Molecular profiling and bioactive potential of an endophytic fungus Aspergillus sulphureus isolated from Sida acuta: a medicinal plant.

CONTEXT: Sida acuta Burm.f. (Malvaceae) extracts are reported to have applications against malaria, diuretic, antipyretic, nervous and urinary diseases. No fungal endophytes of S. acuta are reported. OBJECTIVE: Isolation, identification and evaluation of antibacterial, antioxidant, anticancer and haemolytic potential of fungal endophytes from the ethnomedcinal plant S. acuta. MATERIALS AND METHODS: Sida acuta stem segments were placed on PDA medium to isolate endophytic fungi. The fungus was identified by genomic DNA analysis and phylogenetic tree was constructed using ITS sequences (GenBank) to confirm species. The antibacterial efficacy of Aspergillus sulphureus MME12 ethyl acetate extract was tested against Gram-positive and Gram-negative pathogenic bacteria. DPPH free radical scavenging activity, anticancer and DNA fragmentation against EAC cells, and direct haemolytic activity (100-500 µg/mL) using human erythrocytes were determined. RESULTS AND DISCUSSION: The ethyl acetate extract of A. sulphureus (Fresen.) Wehmer (Trichocomaceae) demonstrated significant antibacterial potential against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella typhi compared to streptomycin. MIC against test pathogens was in the range of 15.6-62.5 µg/mL. The antioxidant results revealed significant RSA from 12.43% to 62.02% (IC50 = 350.4 µg/mL, p ≤ 0.05). MME12 offered considerable inhibition of EAC proliferation (23% to 84%, IC50 = 216.7 µg/mL, p ≤ 0.05) supported by DNA fragmentation studies. The extract also offered insignificant haemolysis (5.6%) compared to Triton X-100. CONCLUSIONS: A single endophytic fungus, A. sulphureus MME12 was isolated and identified using molecular profiling. The above-mentioned findings support the pharmacological application of A. sulphureus MME12 extract and demand for purification of the active principle(s).

Albizia lebbeck seed methanolic extract as a complementary therapy to manage local toxicity of Echis carinatus venom in a murine model.

CONTEXT AND OBJECTIVE: Viperid venom-induced chronic local-toxicity continues even after anti-snake venom treatment. Therefore, traditional antidote Albizia lebbeck L. (Fabaceae) seed extract was tested against Echis carinatus S. (Viperidae) venom (ECV)-induced local toxicity to evaluate its complementary remedy. MATERIALS AND METHODS: Soxhlet extraction of A. lebbeck seeds was performed with the increasing polarity of solvents (n-hexane to water); the extract was screened for phytochemicals (alkaloids, anthraquinones, flavonoids, glycosides, phenolics, saponins, steroids and tannins). In preliminary in vitro analysis, A. lebbeck methanolic extract (ALME) demonstrated significant inhibition of ECV proteases, the major enzyme-toxin responsible for local- toxicity. Therefore, in vitro neutralizing potential of ALME was further evaluated against hyaluronidases and phospholipase A2 (1:1-1:100 w/w). In addition, alleviation of ECV induced characteristic local- toxicity [haemorrhage (i.d.) and myotoxicity (i.m.)] was determined in mice. RESULTS: ALME contained high concentrations of phenolics and flavonoids and demonstrated significant in vitro inhibition of ECV protease (IC50 = 36.32 µg, p < 0.0001) and hyaluronidase (IC50 = 91.95 µg, p < 0.0001) at 1:100 w/w. ALME significantly neutralized ECV induced haemorrhage (ED50 = 26.37 µg, p < 0.0001) and myotoxicity by significantly reducing serum creatinine kinase (ED50 = 37.5 µg, p < 0.0001) and lactate dehydrogenase (ED50 = 31.44 µg, p = 0.0021) levels at 1:50 w/w. DISCUSSION AND CONCLUSION: ALME demonstrated significant neutralization of ECV enzymes that contribute in local tissue damage and haemostatic alterations. The study scientifically supports the anecdotal use of A. lebbeck in complementary medicine and identifies ALME as principle fraction responsible for antivenom properties.

