|Year : 2011 | Volume
| Issue : 2 | Page : 152-156
The effect of a flavonoid fractions diosmin + hesperidin on radiation-induced acute proctitis in a rat model
Atakan Sezer1, Ufuk Usta2, Zafer Kocak3, Mehmet Ali Yagci1
1 Department of General Surgery, Faculty of Medicine, Trakya University, Turkey
2 Department of Pathology, Faculty of Medicine, Trakya University, Turkey
3 Department of Radiation Oncology, Faculty of Medicine, Trakya University, Turkey
|Date of Web Publication||12-Jul-2011|
Department of General Surgery, Trakya University, Edirne 22030
Source of Support: None, Conflict of Interest: None
Background: To explore the protective effect of a flavonoid fractions diosmin + hesperidin (Daflon), against radiation-induced acute proctitis in an experimental rat model.
Materials and Methods: Thirty four rats were divided into four groups. The rats in Group 1 received Daflon and underwent irradiation. The rats in Group 2 received no Daflon and underwent irradiation. The rats in Group 3 received Daflon and underwent sham irradiation. The rats in Group 4 received no Daflon and underwent sham irradiation. Daflon emulsion (100 mg/kg/day) was administered via an orogastric feeding tube to the rats in groups 1 and 3 starting from 1 day prior to irradiation until the euthanasia day (day 15 following irradiation). Radiation therapy was delivered on a cobalt-60 unit using a single fraction of 17.5 Gy defined at a depth of 1 cm through an anterior portal. Slides were examined by the same pathologist under a light microscope two times in a blinded manner.
Results: When compared to group 2, the rats of Group 1 showed less glandular distortion and less mucosal inflammation with less infiltration of the crypt epithelia by the inflammatory cells (P < 0.001). A statistically significant increase in all parameters but muscular wall thickness was observed for the rats in Group 2 as compared to the group 3 and 4.
Conclusions: Administration of a dose of 100 mg/kg/day of the diosmin + hesperidin resulted in decreased morphologic inflammatory changes. This drug may have protective effects against radiation-induced acute proctitis.
Keywords: Acute radiation proctitis, Daflon, flavonoid fractions, radiotherapy
|How to cite this article:|
Sezer A, Usta U, Kocak Z, Yagci MA. The effect of a flavonoid fractions diosmin + hesperidin on radiation-induced acute proctitis in a rat model. J Can Res Ther 2011;7:152-6
|How to cite this URL:|
Sezer A, Usta U, Kocak Z, Yagci MA. The effect of a flavonoid fractions diosmin + hesperidin on radiation-induced acute proctitis in a rat model. J Can Res Ther [serial online] 2011 [cited 2020 May 30];7:152-6. Available from: http://www.cancerjournal.net/text.asp?2011/7/2/152/82927
| > Introduction|| |
Radiotherapy for malignant pelvic disease is often mandatory. Acute radiation proctitis is a complication of this treatment which present with bleeding, mucus discharge, urgency, discomfort, anal pain, and incontinence.  The complication rates reaches to 10%−15% among patients treated with pelvic irradiation.  Inflammation, edema, epithelial degeneration, ulceration, crypt disintegration, mucosal edema, absent mitosis, and crypt abscesses are the histopathological alterations caused by radiation exposed to rectum. ,
The overall management of radiation proctitis has been unsatisfactory, as only a few experimental or clinical treatment modalities were experienced consist of topical butyrate, steroids, sucralfates, short-chain fatty acids, hyperbaric oxygen, mesalazine, formalin, and anti-inflammatory agents. ,,,,,,, Salt-water baths, stool conditioners, and analgesics may provide symptomatic relief.
