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10.5005/jp-journals-10011-1221 Thriveni R et al RESEARCH ARTICLE Effects of Natural Beta-carotene and Commercially Available Combination of Beta-carotene, Vitamin E, Vitamin C and Minerals: A Prospective Comparative Study 1 Thriveni R, 2Praveen AH, 3Mubeen 1 Reader, Department of Oral Medicine and Radiology, AME’s Dental College and Hospital, Raichur, Karnataka, India 2 3 Reader, Department of Oral and Maxillofacial Surgery, AME’s Dental College and Hospital, Raichur, Karnataka, India Professor and Head, Department of Oral Medicine and Radiology, Government Dental College and Research Centre Bengaluru, Karnataka, India Correspondence: Thriveni R, Reader, Department of Oral Medicine and Radiology, AME’s Dental College and Hospital, Raichur Karnataka, India, e-mail: [email protected] ABSTRACT Aim and objective: The purpose of this study was to determine effects of natural beta-carotene and revox in, reducing the oral symptoms of submucous fibrosis patients, thickness of collagen fibers in submucous fibrosis patients and to correlate the changes in epithelium and connective tissue. Materials and methods: The study population consisted of 50 patients with submucous fibrosis divided in two groups randomly. For patients in group A recived intralesional injection of 0.5 ml hyaluronidase 1500 IU and 0.5 ml hydrocortisone acetate 25 mg/ml were injected in buccal mucosa bilaterally in alternative week and natural beta-carotene capsule twice daily for 12 weeks along with ice cream stick mouth exercise. For group B patients intralesional injections and capsule revox were given twice daily for 12 weeks along with ice cream stick mouth exercise. Results: There is statistical significant improvement in mouth opening, reduction of burning sensation and decrease in thickness of collagen bundles were seen in all patients but there was early response in group A than group B. Conclusion: Our preliminary findings suggest that both groups showed statistical significant results with early response in group A. The clinical comparison and effectiveness of natural beta-carotene and revox is not statistical significant. Keywords: Antioxidants, Beta-carotene, Hyaluronidase, Management of submucous fibrosis, Oral submucous fibrosis, Tobacco. INTRODUCTION Oral submucous fibrosis is a premalignant and crippling condition of the oral mucosa, which is found predominantly in native Asian and Asians settled in other countries.1 The characteristic features of the disease include submucosal fibrosis causing to secondary atrophic changes in the epithelium leading to pale whitish mottled mucosa which feels board like, fibrous bands in buccal and labial region and restricting mouth opening. The atrophic epithelium becomes sensitive and vulnerable to carcinomatous changes.2,3 The exact etiology of oral submucous fibrosis is not well-understood, however, various factors like genetic, autoimmune, nutritional and environmental agents are being studied. Among the environmental factors chewing areca nut is the most important and persistent finding.1 There have been reports for successful treatment of oral submucous fibrosis with local injection of placentrex, hyaluronidase and dexamethesone. In resistant cases, surgical excision of bands with submucosal placement of fresh human placental grafts was found to be successful.4 554 The concept of antioxidant supplements could prevent or even cure cancer. In oral submucous fibrosis systemic and topical administration of beta-carotene will improve the integrity of the epithelium as well as induce redifferentiation of dysplastic epithelium. Regular intake of beta-carotene combined with routine measures will considerably reduce the risk of malignant transformation.5 Hence, this study aims at the assessment and influence of natural beta-carotene and combination of beta-carotene and other antioxidants along with local injections of hyaluronidase and hydrocortisone in reducing clinical symptoms of burning, improvement of mouth opening and its effects on collagen. AIMS AND OBJECTIVES 1. To compare the effects of natural beta-carotene and revox (beta-carotene, vitamin E, vitamin C and minerals) in reducing the oral symptoms of submucous fibrosis. 