Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities.

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

A-kinase anchor protein 12 (AKAP12) inhibits cell migration in breast cancer.

A-kinase anchor protein 12 (AKAP12) also known as Gravin and SSeCKS, is a novel potent scaffold protein for many key signaling factors, such as protein kinase C (PKC), PKA, cyclins as well as F-actin. AKAP12 expression is known to be suppressed in several human malignancies including breast, prostate, gastric and colon cancers. In this study, we evaluated the role of AKAP12 in the migration of breast cancer cells, an important cellular process in cancer progression. AKAP12 gene expression was analyzed in human breast cancer tissues using the Gene expression-based Outcome for Breast cancer Online (GOBO) database and TissueScan array, followed by relapse free survival (RFS) analysis with the Kaplan-Meier Plotter. AKAP12 protein was then analyzed in normal MCF10A breast cell line and six different breast cancer cell lines (AU565, Hs578T, MCF7, MDA-MB-231, T47D and ZR751). After which, siRNA-mediated knockdown of AKAP12 was carried out in MCF10A, MDA-MB-231 and Hs578T cells, followed by phenotypic assays. AKAP12 was observed to be reduced in breast cancer tissues as analyzed by GOBO and TissueScan array. Kaplan Meier survival analysis revealed that patients with AKAP12 gene expression had a higher RFS survival. There was also decreased AKAP12 protein expression in breast cancer cell lines compared to MCF10A normal epithelial breast cell line. Knockdown of AKAP12 in both MCF10A cells and Hs578T cells induced cell migration but did not alter cell proliferation. Moreover, siAKAP12 in aggressive MDA-MB-231 breast cancer cells led to an increase in cell migration. Immunofluorescence analysis of AKAP12 depleted MCF10A cells also revealed formation of thick stress fibers which could affect cell migration. Hence, the findings in this study suggest that AKAP12 is a potential metastasis suppressor in breast cancer.

Identification of natural peptides as a new class of antimalarial drugs by in silico approaches.

Malaria is one of the most widespread and serious parasitic diseases worldwide. Currently available antimalarial drugs have side effects, and many strains of Plasmodia have developed resistance to such drugs. The present review examines the use of annexins and of natural peptides from snake venom as a new class of anti-malarial agents, with the key property of reducing inflammation. Severe cases of malaria manifest elevated serum levels of liver enzymes, inflammation, fibrin deposition, apoptosis, and reduction in peripheral CD8+ T cells. The annexin-A1/5 proteins trigger inflammation via increased expression of diverse cytokines (tumor necrosis factor alpha, interleukin-1 beta, interleukin-10), however, by shielding microbial phospholipids they prevent injury via damage-associated molecular patterns (DAMPs). Here, we also review an in silico-based bioengineering approach that may allow for a better design, synthesis and characterization of novel peptides from snake venom as a more effective approach to treatment due to their improved antimalarial activity.

ANXA1 inhibits miRNA-196a in a negative feedback loop through NF-kB and c-Myc to reduce breast cancer proliferation.

MiRNAs are endogenous ~22 nt RNAs which play critical regulatory roles in a wide range of biological and pathological processes, which can act as oncogenes or tumor suppressor genes depending on their target genes. We have recently shown that ANXA1 inhibits the expression of miRNAs including miR196a. Here, we show that miR196a was highly expressed in ER+ MCF-7 breast cancer cells when compared to normal mammary gland cells, with expression levels negatively correlating to ANXA1. ANXA1 inhibits the biogenesis of oncogenic miR-196a by suppressing primary-miR196a indirectly through the stimulation of c-myc and NFkB expression and activity in breast cancer cells. In a negative feedback loop, miR-196a directly inhibits ANXA1 and enhances breast cancer cell proliferation in vitro. Finally, miR196a promotes breast tumor growth in vivo. This study reports a novel regulatory circuit between ANXA1, NF-kB, c-myc and miR-196a which regulates breast cancer cell proliferation and tumor growth.

