Advertisement

Biologia

pp 1–11 | Cite as

Hemostatically potent small molecular weight serine protease from Maclura spinosa (Roxb. ex Willd.) accelerates healing of subcutaneous dermal wounds in Swiss albino mice

  • Venkatesh Bommalapura Kulkarni
  • Raghu Ram Achar
  • Maheshwari Mahadevappa
  • Dinesh Sosalagere Manjegowda
  • Priya Babu Shubha
  • Sharanappa Puttappa
  • Shivananju Nanjunda SwamyEmail author
Original Article
  • 8 Downloads

Abstract

Latex of Maclura spinosa has been used by Kollamalayali tribal community of Western Ghats for curdling of milk. In our previous study we have reported the presence of multiple proteases in the latex, among which the serine protease possesses potent role in hemostasis. With a view to further characterize these proteases the current study was taken up. Maclura spinosa latex contains a serine protease which is resolved using molecular size exclusion column chromatography and an anion exchanger resin in a consecutive manner. The specific activity of the enzyme Maclura spinosa latex protease (MSLP) was found to be 56.15 units/mg and recovery to be 2.68% with a fold purity of 0.41. Being a typical serine protease, MSLP is significantly inhibited by PMSF up to 72.97%. The optimum temperature and pH for the enzyme were found to be 50 °C and 8 respectively. Excision wound healing assay in Swiss albino mice using MSLP, showed accelerated wound closure up to 89.35 ± 1.209% compared to 91.938 ± 1.649% shown by positive control. Further PMSF treated MSLP sample did not show considerable wound healing which confirms exclusive involvement of serine proteases. Examination of biochemical markers viz, hydroxy proline content in healing tissue and catalase activity of fatty tissue also ascertain the potential of MSLP in wound healing. Histopathological studies of healing tissue provide confirmatory evidence with dense collagenation of tissue and fibroblast proliferation rendered by MSLP in the respective treated subjects.

Keywords

Maclura spinosa Maclura spinosa latex protease Serine protease Hemostasis Swiss albino mice Wound healing 

Abbreviations

MSL

Maclura spinosa latex

MSLP

Maclura spinosa latex protease

PMSF

phenyl methyl sulphonyl fluoride

IAA

iodo acetic acid

DEAE-sephadex

diethyl-aminoethyl- sephadex

p-DMAB

para-dimethyl amino benzaldehyde

TRIS

trisaminomethane

MM plot

Michealis–Menten plot

LB plot

Lineweaver–Burk plot

Vmax

Maximal Velocity

Km

Michaelis Constant

SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

Notes

Acknowledgements

The authors wish to thank the management of JSS Mahavidyapeetha, JSS Science and Technology University and JSS Research Foundation, Mysuru for their continued support in this endeavor. The authors acknowledge, TEQIP-III (Technical Education Quality Improvement Programme), a World-Bank-assisted project under the aegis of the Government of India for their financial support.

Compliance with ethical standards

Conflict of interest

The authors report no conflicts of interest associated with this manuscript.

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.