2. To compare the effects of natural beta-carotene and revox (beta-carotene, vitamin E, vitamin C and minerals) on the thickness of collagen fibers in submucous fibrosis. JAYPEE JIAOMR Effects of Natural Beta-carotene and Commercially Available Combination of Beta-carotene, Vitamin E, Vitamin C and Minerals 3. To correlate the changes brought about by the natural betacarotene and revox (beta-carotene, vitamin E, vitamin C and minerals) in epithelium and connective tissue. MATERIALS AND METHODS This study was planned and conducted in the Department of Oral Medicine and Radiology, Government Dental College and Hospital, Bengaluru. Selection of Patients The patients for the study were selected from the outpatients who visited the Department of Oral Medicine and Radiology, Government Dental College and Hospital, Bengaluru. A detailed case history about habits was taken followed by a thorough clinical examination. The diagnosis was made based on the WHO definitions for oral submucous fibrosis.6 Fifty patients who were suffering from oral submucous fibrosis were selected for this study in the age groups of 20 to 40 years. Recording the Data and Clinical Examination After explaining about the study to the subject, an informed consent was obtained and a structured proforma was administered and detailed case history of the patients was recorded. Inclusion Criteria 1. Patients with a habit of chewing betel nut and other commercially available products containing betel nut, tobacco, etc. for more than 1 year. 2. Symptoms of burning sensation in the mouth for spicy food and without spicy food, patient with progressive difficulty in opening the mouth, patients with inability to protrude the tongue and stiffness of the oral mucosa. 3. Patients with mucosal signs of blanching, palpable vertical fibrous bands in the buccal mucosa. Exclusion Criteria 1. Patients who are undertreatment for submucous fibrosis previously. 2. Patients with submucous fibrosis having diabetics, hypertension and pregnant ladies were excluded from the study. 3. Patients with total trismus, chronic ulcers and difficulty in swallowing were excluded. 4. Patients with temparomandibular joint problems and pericoronitis of the lower third molars. Patients who were selected for the study were categorized in different grades according to the grading of Gupta Dinesh Chandra et al.7 Biopsy of the Lesion After thorough clinical examination, the patients were subjected for biopsy. Right buccal mucosa was selected as a standard site for biopsy in all patients. Punch biopsy was done under 2% local anesthesia and subjected to following staining methods. 1. Hematoxyline and Eosine stain 2. Van-Gieson’s stain 3. Masseon‘s Trichrome stain 4. Verhoff’s stain Study Duration and Treatment Procedure The treatment plan was followed for a period of 3 months. All the patients with palpable fibrous bands in the buccal mucosa were randomly divided into two groups. For all the patients interincisal distance measured with the help of slide calipers before and after treatment (Figs 1 and 2) and biopsy was done before treatment and 3 weeks after treatment (Figs 3 and 4). Patients were recalled every week on appointment basis for 3 months. For patients in group A intralesional injection of hyaluronidase 1500 IU mixed in 1 ml of lignocaine solution and injected about 0.5 ml in the buccal mucosa bilaterally and intralesional injection of hydrocortisone acetate 25 mg/ml about 0.5 ml in the buccal mucosa bilaterally was given alternatively per week with natural beta-carotene capsules for 12 weeks and ice cream stick mouth exercise with increase in one stick every alternate day. For group B patients along with intralesional injection of hyaluronidase and hydrocortisone as before, a capsule revox was given for 12 weeks along with ice cream stick mouth exercise. After systemic administration of natural beta-carotene and revox and intralesional injection of hydrocortisone and hyaluronidase, no patient reported back with symptoms of vomiting, gastritis, diarrhea and irritation or swelling at the site of injection. OBSERVATION AND RESULTS The study was conducted on 50 patients of both the genders of different age groups. All patients with submucous fibrosis showed involvement of right and left buccal mucosa and other parts of the oral cavity. Among the study group of 50 patients, few patients had difficulty in mouth opening, few had burning sensation and some patients had both as shown in Graphs 1 and 2 and Table 1. During the period of treatment, in all the patients’ reduction of burning sensation was seen in different stages which were shown in Table 2, Graphs 3 and 4. In this study comparison of groups A and B was done. A test of significance students t-test resulted in a value p > 0.05 (t = 0.916) which is not significant (because p < 0.05), this indicates both the groups were similar before the treatment. Different treatment modalities were carried out for both the groups, after treatment a test of significant paired t-test resulted in a value for mouth opening parameter p < 0.001 (group A t = 16.4754 and group B 18.3351) and for histological parameters p < 0.001 (group A 8.8092 and group B 11.111) Journal of Indian Academy of Oral Medicine and Radiology, October-December 2011;23(4):554-58 555 Thriveni R et al Fig. 1: Preoperative picture