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Effects of berberine gelatin on recurrent aphthous stomatitis: a randomized, placebo-controlled, double-blind trial in a Chinese cohort
Vol. 115 No. 2 February 2013
Effects of berberine gelatin on recurrent aphthous stomatitis: a randomized, placebo-controlled, double-blind trial in a Chinese cohort Xiao-Wen Jiang, PhD, DDS,a,b Yi Zhang, MD,c Ya-Li Zhu, MD,b Hao Zhang, MD,b Kun Lu, MD,b Fen-Fang Li, MD,b and Hai-Yan Peng, MDb The First People’s Hospital of Chenzhou and University of South China, Hunan, China Objective. Recurrent aphthous stomatitis (RAS) is a common oral mucosal disease, yet effective therapeutic approaches are lacking. This study aimed to determine the effects of application of berberine gelatin in the treatment of minor RAS (MiRAS). Methods. A randomized, double-blind, placebo-controlled, clinical trial was performed. The gelatin containing berberine (5 mg/g) or vehicle only was applied 4 times per day for 5 days. Clinical evaluation included pain level, size, erythema, and exudation of certain ulcers on days 1, 2, 4, and 6. Results. A total of 84 subjects fulfilled the study without obvious side effects. Berberine gelatin treatment reduced the ulcer pain score compared with placebo gelatin (P ⬍ 0.05). Ulcer size was significantly reduced (P ⬍ 0.05) and lower erythema (P ⬍ 0.05) and exudation (P ⬍ 0.05) levels were associated with berberine treatment. Conclusions. Berberine gelatin may be a safe and effective treatment for MiRAS. (Oral Surg Oral Med Oral Pathol Oral Radiol 2013;115:212-217)
Recurrent aphthous stomatitis (RAS), which clinically manifests as distinct and shallow ulceration with a necrotic center covered by a pseudomembrane and surrounded by an erythematous halo, is among the most prevalent oral mucosal lesions affecting the general population.1 Minor RAS (MiRAS) is the most common form, consisting of 70% to 87% RAS.2,3 Although RAS is an episodic and self-limited affection, the ulceration usually severely interferes with eating, speaking, and swallowing. Because no definitive etiology has been identified, there is no uniformly effective and reliable therapy in the termination of this potentially debilitating disorder.3 Available systemic or topical treatments currently are aimed at alleviating pain, decreasing functional disability, and promoting ulcer healing. Some systemic medications, although effective, have side effects that limit their extensive or long-term application. Therefore, topical agents (including glucocorticoids, antibiotics, analgesics, astringents, and laser therapy) remain the mainstay of therapy.3 Competing interests. The authors state that they have no conflict of interest. a Department of Stomatology, the First People’s Hospital of Chenzhou, Chenzhou 423000, Hunan, China. b Institute of Translation Medicine on University of South China, Chenzhou 423000, Hunan, China. c Department of Neurology, the First People’s Hospital of Chenzhou, Chenzhou 423000, Hunan, China. Received for publication July 11, 2012; returned for revision Sept 2, 2012; accepted for publication Sept 9, 2012. © 2013 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2012.09.009
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Berberine, an isoquinoline alkaloid originally isolated from medicinal herbs, such as Hydrastis canadensis, Coptidis rhizoma, and Berberis vulgaris, is an antimicrobial drug routinely prescribed in traditional Eastern medicine.4 Recently, several investigations have suggested that berberine possesses a wide range of pharmacologic and biological activities, including antiinflammatory,5-7 antimicrobial,8 antitumor,9 and antipyretic10 properties. We hypothesized that berberine may be an effective and favorable treatment candidate for MiRAS because of its multiple bioactivities. The objective of this randomized, double-blind, placebocontrolled clinical trial was to investigate the efficacy and safety of berberine gelatin in the treatment of MiRAS.
MATERIAL AND METHODS Materials Berberine gelatin (10 g) containing 50 mg berberine (98% HPLC, Biopurify Science and Technology Development Co. Ltd., Chengdu, China), hydroxypropyl methyl cellulose, polyethylene glycol 400, glycerine, and flavoring additives was used. The placebo agent (10 g) contained the above-mentioned excipients with
Statement of Clinical Relevance In this placebo-controlled, double-blind trial, berberine gelatin was effective in alleviating pain intensity and promoting oral aphthous healing without notable side effects.