References

  1. Aderounmu A, Omonisi A, Akingbasote J, Makanjuola M, Bejide R, Orafidiya L, Adelusola K (2013) Wound-healing and potential anti-Keloidal properties of the latex of Calotropis Procera (Aiton) Asclepiadaceae in rabbits. Afr J Tradit Complement Altern Med 10:574–579.  https://doi.org/10.4314/ajtcam.v10i3.28 Google Scholar
  2. Aebi H (1974) Catalase. Pp. 673–684. Methods of enzymatic analysis, Elsevier.  https://doi.org/10.1016/B978-0-12-091302-2.50032-3
  3. Angely CJ, Coppola DM (2010) How does long-term odor deprivation affect the olfactory capacity of adult mice? Behav Brain Funct BBF 6:26.  https://doi.org/10.1186/1744-9081-6-26 CrossRefGoogle Scholar
  4. Bonnans C, Chou J, Werb Z (2014) Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15:786–801.  https://doi.org/10.1038/nrm3904 CrossRefGoogle Scholar
  5. Bowden LG, Byrne HM, Maini PK, Moulton DE (2016) A morphoelastic model for dermal wound closure. Biomech Model Mechanobiol 15:663–681.  https://doi.org/10.1007/s10237-015-0716-7 CrossRefGoogle Scholar
  6. Bryan N., Ahswin H., Smart N., Bayon Y., Wohlert S. & Hunt J.A. 2012. Reactive oxygen species (ROS)--a family of fate deciding molecules pivotal in constructive inflammation and wound healing. Eur. Cell. Mater. 24: 249–265. Doi:  https://doi.org/10.22203/eCM.v024a18
  7. Chakraborti S, Dhalla NS (2017) Proteases in physiology and pathology. Springer, 619 pp.  https://doi.org/10.1007/978-981-10-2513-6
  8. De Wet H, Nciki S, van Vuuren SF (2013) Medicinal plants used for the treatment of various skin disorders by a rural community in northern Maputaland, South Africa. J Ethnobiol Ethnomedicine 9:51.  https://doi.org/10.1186/1746-4269-9-51 CrossRefGoogle Scholar
  9. Eagle H (1937) Studies in blood coagulation: V. the coagulation of blood by proteolytic enzymes (TRYPSIN, PAPAIN). J. Gen. Physiol. 20: 543–560. Doi:  https://doi.org/10.1085/jgp.20.4.543.  https://doi.org/10.1085/jgp.20.4.543
  10. Eming S.A., Martin P. & Tomic-Canic M. 2014. Wound repair and regeneration: mechanisms, signaling, and translation. Sci. Transl. Med. 6: 265sr6. doi:  https://doi.org/10.1126/scitranslmed.3009337
  11. Frank S. & Kämpfer H. 2003. Excisional wound healing: an experimental approach. pp. 003–015. Wound healing, Humana Press, New Jersey. doi:  https://doi.org/10.1385/1-59259-332-1:003
  12. Geetha S, Lakshmi G, Ranjithakani P (1996) An ethnic method of milk curdling using plants. Anc Sci Life 16:60–61Google Scholar
  13. Gillitzer R, Goebeler M (2001) Chemokines in cutaneous wound healing. J Leukoc Biol 69:513–521Google Scholar
  14. Guo S, DiPietro LA (2010) Factors affecting wound healing. J Dent Res 89:219–229CrossRefGoogle Scholar
  15. Hartley BS (1960) Proteolytic enzymes. Annu Rev Biochem 29:45–72.  https://doi.org/10.1146/annurev.bi.29.070160.000401 CrossRefGoogle Scholar
  16. Henry RL, Steiman RH (1968) Mechanisms of hemostasis. Microvasc Res 1:68–82.  https://doi.org/10.1016/0026-2862(68)90007-1 CrossRefGoogle Scholar
  17. Hershko A, Ciechanover A (1992) The ubiquitin system for protein degradation. Annu Rev Biochem 61:761–807.  https://doi.org/10.1146/annurev.bi.61.070192.003553 CrossRefGoogle Scholar
  18. Khan AR, James MN (1998) Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci Publ Protein Soc 7:815–836.  https://doi.org/10.1002/pro.5560070401 CrossRefGoogle Scholar
