Summary and conclusion Summary and conclusion

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1 Summary and conclusion Wound healing is a complex biological processes aiming at restoration of structural organization and functional status of the injured tissue. The events of wound healing are categorized into four sequential yet overlapping phases hemostasis, inflammation, proliferative phase and remodeling phase. The events of healing are controlled and coordinated by extracellular matrix, hemostatic system, inflammatory cells/mediators and fibrinolytic system. In spite of the prompt response from the host, negative factors such as microbial infection, prolonged inflammation and increased wound exudates hinder the process of healing, leading to non-healing, chronic wounds which affect the socio-economic status of an individual. To overcome the complications associated with wound healing and to minimize the time required for healing, management of wounds is a pre-requisite. Conventional wound care strategies involve the use of antibiotics, anti-inflammatory agents, in combination with debridement procedures to facilitate the efficient wound healing process. These strategies are often associated with adverse effects. In extreme cases, surgical interventions including grafting is the last resort; the complication of graft rejection due to histocompatibility becomes a major drawback of grafting. Alternatively, wound management through herbal medicines involves hemostasis, disinfection, debridement along with maintenance of optimal moisture levels in wound environment to aid normal healing process. Herbal medicines are preferred to conventional medicines, for their easy availability, higher efficacy and reduced adverse reactions. Among herbal medicines, latex and latex derived components are widely used in folk medicinal preparations to promote healing of wounds. In this regard, an investigation was aimed at evaluating the role of plant latex proteases in wound healing. Further, the mechanisms by which latex proteases promote healing of wounds were evaluated to provide scientific basis for ethnopharmacological use of plant latex as wound healing agents. In addition, the toxicity of latex proteases was compared with snake venom proteases. Assessment of toxicity in terms of dose and route of administration is an integral component in providing biochemical validation for pharmacological use of latex as wound healing agent. Overall, in the first chapter, the basic aspects of wound healing, complications associated with wound healing, conventional and herbal wound management strategies have been discussed. Further, the importance of latex in promoting the healing of wounds and the biochemical basis 127

2 has been provided. Finally, the toxicity of latex in general and latex proteases in particular has been outlined to assess the possible adverse effects of latex components. Latex plants selected for the initial screening were Wrightia tinctoria, Thevetia neriifolia (Apocyancaeae), Calotropis gigantea, Pergularia extensa (Asclepiadaceae), Synadenium grantii, Euphorbia tirucalli, Euphorbia antiquorum (Euphorbiaceae), Carica papaya (Caricaceae) and Acrhas zapota (Sapotaceae). Based on the initial experiments and available literature, few plants were selected for wound healing and toxicity studies. Platelet aggregation studies were performed using Euphorbiaceae plant latices. Latex is extensively used to stop bleeding from fresh wounds and the property is attributed to protease present in latex. The mechanisms of few cysteine proteases from latex in exhibiting procoagulant effect are known. Whereas, the mechanisms of other cysteine proteases, including latex serine proteases are not reported. In this regard, second chapter was aimed at determining the activity, nature of proteases, their effect on re-calcification of PPP and citrated whole blood. Latices were processed (dilution, freezing, thawing, centrifugation and dialysis) to get protein rich fraction. Processed latex fractions were abbreviated as W. tinctoria latex proteases (WTLP), S. grantii latex proteases (SGLP), C. gigantea latex proteases (CGLP), P. extensa latex proteases (PELP), E. tirucalli latex proteases (ETLP), E. antiquorum latex proteases (EALP). The latex proteases showed moderate to high specific activities towards casein in the order (WTLP>PELP>SGLP>CGLP>EALP>ETLP). Studies with specific protease inhibitors revealed the presence of cysteine proteases in Asclepiadaceae family latices and serine proteases in Apocyanaceae and Euphorbiaceae plant latices. In all the three plant families, aspartate and metalloproteases are absent. Irrespective of the protease type and plant family, proteases from all the latices showed procoagulant effect towards platelet poor plasma and citrated whole blood. The results were compared with trypsin and papain, which showed similar results. The mechanism of procoagulant action was further evaluated by platelet aggregation studies, using PRP as well as washed platelets. Serine proteases from Apocyanaceae family latices, cysteine proteases from Asclepiadaceae and papain did not induce aggregation in PRP. In contrast, serine proteases from 128