Fig. 2: Postoperative picture Fig. 4: Postoperative (H&E stain) To know the effect of natural beta-carotene and revox in improvement of mouth opening, a test significance of ANOVA has carried out which shows: Group A: Grade III 1.33 Grade IV 1.59 Group B: Grade III 1.3882 Grade IV 1.7625 Which is not significant (because p > 0.05) indicates both the treatment in both grades effected similarly. To know the effect of natural beta-carotene and revox in reducing the thickness of collagen bundles a test of significance of ANOVA was done with a resultant mean values as follows: Group A: Grade III 1.02 Grade IV 0.68 Group B: Grade III 1.0324 Grade IV 0.6875 Which indicates both the treatments affected in a similar way in both the grades,but respect of natural beta-carotene or revox grade III, patients responded well than grade IV (p < 0.01). DISCUSSION Fig. 3: Preoperative (H&E stain) which was highly significant indicating both the groups responding very well for the treatment. To compare the effectiveness of treatment in between the groups, after treatment students t-test was done which resulted in a value p > 0.05 (group A, t = 1.507 and group B, t = 1.396 and for mouth opening parameters t = 0.1171) which is not significant indicates both the groups showed similar response. 556 Oral submucous fibrosis has been attributed to the habit of betel nut chewing which is predominant among the Indian population.8 It is well documented that oral submucous fibrosis is a precancerous condition9,10 and consequently possess a challenge to medical profession in general and oral medicine specialist in particular. In our study, there was predominant occurrence in males than females with age group of 20 to 40 years which is because of use of commercially available packed pan masala among young males. Study reports support the hypothesis that the relative risk of developing submucous fibrosis directly proportional to exposure to various harmful substance in a given time to that of total duration of exposure.4 In this study, intralesional injection of hydrocortisone acetate 25 mg/ml and hyaluronidase was injected in the buccal mucosa bilaterally in alternative week for 3 months. Histopathological examination of biopsy specimen after treatment for all patients showed remarkable reduction of JAYPEE JIAOMR Effects of Natural Beta-carotene and Commercially Available Combination of Beta-carotene, Vitamin E, Vitamin C and Minerals Graph 1: Symptoms of group A patients Graph 2: Symptoms of group B patients Table 1: Symptoms Groups Total Patient A B Difficulty in mouth opening Burning sensation Both Male Female Total Male Female Total Male Female Total 5 5 1 1 6 6 2 1 1 1 3 2 14 16 2 1 16 17 Graph 4: Reduction of burning sensation in group B patients Graph 3: Reduction of burning sensation in group A patients Table 2: Reduction of burning sensation Groups Total reduction of burning sensation 2nd week Total Patients A B 3rd week 4th week 5th week 6th week Male Female Male Female Male Female Male Female Male Female 4 0 0 0 5 0 0 0 7 10 3 0 0 6 0 1 0 1 0 1 inflammatory cells, thickness of collagen bundles, which is related to effect of corticosteroids and hyaluronidase. The mode of action of hyaluronidase is thought to be destruction of collagen11 because it has been seen that breaking down of hyaluronic acid and collagen fibers in patients with oral submucous fibrosis was attacked rapidly by hyaluronidase when compare to the normal patients. It lowers the viscosity of intercellular cementing substance and also decreases collagen formation. The locally given steroids act by opposing the acting of soluble factors released by sensitized lymphocytes following activation of nonspecific antigens and they function as immunosuppressive agents. Also the inflammatory reaction at Journal of Indian Academy of Oral Medicine and Radiology, October-December 2011;23(4):554-58 557 Thriveni R et al the site is suppressed thereby fibrosis is prevented by a decreased in fibroblastic proliferation and decreased in deposition of collagen.7 In our study we have found statistical significant improvement in the burning sensation and mouth opening in all patients especially in grade III patients. This could be explained because of various factors including intralesional injection of corticosteroids and hyaluronidase along with systemic administration of spirulina (natural beta-carotene) and revox. Spirulina is a blue-green algae which contains natural antioxidants, such as beta-carotene, vitamin E, selenium, vitamin C, etc.12,13 They act by effectively quench the hazardous free radicals, which initiate carcinogenesis. Beta-carotene also has immunoregulating properties that might retard the development of cancer cells and also increases the number of circulating lymphocytes, enhances the