Flavonoids are a group of plant extracts with structural, biochemical, and pharmacological usage. The anti-inflammatory, immunomodulatory, and anti-oxidant specialties are the multifactorial effects of flavonoids in vivo and in vitro. , The present experimental study was thus aimed at assessing the efficacy of micronized, purified, flavonoid fractions (Daflon, 500 mg; Servier, Turkey) which is cheap and easy attainable drug, in the management of acute radiation proctitis in a rat model.
| > Materials and Methods|| |
The study was performed at the laboratory of "The Unite of the Laboratory Animals for Experimental Studies of Trakya University Medical School" in accordance with the guidelines for the care and use of the laboratory animals established by the "Animal Ethics committee of Trakya University" following the approval of the design by the "Animal Ethics committee of Trakya University". Thirty four female animals Sprague-Dawley rats (weights: 250 ± 30 g and ages: between 11 and 13 weeks) were divided into four groups. The animal room was maintained at a temperature of 22 ± 2 0 C and a relative humidity of 55 ± 15% with a 12-h light-dark cycle. Tap water was freely available throughout the acclimatization and experiment periods.
The rats in group 1 received Daflon and underwent irradiation (10 rats). The rats in group 2 received no Daflon and underwent irradiation (eight rats). The rats in group 3 received Daflon and underwent sham irradiation (eight rats). The rats in group 4 received no Daflon and underwent sham irradiation (eight rats).
Daflon (Micronized, purified, flavonoid fraction 500 mg; Servier, Turkey) was commercially purchased. An emulsion (500 mg Daflon in 10 ml distilled water) were prepared and 100 mg/kg/day which corresponds to 0.5 ml Daflon emulsion was administered via an orogastric feeding tube to the rats in groups 1 and 3 starting from one day prior to irradiation until the euthanasia day (day 15 following irradiation). For the rats in groups 2 and 4, same feeding protocol with 0.9% sodium chloride was performed.
Each rat was anesthetized using 5 mg/kg of xylazine (Rompun, Bayer Türk Kimya Sanayi Limited Şirketi, İstanbul, Turkey) and 30 mg/kg of ketamine hydrochloride (Ketalar, Pfizer İlaçları Limited Şirketi, İstanbul) intramuscularly prior to irradiation. Then, each rat was restrained and taped by the tail and legs on an acryl plate at a supine position. Lead shielding (5 half value layer) was used to cover the rat except for a 3 × 4 cm area of lower pelvis containing 2 cm length of rectum in the middle of the field. Irradiation was delivered on a cobalt-60 unit with the distance of 80 cm from the source to surface using a single fraction of 17.5 Gy defined at a depth of 1 cm through an anterior portal.
Prior to euthanasia on day 15 following irradiation or sham irradiation, euthanasia was performed by cardiac exsanguination under a surgical plane of ketamine and xylazine anesthesia. The rectum specimens excised for histopathological evaluation.
Representative sections of all rectal segments were embedded into paraffin after fixation of the tissues in 10% neutral buffered formalin. Five micrometer sections were obtained from each paraffin block and stained with hematoxylin and eosin. Slides were examined by the same pathologist under a light microscope (Nikon Eclipse E600) two times in a blinded manner. Inflammatory infiltrate in lamina propria (Inflammation), infiltration of crypt epithelium by leucocytes (cryptitis), distortion of the crypts (Crypt distortion) and regenerative/reparative atypia of the epithelial cells (reactive atypia) were examined and were degreed from 0 to 3 according to the severity of the lesions, where; 0 means no lesion, 1 means mild lesion, 2 means moderate lesion and 3 means severe lesion . Crypt abscess was noted as positive or negative for each slide. Thicknesses of mucosa and muscularis propria were measured from their thinnest portions across the diameters of the lumina of the bowels by using an image analyzer attached to a video-microscope (Zeiss Axioplan 2 imaging).
The numeric results were expressed as mean and categorical results were expressed as a number. The Chi-square test was used to compare the differences of categorical variables (inflammation, cryptitis, crypt distortion, and reactive atypia) among the groups. Mean values for mucosa and muscular wall thickness were compared using the one-way analysis of variance (ANOVA). A P-value <0.05 was considered as statistically significant. Statistica 7.0 (StatSoft Inc. Tulsa, OK, USA) statistical software was used for statistical analyses.
| > Results|| |
No mortality was observed in groups 1, 2, and 4. Two rats died within 2 h after the procedure in Group 3 and autopsy was performed to clarify the deaths. The main causes of the deaths were iatrogenic tracheal intubations and respiratory insufficiency due to aspiration of Daflon during the administration via feeding tube. The rectums of these rats were not available for the histopathological evaluation.