3 Euphorbiaceae and trypsin strongly induced aggregation in PRP. Further, serine proteases from Euphorbiaceae plant latices also induced platelet aggregation in washed platelets. In conclusion, cysteine proteases from plant latices interfere in blood coagulation cascade and serine proteases interfere with platelet function to facilitate the formation of clot. Based on the procoagulant nature of latex proteases, their interference in subsequent phases of wound healing was determined. Further, their effect on wound healing was evaluated. MMP-like activity of latex proteases was determined by substrate gel assay. Latex protein fractions efficiently hydrolyzed gelatin, evident by the clear zones of hydrolysis against the dark blue background. The gelatinolytic activity of latex proteases is indicative of their action towards ECM proteins, similar to matrix metalloproteases, which are mediate vital events of wound healing such as evacuation of devitalized tissue and tissue remodeling. Similar results were obtained with the reference proteases trypsin and papain. Wound healing potential of latex proteases was determined by murine excision wound model. Proteases from plant latices were applied topically on the wound and healing activity was monitored daily. The extent of healing in latex protease treated groups was higher in comparison to the control group in which the wounds were untreated, with significant healing (more than 2 fold) during initial 4 days of topical application (p<0.005). The extent of healing in trypsin and papain was high, with 1.5 fold increase during initial 4 days. In contrast, Neosporin treated group did not show significant healing during initial 4 days, but showed better healing activity after 12 days, compared to saline treated wound. The wound contraction rate in wounds treated with crude latex, dewaxed latex, heat denatured latex proteases, inhibitor pre-treated proteases and wounds treated with inhibitor alone did not show significant healing and were comparable to saline treated group. Collagen content in the granulation tissue is an important biochemical parameter to monitor the extent of healing. Processed granulation tissue from protease treated groups showed higher collagen content compared to the control group, with significant increase during initial 4 days of topical application. Further, collagen content was also high in trypsin and papain treated granulation tissues; similar results were observed in Neosporin treated group. 129

4 On the basis of wound healing potential, ethnopharmacological use and nontoxic nature, WTLP were used to determine the wound healing activity in mice excision wound model. WTLP demonstrated caseinolytic activity in a dose dependent manner with high specific activity, was thermostable and was also active over wide range of ph. Proteolytic activity of WTLP was further studied using gelatin and collagen (type I and type IV) (zymogram method and SDS-PAGE analysis respectively). WTLP hydrolyzed gelatin, evident by three clear bands. Further, WTLP also hydrolyzed all subunits of type I and type IV collagen in concentration dependent response. The healing rate in WTLP treated group was significantly higher (> 2 fold) than control and HD-WTLP groups (49% vs. 18% and 20 %; p<0.01) during initial 3 days respectively. Similarly, the percentage of wound closure in Neosporin treatment group was also significantly higher (38%, p<0.01) compared to control and HD- WTLP. The percentage of wound closure remained high in WTLP alone and Neosporin treatment group throughout the period of investigation. However, no significant difference was observed between WTLP and Neosporin treatment groups demonstrating their comparable effectiveness. This data indicates the role for WTLP in augmenting the wound healing process following topical application. Microscopic observation of the sections from healed tissues on day 6 post-wounding revealed the early granulation tissue containing numerous blood vessels in case of WTLP and Neosporin treated mice; in contrast HD-WTLP treated tissue consisted of poorly laid granulation tissue and lesser number of blood vessels. Further, tissue sections from day 9 granulation tissue demonstrated the recovery of skin structure and tissue continuity, with prominent regeneration of epidermal and dermal layers in mice applied with WTLP and Neosporin. On the contrary, less ordered skin structures were observed in HD-WTLP treated mice with less conspicuous dermal and epidermal layers. To confirm the role of serine proteases from W. tinctoria latex in wound healing process, similar wound healing experiments were carried out following pretreatment of WTLP with serine protease inhibitor, PMSF, which failed to enhance the wound contraction rate compared to control throughout the study period. Further, the contraction rate of wounds treated with PMSF alone was almost comparable to the control group, indicating that PMSF does not interfere with normal healing process. These findings further confirm the direct involvement of WTLP in enhanced wound healing activity. Collagen content of granulation tissues in WTLP and Neosporin 130