Local and systemic toxicity of Echis carinatus venom: neutralization by Cassia auriculata L. leaf methanol extract.

Viper bites cause high morbidity and mortality especially in tropical and subtropical regions, affecting a large number of the rural population in these areas. Even though anti-venoms are available, in most cases they fail to tackle viper venom-induced local manifestations that persist even after anti-venom administration. Several studies have been reported the use of plant products and approved drugs along side anti-venom therapy for efficient management of local tissue damage. In this regard, the present study focuses on the protective efficacy of Cassia auriculata L. (Leguminosae) against Echis carinatus venom (ECV) induced toxicity. C. auriculata is a traditional medicinal plant, much valued in alternative medicine for its wide usage in ayurveda, naturopathy, and herbal therapy. Further, it has been used widely by traditional healers for treatment of snake and scorpion bites in the Western Ghats of Karnataka, India. In the present study, C. auriculata leaf methanol extract (CAME) significantly inhibited enzymatic activities of ECV proteases (96 ± 1 %; P = 0.001), PLA2 (45 ± 5 %; P = 0.01) and hyaluronidases (100 %; P = 0.0003) in vitro and hemorrhage, edema and myotoxicity in vivo. Further, CAME effectively reduced the lethal potency of ECV and increased the survival time of mice by ~6 times (17 vs 3 h). These inhibitory potentials of CAME towards hydrolytic enzymes, mortal and morbid symptoms of ECV toxins clearly substantiates the use by traditional healers of C. auriculata as a folk medicinal remedy for snakebite.

Rosmarinic acid mediated neuroprotective effects against H2O2-induced neuronal cell damage in N2A cells.

AIMS: Oxidative stress plays a key role in several ailments including neurodegenerative conditions. The aim of the study was to demonstrate the effect of rosmarinic acid (RA) in preventing oxidative stress related death of neuronal cell lines. MAIN METHODS: In the present study, we demonstrated direct neuroprotective effect of RA using H2O2-induced oxidative challenge in N2A mouse neuroblastoma cells. The mechanism of neutralization of H2O2-induced toxicity by RA was evaluated using MTT, lactate dehydrogenase, mitochondrial membrane potential (MMP), intracellular ROS, and comet assays. Up-regulation of brain neuronal markers at molecular level was performed by RT-PCR. KEY FINDINGS: Results presented in the paper indicate that H2O2-induced cytotoxicity in N2A cells was suppressed by treatment with RA. Moreover, RA is very effective in attenuating the disruption of lactate dehydrogenase, mitochondrial membrane potential and intracellular ROS. Pretreatment with RA significantly prevents genotoxicity (3.7-fold, p<0.01) and promotes the up-regulation of tyrosine hydroxylase (TH) (4.5-fold, p<0.01), and brain-derived neurotrophic factor (BDNF) genes (5.4-fold, p<0.01) against H2O2-induced cytotoxicity in N2A cells. SIGNIFICANCE: Our results revealed that N2A cells are suitable cellular models to evaluate neuroprotective effects of RA, and suggest that RA may potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress.