A brief update on potential molecular mechanisms underlying antimicrobial and wound-healing potency of snake venom molecules.

Infectious diseases remain a significant cause of morbidity and mortality worldwide. A wide range of diverse, novel classes of natural antibiotics have been isolated from different snake species in the recent past. Snake venoms contain diverse groups of proteins with potent antibacterial activity against a wide range of human pathogens. Some snake venom molecules are pharmacologically attractive, as they possess promising broad-spectrum antibacterial activities. Furthermore, snake venom proteins (SVPs)/peptides also bind to integrins with high affinity, thereby inhibiting cell adhesion and accelerating wound healing in animal models. Thus, SVPs are a potential alternative to chemical antibiotics. The mode of action for many antibacterial peptides involves pore formation and disruption of the plasma membrane. This activity often includes modulation of nuclear factor kappa B (NF-κB) activation during skin wound healing. The NF-κB pathway negatively regulates the transforming growth factor (TGF)-ß1/Smad pathway by inducing the expression of Smad7 and eventually reducing in vivo collagen production at the wound sites. In this context, SVPs that regulate the NF-κB signaling pathway may serve as potential targets for drug development.

Ageing and the telomere connection: An intimate relationship with inflammation.

Telomeres are the heterochromatic repeat regions at the ends of eukaryotic chromosomes, whose length is considered to be a determinant of biological ageing. Normal ageing itself is associated with telomere shortening. Here, critically short telomeres trigger senescence and eventually cell death. This shortening rate may be further increased by inflammation and oxidative stress and thus affect the ageing process. Apart from shortened or dysfunctional telomeres, cells undergoing senescence are also associated with hyperactivity of the transcription factor NF-κB and overexpression of inflammatory cytokines such as TNF-α, IL-6, and IFN-γ in circulating macrophages. Interestingly, telomerase, a reverse transcriptase that elongates telomeres, is involved in modulating NF-κB activity. Furthermore, inflammation and oxidative stress are implicated as pre-disease mechanisms for chronic diseases of ageing such as neurodegenerative diseases, cardiovascular disease, and cancer. To date, inflammation and telomere shortening have mostly been studied individually in terms of ageing and the associated disease phenotype. However, the interdependent nature of the two demands a more synergistic approach in understanding the ageing process itself and for developing new therapeutic approaches. In this review, we aim to summarize the intricate association between the various inflammatory molecules and telomeres that together contribute to the ageing process and related diseases.

Targeting transcription factor STAT3 for cancer prevention and therapy.

Signal Transducers and Activators of Transcription (STATs) comprise an important class of transcription factors that have been implicated in a wide variety of essential cellular functions related to proliferation, survival, and angiogenesis. Among various STAT members, STAT3 is frequently overexpressed in tumor cells as well as tissue samples, and regulates the expression of numerous oncogenic genes controlling the growth and metastasis of tumor cells. The current review briefly discusses the importance of STAT3 as a potential target for cancer therapy and also provides novel insights into various classes of existing pharmacological inhibitors of this transcription factor that can be potentially developed as anti-cancer drugs.

Emodin suppresses migration and invasion through the modulation of CXCR4 expression in an orthotopic model of human hepatocellular carcinoma.