5 treated groups was significantly higher than control groups on days 3 and 6 (p< 0.01). Further, on day 9, the collagen content in WTLP group was 1.5 fold (2444 ± 100 vs ± 121 μg/100 mg tissue) higher than the control respectively, demonstrating the wound healing potential of topical application of WTLP. The findings were further supported by variation in catalase and MMPs, which followed normal course of expression throughout the experimental period. Both the enzyme activities were very significantly high in the WTLP treated groups compared with control group on day 3, correlating with the enhanced wound contraction rate. Particularly, the catalase activity in WTLP group was 5.6 fold higher (16.7 ± 1.3 vs. 3 ± 0.3) than control wound and HD-WTLP treated granulation tissue. Similarly the MMP activity in the tissue homogenate on day 3 was significantly higher in WTLP and Neosporin treated granulation tissues compared to other groups. These findings indicate the early onset of inflammatory phase, enabling the appropriate clearance of devitalized tissue and efficient scavenging of free radicals in the wound vicinity, facilitating the progression of healing towards repair phase. However, on days 6 and 9 no significant differences in the activities of these enzymes were observed, indicating the resolution of inflammatory phase, a pre-requisite of the healing process. These results further justify the direct involvement of WTLP in wound healing process and its application in traditional medicine. On the basis of previous studies, in fifth chapter, we compared pharmacological and toxicological properties of serine proteases from W. tinctoria latex extract, cysteine proteases from P. extensa latex extract and metalloproteases from Echis carinatus venom, with special reference to ECM and hemostasis. Plant latex and snake venom serve as efficient weapons of self-defense system of latex bearing plants and venomous snakes respectively. Latex extracts and venom are rich sources of proteolytic enzymes, which are responsible for most of the observed toxicological and pharmacological properties. Proteolytic activity was determined using casein, gelatin, fibrin and fibrinogen. Nature of proteases was determined using specific protease inhibitors. Further, hemorrhagic and myotoxic activities were evaluated in mice. W. tinctoria latex extract (WTLE), P. extensa latex extract (PELE) and E. carinatus venom (ECV) showed comparable proteolytic activity towards various protein substrates. Further, proteases from either source efficiently hydrolyzed fibrinogen and were able to reduce the citrated plasma clotting time. Although PELE 131

6 and ECV exhibited procoagulant activity, both were able to hydrolyze fibrin clot. PELE induced hemorrhage upon intradermal injection of 5 μg protein comparable to 2 μg of ECV. Interestingly, WTLE did not induce hemorrhage, even at higher concentrations. Further, PELE exhibited myotoxic activities following intramuscular injection, evident by distorted skeletal muscle tissue and marked elevation of serum creatine kinase levels (p=0.0002) which is comparable with ECV (p=0.0009). Both hemorrhagic and myotoxic activities of PELE and ECV are completely inhibited by specific cysteine protease inhibitor and metalloprotease inhibitor, respectively. This comparative data indicate that plant latex cysteine proteases exhibited toxic activities similar to snake venom metalloproteases. Therefore, careful assessment of toxicities is pre-requisite in exploiting these proteases for therapeutic applications for treatment and management of chronic inflammatory conditions and clinical conditions associated with abnormal clotting and bleeding. Major outcome of the thesis: Plant latex proteases are procoagulant in nature Latex proteases facilitate the formation of clot in PPP and citrated whole blood Mechanism of clot induction by serine proteases of Euphorbiaceae plant latex is through platelet aggregation (in PRP and washed platelets) Aggregation of platelets by these serine proteases in washed platelets (without fibrinogen supplementation) indicates the action of proteases through PAR on platelets Plant latex proteases exhibit positive effect of wound healing Latex proteases promote healing of fill thickness excision wound which was substantiated by wound contraction rate and collagen content in granulation tissue Significant healing was found in wounds treated with proteases from W. tinctoria, C. gigantea and P. extensa Latex proteases exhibited plasmin-like and MMP-like activities, supporting the enhanced wound contraction rate 132

7 Enhanced healing during initial 3 days of treatment is due to the efficient removal of devitalized tissue by latex proteases to promote healing of wound Ethnopharmacological use of W. tinctoria latex to cure wounds is scientifically validated W. tinctoria latex contains thermostable proteases of serine superfamily W. tinctoria latex proteases (WTLP) hydrolyzed gelatin and collagen indicating the role in debridement, a vital process for wound healing process WTLP facilitated the enhanced wound contraction rate following topical application and the role of proteases was justified by heat inactivation and inhibition studies Wound contraction was supported by enhanced collagen content, increased MMP and catalase activities in WTLP treated granulation tissues compared to control Application of crude latex of W. tinctoria did not cause any toxic symptoms supporting the folk medicinal usage of W. tinctoria latex to cure mouth and gastric ulcers (orally consumed) Plant latex and snake venom proteases exhibit comparable pharmacological and toxicological properties Serine and cysteine proteases from plant latex and snake venom metalloproteases (SVMPs) exhibit comparable pharmacological properties Latex cysteine proteases and SVMPs induce hemorrhagic and myotoxic activities, evidenced by photographic, histological and biochemical parameters Latex serine proteases did not induce hemorrhage ad myotoxicity even at higher concentrations Role of latex cysteine proteases and SVMPs in inducing hemorrhage and myotoxicity are confirmed by inhibition studies with specific protease inhibitors 133

8 Pharmacological or toxicological property of plant latex or snake venom are dependent on the nature of proteases Although toxic when injected intradermally, cysteine proteases from latex promoted wound healing following topical application on wounds The properties of latex cysteine proteases in exhibiting toxicological or pharmacological actions depend on the route of administration 134