Syringol isolated from Eleusine coracana (L.) Gaertn bran suppresses inflammatory response through the down-regulation of cPLA2, COX-2, IκBα, p38 and MPO signaling in sPLA2 induced mice paw oedema.

Eleusine coracana (L.) Gaertn (E. coracana) is one of the highest consuming food crops in Asia and Africa. E. coracana is a plant with several medicinal values including anti-ulcerative, anti-diabetic, anti-viral and anti-cancer properties. However, the anti-inflammatory property of E. coracana remains to be elucidated. Therefore, the objective of present study was to investigate the potential in isolated molecule from E. coracana via a combination of in vitro, in vivo and in silico methods. In this study, we have isolated, purified and characterized an anti-inflammatory molecule from E. coracana bran extract known as syringol. Purification of syringol was accomplished by combination of GC-MS and RP-HPLC techniques. Syringol significantly inhibited the enzymes activity of sPLA2 (IC50 = 3.00 µg) and 5-LOX (IC50 = 0.325 µg) in vitro. The inhibition is independent of substrate concentration, calcium ion concentration and was irreversible. Syringol interacts with purified sPLA2 enzymes as evidenced by fluorescence and molecular docking studies. Further, the syringol molecule dose dependently inhibited the development of sPLA2 and λ-carrageenan induced edema. Furthermore, syringol decreases the expression of cPLA2, COX-2, IκBα, p38 and MPO in edematous tissues as demonstrated by western blots. These studies revealed that syringol isolated from E. coracana bran may develop as a potent anti-inflammatory molecule.

Drupin, a thrombin-like protease prompts platelet activation and aggregation through protease-activated receptors.

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro-coagulant activities of PLPs, an in-depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from the Ficus genus tested, Ficus drupacea exhibited potent pro-coagulant and thrombin-like activity. Drupin, a thrombin-like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro-coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen-activated protein kinases and the nuclear factor-κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2  leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease-activated receptors in drupin-induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin-induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro-coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.

Thrombin-like serine protease, antiquorin from Euphorbia antiquorum latex induces platelet aggregation via PAR1-Akt/p38 signaling axis.

Plant latex proteases (PLPs) are pharmacologically essential and are integral components of traditional medicine in the management of bleeding wounds. PLPs are known to promote blood coagulation and stop bleeding by interfering at various stages of hemostasis. There are a handful of scientific reports on thrombin-like enzymes characterized from plant latices. However, the role of plant latex thrombin-like enzymes in platelet aggregation is not well known. In the present study, we attempted to purify and characterize thrombin-like protease responsible for platelet aggregation. Among tested plant latices, Euphorbia genus latex protease fractions (LPFs) induced platelet aggregation. In Euphorbia genus, E. antiquorum LPF (EaLPF) strongly induced platelet aggregation and attenuated bleeding in mice. The purified thrombin-like serine protease, antiquorin (Aqn) is a glycoprotein with platelet aggregating activities that interfere in intrinsic and common pathways of blood coagulation cascade and alleviates bleeding and enhanced excision wound healing in mice. In continuation, the pharmacological inhibitor of PAR1 inhibited Aqn-induced phosphorylation of cPLA2, Akt, and P38 in human platelets. Moreover, Aqn-induced platelet aggregation was inhibited by pharmacological inhibitors of PAR1, PI3K, and P38. These data indicate that PAR1-Akt/P38 signaling pathways are involved in Aqn-induced platelet aggregation. The findings of the present study may open up a new avenue for exploiting Aqn in the treatment of bleeding wounds.

Serine protease from Tricosanthus tricuspidata accelerates healing of Echis carinatus venom-induced necrotic wound.

Echis carinatus (EC) envenomation causes severe immune response by the accumulation of tissue debris in the form of DAMPs resulting in chronic inflammation and progressive tissue necrosis at the bitten site. Clearing of tissue debris is a prerequisite to enhance the healing of venom-induced necrotic wounds. Tricosanthus tricuspidata is a medicinal plant used extensively for the treatment of snake bite-induced toxicities. The active component responsible for the observed pharmacological action is a serine protease, tricuspidin. The topical application of tricuspidin was able to neutralize ECV-induced mouse footpad tissue necrosis and open wound in rabbits. Tricuspidin exerted its healing action via proteolytic activity as a consequence of upregulation of MMP-8 and down regulation of MMP-9. Further, tricuspidin reduced ECV-induced inflammation by decreasing the expression of TNF-α, IL-6 and MPO, and by increasing the level of VEGF-A and TGF-ß1. The modulation of ECV induced immune/inflammatory mediators by tricuspidin was found to be more effective than trypsin. Moreover, tricuspidin and trypsin activated MAPKs via protease activated receptors-2 (PAR-2). These data indicate that the proteolytic activity of tricuspidin directly involved in the healing of ECV-induced chronic wound.

Plant DNases are potent therapeutic agents against Echis carinatus venom-induced tissue necrosis in mice.

Echis carinatus envenomation leads to severe tissue necrosis at the bitten site by releasing DNA from immune cells that blocks the blood flow. An earlier report has shown that exogenous DNase 1 offers protection against such severe local tissue necrosis. Tricosanthus tricuspidata is a medicinal plant and the paste prepared from its leaves has been used extensively for the treatment of snakebite-induced tissue necrosis. Most studies including reports from our laboratory focused on plant secondary metabolite as therapeutic molecules against snakebite envenomation. However, the involvement of hydrolytic enzymes including DNase in treating snake venom-induced tissue necrosis has not been addressed. Several folk medicinal plants used against snakebite treatment showed the presence of DNase activity and found to be rich in T. tricuspidata. Further, purified T. tricuspidata DNase showed a single sharp peak in reversed-phase high-performance liquid chromatography (RP-HPLC) with an apparent molecular mass of 17 kDa. T. tricuspidata DNase exhibited potent DNA degrading activity performed using agarose gel electrophoresis, spectrophotometric assay, and DNA zymography. In addition, purified DNase from T. tricuspidata was able to neutralize E. carinatus venom-induced mouse tail tissue necrosis and normalized elevated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels 30 minutes post venom injection. T. tricuspidata DNase was also able to reverse E. carinatus venom-induced histopathological changes and collagen depletion in mice tail tissue. All these observed pharmacological actions of T. tricuspidata DNase were inhibited by sodium fluoride (NaF). This study provides scientific validation of the traditional use of T. tricuspidata leaf paste in the healing of snakebite-induced tissue necrosis and might be exploited to treat snake venom-induced local toxicity.