  19. Konno K (2011) Plant latex and other exudates as plant defense systems: roles of various defense chemicals and proteins contained therein. Phytochemistry. 72:1510–1530.  https://doi.org/10.1016/j.phytochem.2011.02.016 CrossRefGoogle Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680–685.  https://doi.org/10.1038/227680a0 CrossRefGoogle Scholar
  21. Landén NX, Li D, Ståhle M (2016) Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci 73:3861–3885.  https://doi.org/10.1007/s00018-016-2268-0 CrossRefGoogle Scholar
  22. Lehninger AL (1950) Role of metal ions in enzyme systems. Physiol Rev 30:393–429.  https://doi.org/10.1152/physrev.1950.30.3.393 CrossRefGoogle Scholar
  23. Li J, Chen J, Kirsner R (2007) Pathophysiology of acute wound healing. Clin Dermatol 25:9–18.  https://doi.org/10.1016/j.clindermatol.2006.09.007 CrossRefGoogle Scholar
  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  25. McCarty SM, Cochrane CA, Clegg PD, Percival SL (2012) The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing. Wound Repair Regen 20:125–136.  https://doi.org/10.1111/j.1524-475X.2012.00763.x CrossRefGoogle Scholar
  26. McDonald JK (1985) An overview of protease specificity and catalytic mechanisms: aspects related to nomenclature and classification. Histochem J 17:773–785.  https://doi.org/10.1007/BF01003313 CrossRefGoogle Scholar
  27. Murata J, Satake M, Suzuki T (1963) Studies on snake venom. XII Distribution of proteinase activities among Japanese and Formosan snake venoms. J Biochem 53:431–443CrossRefGoogle Scholar
  28. Muruganandam S., Kamaraj E., Kadirvelmurugan V. & Ravikumar S. 2016. Ethnobotany_of_Gingee_Hills,_Villupuram_District,_ Tamil_Nadu,_India. 4Google Scholar
  29. Nayak S, Nalabothu P, Sandiford S, Bhogadi V, Adogwa A (2006) Evaluation of wound healing activity of Allamanda cathartica. L. and Laurus nobilis. L. Extracts on rats. BMC complement. Altern. Med 6:12.  https://doi.org/10.1186/1472-6882-6-12 Google Scholar
  30. Neuman RE, Logan MA (1950) The determination of Hydroxyproline. J Biol Chem 184:299–306Google Scholar
  31. Niamke S, Kouame LP, Kouadio JP, Koffi D, Faulet BM, Dabonne S (2006) Effect of some chemicals on the accuracy of protein estimation by the Lowry method. Biokemistri. 17.  https://doi.org/10.4314/biokem.v17i2.32591
  32. Pastar I, Stojadinovic O, Yin NC, Ramirez H, Nusbaum AG, Sawaya A, Patel SB, Khalid L, Isseroff RR, Tomic-Canic M (2014) Epithelialization in wound healing: a comprehensive review. Adv Wound Care 3:445–464.  https://doi.org/10.1089/wound.2013.0473 CrossRefGoogle Scholar
  33. Rajesh R, Shivaprasad HV, Gowda CDR, Nataraju A, Dhananjaya BL, Vishwanath BS (2007) Comparative study on plant latex proteases and their involvement in hemostasis: a special emphasis on clot inducing and dissolving properties. Planta Med 73:1061–1067.  https://doi.org/10.1055/s-2007-981575 CrossRefGoogle Scholar
  34. Reinke JM, Sorg H (2012) Wound repair and regeneration. Eur Surg Res Eur Chir Forsch Rech Chir Eur 49:35–43.  https://doi.org/10.1159/000339613 Google Scholar
  35. Rubinstein HM (1957) Coagulant action of proteolytic enzymes. Nature. 180:1202–1203.  https://doi.org/10.1038/1801202a0 CrossRefGoogle Scholar
  36. Saha SK, Moriya M, Ohinata H, Kuroshima A (1994) Lipid interference with Fluorometric assay of DNA in adipose tissues under various conditions. Jpn J Physiol 44:421–431.  https://doi.org/10.2170/jjphysiol.44.421 CrossRefGoogle Scholar
  37. Shah J, Mohd Y, Omar E, Pai DR, Sood S (2012) Cellular events and biomarkers of wound healing. Indian J Plast Surg Off Publ Assoc Plast Surg India 45:220–228.  https://doi.org/10.4103/0970-0358.101282 Google Scholar