Accumulating evidence(s) indicate that CXCL12-CXCR4 signaling cascade plays an important role in the process of invasion and metastasis that accounts for more than 80% of deaths in hepatocellular carcinoma (HCC) patients. Thus, identification of novel agents that can downregulate CXCR4 expression and its associated functions have a great potential in the treatment of metastatic HCC. In the present report, we investigated an anthraquinone derivative, emodin for its ability to affect CXCR4 expression as well as function in HCC cells. We observed that emodin downregulated the expression of CXCR4 in a dose-and time-dependent manner in HCC cells. Treatment with pharmacological proteasome and lysosomal inhibitors did not have substantial effect on emodin-induced decrease in CXCR4 expression. When investigated for the molecular mechanism(s), it was observed that the suppression of CXCR4 expression was due to downregulation of mRNA expression, inhibition of NF-κB activation, and abrogation of chromatin immunoprecipitation activity. Inhibition of CXCR4 expression by emodin further correlated with the suppression of CXCL12-induced migration and invasion in HCC cell lines. In addition, emodin treatment significantly suppressed metastasis to the lungs in an orthotopic HCC mice model and CXCR4 expression in tumor tissues. Overall, our results show that emodin exerts its anti-metastatic effect through the downregulation of CXCR4 expression and thus has the potential for the treatment of HCC.

Zinc supplementation prevents cardiomyocyte apoptosis and congenital heart defects in embryos of diabetic mice.

Oxidative stress induced by maternal diabetes plays an important role in the development of cardiac malformations. Zinc (Zn) supplementation of animals and humans has been shown to ameliorate oxidative stress induced by diabetic cardiomyopathy. However, the role of Zn in the prevention of oxidative stress induced by diabetic cardiac embryopathy remains unknown. We analyzed the preventive role of Zn in diabetic cardiac embryopathy by both in vivo and in vitro studies. In vivo study revealed a significant decrease in lipid peroxidation, superoxide ions, and oxidized glutathione and an increase in reduced glutathione, nitric oxide, and superoxide dismutase in the developing heart at embryonic days (E) 13.5 and 15.5 in the Zn-supplemented diabetic group when compared to the diabetic group. In addition, significantly down-regulated protein and mRNA expression of metallothionein (MT) in the developing heart of embryos from diabetic group was rescued by Zn supplement. Further, the nuclear microscopy results showed that trace elements such as phosphorus, calcium, and Zn levels were significantly increased (P<0.001), whereas the iron level was significantly decreased (P<0.05) in the developing heart of embryos from the Zn-supplemented diabetic group. In vitro study showed a significant increase in cellular apoptosis and the generation of reactive oxygen species (ROS) in H9c2 (rat embryonic cardiomyoblast) cells exposed to high glucose concentrations. Supplementation with Zn significantly decreased apoptosis and reduced the levels of ROS. In summary, oxidative stress induced by maternal diabetes could play a role in the development and progression of cardiac embryopathy, and Zn supplementation could be a potential therapy for diabetic cardiac embryopathy.

Carboxylate microsphere-induced cellular toxicity in human lung fibroblasts.

Carboxylate microspheres (CMs) are mainly used in industrial, biomedical and various household products. In this study, we assessed the cytotoxic effects of CMs on human MRC-5 lung fibroblasts by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Oxidative stress was determined by measurements of reactive oxygen species and antioxidant (superoxide dismutase and catalase) levels and proinflammatory cytokines quantified by enzyme-linked immunosorbent assay. Morphological changes were examined by light microscopy, confocal microscopy and transmission electron microscopy. The lung fibroblasts were exposed to increasing concentrations of CMs (0.1-1000 µmol/L) for 24 h. The results showed significant changes in cell morphology with induction of cytotoxicity and oxidative stress observed in 10-1000 µmol/L concentrations of CM-treated fibroblasts. Ultrastructural examination revealed the presence of CMs inside the cytoplasm of treated lung fibroblasts. CMs also induced elevated interleukin (IL)-1, IL-6, IL-8, IL-10 and tumor necrosis factor α levels at higher concentrations. We have demonstrated that CMs significantly reduce cell viability in a dose-dependant manner in lung fibroblasts at 0.1-1000 µmol/L doses. The findings suggest that high doses of CMs have the potential to induce cellular toxicity to the lung in vitro.

Identification of a novel Calotropis procera protein that can suppress tumor growth in breast cancer through the suppression of NF-κB pathway.

