PHYTOCHEMICAL COMPOSITION, ANTIOXIDANT AND IN-VITRO ANTI-INFLAMMATORY ACTIVITY OF ETHANOL EXTRACT OF RUTA GRAVEOLENS L. LEAVES

Objective: The uses of medicinal herbs in prevention and treatment of numerous diseases attracting attention of scientists worldwide. It is necessary to bring vital healthcare to the people and the better substitute for various infectious diseases. Ruta graveolens is traditionally used as a medicinal plant as well as a flavoring agent in food. In the present study R. graveolens leaves extracts were evaluated for phytochemical and anti-inflammatory activity.Methods: The phytochemical, antioxidant (DPPH), ferrous reducing antioxidant property, Anti-lipid peroxidation and superoxide scavenging activity of extracts were assayed by spectrophotometerically. Anti-inflammatory activities; Human plural fluid phospholipase A2 (HPF-PLA2) inhibition by egg yolk plate method and lipoxigenase (LOX) inhibition were assayed by IN VITRO method.Results: The phenolics were significantly higher than the flavonoids, saponins and alkaloids contents in all the extracts. Among extracts of R. graveolens, ethanol and water extracts exhibits more phytochemicals. The ethanol extract had the highest antioxidant activity followed by the aqueous extract. The ethanol extract shows highest DPPH free radical scavenging, ferric reducing, superoxide scavenging and anti-lipid peroxidation activity and IC50 was 3.27±0.03, 3.58±0.05, 3.87±0.04 and 4.77±0.04 µg/µl respectively at dose-dependent manner. Further ethanol and water extracts were subjected for LOX inhibition in the concentration dependent manner and IC50 values are 4.25±0.05, 5.15±0.05 µg/µl for 5-LOX and 4.15±0.04, 4.66±0.05 µg/µl for 15-LOX respectively.Conclusion: The result shows R. graveolens has strong antioxidant property and anti-inflammatory activity. It suggests that the strong correlation between antioxidant activity and the phytochemical contents of the extracts.Keywords: HPF, Human Pleural Fluid, Anti-inflammatory, sPLA2, Secretory Phospholipase A2, HPF-PLA2, Human Pleural Fluid-PLA2. LOX Lipoxigenase, Lipid peroxidation, Anti-inflammation

HUMAN SECRETORY PHOSPHOLIPASE A2 (sPLA2) INHIBITION BY AQUEOUS EXTRACT OF MACROTYLOMA UNIFLORUM (SEED) AS AN ANTI-INFLAMMATORY ACTIVITY

Objective: Macrotyloma uniflorum (Horse gram) is an important legume widely consumed in the tropical south Asian countries including India. The present investigation is the elucidation of anti-inflammatory activity of M. uniflorum as it has several medicinal properties. The M. uniflorum was evaluated for inhibition of human secretory phospholipase A2 (sPLA2) as a function of anti-inflammatory activity.Methods: The total phenols, antioxidant (DPPH scavenging), Anti-lipid peroxidation, PLA2 inhibition and lipoxygenase (5 & 15-LOX) inhibition activity of aqueous extracts of M. uniflorum coat and pulp were assayed by in vitro method. The aqueous extract of M. uniflorum seed coat was subjected to inhibit PLA2 enzymes from human inflammatory fluids (Human Synovial Fluid and Human Pleural Fluid) and snake venoms (Naja naja and Vipera russllii) using [14]C labeled E. coli by in vivo method. A further effect of substrate and calcium concentration on inhibition of VRV-PLA2 in presence and absence of M. uniflorum coat extract were assayed.Results: Aqueous coat extract of M. uniflorum shows higher phenolics and biological activity and inhibited all sPLA2 enzymes in concentration dependent manner. The IC50 values are found to be in the range of 11.42-20.88μg and IC50 values for 5-LOX and 15-LOX is 25.92μg and 32.47μg respectively. The extract effectively neutralized indirect hemolytic activity and showed similar potency in neutralizing the in vivo sPLA2 induced mouse paw edema.Conclusion: These findings suggest that, the active compound/s in extracts of M. uniflorum individually or synergistically responsible for observed sPLA2 inhibition.Â

Effect of dairy pond sludge/supernatant application on ryegrass dry matter yield and phosphorus fractions in soil

Pasture yield in dairy grazing systems is critical to supplying sufficient feed for milking cows and maintaining productivity. In the Australian dairy industry, ryegrass and clover are common grasses used in grazed pastures. Dairy shed effluent (DSE), the wastewater produced from washing down the dairy holding yards during and after milking, is generally managed through application to pasture as a fertilizer substitute/supplement following partial treatment in stabilization ponds. The aim of this study is to assess the benefits of applying sludge and supernatant collected from two-stage DSE pond systems to ryegrass pasture. A pot experiment was conducted which involved applying pond sludges and supernatant to soil seeded with ryegrass. The application rates of the pond by-products were set according to their labile (plant available) phosphorus content. Ryegrass yield and leachate generated from each of the pots were recorded, and samples were collected for analysis of nutrients and other parameters. The ryegrass grown in soil treated with pond sludge and supernatant yielded greater dry matter (DM) with higher nutrient content than untreated control pots. In addition, pots treated with pond sludge exhibited lower rates of phosphorus leaching from the soil compared with pots treated with supernatant. Thus, pond sludge retained more plant available phosphorus in soil than both the control and pond supernatant treatment. The potassium to calcium/magnesium ratios in the ryegrass in the pots treated with pond sludge and supernatant were below the recommended upper limit for grazing. Therefore, the application of pond sludges on the dairy paddocks was found to be superior to applying supernatant in terms of utilization and conservation of phosphorus within the dairy farm and presents low risks of groundwater pollution and grass tetany