Topical application of serine proteases from Wrightia tinctoria R. Br. (Apocyanaceae) latex augments healing of experimentally induced excision wound in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Wrightia tinctoria R. Br. (Apocyanaceae) is a folk medicinal plant known to have immunomodulatory, anti-inflammatory and antihemorrhagic potential. Wrightia tinctoria latex is used for treatment of various clinical conditions including psoriasis, blisters, mouth ulcers, and extensively for topical application on fresh wounds to promote accelerated healing. AIMS OF THE STUDY: To investigate the wound healing potential of Wrightia tinctoria latex proteases using a mouse model. MATERIALS AND METHODS: Proteolytic activity of Wrightia tinctoria latex proteases (WTLP) was determined on various substrates (casein, gelatin and collagen (type-I and IV)). The thermal stability and the class of proteases present in WTLP were determined using heat treatment and specific protease inhibitors, respectively. Excision wound model in mice was used to evaluate the healing potential of WTLP application (twice daily, 10mg/kg). Neosporin, a standard drug, was used for comparison. The progression of healing was monitored using physical (wound contraction), biochemical (collagen content, catalase and MMP activity) and histological examinations. RESULTS: WTLP contains thermostable serine proteases, which are completely inhibited by PMSF. WTLP showed strong caseinolytic, gelatinolytic and collagenolytic activity. The excision wound healing rate upon WTLP treatment was significantly higher than (>2-fold) the control group (49% vs. 18%, (**)p<0.01) on day 3 and throughout the study. PMSF pre-treated and heat denatured WTLP failed to promote wound healing. In addition, serial biochemical analysis of the granulation tissue demonstrated 1.5-fold more (2444 ± 100 vs. 1579 ± 121 µg/100mg tissue) hydroxyproline content and 5.6-fold higher catalase activity (16.7 ± 1.3 vs. 3 ± 0.3 units/mg) compared to controls. Further, the enhanced collagen content and matrix metalloproteinase activity correlated with wound contraction rate following WTLP and Neosporin treatment. Histological analysis on day 9 confirmed complete epithelialization, re-establishment of skin structure and accelerated wound healing following WTLP treatment. CONCLUSIONS: The thermostable serine proteases of Wrightia tinctoria latex are directly involved in the wound healing process. Our findings provide a biochemical basis for the role of WTLP in the enhancement of wound healing. The study supports traditional topical application of Wrightia tinctoria latex on fresh wounds to promote accelerated healing.

Vanillic acid as a novel specific inhibitor of snake venom 5'-nucleotidase: a pharmacological tool in evaluating the role of the enzyme in snake envenomation.

Vanillic acid has been investigated for its inhibitory effect on Naja naja, Daboia russellii, and Trimeresurus malabaricus venom 5'-nucleotidase activity. Trimeresurus malabaricus venom 5'-nucleotidase activity was 1.3- and 8.0-fold higher than that of N. naja and D. russellii venoms, respectively. Substrate specificity studies showed that for all the venoms tested, 5'-AMP was the preferred substrate for 5'-nucleotidase. This indicates the central role of adenosine in snake envenomation. Vanillic acid selectively and specifically inhibited 5'-nucleotidase activity among several enzymes present in the three venoms tested. The inhibitor was competitive, as the inhibition was relieved by increased substrate concentration. It appears that the COOH group in vanillic acid is the determining factor for inhibition as vanillin, a structurally similar compound with respect to vanillic acid, had no inhibitory activity. This study for the first time exemplifies vanillic acid as a pharmacological tool in evaluating the role of 5'-nucleotidase in snake envenomation.

Activated effector and memory T cells contribute to circulating sCD30: potential marker for islet allograft rejection.

T-cell activation up-regulates CD30 resulting in an increase in serum soluble CD30 (sCD30). CD4+ T cells, a major source for sCD30, play a significant role in the pathogenesis of rejection. In this study, sCD30 was measured pre- and posttransplant in mouse islet allograft models and human islet allograft recipients. sCD30 was measured by ELISA in diabetic C57BL/6, CD4Knockout (KO) and CD8KO islet allograft recipients. sCD30 increased significantly prior to rejection (1.8 +/- 1 days) in 80% of allograft recipients. Sensitization with donor splenocytes, or a second graft, further increased sCD30 (282.5 +/- 53.5 for the rejecting first graft vs. 374.6 +/- 129 for the rejecting second graft) prior to rejection suggesting memory CD4+ T cells contribute to sCD30. CD4KO failed to reject islet allograft and did not demonstrate sCD30 increase. CD8KO showed elevated (227 +/- 107) sCD30 (1 day) prior to rejection. High pretransplant sCD30 (>20 U/ml) correlated with poor outcome in human islet allograft recipients. Further, increase in sCD30 posttransplant preceded (3-4 months) loss of islet function. We conclude that sCD30 is released from activated CD4 T cells prior to islet allograft rejection and monitoring sCD30 can be a valuable adjunct in the follow-up of islet transplant recipients.