Breast cancer is the most common cancer among women. To date, improvements in hormonal and cytotoxic therapies have not yet led to a sustained remission or cure. In the present study, we investigated the in vitro and in vivo antitumor activities of a novel Calotropis procera protein (CP-P) isolated from root bark. CP-P protein inhibited the proliferation and induced apoptosis of breast cancer cells through the suppression of nuclear factor kappaB (NF-kB) activation. CP-P, when administered individually or in combination with cyclophosphamide (CYC, 0.2 mg/kg) to rats with 7, 12-dimethyl benz(a)anthracene (DMBA)-induced breast cancer decreased tumor volume significantly without affecting the body weight. To elucidate the anticancer mechanism of CP-P, antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST) and non-enzymatic antioxidant - reduced glutathione (GSH), vitamin E and C generation in the breast were analyzed by various assays. SOD, CAT, GST, GSH, vitamin E and C levels were high in combination-treated groups (CP-P+CYC) versus the CYC alone-treated groups. Also, the combination was more effective in down-regulating the expression of NF-kB-regulated gene products (cyclin D1 and Bcl-2) in breast tumor tissues. Our findings indicate that CP-P possesses significant antitumor activity comparable to a commonly used anticancer drug, cyclophosphamide, and may form the basis of a novel therapy for breast cancer.

Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis.

Complications of acute respiratory distress syndrome (ARDS) are common among critically ill patients infected with highly pathogenic influenza viruses. Macrophages and neutrophils constitute the majority of cells recruited into infected lungs, and are associated with immunopathology in influenza pneumonia. We examined pathological manifestations in models of macrophage- or neutrophil-depleted mice challenged with sublethal doses of influenza A virus H1N1 strain PR8. Infected mice depleted of macrophages displayed excessive neutrophilic infiltration, alveolar damage, and increased viral load, later progressing into ARDS-like pathological signs with diffuse alveolar damage, pulmonary edema, hemorrhage, and hypoxemia. In contrast, neutrophil-depleted animals showed mild pathology in lungs. The brochoalveolar lavage fluid of infected macrophage-depleted mice exhibited elevated protein content, T1-α, thrombomodulin, matrix metalloproteinase-9, and myeloperoxidase activities indicating augmented alveolar-capillary damage, compared to neutrophil-depleted animals. We provide evidence for the formation of neutrophil extracellular traps (NETs), entangled with alveoli in areas of tissue injury, suggesting their potential link with lung damage. When co-incubated with infected alveolar epithelial cells in vitro, neutrophils from infected lungs strongly induced NETs generation, and augmented endothelial damage. NETs induction was abrogated by anti-myeloperoxidase antibody and an inhibitor of superoxide dismutase, thus implying that NETs generation is induced by redox enzymes in influenza pneumonia. These findings support the pathogenic effects of excessive neutrophils in acute lung injury of influenza pneumonia by instigating alveolar-capillary damage.

Therapeutic potential of peptides with neutralizing ability towards the venom and toxin (CaTx-I) of crotalus adamanteus.

The CaTx-I (PLA2) toxin of Crotalus adamanteus venom is responsible for most of the symptoms observed during envenomation. Synthetic peptides were designed and screened for venom (0.8 µg/ml) and CaTx-I (0.1 µM) inhibition at varying doses of the peptide (10000- 0.0001 µM) using a Cayman chemical human secretory phospholipase A2 (sPLA2, Type II) assay kit. Further, in vitro neutralization studies were evaluated by a fixed dose of peptide (1 µM) against venom (0.8 µg/ml) and toxin (0.1 µM). Among the linear peptides (PIP-18, cyclic C and PIP59-67) that showed potent neutralizing effects against the venom/toxin of C. adamanteus. PIP-18 [IC50, 1.23 µM] and cyclic C [IC50, 1.27 µM] peptides possessed the strongest inhibitory effect against CaTx-I. A fixed dose of CaTx-I (75 µg/kg) was administered intraperitoneally (i.p.) into mice followed by an i.p. injection of peptides PIP-18 and cyclic C at (6 µg/mouse), venom (150 µg/kg) and toxin CaTx-I alone served as references. Mice treated with PIP-18 and cyclic C showed a very strong neutralizing effect and markedly reduced mortality compared to the control after 24 h. The CA venom and CaTx-I injected mice showed severe toxicity after 24 h. Peptides PIP-18 and cyclic C were non-hemolytic at 100 µM. They produced a significant decrease in lipid peroxidase (LPx) and enhancement of superoxide dismutase (SOD), catalase (CAT) and Glutathione-s-transferase (GST) levels indicating their antioxidant property against venom-induced changes in mice. This study confirmed the potent snake venom neutralizing properties of peptides.

