Amino Acids

, Volume 45, Issue 1, pp 179–189

Efficacy of l-proline administration on the early responses during cutaneous wound healing in rats

  • Thangavel Ponrasu
  • Sankar Jamuna
  • Arulanandham Mathew
  • Karuppanan Natarajan Madhukumar
  • Moorthy Ganeshkumar
  • Kuttalam Iyappan
  • Lonchin Suguna
Original Article


Proline (Pro) plays a versatile role in cell metabolism and physiology. Pro and hydroxypro are major imino acids present in collagen, an important connective tissue protein, essential for wound healing, which is a primary response to tissue injury. This study explains the role of l-pro on cutaneous wound healing in rats when administered both topically and orally. Open excision wounds were made on the back of rats, and 200 μl (200 mg) of pro was administered topically and orally once daily to the experimental rats until the wounds healed completely. The control wounds were left untreated. Granulation tissues formed were removed after day 4 and 8 of post excision wounding, and biochemical parameters such as total protein, collagen, hexosamine, and uronic acid were estimated. Levels of enzymatic and non-enzymatic antioxidants such as catalase, superoxide dismutase, glutathione peroxidase, ascorbic acid, and reduced glutathione were evaluated along with lipid peroxides in the granulation tissues. Tensile strength and period of epithelialization were also measured. It was observed that the treated wounds healed very fast as evidenced by augmented rates of epithelialization and wound contraction, which was also confirmed by histological examinations. The results strappingly authenticate the beneficial effects of the topical administration of l-proline in the acceleration of wound healing than the oral administration and control.