The histopathological evaluation of the rectum by groups is shown in [Table 1]. When compared to group 2, the rats of group 1 showed less glandular distortion and less mucosal inflammation with less infiltration of the crypt epithelia by the inflammatory cells [Figure 1] and [Figure 2]. The decrease of the inflammation in the treatment group was statistically significant (P < 0.001).
|Figure 1: Bowel wall of the group 1 with mild pathological changes in the mucosa. There is mild thickening in the mucosa, however muscularis propria is as thick as the muscularis propria of the control group (H&E, ×50).|
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|Figure 2: Mild infl ammation in the lamina propria of the group 1 with mild glandular distortion. Note the crypt abscess in the lower left (H&E, ×50)|
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|Table 1: Results and comparison of the histopathological scores of the rectal specimens by groups|
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A statistically significant increase in all parameters but muscular wall thickness was observed for the rats in group 2 as compared to the group 3 and 4. There was severe mucosal inflammation in the bowels of rats of group 2 [Figure 3]. The inflammation was mainly in the lamina propria, infiltrating the crypt epithelia leading to cryptitis and severe crypt abscesses. The mucosal glands revealed severe distortion and the epithelial cells revealed severe reactive/regenerative atypia with nuclear widening, hyperchromasia, irregularity, pleomorphism with prominent nucleoli and loss of cytoplasmic mucin [Figure 4]. The histological appearances of the rats of group 3 and group 4 were almost similar and there was no mucosal inflammatory infiltration in the bowels of those groups of rats [Figure 5] and [Figure 6].
|Figure 3: Severe distortion and crybriformity in the glandular structures in the bowel wall of group 2. Note the increased thickness of the mucosa when compared to the muscularis propria (H&E, ×50)|
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|Figure 4: Prominent mucosal infl ammation with severe cryptitis and crypt abscesses in more than one crypt lumina. There is severe glandular distortion, even cribriformity in the mucosal glands. Reactive cytologic atypia is characterized by nuclear pleomorphism, widening and irregularity, existence of prominent nucleoli and also loss of cytoplasmic mucin (H&E, ×50)|
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|Figure 5: Bowel wall of the group 3 almost without any pathology (H&E, ×50)|
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| > Discussion|| |
Histopathological alterations in rectum during pelvic irradiation consist of hyperemia, edema, extensive inflammatory cell infiltration in mucosa, cryptitis, and crypt abscess. ,, In the current report, a prominent mucosal inflammation with severe cryptitis and crypt abscesses was observed for the rats in group 2 (underwent irradiation only). There were also severe distortions and crybriformity in the glandular structures in the bowel wall. Increased thickness of the mucosal wall compared to the muscularis propria wall was also observed as a radiation effects in group 2. Reactive cytological atypia characterized by nuclear pleomorphism, widening and irregularity of prominent nucleoli, and also loss of cytoplasmic mucin were other histopathological alterations noted in group 2.
Flavonoids are phenolic compounds characterized by their diaryl nucleus. They are structurally similar to steroid hormones.  Daflon as a flavonoid drug is a micronized purified flavonoid fraction consisting of 90% diosmin and 10% hesperidin.  The most common indications of Daflon are venous insufficiency and hemorrhoidal diseases in which many studies have demonstrated the potential effects on perivascular edema and inflammatory disease. ,
The inflammatory process plays a crucial role in the pathogenesis and progression of irradiation-induced cellular damage. Arachidonic acid metabolism results in products that play a role in the inflammatory reaction.  The improvement of histological injury consist of morphologic changes in the superficial epithelium, glands, and stroma of the lamina propria in the rectal mucosa during pelvic radiotherapy may be achieved by inhibition of pro or inflammatory processes. Flavonoids have been found to reduce the activity of key enzymes of the arachidonic acid metabolism. , Moreover, Daflon as a flavonoid drug has a strong anti inflammatory effect and decreases the release of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule (ICAM-1) which leads to inhibition of leukocyte activation, adhesion, and migration. In the current study, there was a statistically significant decrease in inflammation [Figure 1] and [Figure 3] for the rats received Daflon (group 1) as compared to the rats received no Daflon (group 2).