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the exception of berberine and had a similar shape, color, and flavor. All gelatin, packed in soft tubes, was produced by the pharmaceutical department of the First People’s Hospital of Chenzhou, University of China South. Subjects and study design This randomized, double-blind, placebo-controlled clinical trial was conducted under the approval of the Institutional Ethics Committee/Institutional Review Board of the First People’s Hospital of Chenzhou, University of South China. The clinical diagnosis was based on the patients’ medical history and clinical appearance.11 To exclude potentially confounding systemic diseases, all patients underwent careful examination by dermatologists, gastroenterologists, ophthalmologists, and hematologists before enrollment. All patients were selected according to inclusion and exclusion criteria. The inclusion criteria were as follows: (1) male or female individuals aged 18 to 50 years, who could exactly follow the study protocol and sign the informed consent form voluntarily; (2) a history of RAS for a minimum of 6 months with an average of 1 to 2 outbreaks per month and 1 to 5 minor aphthae per outbreak; (3) agreement not to use analgesics or other drugs curing oral ulcer during the study; (4) 1 to 3 aphthous ulcerations with a size ⬎5 and ⬍10 mm in diameter; for more than 1 ulceration, a distance between ulcerations of more than 1 cm; (5) time of ulceration onset within 48 hours; and (6) ulcers in locations that are easily accessible for evaluation and treatment. Exclusion criteria were as follows: (1) history of allergies to berberine; (2) pregnancy or lactation; (3) ulcers as a manifestation of a systemic disease process such as Behçet’s disease, ulcerative colitis, Crohn’s disease, or acquired immune deficiency syndrome; (4) taking systemic nonsteroidal anti-inflammatory drugs, immunomodulatory agents, or systemic antibiotics within 2 weeks; (5) treatment with any oral topical medication or invasive dental procedures within 2 weeks; (6) normal resolution of aphthous ulcers expected in ⬍5 days without any treatment; (7) use of toothpaste containing anti-inflammatory drugs; (8) ulceration secondary to hematinic deficiency, vitamin deficiency, or nutritional deficiency; and (9) ulceration caused by drugs such as nicorandil or methotrexate. Patients were then assigned to a berberine group or a control group using a computer-generated random numbers list. Both the investigators and the participants were blind to the group assignment until study completion. In this trial, only 1 ulcer was selected for analysis. For patients with more than 1 ulcer, the most recent ulcer was selected. The pain level was evaluated by the
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Table I. Classification of erythema and exudation levels Classification
Erythema
Exudation
0 1 2 3
No erythema Light red/pink Red but not dark in color Very red, dark in color
No exudation Light exudation Moderate exudation Heavy exudation, with pseudomembrane
subjects following instructions by the investigators, and the size of the ulcers and the erythema and exudation were measured and recorded by the investigator before the first administration and at each evaluation thereafter. To evaluate pain, a visual analog scale consisting of a 100-mm horizontal line between the poles of “no pain” to “unbearable pain” was used. Subjects were instructed to mark the line with a vertical line at the point that best represented the present pain level of the appointed ulcer. Then, the length between the vertical line and the pole representing no pain was measured and recorded to represent the ulcer pain score. To measure the size of the ulcers, the investigators measured the distance between 2 opposite outside edges of the ulcer border with millimeter markings on a calibrated dental probe. The 2 measurements approximately 90 degrees from each other were obtained 3 times, and the largest distance was used as 1 of the measurements. The 2 measurements were then multiplied to represent the cross-sectional area of the ulcer. The degree of erythema and exudation was evaluated and recorded by the investigators on a 4-point scale (Table I) ranging from 0 to 3 based on the methods of Liu et al.12 and Babaee et al.13 All subjects were instructed to dry the ulcer by patting it with clean gauze, squeeze 1 cm of the drug onto a sterile cotton swab, and dab the gelatin onto the ulcer. They were observed for 30 minutes for any possible signs of acute hypersensitivity reactions or other adverse events at the first administration and advised to avoid drinking or eating in the 30 minutes after each administration. The subjects were requested to apply the drug 4 times a day (after meals and before bedtime) for 5 days. The baseline parameters were taken and recorded at the first visit (day 1). The evaluations were made at the same time on the morning of days 2, 4, and 6 before gelatin administration. Participants were requested to bring the soft tube containing the gelatin to every visit to ensure the drugs were used properly. Further, periodic telephone interviews were made to supervise the administration on schedule. Side effects after gelatin administration were recorded, and the oral mucosa was inspected by the investigator at each visit. Subjects were also informed that possible side effects may occur, at which time they should
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terminate use of the gelatin and inform the investigator immediately. To compare the therapeutic effect statistically at different time points, the data were calculated using the following formula [(visit 1–visit 2, visit 4 or visit 6)/visit1] ⫻100% (visitx referring to the values measured at day x) according to previous studies.14 Statistical methods Background and demographic data were summarized with descriptive statistics. Group differences between the berberine group and the control group were evaluated. Comparisons of ulcer history and baseline values of ulcers were performed with the Mann–Whitney test. To evaluate efficacy, the 2 test and the Mann–Whitney test were used. All data were analyzed by SPSS software (SPSS 13.0 for Windows; SPSS, Chicago, IL) and the level of significance was established at a P value less than 0.05.
