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.

Inhibition of testicular and Vipera russelli snake venom hyaluronidase activity by Butea monosperma (Lam) Kuntze stem bark.

This study was carried out to investigate the anti-fertility and anti-venom activities of the extract of the stem bark of Butea monosperma by inhibiting hyaluronidase, which is a spreading factor and plays a role in fertilisation. Among ethanol, methanol and water extracts, the ethanol extract dose-dependently inhibited the ovine, mouse testicular and Vipera russelli snake venom hyaluronidase enzyme activities, with IC50 values 12.00 ± 0.45, 49.40 ± 1.58 µg and 125.42 ± 2.82 µg mL⁻¹, respectively. In a zymogram assay, the extract showed differential inhibition towards hyaluronidase isoform preferentially with low-molecular weight isoforms. The V. russelli snake venom-induced hemorrhage was significantly reduced at 1:05 ratio of venom-to-extract in mouse. The high antioxidant activity and total phenolic content in the ethanolic extract strongly correlated with the hyaluronidase inhibition. The above results justify the traditional use of the stem bark of B. monosperma as a contraceptive and a strong antidote to snake venom.

Phytochemical screening and evaluation of in vitro angiotensin-converting enzyme inhibitory activity of Artocarpus altilis leaf.

This study investigates the effect of Artocarpus altilis leaf extracts on angiotensin-converting enzyme (ACE) activity. Among the extracts tested, hot ethanol extract exhibited a potent ACE-inhibitory activity with an IC50 value of 54.08 ± 0.29 µg mL⁻¹ followed by cold ethyl acetate extract (IC50 of 85.44 ± 0.85 µg mL⁻¹). In contrast, the hot aqueous extracts showed minimum inhibition with the IC50 value of 765.52 ± 11.97 µg mL⁻¹ at the maximum concentration tested. Further, the phytochemical analysis indicated the varied distribution of tannins, phenolics, glycosides, saponins, steroids, terpenoids and anthraquinones in cold and hot leaf extracts. The correlation between the phytochemical analysis and ACE-inhibitory activity suggests that the high content of glycosidic and phenolic compounds could be involved in exerting ACE-inhibitory activity. In conclusion, this study supports the utilisation of A. altilis leaf in the folk medicine for the better treatment of hypertension. Further studies on isolation and characterisation of specific ACE-inhibitory molecule(s) from ethyl acetate, ethanol and methanol extracts of A. altilis leaf would be highly interesting.

Chemical modification of ascorbic acid and evaluation of its lipophilic derivatives as inhibitors of secretory phospholipase A(2) with anti-inflammatory activity.

The halo 6-fatty acid esters of L-ascorbic acid 3a, 3b and 6-fatty acid esters of L-ascorbic acid 5a-g were achieved from L-ascorbic acid 1. Compounds 3a, 3b and 5a-g were evaluated for anti-oxidant, anti-lipid peroxidation, and secretory phospholipase A(2) (sPLA(2)) inhibition in vitro, and sPLA(2) induced mouse paw edema. All the derivatives retained their anti-oxidant property compared to ascorbic acid at 6 × 10(-4)M and are good inhibitors of lipid peroxidation at 1 mg ml(-1) as evaluated by 2, 2-Diphenyl-1-picrylhydrazyl radical and thio-barbituric acid methods, respectively. Compounds 5e and 5f significantly inhibited purified group I sPLA(2) from Naja naja and group II sPLA(2) from Vipera russelli, human synovial fluid and human pleural fluid with IC(50) value ranging from 64 ± 1.95 to 82 ± 1.3 and 48 ± 2.27 to 61 ± 2.23 µM, respectively. The compounds 5e and 5f also showed varying degree of potency in neutralizing indirect hemolytic activity of sPLA(2) at 50 µM concentration, and sPLA(2) induced mouse paw edema at the dose 3 mg/kg. Further docking studies also confirmed that compounds 5e and 5f have maximum interaction with increasing negative energy value. Single molecule possessing both anti-oxidant and anti-inflammatory activities is of great therapeutic significance in inflammatory disorders.

Radical scavenging and angiotensin converting enzyme inhibitory activities of standardized extracts of Ficus racemosa stem bark.

