31, 36.16?±?0.64?µg/mL, respectively, for BuChE. GC-MS reveals that 1,2-benzenedicarboxylic acid, diisooctyl ester is the major compound present in dichloromethane extract of S. wightii. The mode of inhibition exhibited by dichloromethane extract against the cholinesterases was found to be competitive type.

Discussion and conclusion: The presence of high amount of terpenoids could be the possible reason for its potential antioxidant and ChE inhibitory activity.     

Evaluation of proton beam radiation sensitivity of proliferating choroidal endothelial and retinal ganglion cells with clonogenic assay

Context: Proton beam therapy offers the advantage of precise delivery with limited damage to the healthy tissue and is being tested in the management of exudative age-related macular degeneration (AMD). However, the dosages tested are empirical and not based on preclinical studies.

Objective: In this study we evaluated the effects of varying doses of proton beam radiation on choroidal endothelial cells (CECs) and retinal ganglion cells (RGCs) using clonogenic assay to determine differential sensitivity.

Materials and methods: Each cell type has different efficiency to replicate (plating efficiency (PE)). PE of CEC (RF/6A) and RGC (RGC-5) grown in culture flasks was determined by plating 250 cells each (without any treatment) and counting the number of colonies after 13 days. Radiation induced sensitivity was determined by exposing the semi-confluent RF/6A and RGC-5 cells to proton beam at the doses of 0 (control), 2, 4, 8 and 12 cobalt gray equivalent (CGE). The ability of the cells to repair and replicate to form colonies were analyzed 13 days after radiation with crystal violet stain and the survival ratio was calculated. The significance of survival was analyzed using ANOVA (Graphpad Instat.3).

Results: The PE of CEC and RGC was 12.96?±?0.29% and 40.7?±?1.48%, respectively. A survival ratio of CEC at 2, 4, 8 and 12 CGE proton radiation was 66.0?±?8.6%, 44.3?±?6.5%, 7.6?±?0.3% and 1.14?±?0.06% on exposure to 2, 4, 8 and 12 CGE proton radiation, respectively, p?<?0.01). Survival ratio of RGC was 71.1?±?22.4% (p?=?0.05), 40.2?±?7.9%, 8.89?±?2.6% and 0.78?±?0.31% at 2, 4, 8 and 12 CGE dosages (p?<?0.001).

Discussion: CEC showed dose-dependent decrease in survival rate with values attaining significance at all radiation dosages. In contrast, RGC was comparatively radio resistant and were able to replicate at lower doses and sensitive at higher doses after proton beam radiation.

Conclusion: Since CECs proliferate during neovascularization, this clonogenic assay is a useful assay to assess the sensitivity of CEC to radiation. This study identified that CEC were more sensitive to proton beam radiation than RGC at all doses. This may provide a therapeutic window for administration of proton beam radiation in the management of AMD.