PLA2 mediated arachidonate free radicals: PLA2 inhibition and neutralization of free radicals by anti-oxidants--a new role as anti-inflammatory molecule.

PLA2 enzyme catalyses the hydrolysis of cellular phospholipids at the sn-2 position to liberate arachidonic acid and lysophospholipid to generate a family of pro-inflammatory eicosanoids and platelet activating factor. The generation of pro-inflammatory eicosanoids involves a series of free radical intermediates with simultaneous release of reactive oxygen species (superoxide and hydroxyl radicals). Reactive oxygen species formed during arachidonic acid metabolism generates lipid peroxides and the cytotoxic products such as 4-hydroxy nonenal and acrolein, which induces cellular damage. Thus PLA2 catalyzes the rate-limiting step in the production of pro-inflammatory eicosanoids and free radicals. These peroxides and reactive oxygen species in turn activates PLA2 enzyme and further attenuates the inflammatory process. Therefore scavenging these free radicals and inhibition of PLA2 enzyme simultaneously by a single molecule such as antioxidants is of great therapeutic relevance for the development of anti-inflammatory molecules. PLA2 enzymes have been classified into calcium dependent cPLA2 and sPLA2 and calcium independent iPLA2 forms. In several inflammatory diseases sPLA2 group IIA is the most abundant isoform identified. This isoform is therefore targeted for the development of anti-inflammatory molecules. Many secondary metabolites from plants and marine sponges exhibit both anti-inflammatory and antioxidant properties. Some of them include flavonoids, terpenes and alkaloids. But in terms of PLA2 inhibition and antioxidant activity, the structural aspects of flavonoids are well studied rather than terpenes and alkaloids. In this line, molecules having both anti-oxidant and PLA2 inhibitions are reviewed. A single molecule with dual activities may prove to be a powerful anti-inflammatory drug.

Group IIA secretory PLA2 inhibition by ursolic acid: a potent anti-inflammatory molecule.

Ursolic acid (3beta-hydroxy-urs-12-en-28-oic acid) isolated from many medicinal plants has diverse pharmacologically important properties, including strong anti-inflammatory activity. However its interaction with pro-inflammatory PLA2 is not known. Ursolic acid inhibited secretory PLA2 (sPLA2) enzymes purified from Vipera russelli, Naja naja venom and human pleural fluid and synovial fluid. IC50 values determined for these enzymes ranged from 12 to 18 microM. Group II secretory PLA2 from both venoms & human inflammatory source were found to be sensitive to inhibition in comparison with group I cobra venom sPLA2. Variation in Ca2+ concentration from 2.5-15 mM did not alter the level of inhibition. Similarly sPLA2 inhibition by ursolic acid is independent of substrate concentration. Ursolic acid interacts with purified venom sPLA2 enzymes and enhances relative fluorescence intensity in a dose dependent manner. In the presence of ursolic acid apparent shift in the far UV-CD spectra of sPLA2 was observed, indicating a direct interaction with the enzyme and formation of enzyme-ursolic acid complex. This complex results in irreversible inhibition of sPLA2 as evident by dialysis study. Inhibition of sPLA2 induced mouse paw edema and indirect hemolytic activity confirmed its sPLA2 inhibitory activity in vivo and in situ respectively. These studies revealed that the strong anti-inflammatory activity of ursolic acid is by inhibiting sPLA2 enzymes.

Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot.

