SUMMARY.This study was designed to evaluate the efficacy of micronized flavonoid fraction (Daflon) in an experimental burn injury model. Thirty Wistar-Maller rats underwent burn injury by the standard Walker-Mason method. The rats were randomly divided into three groups. The first group received no treatment (control), the second group received Daflon 60 mg/kg/day orally, and the third group received Daflon 60 mg/kg/day two times topically. The surface area measurements and histopathological evaluations on days 4 and 12 of the locally and systemically treated groups were compared with each other and with the control group. The study showed that both systemic and local use of Daflon improved burn healing.
The micronized purified flavonoid fraction Daflon is composed of 90% diosmin and 10% hesperidin. It mainly increases vascular tone and protects vascular endothelium. A considerable number of clinical and experimental studies have demonstrated that the drug is effective on both diseased and intact vasculature, increasing vascular tone and decreasing oedema.1 Recent studies have disclosed that it has anti-inflammatory effects. These properties of the drug suggest that it may also be beneficial in wound healing.2 The objective of the present study, which concerns a standard thermal injury model, is to determine the beneficial effects of flavonoids possessing an anti-oedema effect on wound healing. This study is the first to use Daflon in experimental burn injury models with respect to med-line scanning covering the period between the years 1970 and 2000.
Thirty Wistar-Maller rats weighing 200-250 mg were used. Each rat was anaesthesized with sodium phenobarbital 50 mg/kg intraperitoneally. The burn injury was inflicted by the standard method described by Walker and Mason.3 The back region of the rat was shaved using a razorblade. The rat was placed in a plastic mould with a window exposing 3 cm of body surface. A full-thickness injury was inflicted on the back skin of the rat by immersing the mould containing the animal for 10 sec in 100 °C water.
The rats were divided into three groups:
Group 1: This was the control group, receiving no treatment.
Group 2: This group received micronized purified flavonoid fraction at a dose of 60 mg/kg/day orally.
Group 3: This group received micronized purified flavonoid fraction at a dose of 60 mg/kg/day two times topically, mixed with sterile Vaseline.
The rats were placed in three cages. Each group of rats was caged separately. Wound dimension was measured on days 4 and 12. On day 12 of the study, biopsies obtained from wounded areas of the rats were subjected to pathological examination. The specimens were fixed in 10% formaldehyde, following routine checks, sectioned (width, 4-6 micron), and dyed with haematoxylin and eosin. The histopathological examination results were categorized as follows:
Score 1 (poor): existence of purulent exudate plus granulation tissue;
Score 2 (mild): existence of granulation tissue alone;
Score 3 (good): existence of wound-repair collagen.
The comparisons of wound healing on days 4 and 12 were analysed by the paired Student-t test, the one-way Anova-Bonferroni test for multiple comparisons, and the ¯2 test.
After evaluation of the wound surface areas (Figs. 1,2), the results of the locally or systemically treated groups proved to be significantly better than those of the control group on days 4 and 12 (F = 19.2, p = 0.000; F = 5.14, p = 0.013, respectively) (Table I). Evaluations of wounds on day 12 showed that they had recovered when compared with wounds on day 4 in all groups; however, the best results were observed in the systemic group (61% reduction of the day 4 surface area versus 52% in the topical and 32% in the control groups). There was no statistical difference between rats treated orally and those treated topically with regard to wound surface measurements. Specimens obtained from the wounded region of the rats were subjected to histopathological examination and scored on 12 day (Table II). The treatment pooled group demonstrated a better healing trend than the control group (¯ = 5.513; p = 0.064).
| <% immagine "Fig. 1","gr0000017.jpg","Existence of wound repair collagen on day 12. Score 3 (Trichrome x 100).",230 %> | <% immagine "Fig. 2","gr0000018.jpg","Granulation tissue on day 4. Neovascularization intermingled with lymphocytes, plasma cells, and polymorphonuclear leukocytes in left corner (x 400, H & E).",230 %> |
Daflon, a very common treatment of choice in venous disorders, exerts its anti-inflammatory effects by way of diverse mechanisms. One of these is the reduction of the effects of histamine, bradykinin, and leukotrienes which increase vascular permeability during inflammatory states, thus preventing the escape of macromolecules from the microcirculation.4 A study by Di Perri and Auteri demonstrated that both in vivo and in vitro micronized flavonoid fraction decreased activation of the complement system.5 This activity mechanism constitutes one of the drug’s anti-inflammatory functions, and it consequently may be useful in wound healing. In another study, by Lonchampt et al., the effects of the drug both in vivo and in vitro on oxygen radicals were investigated, and a protective role for the drug against oxygen radicals was documented.6 Recent studies have suggested a positive activity for micronized flavonoid fraction on the microcirculation.7-9 The necessity of an adequate microcirculation in wound healing is of utmost importance. It has been demonstrated that micronized diosmin prevents the adherence of leukocytes to endothelial cells, thereby diminishing capillary permeability10,11 and leading to diminution of oedema in the wound area. In the present study, both systemic and local administration of Daflon in thermal burns - as confirmed by surface area measurements of the wounds on days 4 and 12 - proved to be beneficial (p = 0.000) (Table I). The speed of healing in the two treatment groups was almost double that of control (52% and 62% versus 32%). Probably owing to the relatively low number of animals, there was no clear statistical significant difference; however, the quality of healing appeared to be better in treated animals (Table II). This positive activity may be considered to be due to the drug’s role in the reduction of oedema and regulation of the microcirculation. In burns, depending on the leukocytes accumulated by infectious agent or agents in the medium, there is an overproduction of arachidonic acid metabolites and oxygen radicals.12 The anti-oxidant and anti-inflammatory properties of micronized flavonoid fraction may therefore contribute to burn healing. Flavonoids may also play a role to challenge bacterial growth located over thermally burned grounds, although until now there has been no previous study promoting this suggestion. We found that 50% of untreated animals had purulent exudation versus only 15% in the treated group (Table II).
In conclusion, Daflon proved to be capable of accelerating and improving the healing of burns. Although there was no clear difference between topical and systemic administration, our feeling is that systemic administration may be better.
RESUME. Le but de cette étude était d’évaluer l’efficacité de la fraction flavonoïde micronisée (Daflon) dans un modèle expérimental de lésion. Trente rats Wistar-Millar ont subi une brûlure selon la méthode type de Walker-Mason. Les rats ont été divisés au hasard en trois groupes. Les rats du premier groupe n’ont reçu aucun traitement (groupe témoin), le deuxième groupe a reçu par voie orale Daflon 60 mg/kg par jour, et le troisième groupe a reçu Daflon 60 mg/kg par jour deux fois en manière topique. Les évaluations de la superficie et la détermination histopathologique effectuées le jour 4 et le jour 12 dans les groupes traités en manière topique et systémique ont été comparées les une avec les autres et avec celles du groupe témoin. L’étude démontre que l’emploi soit systémique soit topique améliore la guérison des brûlures.