l-Proline Collagen Excision wound Tensile strength Epithelialization 


  1. Aebi H, Scherz B, Ben-Yoseph Y, Wyss SR (1975) Dissociation of erythrocyte catalase into subunits and their re-association. Experientia 31(4):397–399CrossRefPubMedGoogle Scholar
  2. Aoki M, Komatsu M, Ochiai M, Watanabe F, Igarashi M, Nonomura T, Tomita S, Umechara N, Nakai N, Morishita K (2010) Healing effects of oral administration of L-citrulline or l-ornithine in a rat pressure-ulcer model—Comparison with l-arginine. Jpn Pharm Ther 38(9):807–816Google Scholar
  3. auf dem Keller U, Kumin A, Braun S, Werner S (2006) Reactive oxygen species and their detoxification in healing skin wounds. J Invest Dermatol Symp Proc 11:106–111CrossRefGoogle Scholar
  4. Bae SR, Park C, Choi JC, Poo H, Kim CJ, Sung MH (2010) Effects of ultra high molecular weight poly-gamma-glutamic acid from Bacillus subtilis (chungkookjang) on corneal wound healing. J Microbiol Biotechnol 20(4):803–808PubMedGoogle Scholar
  5. Baeuerle PA, Henkel T (1994) Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 12:141–179CrossRefPubMedGoogle Scholar
  6. Barbul A (2008) Proline precursors to sustain Mammalian collagen synthesis. J Nutr 138(10):2021S–2024SPubMedGoogle Scholar
  7. Beer HD, Longaker MT, Werner S (1997) Reduced expression of PDGF and PDGF receptors during impaired wound healing. J Invest Dermatol 109:132–138CrossRefPubMedGoogle Scholar
  8. Bitter T, Muir HM (1962) A modified uronic acid carbazole reaction. Anal Biochem 4:330–334CrossRefPubMedGoogle Scholar
  9. Blakytny R, Jude E (2006) The molecular biology of chronic wounds delayed healing in diabetes. Diabet Med 23:594–608CrossRefPubMedGoogle Scholar
  10. Chithra P, Sajithlal GB, Chandrakasan G (1998) Influence of Aloe vera on the glycosaminoglycans in the matrix of healing dermal wounds in rats. J Ethnopharmacol 59:179–186CrossRefPubMedGoogle Scholar
  11. Chojkier M, Houglum K, Herruzo JS, Brenner DA (1989) Stimulation of collagen gene expression by ascorbic acid in cultures human fibroblasts: a role for lipid peroxidation. J Biol Chem 264:16957–16962PubMedGoogle Scholar
  12. Daoud AS, Fritz KE, Jarmolych J, Augustyn J, Nawhinney TP (1977) Producton of glycosaminoglycans, collagen, and elastic tissue by aortic medial plants. Adv Exp Med Biol 82:928–933PubMedGoogle Scholar
  13. David Stain H, Keiser HR (1970) Collagen metabolism in granulating wounds. J Surg Res 11:277–283CrossRefGoogle Scholar
  14. Debats IBJG, Wolfs TGAM, Gotoh T, Cleutjens JPM, Peutz-Kootstra CJ, Van der Hulst RRWJ (2009) Role of arginine in superficial wound healing in man. Nitric Oxide 21:175–183CrossRefPubMedGoogle Scholar
  15. Degjim Z, Çelebi N, Sayan H, Babul A, Erdog¡an D, Take G (2002) An investigation on skin wound healing in mice with a taurine-chitosan gel formulation. Amino Acids 22:187–198CrossRefGoogle Scholar
  16. Dioguardi FS (2008) Nutrition and skin. Collagen integrity: a dominant role for amino acids. Clin Dermatol 26(6):636–640CrossRefPubMedGoogle Scholar
  17. Dunphy JE, Udupa KN (1955) Chemical and histochemical sequences in the normal healing of wounds. N Engl J Med 253:847–851CrossRefPubMedGoogle Scholar
  18. Elson LA, Morgan WTJ (1933) A colorimetric method for the determination of glucosamine and chondrosamine. Biochem J 27:1824–1828PubMedGoogle Scholar
  19. Girodon F, Blache D, Monget AL, Lombart M, Brunet-Lecompte P, Arnaud J, Richard MJ, Galan P (1997) Effect of a two-year supplementation with low doses of antioxidant vitamins and/or minerals in elderly subjects on levels of nutrients and antioxidant defense parameters. J Am Coll Nutr 16(4):357–365PubMedGoogle Scholar
  20. Grant ME, Prockop DJ (1972) The biosynthesis of collagen. N Engl J Med 286:194–199CrossRefPubMedGoogle Scholar
  21. Guoyao Wu, Bazer Fuller W, Burghardt Robert C, Johnson Gregory A, Kim Sung Woo, Knabe Darrell A, Li Peng, Li Xilong, McKnight Jason R, Carey Satterfield M, Spencer Thomas E (2011) Proline and hydroxyproline metabolism: implications for animal and human nutrition. Amino Acids 40:1053–1063CrossRefGoogle Scholar
  22. Gupta A, Kumar R, Pal K, Singh V, Banerjee PK, Sawhney RC (2006) Influence of sea buckthorn (Hippophae rhamnoides L.) flavones on dermal wound healing in rats. Mol Cell Biochem 290:193–198CrossRefPubMedGoogle Scholar
  23. Hu CA, Khalil S, Zhaorigetu S, Liu Z, Tyler M, Wan G, Velle D (2008) Human D1-pyrroline-5- carboxylate synthase: function and regulation. Amino Acids 35:665–672CrossRefPubMedGoogle Scholar
  24. Iba Y, Shibata A, Kato M, Masukawa T (2004) Possible involvement of mast cells in collagen remodeling in the late phase of cutaneous wound healing in mice. Int Immunopharmacol 4:187–190CrossRefGoogle Scholar
  25. Kaul S, Sharma SS, Mehta IK (2008) Free radical scavenging potential of l-proline: evidence from in vitro assays. Amino Acids 34:315–320CrossRefPubMedGoogle Scholar
