S. Devaraja

An overview on genus garcinia: phytochemical and therapeutical aspects

The genus Garcinia belongs to the family Clusiaceae and has been involved in ayurvedic preparations to medicate various pathophysiological disorders. The bioactive molecules like hydroxycitric acid (HCA), flavonoids, terpenes, polysaccharides, procyanidines and polyisoprenylated benzophenone derivatives like garcinol, xanthochymol and guttiferone isoforms have been isolated from the genus Garcinia. The genus has received the attention of pharmaceutical industries due to their immense remedial qualities. The HCA has been known for its hypolipidemic property. The polyisoprenylated benzophenone and xanthone derivatives are known for their antioxidant, apoptotic, anti-cancer, anti-inflammatory, anti-bacterial, anti-viral, anti-fungal, anti-ulcer, anti-protozoal, and HAT inhibiting properties. Future studies on the synthesis of therapeutically important products and their analogs and evaluation of their safety and efficacy would be of great interest. Though the genus includes more than 300 species, we have made an effort to conceive the curative qualities of bioactive compounds of selected plants to the best of our knowledge.

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide.

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.

Vipera russelli venom-induced oxidative stress and hematological alterations: Amelioration by crocin a dietary colorant

Snakebite is a serious medical and socio‐economic problem affecting the healthy individuals and agricultural and farming populations worldwide. In India, Vipera russelli snakebite is common, ensuing high morbidity and mortality. The venom components persuade multifactorial stress phenomenon and alter the physiological setting by causing disruption of the blood cells and vital organs. The present study demonstrates the anti‐ophidian property of Crocin (Crocus sativus), a potent antioxidant against viper venom‐induced oxidative stress. The in vivo oxidative damage induced by venom was clearly evidenced by the increased oxidative stress markers and antioxidant enzymes/molecules along with the proinflammatory cytokines including IL‐1β, TNF‐α and IL‐6. Furthermore, venom depleted the hemoglobin, hematocrit, mean corpuscular volume and platelet count in experimental animals. Crocin ameliorated the venom‐induced oxidative stress, hematological alteration and proinflammatory cytokine levels. At present, administration of antivenom is an effective therapy against systemic toxicity, but it offers no protection against the rapidly spreading oxidative damage and infiltration of pro‐inflammatory mediators. These pathologies will continue even after antivenom administration. Hence, a long‐term auxiliary therapy is required to treat secondary as well as neglected complications of snakebite. Copyright

A low molecular weight serine protease: Purification and characterization from Hippasa agelenoides (funnel web) spider venom gland extract

Despite the long history [Kaiser, E., 1956. Enzymatic activity of spider venoms. In: Buckley, E.E., Porges, N. (Eds.), Venoms. American Association for the Advancement of Science, Washington, DC, pp. 91-93] on proteolytic activity, no study so far claims the isolation of a serine protease from the spider venom/venom gland extract. Therefore, the present study describes the isolation and characterization of a low molecular weight serine protease from Hippasa agelenoides venom gland extract. The protease (Hag-protease) was purified to homogeneity using the combination of gel-permeation and ion-exchange chromatography. The molecular mass was found to be 16.350 kDa by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Hag-protease was optimally active at pH 7.5 and temperature of 37 degrees C. PMSF abolished the enzyme activity while EDTA, EGTA, IAA, 1, 10-phenanthrolene did not. It hydrolyzed proteins such as casein, fibronectin and collagen type-I dose dependently but did not degrade gelatin and collagen type-IV. The isolated protease was non-lethal and devoid of hemorrhagic, myotoxic and edema forming activities. The light microscopy of Hag-protease treated skin tissue sections at the site of injection showed extensive damage of extracellular matrix (ECM) of hypodermis without causing any damage to blood vessels and capillaries. Similar damage of ECM of muscle tissue sections without affecting myocytes was noticed. Hag-protease was found to be procoagulant in property when studied plasma recalcification time.

