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Quercetin incorporated collagen matrices for dermal wound healing processes in rat
Biomaterials 24 (2003) 2767–2772
Quercetin incorporated collagen matrices for dermal wound healing processes in rat K. Gomathi, D. Gopinath, M. Rafiuddin Ahmed, R. Jayakumar* Bio-organic Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India Received 5 July 2002; accepted 9 November 2002
Abstract We have been developing antioxidants incorporated collagen matrix as a novel biomaterial for various biomedical applications. In this study we made use of quercetin incorporated collagenous matrix for dermal wound healing in rat. Quercetin incorporated collagen (QIC) treated groups were compared with control and collagen (CS) treated animals. QIC treated animal showed a better healing when compared to control and CS treated wound. The biochemical parameters like hydroxyproline, protein, uronic acid content in the healing wound, revealed that there is an increase in proliferation of cells in quercetin treated groups when compared to CS group and there is considerable increase in wound contraction when compared to CS treated group. In addition we adapted the antioxidant assay using 2,20 -azobisisobutryonitrile (AIBN) to assess in vitro antioxidant activity of QIC. The antioxidant studies indicate QIC quench the radicals more efficiently. These results suggested that quercetin incorporated collagen matrix could be a novel dressing material for dermal wound healing. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Quercetin; Collagen; Wound healing; Antioxidant enzymes
1. Introduction Wound healing is a highly specified process [1]. The healing process starts with the formation of granulation tissue and ends with scar formation. In most of the cases, the complication in wound healing is due to inflammation. Inflammation results in a continuous generation of reactive species, such as the superoxide radical or the non-radical hydrogen peroxide [2]. An imbalance between oxygen species and the antioxidant defence mechanisms of a cell, leading to an excessive production of oxygen metabolites, leads to condition of ‘‘oxidative stress’’. Oxidative stress results in lipid peroxidation, DNA breakage, and enzyme inactivation, including free radical scavenger enzymes [3]. Evidence for the potential role of oxidants in the pathogenesis of many diseases suggests that antioxidants may be of therapeutic use in these conditions [4]. Flavonoids, a group of naturally occurring benzo-g-pyrone deriva-
*Corresponding author. Fax: +91-44-4911589. E-mail address: [email protected] (R. Jayakumar).
tives, have been shown to possess several biological properties including hepatoprotective, antithrombotic, antiinflammatory, and antiviral activities, many of which may be related, partially at least to their antioxidant and free radical scavenging ability [5]. Quercetin is one such flavonoids which delay oxidant injury and cell death by scavenging oxygen radicals [6], protecting against lipid peroxidation [7] and thereby terminating the chain-radical reaction, chelating metal ions, to form inert complexes that cannot take part in the conversion of superoxide radicals and hydrogen peroxide into hydroxyl radicals. Studies of the topical application of compounds with free radical scavenging properties on patients have been shown to significantly improve wound healing and protect tissue from oxidative damage [8] however the mode of application of quercetin is matter of concern, as high concentrated application may result in toxic response in the wound [9]. Hence it is proposed to incorporate quercetin in the collagen matrix to ensure the slow release and better activity of the quercetin. Further collagen will provide a template for new cell proliferation in growth [10]. Biodegradable of collagen material follows a course that is identical to normal wound healing; implanted
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collagen will be degraded through native enzymatic pathways without any toxic response [10]. In this study, we specifically focused on quercetin incorporated collagenous matrix for dermal wound healing in rats.
2. Materials and methods 2.1. Preparation of collagen films Collagen was isolated from Bovine Achilles tendon, using 0.5 m acetic acid 5% NaCl. Purified collagen was placed in glass trays and allowed to air dry under a laminar hood. 2.2. Preparation of quercetin incorporated collagen films Quercetin was purchased from Merck chemicals (India) and used as such. Quercetin was added to the collagen solution at a concentration of 1 mm. The concentration of collagen in the solution was estimated following the method of Neuman and Logan [11]. The solution was placed in glass trays and allowed to air dry under a laminar flow hood protected from light. The collagen was tested for sterility by the direct inoculation method. If no evidence of microbial growth was found, the preparation being examined was considered to pass the test for sterility. 2.3. Micro-shrinkage temperature Micro-shrinkage temperature of collagen films were measured using the method developed by Nutting et al. [12].
2.6. Wound formation Fifteen male albino wistar rats weighing 180–200 g were used. The animals were maintained as per the standard guidelines. Animals were divided in to three groups, each group comprising of 5 rats each. Group I: Control (CR group). Group II: Collagen sheet group (CS group). Group III: Quercetin incorporated collagen group (QIC group). Rats were given a dose of thiopentone sodium 40 mg/ kg body weight intraperitoneally. Using scalpel blade full thickness wounds measuring 2 2 cm were created to the depth of loose subcutaneous tissues. The collagen films were applied on the excised wounds. Animals were sacrificed on day 7 subsequent to wound creation by administering an over dose of anesthetic ether. All the assays were performed on the granulation tissue alone. The following formula was used to calculate the percentage of wound contraction. Wound contraction Wound area day 0 wound area day 7% 100: ¼ Wound area day Hydroxyproline was measured using the method of Neuman and Logan [11]. Uronic acid was determined by Bitter and Miur method [16]. Total protein was assayed using method of Lowry et al. [17]. Superoxidedismutase was assayed using method of Misra and Fridovich [18]. Catalase was assayed using method of Bergmeyer [19].
2.4. Determination of antioxidant potential of quercetin and incorporated quercetin
2.7. Statistical analysis
Antioxidant potential of quercetin and incorporated quercetin was determined by using the method of Ignjatovic et al. [13] and Binyang et al. [14].
Data are expressed as means7SEM, and differences were evaluated by student’s ‘t’ test. A p value of o0.05 was considered statistically significant.
2.5. Antioxidant efficiency of QIC by lipid peroxidation method 3. Results Oleic acid in hexane (0.1 m) was treated with cellulose paper. Radical initiator 2,20 azobisisobutyronitrile (AIBN), was added to this paper, and monitored for the absorbance at l234 for 30 min, and then quercetin incorporated collagen film was placed over the cellulose paper and monitored for the absorbance for another 30 min. For quercetin without collagen, oleic acid solution (0.1 m) was treated with radical initiator and absorbance was monitored for 30 min and then added a small amount of quercetin dissolved in ethanol (0.1 mm) [15].
3.1. Micro-shrinkage temperature Micro-shrinkage temperature was performed to assess the hydrothermal stability of the quercetin incorporated collagen matrix. Shrinkage temperature for normal collagen is 45 C and for quercetin incorporated collagen it is 50 C. The present investigation shows that there is a slight increase in the shrinkage temperature than the control collagen films prepared under similar conditions.
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3.2. Antioxidant potential of quercetin and incorporated quercetin To compare the antioxidant activity of control quercetin and incorporated quercetin in the membrane the UV visible absorption spectroscopic measurements were performed in the pH range of 5–9. Quercetin solution in the acidic medium at pH 5, the absorption peak appears at lmax 370 nm. Further increase in pH to7 the absorption decreases with red shift of lmax 381 and a new absorption band appears at lmax 321. On further increasing the pH to 8 the major peak absorption maximum shows hyperchromic and bathochromic shift. The new absorption band that appeared at pH 7 becomes more intense after attaining the pH 8 (Fig. 1b). The intensity of this peak further increased at pH 9 (Fig. 1b). In the quercetin incorporated collagen films at pH 5–8 the peak shows the absorption maximum at lmax 381 nm with regular hypochromic shift (Fig. 1a). On further increase of pH the absorption maximum attains lmax 395 nm with significant hypochromic and bath-
Fig. 2. Structural transformation of quercetin, AH+: cationic; A: neutral; A: anionic.
ochromic shift. Fig. 2 shows the structural transformations of quercetin in different pH. 3.3. Lipid peroxidation inhibition studies In vitro assay for lipid peroxidation shows that after an addition of radical initiator 2,20 azobisisobutyronitrile (AIBN) the absorbance steadily increases, by adding quercetin incorporated collagen film the increase in the intensity totally held-up. This is due to the scavenging action of quercetin in the collagen film (Fig. 3a). It should be noted that the scavenging action is not hindered by collagen as the quercetin without collagen shows same scavenging action (Fig. 3b). Ethanol treated as control and found the absorption is not altered (Fig. 3c). 3.4. Biochemical investigations
Fig. 1. Antioxidant potential in different pH: (a) Quercetin incorporated collagen matrix, (b) quercetin in solution.
Visual inspection of the wound showed that all the animals had well formed granulation tissue by day 7. Among the experimental groups, animals having quercetin incorporated collagen matrices showed more significant (po0:01) wound contraction than
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animals having only collagen films. The wound contraction was more in collagen group, which was statistically non-significant, than the CR group (Table 1a). A highly significant increase in protein content was observed in the QIC group (po0:001) compared to CS and CR groups (Table 1c). Hydroxy proline content was increased in QIC group when compared to CS and CR
group (Table 1b). The uronic acid content of the CS groups are more than the CR group (Table 1d), whereas decreased uronic acid content was observed in the QIC group. There was a highly significant decrease (po0:01) in the antioxidant enzyme superoxide dismutase (Table 1e) in the QIC group than the CS and CR groups. The rate of catalase activity was slightly greater in the QIC group than the CS and CR groups (Table 1f).
4. Discussion
Fig. 3. Lipid peroxidation inhibition studies: (a) Quercetin incorporated collagen, (b) quercetin in solution, (c) ethanol.
Micro-shrinkage temperature findings shows that quercetin incorporated collagen possess the greater thermal stability than normal collagen. This may be due to the stabilizing action of quercetin on collagen. Earlier researchers reported that catechin treated rat tendon collagen also shows an increase in the thermal shrinkage temperature [20]. This shows that flavonoids have the ability to increase the shrinkage temperature by stabilising the collagen matrix. Antioxidant potential of quercetin and QIC were analysed by spectrophotometric methods by varying pH. Quercetin and incorporated quercetin does not show similar spectral changes. In quercetin the deprotonation of ring B and ring A starts at pH 7 and shows absorption maxima at 381 and 321 nm. On further increase of pH, the deprotonation attains the equilibrium. Earlier data [13] shows that quercetin was reported to have maximum antioxidant activity at pH 7 (due to deprotonation) our results agrees with this observation. However in the case of QIC at pH 5 the spectrum shows absorption maxima at 381 nm. This peak is due to deprotonation of ring B. The absorption maxima at 330 nm indicate the deprotonation of ring A even at pH 5. Quercetin in the collagen matrix shows spectroscopic characteristic similar to O-quinoid like structure [21]. On further increase of pH to 7, the antioxidant potential remains same as that of pH 5. Normal quercetin attains the maximum antioxidant activity at pH 7 with lmax 381 nm. An incorporated quercetin, at pH 5 itself attains the lmax 381 nm
Table 1 Biochemical parameters in rats with full thickness wounds on day 7 S. no.
a. b. c. d. e. f.
Biochemical parameters
Wound contraction (%) Hydroxy proline content of granulation tissue (mg/100 mg tissue) Protein content of granulation tissue (mg/g tissue) Uronic acid content of granulation tissue (mg/100 mg tissue) Superoxide dismutase activity of granulation tissue (units/g tissue) Catalase activity of granulation tissue (units/g tissue)
Treatments groups Control (CS)
Collagen treated (CR)
Quercetin incorporated collagen (QIC)
4374.95 0.780570.066 5670.036 325751 3172.95 1.912570.6
3971.75 0.853370.074 5870.3 398764 1072.48 1.770.6
2071.77 1.83670.366 7671.85 22577 570.5 2.5570.7
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indicating structure A could be stabilized in the collagen matrix (Fig. 2). The antioxidant activity of flavonoids appears to be altered by their microenvironment in the membrane [22]. Time dependence absorbance study clearly indicates the scavenging action of quercetin against peroxy radicals (Fig. 3). Oleic acid impregnated cellulose paper on treatment with radical initiator (AIBN), results in conjugated diene (oxidized form) that absorbs at 234 nm [23] the quercetin incorporated collagen (radical scavenger) film was placed over the impregnated cellulose, quercetin momentarily stops the peroxidation of oleic acid [24,25]. The immediate decrease in absorbance after addition of the incorporated quercetin was due to the reaction of quercetin with free radicals, preserving the unsaturated fatty acid chain from further peroxidation .To confirm the antioxidant activity of the incorporated quercetin, kinetic study was carried out for normal quercetin in ethanol medium as positive control and ethanol was used as an negative control (Figs. 3b and c). Marginally high protein content of treated group than the control group may be due to either cellular infiltration or increase in collagen synthesis. The increase hydroxy proline content (Table 1b) in the QIC group agrees with the increase in protein content, which is predominantly due to enhanced collagen synthesis in the QIC group. Wound contraction is mediated by specialized fibroblasts (myofibroblasts) found with in granulation tissue [25]. These cells are known to contract collagen gel, which were newly synthesized in the site of healing wound. Increased wound contraction in QIC may be due to the enhanced activity of fibroblast in the QIC group [26,27]. The increase in hydroxy proline content indicates that there was an enhanced production of collagen. Cohen et al. found that the increase in collagen synthesis will lead to decrease in hyaluronic acid [28]. This may be the reason for decrease in uronic acid content in QIC group. Earlier reports shows that when there is increase in oxidative stress, the stress itself induces gene expression of SOD by activating the transcription factor NF-kb [29,30]. In the QIC group the decrease in the SOD level is due to the antioxidant activity of quercetin. This is because quercetin scavenges the superoxide radical resulting in decrease in oxidative stress. Quercetin converts the superoxide radical to hydrogen peroxide when there is increase in hydrogen peroxide it stimulates the expression of catalase [31]. This may be the reason for the enhanced activity of catalase in QIC group. Substantial healing of dermal wound with collagen matrix has been reported. We have found that quercetin incorporated collagen matrix heals the wound and scavenges the free radicals effectively than normal collagen matrix.
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Acknowledgements We are grateful to Dr. T. Ramasami, Director, CLRI, Chennai for his kind permission to publish this work. We are thankful to Mr. V. Elango, Department of Biochemistry, CLRI for assisting in the animal experiments. The authors D.G.N. and M.R.A. acknowledge the CSIR for financial support in the form of Senior Research Fellowships.
References [1] Burek AA, Vitello WA, Laughlin RT. Advances in wound healing. Curr Opin Orthop 2000;11:92–8. [2] Pawlak W, Kedziora J, Zolynski K, Kedziora-Kornatowska K, Blaszczyk J, Witkowski P. Free radical’s generation by granulocytes from men during bed rest. J Gravit Physiol 1998;5(1):131–2. [3] Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 1996;313:17–29. [4] Skaper SD, Fabris M, Ferrari V, Carbonare MD, Leon A. Quercetin protects cutaneous tissue associated cell types including sensory neurons from oxidative stress induces by glutathione depletion: cooperative effects of ascorbic acid. Free Radic Biol Med 1997;22(4):669–78. [5] Chen Y, Zheng R, Jia Z, Ju Y. Flavinoids as super oxide scavengers and antioxidants. Free Radic Biol Med 1990;9:19–21. [6] Jovanovic SV, Steenken S, Tosic M, Majanovic A, Simic MG. Flavinoids as antioxidants. J Am Chem Soc 1994;116: 4846–51. [7] Dechameux T, Dubois F, Beauloye C, Wattiaux-de Coninck S, Waniaux R. Effect of various flavinoids on lysosomes subjected to an oxidative stress. Biochem Pharmacol 1992;44:1243–8. [8] Martin A. The use of antioxidants in healing. Dermatol Surg 1996;22:156–60. [9] Gaspar J, Rodriguer A, Zatres A, Silva F, Costa S, Montessori MJ, Monteiro C, Rueff J. On the mechanisms of genotoxicity and metabolism of quercetin. Mutagenesis 1994;9:445–9. [10] Pachence JM. Collagen based device for soft tissue repair. J Biomed Mater Res (Appl Biomater) 1996;33:35–40. [11] Neuman RE, Logan MA. The determination of hydroxy proline. J Biol Chem 1950;184:299–306. [12] Nutting GC, Borasky R. Microscopic method for determining shrinkage temperature of collagen and leather. J Am Leather Chem Assoc 1948;44:831. [13] Ignjatovic LM, Jasmina M, Markovic D, Markovic DA, Baranac JM. The spectroabsorphotometric and voltammetric behavior of malvin in buffered solutions and its antioxidant properties. J Serb Shem Soc 2002;67(1):53–60. [14] Binyang AK, Fumiyo K. Electrochemical behaviour of quercetin and kaempferol in neutral buffer solution. Anal Sci 2001;17: 987–9. [15] Nostro PL, Capuzzi G, Romani A, Mulinacci N. Self assembly and antioxidant properties of octanoyl-6-o-ascorbic acid. Langmuir 2000;16:1744–50. [16] Bitter T, Muir H. A modified uronic acid and carbazole reaction. Anal Biochem 1962;4:330–4. [17] Lowry OH, Roseborough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. [18] Misra HP, Fridovich I. The role of superoxide anion in the auto oxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247:3170–5.
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[19] Bergmeyer HV, Gowehn K, Grassel M. In: Bergmeyer HU, editor. Methods of enzymatic analysis. New York: Academic Press, 1974. p. 438. [20] Rao CN, Rao VH, Sanjeevi R. Influence of bioflavanoids on the metabolism and cross linking of collagen. Indian J Biochem Biophys 1981;18:224. [21] Merodiewa D, Jaswal AK, Cenas N, Dickancaite E, Aguilar JS. Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic Biol Med 1999;26:107–16. [22] Saija A, Scalese M, Lanza M, Marzollo D, Bonina F, Castelli F. Flavanoid is an antioxidant agents: importance of their interactions with biomembranes. Free Radic Biol Med 1995;19(4): 481–6. [23] Polazza P, Krinsky NI. The inhibition of radical initiated peroxidation of microsomal lipids by both alpha-tocopherol and beta-carotene. Free Radic Biol Med 1991;11(4):407–14. [24] Iishikawa T, Takayi H, Kanou M, Kawai S, Ohashi H. Radical capturing reaction of 5,7,30 40 tetra methyl quercetin with the AIBN radical initiator. Biosci Biotechol Biochem 2000;64(1): 173–7.
[25] Kato K, Terao S, Shimamoto N, Hirata M. Studies on scavenges of active oxygen species. 1. synthesis and biological activity of 2-oalkyl ascorbic acids. J Med Chem 1988;31:793. [26] Moulin V, Auger FA, Garel D, Germain L. Role of wound healing myofibroblasts on re-epithelization of human skin. Burns 2000;26:3–12. [27] Chen CC, Mo FF, Lau LF. The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts. J Biol Chem 2001;276(50):47329–37. [28] Cohen IK, Haynes JH. Wound healing and skin physiology. In: Almeyer, editor. London: Springer, 1995. p. 27. [29] Musonda CA, Chipman JK. Quercetin inhibits hydrogen peroxide (H2O2)-induced NF-kB DNA binding activity and DNA damage in Hepg2 cells. Carcinogenesis 1998;19:1583–9. [30] Wang X, Martindale JL, Liu Y, Holbrook NJ. The cellular response to oxidative stress: influences of mitogen activated protein kinase signalling pathways on cell survival. Biochem J 1998;333:291–300. [31] Bae GU, Seo DW, Kwon HK, Lee HY, Hong S, Lee ZW, Ha KS, Lee HW, Han JW. Hydrogen peroxide activates p70sek signaling pathway. J Biol Chem 1999;374(46):32596–602.
Quercetin incorporated collagen matrices for dermal wound healing processes in rat K. Gomathi, D. Gopinath, M. Rafiuddin Ahmed, R. Jayakumar* Bio-organic Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India Received 5 July 2002; accepted 9 November 2002
Abstract We have been developing antioxidants incorporated collagen matrix as a novel biomaterial for various biomedical applications. In this study we made use of quercetin incorporated collagenous matrix for dermal wound healing in rat. Quercetin incorporated collagen (QIC) treated groups were compared with control and collagen (CS) treated animals. QIC treated animal showed a better healing when compared to control and CS treated wound. The biochemical parameters like hydroxyproline, protein, uronic acid content in the healing wound, revealed that there is an increase in proliferation of cells in quercetin treated groups when compared to CS group and there is considerable increase in wound contraction when compared to CS treated group. In addition we adapted the antioxidant assay using 2,20 -azobisisobutryonitrile (AIBN) to assess in vitro antioxidant activity of QIC. The antioxidant studies indicate QIC quench the radicals more efficiently. These results suggested that quercetin incorporated collagen matrix could be a novel dressing material for dermal wound healing. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Quercetin; Collagen; Wound healing; Antioxidant enzymes
1. Introduction Wound healing is a highly specified process [1]. The healing process starts with the formation of granulation tissue and ends with scar formation. In most of the cases, the complication in wound healing is due to inflammation. Inflammation results in a continuous generation of reactive species, such as the superoxide radical or the non-radical hydrogen peroxide [2]. An imbalance between oxygen species and the antioxidant defence mechanisms of a cell, leading to an excessive production of oxygen metabolites, leads to condition of ‘‘oxidative stress’’. Oxidative stress results in lipid peroxidation, DNA breakage, and enzyme inactivation, including free radical scavenger enzymes [3]. Evidence for the potential role of oxidants in the pathogenesis of many diseases suggests that antioxidants may be of therapeutic use in these conditions [4]. Flavonoids, a group of naturally occurring benzo-g-pyrone deriva-
*Corresponding author. Fax: +91-44-4911589. E-mail address: [email protected] (R. Jayakumar).
tives, have been shown to possess several biological properties including hepatoprotective, antithrombotic, antiinflammatory, and antiviral activities, many of which may be related, partially at least to their antioxidant and free radical scavenging ability [5]. Quercetin is one such flavonoids which delay oxidant injury and cell death by scavenging oxygen radicals [6], protecting against lipid peroxidation [7] and thereby terminating the chain-radical reaction, chelating metal ions, to form inert complexes that cannot take part in the conversion of superoxide radicals and hydrogen peroxide into hydroxyl radicals. Studies of the topical application of compounds with free radical scavenging properties on patients have been shown to significantly improve wound healing and protect tissue from oxidative damage [8] however the mode of application of quercetin is matter of concern, as high concentrated application may result in toxic response in the wound [9]. Hence it is proposed to incorporate quercetin in the collagen matrix to ensure the slow release and better activity of the quercetin. Further collagen will provide a template for new cell proliferation in growth [10]. Biodegradable of collagen material follows a course that is identical to normal wound healing; implanted
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collagen will be degraded through native enzymatic pathways without any toxic response [10]. In this study, we specifically focused on quercetin incorporated collagenous matrix for dermal wound healing in rats.
2. Materials and methods 2.1. Preparation of collagen films Collagen was isolated from Bovine Achilles tendon, using 0.5 m acetic acid 5% NaCl. Purified collagen was placed in glass trays and allowed to air dry under a laminar hood. 2.2. Preparation of quercetin incorporated collagen films Quercetin was purchased from Merck chemicals (India) and used as such. Quercetin was added to the collagen solution at a concentration of 1 mm. The concentration of collagen in the solution was estimated following the method of Neuman and Logan [11]. The solution was placed in glass trays and allowed to air dry under a laminar flow hood protected from light. The collagen was tested for sterility by the direct inoculation method. If no evidence of microbial growth was found, the preparation being examined was considered to pass the test for sterility. 2.3. Micro-shrinkage temperature Micro-shrinkage temperature of collagen films were measured using the method developed by Nutting et al. [12].
2.6. Wound formation Fifteen male albino wistar rats weighing 180–200 g were used. The animals were maintained as per the standard guidelines. Animals were divided in to three groups, each group comprising of 5 rats each. Group I: Control (CR group). Group II: Collagen sheet group (CS group). Group III: Quercetin incorporated collagen group (QIC group). Rats were given a dose of thiopentone sodium 40 mg/ kg body weight intraperitoneally. Using scalpel blade full thickness wounds measuring 2 2 cm were created to the depth of loose subcutaneous tissues. The collagen films were applied on the excised wounds. Animals were sacrificed on day 7 subsequent to wound creation by administering an over dose of anesthetic ether. All the assays were performed on the granulation tissue alone. The following formula was used to calculate the percentage of wound contraction. Wound contraction Wound area day 0 wound area day 7% 100: ¼ Wound area day Hydroxyproline was measured using the method of Neuman and Logan [11]. Uronic acid was determined by Bitter and Miur method [16]. Total protein was assayed using method of Lowry et al. [17]. Superoxidedismutase was assayed using method of Misra and Fridovich [18]. Catalase was assayed using method of Bergmeyer [19].
2.4. Determination of antioxidant potential of quercetin and incorporated quercetin
2.7. Statistical analysis
Antioxidant potential of quercetin and incorporated quercetin was determined by using the method of Ignjatovic et al. [13] and Binyang et al. [14].
Data are expressed as means7SEM, and differences were evaluated by student’s ‘t’ test. A p value of o0.05 was considered statistically significant.
2.5. Antioxidant efficiency of QIC by lipid peroxidation method 3. Results Oleic acid in hexane (0.1 m) was treated with cellulose paper. Radical initiator 2,20 azobisisobutyronitrile (AIBN), was added to this paper, and monitored for the absorbance at l234 for 30 min, and then quercetin incorporated collagen film was placed over the cellulose paper and monitored for the absorbance for another 30 min. For quercetin without collagen, oleic acid solution (0.1 m) was treated with radical initiator and absorbance was monitored for 30 min and then added a small amount of quercetin dissolved in ethanol (0.1 mm) [15].
3.1. Micro-shrinkage temperature Micro-shrinkage temperature was performed to assess the hydrothermal stability of the quercetin incorporated collagen matrix. Shrinkage temperature for normal collagen is 45 C and for quercetin incorporated collagen it is 50 C. The present investigation shows that there is a slight increase in the shrinkage temperature than the control collagen films prepared under similar conditions.
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3.2. Antioxidant potential of quercetin and incorporated quercetin To compare the antioxidant activity of control quercetin and incorporated quercetin in the membrane the UV visible absorption spectroscopic measurements were performed in the pH range of 5–9. Quercetin solution in the acidic medium at pH 5, the absorption peak appears at lmax 370 nm. Further increase in pH to7 the absorption decreases with red shift of lmax 381 and a new absorption band appears at lmax 321. On further increasing the pH to 8 the major peak absorption maximum shows hyperchromic and bathochromic shift. The new absorption band that appeared at pH 7 becomes more intense after attaining the pH 8 (Fig. 1b). The intensity of this peak further increased at pH 9 (Fig. 1b). In the quercetin incorporated collagen films at pH 5–8 the peak shows the absorption maximum at lmax 381 nm with regular hypochromic shift (Fig. 1a). On further increase of pH the absorption maximum attains lmax 395 nm with significant hypochromic and bath-
Fig. 2. Structural transformation of quercetin, AH+: cationic; A: neutral; A: anionic.
ochromic shift. Fig. 2 shows the structural transformations of quercetin in different pH. 3.3. Lipid peroxidation inhibition studies In vitro assay for lipid peroxidation shows that after an addition of radical initiator 2,20 azobisisobutyronitrile (AIBN) the absorbance steadily increases, by adding quercetin incorporated collagen film the increase in the intensity totally held-up. This is due to the scavenging action of quercetin in the collagen film (Fig. 3a). It should be noted that the scavenging action is not hindered by collagen as the quercetin without collagen shows same scavenging action (Fig. 3b). Ethanol treated as control and found the absorption is not altered (Fig. 3c). 3.4. Biochemical investigations
Fig. 1. Antioxidant potential in different pH: (a) Quercetin incorporated collagen matrix, (b) quercetin in solution.
Visual inspection of the wound showed that all the animals had well formed granulation tissue by day 7. Among the experimental groups, animals having quercetin incorporated collagen matrices showed more significant (po0:01) wound contraction than
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animals having only collagen films. The wound contraction was more in collagen group, which was statistically non-significant, than the CR group (Table 1a). A highly significant increase in protein content was observed in the QIC group (po0:001) compared to CS and CR groups (Table 1c). Hydroxy proline content was increased in QIC group when compared to CS and CR
group (Table 1b). The uronic acid content of the CS groups are more than the CR group (Table 1d), whereas decreased uronic acid content was observed in the QIC group. There was a highly significant decrease (po0:01) in the antioxidant enzyme superoxide dismutase (Table 1e) in the QIC group than the CS and CR groups. The rate of catalase activity was slightly greater in the QIC group than the CS and CR groups (Table 1f).
4. Discussion
Fig. 3. Lipid peroxidation inhibition studies: (a) Quercetin incorporated collagen, (b) quercetin in solution, (c) ethanol.
Micro-shrinkage temperature findings shows that quercetin incorporated collagen possess the greater thermal stability than normal collagen. This may be due to the stabilizing action of quercetin on collagen. Earlier researchers reported that catechin treated rat tendon collagen also shows an increase in the thermal shrinkage temperature [20]. This shows that flavonoids have the ability to increase the shrinkage temperature by stabilising the collagen matrix. Antioxidant potential of quercetin and QIC were analysed by spectrophotometric methods by varying pH. Quercetin and incorporated quercetin does not show similar spectral changes. In quercetin the deprotonation of ring B and ring A starts at pH 7 and shows absorption maxima at 381 and 321 nm. On further increase of pH, the deprotonation attains the equilibrium. Earlier data [13] shows that quercetin was reported to have maximum antioxidant activity at pH 7 (due to deprotonation) our results agrees with this observation. However in the case of QIC at pH 5 the spectrum shows absorption maxima at 381 nm. This peak is due to deprotonation of ring B. The absorption maxima at 330 nm indicate the deprotonation of ring A even at pH 5. Quercetin in the collagen matrix shows spectroscopic characteristic similar to O-quinoid like structure [21]. On further increase of pH to 7, the antioxidant potential remains same as that of pH 5. Normal quercetin attains the maximum antioxidant activity at pH 7 with lmax 381 nm. An incorporated quercetin, at pH 5 itself attains the lmax 381 nm
Table 1 Biochemical parameters in rats with full thickness wounds on day 7 S. no.
a. b. c. d. e. f.
Biochemical parameters
Wound contraction (%) Hydroxy proline content of granulation tissue (mg/100 mg tissue) Protein content of granulation tissue (mg/g tissue) Uronic acid content of granulation tissue (mg/100 mg tissue) Superoxide dismutase activity of granulation tissue (units/g tissue) Catalase activity of granulation tissue (units/g tissue)
Treatments groups Control (CS)
Collagen treated (CR)
Quercetin incorporated collagen (QIC)
4374.95 0.780570.066 5670.036 325751 3172.95 1.912570.6
3971.75 0.853370.074 5870.3 398764 1072.48 1.770.6
2071.77 1.83670.366 7671.85 22577 570.5 2.5570.7
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indicating structure A could be stabilized in the collagen matrix (Fig. 2). The antioxidant activity of flavonoids appears to be altered by their microenvironment in the membrane [22]. Time dependence absorbance study clearly indicates the scavenging action of quercetin against peroxy radicals (Fig. 3). Oleic acid impregnated cellulose paper on treatment with radical initiator (AIBN), results in conjugated diene (oxidized form) that absorbs at 234 nm [23] the quercetin incorporated collagen (radical scavenger) film was placed over the impregnated cellulose, quercetin momentarily stops the peroxidation of oleic acid [24,25]. The immediate decrease in absorbance after addition of the incorporated quercetin was due to the reaction of quercetin with free radicals, preserving the unsaturated fatty acid chain from further peroxidation .To confirm the antioxidant activity of the incorporated quercetin, kinetic study was carried out for normal quercetin in ethanol medium as positive control and ethanol was used as an negative control (Figs. 3b and c). Marginally high protein content of treated group than the control group may be due to either cellular infiltration or increase in collagen synthesis. The increase hydroxy proline content (Table 1b) in the QIC group agrees with the increase in protein content, which is predominantly due to enhanced collagen synthesis in the QIC group. Wound contraction is mediated by specialized fibroblasts (myofibroblasts) found with in granulation tissue [25]. These cells are known to contract collagen gel, which were newly synthesized in the site of healing wound. Increased wound contraction in QIC may be due to the enhanced activity of fibroblast in the QIC group [26,27]. The increase in hydroxy proline content indicates that there was an enhanced production of collagen. Cohen et al. found that the increase in collagen synthesis will lead to decrease in hyaluronic acid [28]. This may be the reason for decrease in uronic acid content in QIC group. Earlier reports shows that when there is increase in oxidative stress, the stress itself induces gene expression of SOD by activating the transcription factor NF-kb [29,30]. In the QIC group the decrease in the SOD level is due to the antioxidant activity of quercetin. This is because quercetin scavenges the superoxide radical resulting in decrease in oxidative stress. Quercetin converts the superoxide radical to hydrogen peroxide when there is increase in hydrogen peroxide it stimulates the expression of catalase [31]. This may be the reason for the enhanced activity of catalase in QIC group. Substantial healing of dermal wound with collagen matrix has been reported. We have found that quercetin incorporated collagen matrix heals the wound and scavenges the free radicals effectively than normal collagen matrix.
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Acknowledgements We are grateful to Dr. T. Ramasami, Director, CLRI, Chennai for his kind permission to publish this work. We are thankful to Mr. V. Elango, Department of Biochemistry, CLRI for assisting in the animal experiments. The authors D.G.N. and M.R.A. acknowledge the CSIR for financial support in the form of Senior Research Fellowships.
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