RESULTS A total of 87 subjects were recruited in this study. Only 2 subjects in the berberine group and 1 subject in the control group dropped out because of a conflicting schedule and not following the instructions. We evaluated demographic and efficacy data without those subjects, which did not influence the interpretation or conclusions. Therefore, 42 subjects in the berberine group and 42 subjects in the control group fulfilled the study.
Table II. Sociodemographic and basic clinical characteristics of the population
Age (years) Mean ⫾ SD Range Gender Male Female Education Primary school High school College graduate Tobacco Nonsmoker Smoker Allergy history Yes No Ulcer location Cheek Tongue Lip Number of ulcers 1 2 3
Berberine group (n ⫽ 42) (n, %)
Control group (n ⫽ 42) (n, %)
30.86 ⫾ 7.53 19-51
31.19 ⫾ 6.00 20-45
0.638
14 (33.33) 28 (66.67)
10 (23.81) 32 (76.19)
0.337
1 (2.38) 24 (57.14) 17 (40.48)
2 (4.76) 28 (66.67) 12 (28.57)
0.226
4 (9.52) 38 (90.48)
3 (7.14) 39 (92.86)
0.695
0(0) 42 (100)
0(0) 42 (100)
1
18 (42.86) 8 (19.04) 16 (38.10)
24 (57.14) 4 (9.52) 14 (33.34)
0.316
30 (71.43) 10 (23.81) 2 (4.76)
27 (88.10) 8 (9.52) 7 (2.38)
0.328
P
Demographics The demographics of the berberine group and the control group were similar, including age, gender, known allergies, and baseline values of ulcer location and number (Table II). The mean age of the subjects was approximately 31 years. Of the subjects, 71.4% were female and 28.6% were male. Moderation of pain The mean ulcer pain scores of the 2 groups matched well (P ⬎ 0.05) at trial entry, but later significant relief was observed in the berberine group (Figure 1). On day 2, the ulcer pain score of the berberine group decreased by 16.1%, which was greater than that (6.56%) in the control group (P ⬍ 0.05). On day 4, the reduction in ulcer pain score of the berberine group was much greater than that of the control group (44.57% vs 22.60% P ⬍ 0.02). On day 6, pain moderation in the berberine group was 76.65%, which was greater than that (60.87%) in the control group (P ⬍ 0.03). Reduction in ulcer size There was no significant difference between the mean ulcer size of the 2 groups at study entry (P ⬎ 0.05).
Fig. 1. Comparison of ulcer pain score between the berberine and control groups. *Ulcer pain score improvement in the control group was significantly less than in the berberine group, P ⬍ 0.05. #Ulcer pain score in the berberine group did not differ from that in the control group, P ⬎ 0.05.
However, significant group differences appeared at subsequent visits (Figure 2). On day 2, the ulcer size in the berberine group decreased by 35.63%, which was greater than the decrease (14.14%) in the control group (P ⬍ 0.01). On day 4, the ulcer size in the berberine group decreased by 54.07%, which was significantly different from the control group (37.66%, P ⬍ 0.03). On day 6, the berberine group maintained a significantly greater decrease in ulcer
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Fig. 2. Comparison of mean ulcer size between the berberine and control groups. *Ulcer size reduction in the control group was significantly less than in the berberine group, P ⬍ 0.05; # Ulcer size in the berberine group did not differ from that in the control group, P ⬎ 0.05.
size than the control group (70.56% vs 54.35%; P ⬍ 0.005). Reduction in erythema and exudation Both the erythema and the exudation levels improved in the 2 groups (Table III). The group difference demonstrated no statistical significance on day 1 and 2 visits (P ⬎ 0.05, P ⬎ 0.05, respectively); however, the berberine group exhibited significantly lower erythema (P ⬍ 0.01, P ⬍ 0.01, respectively) and exudation (P ⬍ 0.04, P ⬍ 0.05, respectively) levels than the control group on day 4 and 6 visits. Safety evaluation No obviously adverse side effect such as hypersensitivity, infection, or taste sense malfunction occurred. All patients tolerated the gelatin well with a good rating.
