Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity.

BACKGROUND: The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. METHODS: E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC-MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico. RESULTS: The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A(2), hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom. CONCLUSION: For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity. GENERAL SIGNIFICANCE: Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.

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.

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.

Silymarin potentiates the anti-inflammatory effects of Celecoxib on chemically induced osteoarthritis in rats.

Silymarin (SMN) is used as an antioxidant complex to attenuate the pro-oxidant effects of toxic agents. This study was carried out to investigate the effect of SMN, Celecoxib (CLX) individually and in combination on monoiodoacetate (MIA)-induced osteoarthritis (OA) in rat. Forty adult Wistar rats were assigned to control and test groups. Animals in the test group following OA induction were subdivided into 4 subgroups according to the treatment profile: OA(+); received saline normal (5ml/kg, b.w.), OA(+)CLX(+); received CLX (100mg/kg, orally), OA(+)SMN(+), received SMN (50mg/kg, orally), and OA(+)CLX(+)SMN(+), received both CLX and SMN. The animals received test compounds by gastric gavage for 14 consecutive days. Animals in the OA(+) group showed a significant (p<0.01) increase in serum and synovial levels of IL-1ß, while both test compounds reduced the IL-1ß level. Both CLX and SMN lowered the OA-increased level of malondialdehyde by 77% and 79% and nitric oxide by 73% and 76%, respectively, in the synovial tissue. Special safranin O (SO) histopathological staining revealed that CLX and SMN improved the MIA-induced destruction and fibrillation in cartilage surface. CLX and SMN regulated the MIA-up regulated IL-1ß at mRNA level. The combination therapy resulted in an additive effect between CLX and SMN in biochemical, histopathological and molecular assays. These findings suggest that SMN exerts anti-inflammatory effect and also potentiates the anti-inflammatory effect of CLX on MIA-induced OA. The anti-inflammatory property of SMN may attribute to its antioxidant capacity, which affects the proinflammatory mediators at translational and transcriptional level.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents.

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

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.

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.

The anti-snake venom properties of Tamarindus indica (leguminosae) seed extract.

In Indian traditional medicine, various plants have been used widely as a remedy for treating snake bites. The aim of this study was to evaluate the effect of Tamarindus indica seed extract on the pharmacological as well as the enzymatic effects induced by V. russelli venom. Tamarind seed extract inhibited the PLA(2), protease, hyaluronidase, l-amino acid oxidase and 5'-nucleotidase enzyme activities of venom in a dose-dependent manner. These are the major hydrolytic enzymes responsible for the early effects of envenomation, such as local tissue damage, inflammation and hypotension. Furthermore, the extract neutralized the degradation of the Bbeta chain of human fibrinogen and indirect hemolysis caused by venom. It was also observed that the extract exerted a moderate effect on the clotting time, prolonging it only to a small extent. Edema, hemorrhage and myotoxic effects including lethality, induced by venom were neutralized significantly when different doses of the extract were preincubated with venom before the assays. On the other hand, animals that received extract 10 min after the injection of venom were protected from venom induced toxicity. Since it inhibits hydrolytic enzymes and pharmacological effects, it may be used as an alternative treatment to serum therapy and, in addition, as a rich source of potential inhibitors of PLA(2), metalloproteinases, serine proteases, hyaluronidases and 5 cent-nucleotidases, the enzymes involved in several physiopathological human and animal diseases.

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.

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.

Hyaluronidase and protease activities from Indian snake venoms: neutralization by Mimosa pudica root extract.

The aqueous root extract of Mimosa pudica dose dependently inhibited the hyaluronidase and protease activities of Indian snakes (Naja naja, Vipera russelii and Echis carinatus) venom.

Furosemide inhibits 11 beta-hydroxysteroid dehydrogenase in vitro and in vivo.

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) protects the non-selective renal mineralocorticoid receptor from the endogeneous glucocorticoid cortisol. Thus, drugs inhibiting 11 beta-OHSD might enhance urinary loss of potassium. In an attempt to find drugs inhibiting 11 beta-OHSD, 23 commonly used agents known to interfere with the potassium metabolism have been screened for inhibitory effect on 11 beta-OHSD. Furosemide appeared as the only inhibitor. Its inhibition constant (Ki) was 19.5 microM when kidney and 21.3 microM when liver microsomes were used as a source of 11 beta-OHSD. The type of inhibition was competitive. For confirmation that furosemide specifically inhibits 11 beta-OHSD, the complementary DNA (cDNA) of 11 beta-OHSD was transfected into COS-1 cells devoid of spontaneous expression of 11 beta-OHSD. In these cells, oxidation of corticosterone (Ki = 17.4 microM) and reduction of dehydrocorticosterone (Ki = 12.5 microM) was inhibited by furosemide. To establish whether this inhibition also occurs in vivo, the 11 beta-hydroxysteroid prednisolone was administered with and without furosemide to rats. The concentration ratio of prednisolone to its 11-ketometabolite prednisone increased in kidney and liver tissue after furosemide administration, indicating inhibition of 11 beta-OHSD. These data suggest that furosemide modulates in vivo the access of 11 beta-OH glucocorticoids to their target organs.

Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus flavoviridis) venom and their interaction with the alkaloid aristolochic acid.

A basic phospholipase A2 (PLA2) enzyme, TFV PL-X (pI 9.2) and two acidic PLA2 enzymes, TFV PL-Ia (pI 4.9) and TFV PL-Ib (pI 4.5) were purified from Trimeresurus flavoviridis venom on CM-Sephadex C-25 and QAE-Sephadex A-25 columns, respectively. The basic enzyme exists as a monomer, whereas the acidic enzymes are dimers. These enzymes differ in properties such as molecular weight, Km, optimum pH and temperature and pharmacological properties. The basic enzyme hydrolysed purified phospholipids in the order of PC greater than PE greater than PS greater than PI = 0, while for TFV PL-Ia and TFV PL-Ib the order was PC greater than PE greater than PS = PI = 0. TFV PL-X was comparatively more toxic, with an LD50 value of 4.2 micrograms/g (i.p.), while the acidic PLA2 enzymes had LD50 values above 8 micrograms/g (i.p.). All three enzymes induced edema when injected into the mouse foot pad. Aristolochic acid, an alkaloid (8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid) from the medicinal plant Aristolochia radix, interacts with these PLA2 enzymes. It is a competitive inhibitor with varying affinity when PC is used as substrate. Aristolochic acid inhibits direct and indirect hemolytic activity, as well as edema-inducing activity, of TFV PL-X, but fails to neutralize the lethal potency of the enzyme. On the other hand, it inhibits direct and indirect lytic activity of TFV PL-Ia and TFV PL-Ib only at 10-fold higher concentrations and it enhances the edema-inducing activity of these enzymes. Such effects of aristolochic acid indicates that (1) different mechanisms may be involved in the edema-inducing activity of PLA2 enzymes and (2) catalytic and pharmacological sites are separate on the PLA2 molecule.