Latex glycoprotein (LGP) from Synadenium grantii latex was purified by the combination of heat precipitation and gel permeation chromatography. LGP is a heat stable protein even at 80 degrees C showed a sharp single band both in SDS-PAGE as well as in native (acidic) PAGE. LGP is a monomeric protein appears as single band under reducing condition. It is a less hydrophobic protein showed sharp single peak in RP-HPLC with retention time of 13.3 m. The relative molecular mass of LGP is 34.4 kDa. CD spectrum of LGP explains less content of alpha-helix (7%), and high content of beta-pleated sheets (48%) and random coils (46%). The N-terminal sequence of LGP is D-F-P-S-D-W-Y-A-Y-E-G-Y-V-I-D-R-P-F-S. Purified LGP is a fibrinogen degrading protease hydrolyses all the three subunits in the order of Aalpha, Bbeta and gamma. The hydrolytic pattern is totally different from plasmin as well as thrombin. LGP reduces recalcification time from 165 to 30 s with citrated human plasma but did not show thrombin like as well as factor Xa-like activity. Although LGP induces procoagulant activity, it hydrolyses partially cross-linked fibrin clot. It hydrolyses all the subunits of partially cross-linked fibrin clot (alpha- chains, beta-chain and gamma-gamma dimer). LGP is a serine protease, inhibited by PMSF. Other serine protease inhibitors, aprotinin and leupeptin did not inhibit the caseinolytic activity as well as fibrinogenolytic activity. We report purification and characterization of a glycoprotein from Synadenium grantii latex with human fibrino(geno)lytic activity.

Anticoagulant effect of Naja naja venom 5'nucleotidase: demonstration through the use of novel specific inhibitor, vanillic acid.

The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A(2)s (PLA(2)s), 5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5'nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5'nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5'nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5'nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA(2). The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5'nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5'nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5'nucleotidase in mediating N. naja venom anticoagulant effect.

Differential action of proteases from Trimeresurus malabaricus, Naja naja and Daboia russellii venoms on hemostasis.

The action of venom proteases and their role in hemostasis has been compared in the venoms of Trimeresurus malabaricus, Daboia russellii and Naja naja from the Southern region of Western Ghats, India. These venoms exhibit varying amounts of proteolytic activity and also influence hemostasis differently. Casein hydrolyzing activity of T. malabaricus venoms was 16 and 24 fold higher than those of N. naja and D. russellii venoms, respectively. With the synthetic substrate TAME, the highest activity was observed in T. malabaricus venom. N. naja venom did not hydrolyze TAME even at higher concentrations. These variations in proteolytic activity also influenced the coagulation process. T. malabaricus and D. russellii venoms are strongly procoagulant and reduce the re-calcification time from 148 to 14 and 12 s, respectively. Similarly, both T. malabaricus and D. russellii venoms reduce the prothrombin time from 12.5 to 6.0 s. On the other hand, N. naja venom is anticoagulant and prolongs re-calcification time to 600 s and prothrombin time to 42 s. In spite of varied effects on hemostasis, all the venoms hydrolyze fibrinogen. T. malabaricus venom hydrolyses both Aalpha and Bbeta subunits. While D. russellii and N. naja venoms hydrolyse only Aalpha. None of these venoms hydrolyze the gamma subunit of fibrinogen. Inhibition studies with specific protease inhibitors revealed that both N. naja and T. malabaricus venoms contain only metalloproteases. D. russellii venom contained both serine and metalloproteases. Only, T. malabaricus venom exhibited thrombin-like activity and induces fibrin clot formation with purified fibrinogen within 58 s. Even though D. russellii venom exhibits procoagulant activity, it did not show thrombin-like activity and may act on other coagulation factors.

Strong myotoxic activity of Trimeresurus malabaricus venom: role of metalloproteases.