DISCUSSION For the treatment of MiRAS, several topical and systematic agents have been adopted. However, efficacious and safe therapy in termination of the potentially debilitating disorder is limited.3 Therefore, MiRAS remains a challenge for stomatological specialists. Recently, many research-based pharmaceutical companies have been investing time and money in the development of medicinal natural products as a method for addressing the drug resistance and cost-effectiveness of synthetic drugs.15 Berberine is an isoquinoline alkaloid present in some herb materials and has proven to have a range of pharmacologic and biological activities. In this population, by a mechanism that remains unclear, berberine gelatin is associated with relief of pain at-
ORIGINAL ARTICLE Jiang et al. 215
tacks and the promotion of healing without obvious side effects. The precise etiology of RAS remains unknown. The inflammatory process plays an important role in RAS, and neutrophils and mononuclear cell infiltration in the lamina propria are histologic characteristics.16,17 Also, focal immune dysfunction exerts a significant role.18,19 Although it is unlikely that oral bacteria play a primary pathogenic role in the development of RAS, the presence of abnormal oral flora was associated with an aberrant immune and inflammation response.18,20 Because of the potential for multiple etiologies and pathophysiologies, the optimal therapeutic agent should posses multiple bioactivities. Berberine was selected as a possible candidate treatment for MiRAS in this study because of its known anti-inflammation, antimicrobe, and immunomodulation properties. The literature is promising with regard to the direct anti-inflammation properties of berberine. Li et al.21 and Cui et al.22 demonstrated that berberine could diminish inflammatory cell infiltration in animal models. Additional studies9,23,24 reported that berberine could repress proinflammatory responses through activated protein kinase activation in macrophages; downregulate the expression of proinflammatory genes (tumor necrosis factor-␣, interleukin [IL]-1, IL-6, and so on); and upregulate IL-10, which exerted the main negative regulatory feedback on Th1 cytokine production. Berberine was also demonstrated to possess significant antimicrobial activity against a wide range of microorganisms, including Staphylococcus, Streptococcus, Candida, and Salmonella.25,26 Furthermore, berberine is an immunosuppressive that demonstrates a safe drug profile.27,28 Although the exact mechanisms of berberine in the treatment of MiRAS remain to be clarified, the multiple bioactivities may be beneficial in accelerating pain relief and ulcer healing. Interestingly, some curative effect for pain relief was also observed in the control group, which may be because the placebo gelatin provides a protective film to isolate the physical and chemical stimuli and promote the healing process. The double-blind experiment protocol may also cause some beneficial psychotherapy effect. The berberine gelatin could adhere to the oral mucosa to release berberine slowly at the site of the ulcer, resulting in higher drug concentrations and a longerlasting release period. Berberine is easily accessible in China at a low cost. The gelatin is also easy for patients to administer and carry. In view of the many drawbacks of synthetic compounds used as topical agents, berberine, which has a plant origin and no obvious side effects, would be a promising candidate in the treatment of MiRAS.
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Table III. Changes in erythema and exudation Berberine group (n ⫽ 42) Erythema Day 1 Day 2 Day 4 Day 6 Exudation Day 1 Day 2 Day 4 Day 6
Control group (n ⫽ 42)
0
1
2
3
0
1
2
3
P*
0 10 26 31
0 10 13 9
31 17 2 2
9 5 1 0
0 6 10 16
3 5 10 15
29 23 15 8
10 8 7 3
⬎0.05 ⬎0.05 ⬍0.01 ⬍0.01
0 9 24 30
3 11 13 9
27 14 3 2
12 8 2 1
0 9 17 22
4 9 10 10
25 15 9 7
13 9 6 3
⬎0.05 ⬎0.05 ⬍0.04 ⬍0.05
*Mann–Whitney test.
In conclusion, the present study findings demonstrate that the topical application of berberine gelatin could decrease pain intensity and promote ulcer healing with minimal safety concerns. Therefore, in clinical practice, berberine gelatin could be a well-tolerated and safe addition to the treatment armamentarium for MiRAS. This study was supported by the Scientific Research Project of Xiangnan University and the First People’s Hospital of Chenzhou, University of South China, and the Science and Technology Bureau Research Project of Chenzhou Municipal. We gratefully acknowledge Drs. Jiali Tan, Dunting Zhang, and Haiwen Zhou as investigators.
11.
12.
13.
REFERENCES 1. Ship JA. Recurrent aphthous stomatitis. An update. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;81:141-7. 2. Akintoye SO, Greenberg MS. Recurrent aphthous stomatitis. Dent Clin North Am 2005;49:31-47. 3. Barrons RW. Treatment strategies for recurrent oral aphthous ulcers. Am J Health Syst Pharm 2001;58:41-50. 4. Tang J, Feng Y, Tsao S, Wang N, Curtain R, