;;. Indian Journal of Experimental Biology Vol. 43, March, 2005, pp. 254-258 In vivo effect of whole grain flour of finger millet (Eleusine coracana) and kodo millet (Paspalum scrobiculatum) on rat dermal wound healing 1 2 Pras hant S Hegde , Anitha B & T S Chandra'* 'Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India 2Department of Biochemi stry, Central Leather Research Insititute, Adyar, Chennai 600 020, India Received 4 June 2004; revised 25 No vember 2004 Influence of fin ger millet and kodo millet on rat dermal wound healing was assessed by making a 4 cm2 (2 x 2 em) ex­ cision wound on the shaven back of rats under ether anesthesia. Fin ger millet or kodo millet fl our (3 00 mg) as aqueous paste was applied topi call y once dail y for 16 days. The granulati on tissue formed on day 4, 8 and 12 was used to estimate some biochemi cal parameters like protein , DNA, collagen and lipid peroxides. There was significant in crease in protein and coll a­ gen contents and decrease in lipid peroxides. Biophysical parameters like rate of contraction and number of days for epithe­ liali zation were also studied. Rate of contraction was 88-90% in kodo millet and fin ger millet treated rats in comparison to 75% in untreated rats. The number of days for complete closure of wounds was lower for finger millet (13 days) and kodo millet (14 days) treated rats in compari son to untreated (1 6 days) rats. The results implicate a possible therapeutical role fo r fin ger millet and kodo millet in accelerating the process of wound healing. Keywords: Antiox idant, Collagen, Eleusine coracana, Finger millet, Free radicals, Kodo millet, Paspalum scrobiculatum, Wound healing, IPC Code: Int C l7 A6 1B Wound healing and ti ssue repair are complex proc­ fluencing phases such as inflammation, fibroplas ia, esses that involve a seri es of biochemical and cellular collagen synthesis and maturati on, and wound con­ reacti ons beginning with inflammati on and followed traction. These effects may be due to the reported hy­ by repair and remodelling of the injured ti ssue'. The poglycemic effects of the Aloe gd. formati on of crosslinks and development of tensile Similar studies on wounds treated with curcumin strength is mainly contributed by type I collagen in showed earlier re-epithelializati on, improved neovas­ th e wound matrix . However, type III collagen is the cularization, increased migrati on of various cell s in­ 2 earli est form of coll agen detected in the wound . It cluding dermal myofibrobl asts, fibroblasts, and fo rms scaffolding in the early wound healing process macrophages into the wound bed, and a higher coll a­ 5 and subsequently the accumulation of type III colla­ gen content . Plant products have been shown to have gen is overcome by the rapid accumulation of type I a good therapeutic potential as anti-inflammatory collagen (which provides mechanical integrity), until agents and promoters of wound healing, due to th e 3 the normal ratio of type I to type Ill is re-established . presence of active alkaloids, terpenes and 6 8 The earl y type III coll agen has, in the healing process, flavonoids - . important functions such as establishing initial wound Finger millet and kodo millet are rich source of structure, guiding inflammatory cells and fibroblasts polyphenols and exhibit si gni ficant antioxidant activ­ 9 10 into the wound site, providing a matrix for re­ ity · • Owing to known role of antioxidants in establi shment of blood supply. It also acts to regulate wound healing and reports that phenolics like curcu­ 5 coll agen fibre diameter and organi zati on. min exhibit anti-inflammatory acti vity , in the present Earli er studies on positi ve influence of Aloe vera , a study the finger millet and kodo mi llet fl our were tropi cal cactus, on the healing of wounds in diabetic analyzed for wound healing properties on topi cal ap­ rats indicated that it may enhance the process by in- plication. Materials and Methods ' Correspondent author Telepho ne: 9 1-44-22578245; Fax : 9 1-44-22570305 Hydroxyproline, chloramine T, thiobarbituric acid, Email : chandra@ iitm .ac.i n; [email protected] 1,1,3,3, tetra methoxy propane, bovine serum albumin HEGDE et al.: EFFECT OF MILLET FLOUR ON DERMAL WOUND HEALING 255 and calf thymus DNA were obtained from Sigma­ Rate of contraction- The excision wounds were Aldrich Chemical Company, St Louis, USA. All other traced on a paper having a mm scale and the changes chemicals were of analytical grade commercially in wound size were measured planimetrically. Reduc­ available. tion in wound (reflecting the rate of wound contrac­ Experimental set up - A standard full thickness tion) was calculated as per cent of original wound size 2 2 wound (4 cm ) was created on the