Progressive hemorrhage and myotoxicity induced by echis carinatus venom in murine model: neutralization by inhibitor cocktail of n,n,n `,n `-tetrakis (2-pyridylmethyl) ethane-1,2-diamine and silymarin

Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn(2+)MPs), phospholipase A(2)s (PLA(2)s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N', N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn(2+)MPs of ECV (IC50: 6.7 mu M). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA(2)s (IC50: 12.5 mu M) and HYs (IC50: 8 mu M) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity

Wastewater and Stormwater Minimisation in a Coal Mine

This paper presents a case study on the application of cleaner production principles in the mining industry. The water balance prepared for the case study showed that less than 20% of the wastewater generated by the colliery is discharged off-site. The remaining 80% of the wastewater is recycled back into the colliery. Modeling of the stormwater system showed that 75% of the clean runoff becomes contaminated through poor management practices. It was also found that the present system of stormwater management causes the process wastewater management system to fail in wet weather. Improved process and stormwater management systems are proposed. Relatively simple alterations to the operation of the coal wash filtration dams are expected to reduce the periods of inefficient operation of these dams by 95% and the pumping cost by 30%. The use of stormwater diversion channels and retention basins reduces the overflow volumes of the process wastewater treatment dams in 5 year average recurrence interval (ARI) storms by 100%. The paper also includes several recommendations for reducing the production of process wastewater at source and off-site disposal of wastewater

Thrombin like activity of asclepias curassavica L. latex: Action of cysteine proteases

Aim of the study: To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. Materials and methods: A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen. respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. Results: The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Conclusion: Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine. (C) 2009 Elsevier Ireland Ltd. All rights reserved

Malabarase, a serine protease with anticoagulant activity from Trimeresurus malabaricus venom

In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4. kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bβ but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152-341. s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14. s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders

Anti-inflammatory activity of oleanolic acid by inhibition of secretory phospholipase a(2)

Oleanolic acid, a triterpenoid known for its anti-inflammatory properties, is commonly present in several medicinal plants. The present study evaluated the effect of oleanolic acid on sPLA(2), a key enzyme in inflammatory reactions. Oleanolic acid inhibited sPLA(2) activities of human synovial fluid (HSF), human pleural fluid (HPF) and Vipera russelli (VRV-PL-V) and Naja naja (NN-PL-I) snake venoms in a concentration-dependent manner. The IC50 Values of sPLA(2) from these sources ranged from 3.08 to 7.78 mu M. Increasing calcium (Ca2+) concentrations from 2.5 to 15 mM and substrate concentration up to 180 nM did not affect the level of inhibition. Oleanolic acid enhanced the relative intrinsic fluorescence intensity of sPLA(2) (VRV-PL-V). In the presence of oleanolic acid, an apparent shift in the far UV-CD spectrum of sPLA(2) was observed. These studies indicate direct interaction with the enzyme and formation of an sPLA(2)-oleanolic acid complex. The complex formed resulted in irreversible inhibition of sPLA(2). Oleanolic acid inhibited indirect hemolytic activity and mouse paw edema induced by sPLA(2). inhibition of in vitro and in vivo sPLA(2) activity by oleanolic acid explains the observed anti-inflammatory properties of several oleanolic acid-containing medicinal plants

Synthesis and evaluation of Tricyclic Dipyrido Diazepinone derivatives as inhibitors of secretory Phospholipase A2 with Anti-Inflammatory activity

A series of tricyclic dipyrido diazepinone derivatives 6(a-f) bearing different substituents at the tenth position of diazepinone ring were designed and are characterized by 1H NMR, FTIR and X-Ray crystallography studies. The synthesised derivatives are tested in-vitro phospholipase A2 (PLA2) enzyme inhibitory activity and in-vivo antiinflammatory activity against purified group I and group II PLA2 enzymes from the snake venom and human pleural fluid. Compounds bearing aromatic ring with different substituents at different positions shown varied specificity. The 6f derivative with strong electron withdrawing nitro (-NO2) and trifluoromethyl (-CF3) groups at ortho and para positions respectively shown greater inhibitory activity. Inhibitory effect of the compound appeared to be direct interaction with active site and likely competes with substrates as supported by substrate dependent and calcium independent assays. The IC50 value of potent PLA2 inhibitor 6f was 22.1 μM and showed similar potency in the neutralization of in vivo PLA2 induced mouse paw edema and hemolytic activity

Cysteine proteases from the asclepiadaceae plants latex exhibited thrombin and plasmin like activities

In the present study we evaluated the presence of cysteine protease from the latex of four plants Asclepias curassavica L., Calotropis gigantea R.Br., Pergularia extensa R.Br. and Cynanchum puciflorum R.Br. belongs to the family Asclepiadaceae. Cysteine proteases from these plants latex exhibited both thrombin and plasmin like activities. Latex enzyme fraction in a concentration dependent manner induced the formation of clot in citrated blood plasma. Direct incubation of fibrinogen with latex enzyme fraction resulted in the formation of fibrin clot similar to thrombin enzyme. However prolonged incubation resulted in degradation of the formed fibrin clot suggesting plasmin like activity. Latex enzyme fraction preferentially hydrolyzed A alpha and B beta chains of fibrinogen to form fibrin clot. Latex enzyme fraction also hydrolyzed the subunits of fully cross linked fibrin efficiently, the order of hydrolysis was alpha-polymer > alpha-chains > beta-chain and gamma-gamma dimer. Cysteine proteases from all the four Asclepiadaceae plants latex exhibited similar action on fibrinogen and fibrin. This study scientifically validate the use of plant latex in stop bleeding and wound healing by traditional healers all over the world