Trimeresurus malabaricus is an endemic snake found in the Southern region of Western Ghats section of India along with the more widely distributed species like Naja naja and Daboia russelii. T. malabaricus venom is not lethal when injected (i.p.) up to 20 mg/kg body weight in mice, but causes extensive local tissue degeneration. N. naja and D. russelii are highly toxic (i.p.) with minimum local tissue damage in experimental mice. In this study a comparative analysis of local tissue damage of T. malabaricus venom is made with N. naja and D. russelii snake venoms of the Southern regions of Western Ghats. T. malabaricus venom exhibits caseinolytic activity 16 and 24 times more than N. naja and D. russelii venom. Inhibition studies with specific protease inhibitors reveal that the major proteases belong to metalloproteases. T. malabaricus venom hydrolyses gelatin and induces strong hemorrhagic activity in mice. Both N. naja and D. russelii fail to hydrolyze gelatin even at very high concentration and did not induce any hemorrhagic activity. With D. russelii venom small hemorrhagic spot was observed at the site of injection. The hemorrhagic activity of T. malabaricus venom is completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor. The i.m. injection of T. malabaricus venom causes extensive degradation of muscle tissue within 24 h. The light microscopic observation of muscle tissue showed congestion of blood vessels and hemorrhage at the early stage followed by extensive necrosis of muscle fibers. The elevated levels of serum CK and LDH activity further supported the muscle degeneration. Such pathological symptoms were not seen with N. naja and D. russelii snake venom. The hemorrhagic and the muscle necrosis was completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor strongly suggests that the major toxin component in the T. malabaricus venom is metalloprotease and its activity can be easily neutralized using chelating agents and its use in the first aid as chelation therapy is beneficial.

Synthesis and evaluation of trimethoxyphenyl isoxazolidines as inhibitors of secretory phospholipase A2 with anti-inflammatory activity.

A series of trimethoxyphenyl isoxazolidine derivatives, 5a(i-v) and 5b(i-v), bearing different constituents at the 5th position of the isoxazolidine ring were synthesized and evaluated in vitro and in vivo for their inhibitory activity against purified group I and II phospholipase A2 (PLA2) enzymes from snake venom and human inflammatory synovial fluid. Irrespective of modification to the pharmacophore (isoxazolidine ring), they exhibited greater specificity for group II PLA2. The length of alkyl or aryl group at the 5th position, which alters the hydrophobic and aromatic property, was responsible for enhancing the inhibition towards PLA2 enzymes. All of the compounds quench the fluorescent property of the purified PLA2 enzyme, and quenching increases with the increase in length of alkyl or aryl group. The inhibitory effect of compounds appeared to be due to the direct interaction of compounds with the enzyme. Inhibition is substrate-dependent, and the inhibitor likely competes with the substrate for the same binding site of the enzyme. The IC50 value for the most potent interacting inhibitor 5b(v) was 54.8 microM. The most active interacting compounds 5a(v) and 5b(v) from in vitro inhibition of PLA2 activity showed similar potency in in vivo neutralization of PLA2-induced mouse paw edema and hemolytic activity.

Procoagulant activity of Calotropis gigantea latex associated with fibrin(ogen)olytic activity.

The latex of Calotropis gigantea is a rich source of useful components that has medicinal properties and one of the main applications is in controlling bleeding. The crude latex extract contained many proteins, which are highly basic in nature and exhibited strong proteolytic activity. The crude extract hydrolyses casein, human fibrinogen and crude fibrin clot in a dose-dependent manner. The hydrolyzing activity was completely inhibited by IAA indicating they belong to the super family, cysteine proteases. Crude extract hydrolyses Aalpha, Bbeta and gamma subunits of fibrinogen. Among all the subunits the preferential subunit to get hydrolyzed was Aalpha followed by Bbeta and gamma subunit is highly resistant and hydrolyzed at higher protein concentration or over a prolonged incubation time. The crude extract hydrolysis crude fibrin clot strongly compared to trypsin and papain. Pharmacologically the crude extract is hemorrhagic and induces skin hemorrhage at >75 microg and reduces the coagulation time of citrated plasma from 150 to 47 s and promotes blood coagulation. Procoagulation and blood clot hydrolysis are important in wound healing process. This is due to unique cysteine proteases of plant latex and is responsible for the pharmacological actions observed in folk medicine.