  38. Sumithira G, Ashma A, Rajamathanky H, Kavya V, Riyas KM (2017) A review on ethanobotanical uses and pharmacology of plecospermum spinosum 6:5Google Scholar
  39. Thakur R, Jain N, Pathak R, Sandhu SS (2011) Practices in Wound Healing Studies of Plants. Evid-Based Complement Altern Med ECAM:2011.  https://doi.org/10.1155/2011/438056
  40. Travis BL, Ferguson JH (1951) Proteolytic enzymes and platelets in relation to blood coagulation 1. J Clin Invest 30:112–123.  https://doi.org/10.1172/JCI102411 CrossRefGoogle Scholar
  41. Uday P, Achar RR, Bhat PR, Rinimol RV, Bindu J, Nafeesa Z, Swamy NS (2015) Laticiferous plant proteases in wound care. Int J Pharm Pharm Sci 7:44–49Google Scholar
  42. Upadhyay R (2011) Plant latex: a natural source of pharmaceuticals and pesticides. Int J Green Pharm 5:169.  https://doi.org/10.4103/0973-8258.91222 CrossRefGoogle Scholar
  43. van Hinsbergh VWM, Engelse MA, Quax PHA (2006) Pericellular proteases in angiogenesis and vasculogenesis. Arterioscler Thromb Vasc Biol 26:716–728.  https://doi.org/10.1161/01.ATV.0000209518.58252.17 CrossRefGoogle Scholar
  44. Venkatesh B.K., Achar R.R., Sharanappa P., Priya B.S. & Swamy S.N. 2015. Synergistic caseinolytic activity and differential fibrinogenolytic action of multiple proteases of Maclura spinosa (Roxb. ex Willd.) latex. Pharmacogn Mag 11: S457–S461. doi:  https://doi.org/10.4103/0973-1296.168987
  45. Wallace HA, Bhimji SS (2018) Wound healing phases. StatPearls Publishing, Treasure Island (FL), StatPearlsGoogle Scholar
  46. Walsh PN, Ahmad SS (2002) Proteases in blood clotting. Essays Biochem 38:95–111.  https://doi.org/10.1042/bse0380095 CrossRefGoogle Scholar
  47. Wilgus TA, Roy S, McDaniel JC (2013) Neutrophils and wound repair: positive actions and negative reactions. Adv. Wound Care. 2:379–388.  https://doi.org/10.1089/wound.2012.0383 CrossRefGoogle Scholar
  48. Xue M, Le NT, Jackson CJ (2006) Targeting matrix metalloproteases to improve cutaneous wound healing. Expert Opin Ther Targets 10:143–155.  https://doi.org/10.1517/14728222.10.1.143 CrossRefGoogle Scholar
  49. Yariswamy M, Shivaprasad HV, Joshi V, Urs ANN, Nataraju A, Vishwanath BS (2013) Topical application of serine proteases from Wrightia tinctoria R. Br. (Apocyanaceae) latex augments healing of experimentally induced excision wound in mice. J. Ethnopharmacol 7.  https://doi.org/10.1016/j.jep.2013.06.056

Copyright information

© Institute of Molecular Biology, Slovak Academy of Sciences 2019

Authors and Affiliations

  • Venkatesh Bommalapura Kulkarni
    • 1
    • 2
  • Raghu Ram Achar
    • 3
  • Maheshwari Mahadevappa
    • 4
  • Dinesh Sosalagere Manjegowda
    • 5
  • Priya Babu Shubha
    • 6
  • Sharanappa Puttappa
    • 7
  • Shivananju Nanjunda Swamy
    • 1
    Email author
  1. 1.Department of BiotechnologyJSS Science and Technology UniversityMysuruIndia
  2. 2.JSS Research FoundationJSS Technical Institutions CampusMysuruIndia
  3. 3.Biochemistry Division, Faculty of Life SciencesJSS Academy of Higher Education and ResearchMysuruIndia
  4. 4.Department of Pathology, JSS Medical CollegeJSS Academy of Higher Education and ResearchMysuruIndia
  5. 5.K S Hegde Medical AcademyNitte UniversityMangloreIndia
  6. 6.Department of Studies in ChemistryUniversity of MysoreMysuruIndia
  7. 7.Department of Studies in BiosciencesHassanIndia

Personalised recommendations