  26. Kershenobich D, Fierro FJ, Rojkind M (1970) The relationship between the free pool of proline and collagen in human liver cirrhosis. J Clin Invest 49:2246–2249CrossRefPubMedGoogle Scholar
  27. Kivirikko KI, Prockop DJ (1967) Enzymatic hydroxylation of proline and lysine in protocollagen. Proc Natl Acd Sci USA 57:782–789CrossRefGoogle Scholar
  28. Krane SM (2008) The importance of proline residues in the structure, stability and susceptibility to proteolytic degradation of collagens. Amino Acids 35:703–710CrossRefPubMedGoogle Scholar
  29. Lambeth JD (2004) NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4:181–189CrossRefPubMedGoogle Scholar
  30. Levenson SM, Rettura G, Barbul A, Seifter E (1980) Citrulline replaces arginine as dietary essential in rats: ornithine does not. Fed Proc 39:726Google Scholar
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenaol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  32. Ma Y, Kleinbeck K, Kao WJ (2011) Extracellular matrix-derived tripeptide proline-glycine-proline inhibits keratinocyte proliferation and migration. Wound Rep Regen 19(6):718–726CrossRefGoogle Scholar
  33. Martin P, Parkhurst SM (2004) Parallels between tissue repair and embryo morphogenesis. Development 131:3021–3034CrossRefPubMedGoogle Scholar
  34. Martin A, Komada MR, Sane DC (2003) Abnormal angiogenesis in diabetes mellitus. Med Res Rev 23:117–145CrossRefPubMedGoogle Scholar
  35. Misra HP, Fridovich I (1972) The generation of superoxide radical during the auto oxidation of hemoglobin. J Biol Chem 247(21):6960–6962PubMedGoogle Scholar
  36. Moron MS, Depierre JW, Mannervik B (1979) Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta 582(1):67–78CrossRefPubMedGoogle Scholar
  37. Nayak BS, Ramdath DD, Marshall JR, Isitor GN, Eversley M, Xue S, Shi J (2010) Wound-healing activity of the skin of the common grape (Vitis Vinifera) variant Cabernet Sauvignon. Phytother Res 24(8):1151–1157PubMedGoogle Scholar
  38. Nayak BS, Kanhai J, Milne DM, Pereira LP, Swanston WH (2011a) Experimental evaluation of ethanolic extract of Carapa guianensis L. leaf for its wound healing activity using three wound models. J Evid Based Complementary Altern Med. doi:10.1093/ecam/nep160
  39. Nayak BS, Ramdath DD, Marshal JR, Isitor G, Xue S, Shi J (2011b) Wound healing properties of the oils of Vitis vinifera and Vaccinium macrocarpon. Phytother Res 25:1201–1208CrossRefGoogle Scholar
  40. Nayak BS, Ramdeen R, Adogwa A, Ramsubhag A, Marshall JR (2012) Wound-healing potential of an ethanol extract of Carica papaya (Caricaceae) seeds. Int Wound J 9(6):650–655CrossRefPubMedGoogle Scholar
  41. Nicosia RF, Belser P, Bonanno E, Diven J (1991) Regulation of angiogenesis in vitro by collagen metabolism. In Vitro Cell Dev Biol 27A(12):961–966CrossRefPubMedGoogle Scholar
  42. Nithya M, Suguna L, Rose C (2003) The effect of nerve growth factor on the early responses during the process of wound healing. Biochim Biophys Acta 1620:25–31CrossRefPubMedGoogle Scholar
  43. Omaye ST, Turnbull JD, Sauberlich HE (1979) Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods Enzymol 62:3–11CrossRefPubMedGoogle Scholar
  44. Paglia DE, Valentine WN (1984) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 7:158–169Google Scholar
  45. Patel GK, Knight AG (2005) Generalized cutaneous necrosis: a complication of low-molecular-weight heparin. Int wound J 2:267–270CrossRefPubMedGoogle Scholar
  46. Paul RG, Tarlton JF, Purslow PP, Sims TJ, Watkins P, Marshall F, Ferguson MJ, Bailey AJ (1997) Biomechanical and biochemical study of a standardized wound healing model. Int J Biochem Cell Biol 29:211–220CrossRefPubMedGoogle Scholar
  47. Piez KA (1963) The amino acid chemistry of some calcified tissues. Ann N Y Acad Sci 109:256–268CrossRefPubMedGoogle Scholar
  48. Ponrasu T, Suguna L (2012) Efficacy of Annona Squamosa on wound healing in streptozotocin induced diabetic rats. Int Wound J 9(6):613–623CrossRefPubMedGoogle Scholar
  49. Porat S, Rousso M, Shosan S (1980) Improvement of the gliding function of flexor tendons by topically applied enriched collagen solution. J Bone Joint Surg Br 62-B(2):208–213PubMedGoogle Scholar
  50. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179(73):588–590CrossRefPubMedGoogle Scholar
  51. Santos MT, Valles J, Aznar J, Vilches J (1980) Determination of plasma malondialdehyde-like material and its clinical application in stroke patients. J Clin Pathol 33:973–976CrossRefPubMedGoogle Scholar
  52. Savunen TJA, Viljanto JA (1992) Prediction of wound tensile strength: an experimental study. Br J Surg 79:401–403CrossRefPubMedGoogle Scholar
  53. Schiller S, Slover GA, Dorfman A (1961) A method for the separation of acid mucopolysaacharides: its application to the isolation of heparin from the skin of rats. J Biol Chem 236:983–987PubMedGoogle Scholar