proliferation and induction of cytotoxic T cells and increases the number of helper T cells and tumor necrosis factor.5,14 Considerable data support the concept of synergistic effect among the antioxidants, beta-carotene, ascorbic acid and alphatocopherol are free radical scavengers with different subcellular distributions and jointly participate in cancer prevention by immunosurveillance against tumor.5,14 In the recent studies following treatment modalities have been used to treat the patients with oral submucous fibrosis: 1. Interferon gamma—intralesional injection of 0.01 to 10.0 U/ml, three times a day for 6 months.15 2. Pentoxifylline—400 mg three times a day for 7 months.16 3. Lycopene—8 mg twice a day for 2 months.17 In our study, clinical comparative effect of natural betacarotene and revox, is not statistically significant but irrespective of use of those, there is statistical significant improvement in grade III patients than grade IV patients in both the groups. Natural beta-carotene and revox are both freely available in the market and they have almost similar biological effects with no adverse effects. Hence, it becomes the clinician’s choice to select spirulina or revox to improve nutritional status, immune status and prevention of cancer in patients with submucous fibrosis. CONCLUSION Though this a preliminary study but one can draw to certain extent the following conclusion by its results: • There is a statistical significant improvement in the mouth opening, reduction of burning sensation and decrease in the thickness of collagen bundles were seen in all patients, but there was early response in group A than group B patients. • The clinical comparison and effectiveness of natural betacarotene and revox is not statistically significant. This study has given scope for further studies with larger sample size and use of electron microscopic study for tissue 558 changes correlated with blood samples of natural beta-carotene and revox are required to conclude above results. REFERENCES 1. Shah N, Sharma PP. Role of chewing and smoking habits in the etiology of oral submucous fibrosis: A case-control study. J Oral Pathol Med 1988;27:475-79. 2. Haider SM, Merchant AT, Fikree FF, Rahbar MH. Clinical and functional staging of oral submucous fibrosis. British Journal of Oral and Maxillofacial Surgery 2000;38:12-15. 3. Srinivasan M, Jewell SD. Quantitave estimation of PNCA, Cmyc, EGFR and TGF- alpha in oral submucous fibrosis: An immunohistochemical study. Oral Oncology 2000;37:461-67. 4. Gupta Deepak, Sharma SC. Oral submucous fibrosis: A new treatment regimen. J Oral Maxillofac Surg 1988;46:830-33. 5. Kaugars George E, Sol Silverman Jr, John GL Lovas, Thompson Joan S, Brandt Richard B, Singh Vishwa N. Use of antioxidant supplement in the treatment of human oral leakoplakia. Oral. Surg Oral Med Oral Pathol 1996; 81(1):5-14. 6. Jens J Pindborg, Dr Odont, Sirsat Satyavati M. Oral submucous fibrosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1966;22(6):764-79. 7. Gupta Dinesh Chandra, Dolas Rameshwar, Ali Iqbal. Treament modalities in oral submucous fibrosis; How they strnd today? Study of 600 cases. Int J of Oral and Maxillofac Surg 1992;7: 43-47. 8. Murti PR, Bhonsle RB, Gupta PC, Daftary DK, Pindborg JJ, Mehta FS. Etiology of oral submucous fibrosis with special reference to the role of areca nut chewing. J Oral Pathol Med 1992;24:145-52. 9. Maher R, Lee AJ, Warnakulasuriya KAAS, Lewis JA, Jhonson NW. Role of areca nut in the causation of oral submucous fibrosis. A case-control study in Pakistan. J Oral Pathol Med 1994;23:65-69. 10. Pondborg JJ, Murti PR, Bhonsle RB, Gupta PC, Daftary DK, Mehta Fali S. Is submucous fibrosis a precancerous condition in the oral cavity? Int Dent J 1972;22:474-80. 11. Kakar PK, Puri RK, Venkatachalam VP. Oral submucous fibrosis treatment with hyalase. J of Larygol and Otol 1985;99:57-59. 12. Ben Amotz A, Edelstein S, Avron M. Use of the beta-carotene rich alga dunaliella bardawil. As a source of retinol. British Poultry Science 1986;27(4):613-19. 13. Machlin Lawrence J, Bendich Adrianne. Free radical tissue damage: Protective role of antioxidant nutrients. FASEB J 1987;1:441-45. 14. Guerrero Byers Nicole. Epidiologic evidence for vitamin C and vitamin E in cancer prevention. Am J Clin Nutr 1995;62: 1385-92S. 15. Haque M F, Meghji S, Nazir R, Harris M. Interferon gamma (IFN-Gamma) may reverse oral submucous fibrosis. J Oral Pathol Med 2001;30(1):12-21. 16. Rajendran R, Rani V, Shaikh S. Pentoxifylline therapy: A new adjunct in the treatment of oral submucous fibrosis. Indian J Dent Res 2006;17(4):190-98. 17. Kumar A, Bagawadi A, Keluskar V, Singh M. Efficacy of lycopene in the management of oral submucous fibrosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103(2): 207-13. JAYPEE