The present study evaluated the radical scavenging and angiotensin converting enzyme (ACE) inhibitory activity of cold and hot aqueous extracts of Ficus racemosa (Moraceae) stem bark. The extracts were standardized using HPLC. Radical scavenging activity was determined using 1,1-diphenyl-2-picrylhydrazyl radical and angiotensin converting enzyme inhibitory activity using rabbit lung and partially purified porcine kidney ACE. HPLC profiles of cold aqueous extract (FRC) showed the presence of bergenin, an isocoumarin, while hot aqueous extract (FRH) was found to contain ferulic acid, kaempferol and coumarin in addition to bergenin. FRH showed significantly higher (p ≤ 0.01) radical scavenging activity than FRC and butylated hydroxytoluene (BHT), consequently resulting in a significantly lower (p ≤ 0.01) IC50 value than FRC and BHT. Both the extracts exhibited a dose dependent inhibition of porcine kidney and rabbit lung ACE. FRH showed significantly higher (p ≤ 0.01) activity than FRC with lower IC(50) values of 1.36 and 1.91 µg/mL respectively, for porcine kidney and rabbit lung ACE, compared with those of FRC (128 and 291 µg/mL). Further, a significant correlation (r = 0.893; p ≤ 0.05) was observed between radical scavenging activity and ACE-inhibitory activity. This is the first report on the ACE-inhibitory activity of F. racemosa stem bark suggesting its potential to be utilized as a therapeutic alternative for hypertension.

Anti-inflammatory activity of oleanolic acid by inhibition of secretory phospholipase A2.

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 IC (50) values of sPLA (2) from these sources ranged from 3.08 to 7.78 muM. Increasing calcium (Ca (2+)) 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.

Comparative study on plant latex proteases and their involvement in hemostasis: a special emphasis on clot inducing and dissolving properties.

In the present study we compared the clot inducing and dissolving properties of Calotropis gigantea R. Br. (Asclepiadaceae), Synadenium grantii Hook. f. (Euphorbiaceae) and Wrightia tinctoria R. Br. (Apocynaceae) latex extracts. All the three latex extracts hydrolyzed casein, fibrinogen and crude fibrin dose-dependently. The proteolytic action on fibrinogen subunity was in the order of Aalpha > Bbeta > gamma. All extracts exhibited procoagulant activity as assayed by re-calcification time. However, thrombin like activity is restricted to C. gigantea. In addition, the extracts dose-dependently hydrolyzed blood and plasma clots. Furthermore, the hydrolyzing pattern of fibrin in the plasma clot was substantiated by SDS-PAGE. The extracts hydrolyzed all the subunits (alpha polymer, alpha-chains, gamma-gamma dimer and beta-chain) of fibrin efficiently. Both fibrinogenolytic and fibrinolytic activity potency of the extracts were in the order of C. gigantea > S. grantii > W. tinctoria. Among the three latices, C. gigantea is toxic with a minimum hemorrhagic dose (MHD) of > 75 microg, whereas S. grantii and W. tinctoria latex extracts were non-toxic and did not induce any hemorrhagic effect at the tested dose (> 200 microg). The proteolytic activity of C. gigantea latex extract on different substrates was inhibited by IAA. On the other hand, the proteolytic activities of S. grantii and W. tinctoria were inhibited by PMSF. Thus, this study provides the basis for the probable action of plant latex proteases to stop bleeding and effect wound healing as exploited in folk medicine.

Use of Pavo cristatus feather extract for the better management of snakebites: neutralization of inflammatory reactions.

In Indian traditional medicine, peacock feather in the form of ash (Bhasma) or water extract are used against snakebite and to treat various problems associated with lungs. This study was aimed to evaluate the water extract of peacock feather (PCF) against the local tissue damage caused due to snakebite. PCF water extract showed inhibition towards phospholipase A2 enzyme activity from snake venom (Naja naja and Vipera russelii), inflammatory fluids (synovial, pleural, ascites) and normal serum in a dose-dependent manner. Hyaluronidase and proteases are other major enzymes in snake venoms responsible for local tissue damage. PCF water extract inhibited hyaluronidase and proteolytic enzyme activities from Vipera russelii, Naja naja and Trimeresurus malabaricus venom. The active principle is a hydrophilic molecule easily extractable in water or polar solvents. PCF water extract gave positive results for the presence of protein and secondary metabolites like carotenoids and steroids. Analysis of metal ions revealed that iron is the major ion (> 20-fold). Other metal ions detected in smaller amount are copper, chromium, zinc and nickel. The least amount of ion detected is gold. Co-injection of PCF water extract with snake venom and inflammatory PLA2 enzymes neutralize the edema inducing activity of all the PLA2 enzymes studied. Since it inhibits hyaluronidase and proteases enzyme activity from snake venom PCF water extract is a powerful neutralizing agent, which has therapeutic application against venom toxicity.