Radiation therapy induces oxidative stress and it has been shown that free oxygen radicals have an essential role in radiation-induced normal tissue toxicity and in inflammation. The decrease in antioxidant enzyme levels may prevents damage of irradiation to normal tissues and decreases the inflammation.  Flavonoids have been studied for their antioxidant and radical scavenger properties and it has been reported that Daflon is a powerful scavenger of antioxidant enzymes such as superoxide, singlet oxygen and hydroxyl radicals.  Our results show that mucosal damage caused by irradiation may be diminished by using Daflon but we do not know that this effect may be due to the decrease to the levels of free oxygenated radicals.
There is no available therapy has, as yet, proven effective to treat acute radiation proctitis. In a randomized, double-blind study of Vernia et al.,  20 patients presented with acute radiation proctitis treated with topical sodium butyrate for three weeks. Clinical, endoscopic, and histological findings demonstrate the effectiveness of sodium butyrate in acute radiation proctitis. Kochhar et al.  compared oral sulfasalazine plus rectal steroids versus rectal sucralfate in 37 patients in a prospective, randomized, double-blind controlled trial. While the anti-inflammatory group showed clinical improvement compared sucralfate group, endoscopic findings were superior compared to the anti-inflammatory group. Rougier et al.,  worked on the efficiency of local administration of two types of corticosteroid consist of betamethasone enema and hydrocortisone acetate mousse. Over the 4 weeks of treatment the endoscopic appearance improved in a greater proportion of the hydrocortisone group rather than the betamethasone group. Short-chain fatty acids, hyperbaric oxygen, mesalazine, formalin, anti-inflammatory agents, and amiphostine were experienced in the treatment or prevention of radiation proctitis but none of them concluded in a satisfactory result. ,,,,,
| > Conclusion|| |
Administration of a dose of 100 mg/kg of the flavonoid fractions diosmin + hesperidin resulted in decreased morphologic changes and inflammatory response. Therefore this drug, which is cheap and easy attainable, may have some protective effects against radiation-induced acute proctitis. Further experimental and clinical studies are needed to address the role of Daflon on radiation-induced bowel toxicity.
| > Acknowledgments|| |
A.S. was involved in the study design, data collection, and writing and editing all aspects of this manuscript. U.U., Z.K., and M.A.Y. were involved in the study design and editing this manuscript. All of the authors have read and approved the final version of this manuscript.
| > References|| |
|1.||Reis ED, Vine AJ, Heimann T. Radiation damage to the rectum and anus: Pathophysiology, clinical features and surgical implications. Colorectal Dis 2002;4:2-12. |
|2.||Hellman S. Principles of Cancer Menagement: Radiation Therapy. In: DeVita VT, Hellman S, Rosenberg SA, editors. Cancer-Principles and Practice of Oncology. 5 th ed. New York: Lippincott-Raven Press; 1997. p. 307-21. |
|3.||Hovdenak N, Fajardo LF, Hauer-Jensen M. Acute radiation proctitis: A sequential clinicopathologic study during pelvic radiotherapy. Int J Radiat Oncol Biol Phys 2000;48:1111-7. |
|4.||Korkut C, Asoglu O, Aksoy M, Kapran Y, Bilge H, Kiremit-Korkut N, et al. Histopathological comparison of topical therapy modalities for acute radiation proctitis in an experimental rat model. World J Gastroenterol 2006;12:4879-83. |
|5.||Gul YA, Prasannan S, Jabar FM, Shaker AR, Moissinac K. Pharmacotherapy for chronic hemorrhagic radiation proctitis. World J Surg 2002;26:1499-502. |