Therapeutic drug-induced platelet apoptosis: an overlooked issue in pharmacotoxicology

The surfacing of the applied fields of biology such as, biotechnology, pharmacology and drug discovery was a boon to the modern man. However, it had its share of disadvantages too. The indiscriminate use of antibiotics and other biological drugs resulted in numerous adverse reactions including thrombocytopenia. One of the reasons for drug-induced thrombocytopenia could be attributed to an enhanced rate of platelet apoptosis, which is a less investigated aspect. The present essay sheds light on the adverse (pro-apoptotic) effects of some of the commonly used drugs and antibiotics on platelets viz. cisplatin, aspirin, vancomycin and balhimycin. Furthermore, the undesirable reactions resulting from chemotherapy could be attributed at least to some extent to the systemic stress induced by microparticles, which in turn are the byproducts of platelet apoptosis. Thereby, the essay aims to highlight the challenges in the emerging trend of cross-disciplinary implications, i.e., drug-induced platelet apoptosis, which is a nascent field. Thus, the different mechanisms through which drugs induce platelet apoptosis are discussed, which also opens up a new perspective through which the adverse effects of commonly used drugs could be dealt. The drug-associated platelet toxicity is of grave concern and demands immediate attention. Besides, it would also be appealing to examine the platelet pro-apoptotic effects of other commonly used therapeutic drugs.

Attenuation of adjuvant-induced arthritis by dietary sesamol via modulation of inflammatory mediators, extracellular matrix degrading enzymes and antioxidant status

PurposeThe dietary sesamol is one of the important constituent of sesame seed that has been mainly claimed to combat cardiovascular disease and diabetes, which are the major secondary complications of arthritis. Thus, the present study was designed to evaluate the anti-arthritic, anti-inflammatory and anti-stress potentials of sesamol.MethodsArthritis was induced using Freund’s complete adjuvant to hind paw of experimental rats. The physical and biochemical alterations and its recovery by sesamol were assessed by measuring enzymatic and non-enzymatic mediators. Arthritis-induced inflammation, oxidative stress and their protective by sesamol were measured by determining the levels of pro-inflammatory cytokines and oxidative stress markers.ResultsIn the present study, sesamol was demonstrated to alleviate arthritis-induced cartilage degeneration by mitigating augmented serum levels of hyaluronidase and matrix metalloproteinases (MMP-13, MMP-3 and MMP-9). It also protected bone resorption by reducing the elevated levels of bone joint exoglycosidases, cathepsin D and tartarate-resistant acid phosphatases. Sesamol also abrogated the non-enzymatic inflammatory markers (TNF, IL-1β, IL-6, COX-2, PGE2, ROS, and H2O2,) effectively. In addition, sesamol neutralizes arthritis-induced oxidative stress by restoring the levels of reactive oxygen species, lipid and hydro peroxides and sustained antioxidant homeostasis by re-establishing altered activities of superoxide dismutase, catalase and glutathione-s-transferase.ConclusionTaken together, the study demonstrated the anti-arthritic, anti-inflammatory, anti-oxidative stress and chondro-protective potentials of sesamol in vivo. Thus, sesamol could be a single bullet that can fight arthritis as well as the secondary complications of arthritis such as cardio vascular disorders and diabetes.

NETosis and lack of DNase activity are key factors in Echis carinatus venom-induced tissue destruction

Indian Echis carinatus bite causes sustained tissue destruction at the bite site. Neutrophils, the major leukocytes in the early defence process, accumulate at the bite site. Here we show that E. carinatus venom induces neutrophil extracellular trap (NET) formation. The NETs block the blood vessels and entrap the venom toxins at the injection site, promoting tissue destruction. The stability of NETs is attributed to the lack of NETs-degrading DNase activity in E. carinatus venom. In a mouse tail model, mice co-injected with venom and DNase 1, and neutropenic mice injected with the venom, do not develop NETs, venom accumulation and tissue destruction at the injected site. Strikingly, venom-induced mice tail tissue destruction is also prevented by the subsequent injection of DNase 1. Thus, our study suggests that DNase 1 treatment may have a therapeutic potential for preventing the tissue destruction caused by snake venom.