  54. Shen TE, Strecker HJ (1975) Synthesis of proline and hydroxy-proline in human lung (WI-38) fibroblasts. Biochem J 150:453–461PubMedGoogle Scholar
  55. Shetty S, Udupa S, Udupa L (2008) Evaluation of antioxidant and wound healing effects of alcoholic and aqueous extract of Ocimum sanctum linn in rats. Evid Based Complement Altern Med 5:95–101CrossRefGoogle Scholar
  56. Shi HP, Fishel RS, Efron DT, Williams JZ, Fishel MH, Barbul A (2002) Effect of supplemental ornithine on wound healing. J Surg Res 106:299–302CrossRefPubMedGoogle Scholar
  57. Shi HP, Most D, Erfon DT, White MB, Barbul A (2003) Supplemental l-arginine enhances wound healing in diabetic rats. Wound Rep Regen 11:198–203CrossRefGoogle Scholar
  58. Siegel RC (1976) Collagen cross-linking: synthesis of collagen cross-links in vitro with highly purified lysyl oxidase. J Biol Chem 251(18):5786–5792PubMedGoogle Scholar
  59. Simon AR, Belabed L, Naour GL, Marc J, Capron F, Cynober L, Darquy S (2012) Arginine plus proline supplementation elicits metabolic adaptation that favors wound healing in diabetic rats. Am J Regul Integr Comp Physiol 303:R1053–R1061CrossRefGoogle Scholar
  60. Singer AJ, Clark RAF (1999) Cutaneous wound healing. N Eng J Med 34:738–746Google Scholar
  61. Steiling H, Munz B, Werner S, Brauchle M (1999) Different types of ROS-scavenging enzymes are expressed during cutaneous wound repair. Exp Cell Res 247:484–494CrossRefPubMedGoogle Scholar
  62. Sumitra M, Manikandan P, Gayathri VS, Suguna L (2009) Influence of honey on energy metabolism during wound healing in rats. Scholar Res Exch 2009:1–6CrossRefGoogle Scholar
  63. Tanphaichitr V (1994) Thiamin. In: Shils ME, Olson JA, Shike M (eds) Modern nutrition in health and disease. Lea and Febiger, 8th edn. vol. 1. Philadelphia PA, pp 359–365Google Scholar
  64. Tapiero H, Mathe G, Couvreur P, Tew KD (2002) II. Glutamine and glutamate. Biomed Pharmacother 56:446–457CrossRefPubMedGoogle Scholar
  65. Tekin E, Taneri F, Ersoy E, Oguz M, Eser E, Tekin I, Bozkurt S, Onuk E (2000) The effects of glutamine-enriched feeding on incisional healing in rats. Eur J Plast Surg 23:78–81CrossRefGoogle Scholar
  66. Trent JT, Kirsner RS (2004) Leg ulcers secondary to prolidase deficiency. Adv Skin Wound Care 17:468–472CrossRefPubMedGoogle Scholar
  67. Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35:753–759CrossRefPubMedGoogle Scholar
  68. Vervaart P, Knight KR (1996) Oxidative stress and the cell. Clin Biochem Rev 17:3–13Google Scholar
  69. Vogel HG (1971) Studies Antagonistic effect of aminoacetonitrile and prednisolone on mechanical properties of rat skin. Biochim Biophys Acta 252:580–585CrossRefPubMedGoogle Scholar
  70. Woessner JF (1961) The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 93:440–447CrossRefPubMedGoogle Scholar
  71. Wu G, Flynn NE, Knabe DA (2000) Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets. Am J Physiol Endocrinol Metab 279:E395–E402PubMedGoogle Scholar
  72. Wu G, Bazer FW, Hu J, Johnson GA, Spencer TE (2005) Polyamine synthesis from proline in the developing porcine placenta. Biol Reprod 72:842–850CrossRefPubMedGoogle Scholar
  73. Wu G, Bazer FW, Datta S, Johnson GA, Li P, Satterfield MC, Spencer TE (2008) Proline metabolism in the conceptus: implications for fetal growth and development. Amino Acids 35:691–702CrossRefPubMedGoogle Scholar
  74. Wu G, Bazer FW, Burghardt RC, Johnson GA, Kim SW, Li XL, Satterfield MC, Spencer TE (2010) Impacts of amino acid nutrition on pregnancy outcome in pigs: mechanisms and implications for swine production. J Anim Sci 88:E195–E204CrossRefPubMedGoogle Scholar
  75. Zaidi S, Patel A, Mehta N, Patel K, Takiar R, Saiyed H (2005) Early biochemical alterations in manganese toxicity: ameliorating effects of magnesium nitrate and vitamins. Ind Health 43(4):663–668CrossRefPubMedGoogle Scholar
  76. Zardooz H, Rostamkhani F, Zaringhalam J, Shahrivar FF (2010) Plasma corticosterone, insulin and glucose changes induced by brief exposure to isoflurane, diethyl ether and CO2 in male rats. Physiol Res 59:973–978PubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Thangavel Ponrasu
    • 1
  • Sankar Jamuna
    • 2
  • Arulanandham Mathew
    • 3
  • Karuppanan Natarajan Madhukumar
    • 4
  • Moorthy Ganeshkumar
    • 1
  • Kuttalam Iyappan
    • 5
  • Lonchin Suguna
    • 1
  1. 1.Department of BiochemistryCentral Leather Research Institute (Council of Scientific and Industrial Research)Adyar, ChennaiIndia
  2. 2.Department of BiochemistryUniversity of MadrasChennaiIndia
  3. 3.School of BiotechnologyVellore Institute of TechnologyVelloreIndia
  4. 4.Shirmpex Biotech ServicesSozhinganallur, ChennaiIndia
  5. 5.Chemical Engineering DivisionCentral Leather Research Institute, (Council of Scientific and Industrial Research)Adyar, ChennaiIndia

Personalised recommendations