Evaluation of antibacterial activity of proteins and peptides using a specific animal model for wound healing.

Wound healing is a complex process involving the integrated actions of numerous cell types, soluble mediators, and extracellular matrix (ECM). In this study, phospholipase A(2) (PLA(2)) purified from crotalid snake venom was found to express in vitro bactericidal activity against a group of clinical human pathogens. Based on the sequence homology of PLA(2), a series of peptides were derived from the C-terminal region of crotalid PLA(2). These short synthetic peptides were found to reproduce the bactericidal activity of its parent molecule. In vitro assays for bactericidal and cytolytic activities of these peptides showed very high microbicidal potency against Gram-negative and Gram-positive (Staphylococcus aureus) bacteria. Variants of the peptides showed reduced toxicity toward normal human cells, while retaining high bactericidal potency. Here we describe the protocol for evaluating the wound healing process by antibacterial peptides. We evaluated the biological roles of the candidate peptides in skin wound healing, using a specific BALB/c mice model. Peptide-treated animals showed accelerated healing of full-thickness skin wounds, with increased reepithelialization, collagen synthesis, and angiogenesis observed during the healing process. Healing wounds in protein/peptide-treated mice had higher densities of neutrophils, macrophages, and fibrocytes. Along with increased leukocyte infiltration, levels of macrophage-derived chemokine expression were also upregulated. These results demonstrate that the protein/peptide derived from snake venoms promotes healing of skin wounds. The primary mechanism seems to be an increase in leukocyte infiltration, leading to locally elevated synthesis and release of collagen and growth factors.

Venom neutralization by purified bioactive molecules: Synthetic peptide derivatives of the endogenous PLA(2) inhibitory protein PIP (a mini-review).

Envenomation due to snakebite constitutes a significant public health problem in tropical and subtropical countries. Antivenom therapy is still the mainstay of treatment for snake envenomation, and yet despite recent research focused on the prospects of using antivenom adjuncts to aid in serotherapy, no new products have emerged so far for therapeutic use. Various methodologies including molecular biology, crystallography, functional and morphological approaches, etc., are employed in the search for such inhibitors with a view to generate molecules that can stop partially or completely the activities of toxic phospholipase A(2) (PLA(2)) and snake venom metalloproteinase (SvMPs) enzymes at the molecular level. Herein, both natural and synthetic inhibitors derived from a variety of sources including medicinal plants, mammals, marine animals, fungi, bacteria, and from the venom and blood of snakes have been briefly reviewed. Attention has been focused on the snake serum-based phospholipase A(2) inhibitors (PLIs), particularly on the PLI derived from python snake serum (PIP), highlighting the potential of the natural product, PIP, or possible derivatives of it, as a complementary treatment to serotherapy against the inflammation and/or muscle-damaging activity of snake venoms. The data indicate a more efficient pathway for inhibition and blocking the activity of PLA(2)s and matrix metalloproteinases (MMPs), thus representing a feasible complementary treatment for snakebites. Such information may be helpful for interfering on the biological processes that these molecules are involved in human inflammatory-related diseases, and also for the development of new drugs for treatment of snake envenomation.