shaven back of (4 cm ) . male albino Wistar rats, 50-200 g, 3 months old, ob­ Period of epithelialization- The wounds were also tained from Tamilnadu Animal and Veterinary Sci­ inspected for complete epithelialization as indicated ence University, India under light anesthesia. Un­ by shedding of eschar without any raw wound left treated rats served as control (Group I); Group II rats behind. Days required for this sloughing was taken as were treated by topical application of 300 mg of the period of epithelialization. aqueous paste of finger millet (FM) C0-13 flour and Statistical analysis- All experiments were car­ Group III rats were treated by topical application of ried out with 6 rats in each group and results are ex­ 300 mg of aqueous paste of kodo millet (KM) Yam­ pressed as mean ± SD. Statistical analysis was done ban 1 flour. In each group rats were taken in replica­ using Single way Anova and Newman-Keuls Multiple tions (n=6). The granulation tissues formed were re­ Comparison Test. P value of less than 0.05 was con­ moved on day 4, 8 and 12 after wound creation and sidered significant. used to estimate the biochemical and biophysical parameters. Results and Discussion Extraction of total protein and DNA- The wet There was a significant increase in protein and granulation ti ssue samples were extracted in Trichlo­ 11 collagen contents on the day 4 and 8 of healing in FM roacetic acid (5 %) . TCA (5 %; 10 ml) was added to and KM treated rats in comparison to control and a the ti ssue samples (100 mg wet weight) and kept at decrease by day 12 (Table 1). The levels of collagen 90° C for 30 min in a water bath to extract protein and and its types contribute significantly to the wound DNA. The solution was centrifuged at 5000 rpm for healing process. Collagen is the main protein in the I 0 minutes and the supernatant was used for the extracellular matrix and plays an important role in the 16 estimations. healing process even after the wound is closed . Estimation of protein- Total protein was deter­ Though, the major function of collagen is to provide mined by usin g Bovine Serum Albumin (BSA) as strength and integrity to the wound, it also plays a role 12 standard . The total protein contents was expressed in other functions such as homeostasis, re­ as mg/100 mg wet tissue. epithelialization, cell-cell and cell-matrix interac­ 17 20 Estimation of DNA- Tissue DNA content was tions - . determined by using calf thymus DNA as the stan­ Increase in total protein contents correlated well dard1 3. DNA content of tissue was expressed as with increased collagen content in the granulation mg/1 00 mg wet weight. tissue in the millets treated group (Table l).The mil­ Estimation of total collagen- The total collagen lets could have stimulated collagen synthesis or in­ content of the granulation tissue was determined by creased the proliferation of fibroblasts synthesizing the estimation of hydroxyproline. The samples were collagen or both. There was also an increase in DNA washed with physiological saline, cut into small content on the day 4 and 8 of healing in FM and KM pieces, defatted with chloroform: methanol (2: I v/v) treated rats and a decrease on day 12 (Table I). and lyophilized. Lyophilized tissue (5 mg) was There was a significant reduction in lipid peroxides digested with 6N HCl for 24 hr at 110° C. The amount on the day 4, 8, and 12 of healing in FM and KM of hydroxyproline was estimated as per Stegmann and treated rats in comparison to control (Table 1) . The 14 Stalder . Collagen content of tissue was expressed as rate of wound contraction on the day 4, 8 and 12 was mg/100 mg wet weight. also higher in FM and KM treated rats (Table 1). The Estimation of lipid peroxides- Lipid peroxides (as number of days for complete closure of wound de­ malondialdehyde) of the tissue extracts were deter­ creased significantly in the treated rats (Table 1). 15 mined as per Nagababu and Lakshmaiah . The lipid The antioxidant and anti-inflammatory activities in peroxide content was expressed as n mole of extracts of leaves of Hyptis suaveoleni 1 and macro­ MDA/100 mg wet weight. fungus Phellinus rimosus (Berk) Pilat22 and 256 INDIAN J EXP BIOL, MARCH 2005 Tabl e I - Total protein , col lagen, DNA, lipid peroxides content, rate of wo und contraction of granulation tissues and period of ep i­ thelia lization in normal and experimental rats [Values are mean ±SO of six animals in each group] Biochemical parameters Group I Group II Group II I Protein a 3.57 ± 0.12 5.06 ± 0.11 u* 4.92 ± 0.09"' b 7.07 ± 0.08 8.63±0.!0"* 8.45 ± 0.08"* c 6.02 ± 0.09 7.70 ± 0.09"$ 7.59 ± 0.07"$ DNA a 1.55 ± 0.07 1.89 ± 0.07 1.9 1 ± 0 08 b 5.39 ± 0.10 5.98 ± 0.08 5.75±0.16 c 4.26 ± 0.1 3 4.93±0.12 4.66 ± 0.12 Collagen a 2.34 ± 0.