|6.||Vernia P, Fracasso PL, Casale V, Villotti G, Marcheggiano A, Stigliano V, et al. Topical butyrate for acute radiation proctitis: Randomised, crossover trial. Lancet 2000;356;1232-5. |
|7.||Cavciæ J, Turciæ J, Martinac P, Jelinciæ Z, Zupanciæ B, Panijan-Pezeroviæ R, et al . Metronidazole in the treatment of chronic radiation proctitis: Clinical trial. Croat Med J 2000;4:314-8. |
|8.||Kneebone A, Mameghan H, Bolin T, Berry M, Turner S, Kearsley J, et al. The effect of oral sucralfate on the acute proctitis associated with prostate radiotherapy: A double-blind, randomized trial. Int J Radiat Oncol Biol Phys 2001;5:628-35. |
|9.||al-Sabbagh R, Sinicrope FA, Sellin JH, Shen Y, Roubein L. Evaluation of short-chain fatty acid enemas: Treatment of radiation proctitis. Am J Gastroenterol 1996;91:1814-6. |
|10.||Jones K, Evans AW, Bristow RG, Levin W. Treatment of radiation proctitis with hyperbaric oxygen. Radiother Oncol 2006;78:91-4. |
|11.||Freund U, Schölmerich J, Siems H, Kluge F, Schäfer HE, Wannenmacher M. Unwanted side-effects in using mesalazine (5-aminosalicylic acid) during radiotherapy. Strahlenther Onkol 1987;163:678-80. |
|12.||Luna-Perez P, Rodriguez-Ramirez SE. Formalin instillation for refractory radiation-induced hemorrhagic proctitis. J Surg Oncol 2002;80:41-4. |
|13.||Pickelmann S, Nolte D, Leiderer R, Möllmann M, Schütze E, Messmer K. Effects of the phlebotropic drug Daflon 500mg on postischemic microvascular disturbances in striated skin muscle: An intravital microscopic study in the hamster. J Lab Clin Med 1999;134:526-35. |
|14.||Labrid C. Pharmacologic properties of Daflon 500 mg. Angiology 1994;45:524-30. |
|15.||Buell MG, Hardins RK. Proinflammatory effects of local abdominal irradiation on rat gastrointestinal tract. Dig Dis Sci 1989;34:390-9. |
|16.||Gelfand MD, Tepper M, Katz LA, Binder HJ, Yesner R, Floch MH. cute irradiation proctitis in man: Development of eosinophilic crypt abscesses. Gastroenterology 1968;54:401-11. |
|17.||Zand RS, Jenkins DJ, Diamandis EP. Steroid hormone activity of Flavonoids and related compounds. Breast Cancer Res Treat 2000;62:35-49. |
|18.||Launois R. At the crossroads of venous insufficiency and hemorrhoidal disease: Daflon 500 mg. repercussions of venous insufficiency on everyday life. Angiology 1994;45:495-504. |
|19.||Lonchampt M, Guardiola B, Sicot N, Bertrand M, Perdrix L, Duhault J. Protective effect of a purified flavonoid fraction against reactive oxygen radicals. In vivo and in vitro study. Arzneimittelforschung 1989;39:882-5. |
|20.||Jean T, Bodinier MC. Mediators involved in inflammation: Effects of Daflon 500 mg on their release. Angiology 1994;45:554-9. |
|21.||Brown MS, Buchanan RB, Karran SJ. Clinical observations on the effects of elemental diet supplementation during irradiation. Clin Radiol 1980;31:19-20. |
|22.||Kochhar R, Patel F, Dhar A, Sharma SC, Ayyagari S, Aggarwal R, et al. Radiation-induced proctosigmoiditis. Prospective, randomized, double-blind controlled trial of oral sulfasalazine plus rectal steroids versus rectal sucralfate. Dig Dis Sci 1991;36:103-7. |
|23.||Rougier P, Zimmerman P, Pignon JP, Kac J, Crespon B, Zrihen E, et al. Rectites radiques: Efficate comparee de deux types de corticoides adminstre localement. Med Chir Dig 1992;21:91-3. |
|24.||Simone NL, Ménard C, Soule BP, Albert PS, Guion P, Smith S, et al. Intrarectal amifostine during external beam radiation therapy for prostate cancer produces significant improvements in Quality of Life measured by EPIC score. Int J Radiat Oncol Biol Phys 2008;70:90-5. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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