Viper metalloproteinase (Agkistrodon halys pallas) with antimicrobial activity against multi-drug resistant human pathogens.

Metalloproteinases are abundant enzymes in crotalidae and viperidae snake venoms. Snake venom metalloproteinases (SVMPs) comprise a family of zinc-dependent enzymes, which display many different biological activities. A 23.1 kDa protein was isolated from Agkistrodon halys (pallas, Chinese viper) snake venom. The toxin is a single chain polypeptide with a molecular weight of 23146.61 and an N-terminal sequence (MIQVLLVTICLAVFPYQGSSIILES) relatively similar to that of other metalloprotein-like proteases isolated from the snake venoms of the Viperidae family. The antibacterial effect of Agkistrodon halys metalloproteinase (AHM) on Burkholderia pseudomallei (strains TES and KHW), Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacterium) was studied at a concentration 120 microM. Interestingly, we found that the metalloproteinase exhibited antibacterial properties and was more active against S. aureus, P. vulgaris, P. mirabilis and multi-drug resistant B. pseudomallei (strain KHW) bacteria. AHM variants with high bacteriostatic activity (MIC 1.875-60 microM) also tended to be less cytotoxic against U-937 human monocytic cells up to 1 mM concentrations. These results suggest that this metalloprotein exerts its antimicrobial effect by altering membrane packing and inhibiting mechanosensitive targets.

Anti-tumor promoting potential of luteolin against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats.

In this study, we evaluated the anti-tumor potential of luteolin (30mg/kg, p.o.), combined with cyclophosphamide (10mg/kg, i.p.) (LU+CYC) orally administered for 20 days; and CYC individually for 10 days against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in Wistar rats. Combination treatment (LU+CYC) inhibited the incidence rate of tumors and decreased tumor volume significantly without changing the total body weight of the animals. Long-term treatment did not show any apparent toxicity in rats. The CYC-treated group showed potential reduction of tumor volume (74%), severe toxicity, and loss of body weight. In order to elucidate the anticancer mechanism of luteolin, antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) generation in the liver, kidney and breast, as well as protein profiles, were also examined. Biochemical analysis of the combination-treated group showed significant (P<0.01; P<0.05) inhibition of lipid peroxide (LPx) formation (oxygen-free radicals), the level and the activity of SOD, CAT and GPx were found to be very high than the LU and CYC individually treated rats at a 30mg/kg dose. 2D gel electrophoresis analysis revealed that (56kDa) high molecular weight protein was detected in tumors of rats receiving combination treatment than the cancer controls. The biological significance of that protein involved for the dysfunction of cancer cells and induces apoptosis. Histopathological changes also confirmed the formation of tumor tubules and neovascularization after the treatment. Overall, these results suggest that the combination treatment provided antioxidant defense with strong chemopreventive activity against the genesis of DMBA-induced mammary tumors.

Effect of aqueous extract of Tragia involucrata Linn. on acute and subacute inflammation.

Antiinflammatory activity of aqueous extract of Tragia involucrata was tested on carrageenan-induced hind paw oedema and cotton pellet granuloma models in albino rats. In the subacute model, cotton pellet granuloma was produced by implantation of 10 mg sterile cotton in the axilla under ether anaesthesia. The animals were administered an aqueous extract at various concentrations of 50, 100, 200, 300 and 400 mg/kg. Phenyl butazone (80 mg/kg) was used as a standard drug. The paw diameter was measured at different time intervals and the dry granuloma weight was taken after the treatment. The aqueous leaf extract (400 mg/kg) showed the maximum inhibition (84.23%) of oedema at the end of 3 h following carrageenin-induced rat paw oedema. In subacute inflammation, the extract showed 76.25% reduction in granuloma weight. The results prove that the aqueous leaf extract showed highest antiinflammatory activity in acute and subacute inflammation and also support the usage of traditional claims.