Factor Xa-like and fibrin(ogen)olytic activities of a serine protease from Hippasa agelenoides spider venom gland extract

In the recent past, a low molecular mass serine protease, the Hag-protease that caused pro-coagulant activity and as well as local toxicity was isolated and characterized from the Hippasa agelenoides spider venom gland extract (Devaraja et al., Toxicon 52:130–138, 2008). In the current study, the pro-coagulant property has been investigated further and the results are presented. The Hag-protease reduced the re-calcification time of citrated human plasma. It reduced the activated partial thromboplastin time (APTT), and prothrombin time (PT) suggesting its participation in common pathway of blood coagulation. Interestingly, it coagulated the citrated human plasma in the absence of CaCl2 but, it was lacking thrombin-like activity as it did not clot the purified fibrinogen. Strikingly, the enzyme coagulated the factor X deficient congenital human plasma, suggesting the factor Xa-like activity. However, the cumulative augmented activity was observed in presence of CaCl2 and phospholipids. Further, the Hag-protease preferentially hydrolyzed the Aα chain and then the Bβ-chain, but not the γ-chain. As a result, truncated fibrinogen generated was lacking in the polymerization property. It hydrolyzed all the subunits of partially cross-liked fibrin clot (α-polymer, α-chain, β-chain, and γ–γ dimers). Further, at low concentrations, the Hag-protease stimulated the aggregation of human platelets in platelet rich plasma, but at high concentrations caused spontaneous clumping. In contrast, it inhibited the collagen induced aggregation of washed human platelets. In summary, the present study for the first time reporting the factor Xa-like activity of a serine protease especially from the spider venom that exhibited opposing effects on hemostasis, the pro-coagulant activity and the anti-coagulant activity including fibrin(ogen)olytic and platelet aggregation inhibition activities.

Alleviation of viper venom induced platelet apoptosis by crocin (Crocus sativus): implications for thrombocytopenia in viper bites.

Viper envenomations are characterized by prominent local and systemic manifestations including hematological alterations. Snake venom metalloproteinases (SVMPs) and phospholipase A2 (PLA2) plays crucial role in the pathophysiology of hemorrhage by targeting/altering the platelets function which may result in thrombocytopenia. Platelets undergo the classic events of mitochondria-mediated apoptotic pathway due to augmented endogenous reactive oxygen species (ROS) levels. The observed anticoagulant effects during viper envenomations could be due to exacerbated platelet apoptosis and thrombocytopenia. Moreover, antivenin treatments are ineffective against the venom-induced oxidative stress; therefore, it necessitates an auxiliary therapy involving antioxidants which can effectively scavenge the endothelium-generated/endogenous ROS and protect the platelets. The present study explored the effects of viper venom on platelet apoptosis and its amelioration by a phytochemical crocin. The study evaluated the Vipera russelli venom-induced apoptotic events including endogenous ROS generation, intracellular Ca2+ mobilization, mitochondrial membrane depolarization, cyt-c translocation, caspase activation and phosphatidylserine externalization which were effectively mitigated when the venom was pre-treated with crocin. The study highlights one of the less studied features of venom-induced secondary complications i.e. platelet apoptosis and sheds light on the underlying basis for venom-induced thrombocytopenia, systemic hemorrhage and in vivo anticoagulant effect.

Differential Action of Phytochemicals on Platelet Apoptosis: A Biological Overview

Platelets are anuclear blood cells originating from bone megakaryocytes. Despite being anuclear, their number is maintained by apoptosis, a process of programmed cell death. The rate of apoptotic death of platelets is accelerated by oxidative and shear stress, ex vivo storage (blood banking conditions) and certain pathophysiological disorders. These factors initiate apoptotic events through the mitochondria- mediated intrinsic pathway. Besides, apoptotic platelets also release phosphatidylserine-positive membrane fractions called microparticles, which cause fibrin deposition and thrombus formation, and are involved in the promulgation of a host of disease conditions including cardiovascular diseases. In this context, several phytochemicals have been reported to be cardioprotective and work by inhibiting platelet aggregation or by dissolving the fibrin clots. Besides, ample reports focus on the positive effects of phytochemicals on normal physiology of platelets, but do not focus on their adverse effects on platelets. Moreover, platelets are reported to be extremely sensitive to therapeutic components in the blood. For example, resveratrol and thymoquinone are hitherto known compounds to possess proapoptotic effects on platelets. In contrast, cinnamtannin B1 and crocin exhibit antiapoptotic effects. Thus, the current review aims to elucidate the underlying mechanisms through which the phytochemicals mediate their effects on platelet apoptosis. Moreover, the need for scrutiny of therapeutic compounds for their effects on platelet functions before including them in treatment regimen is also being emphasized.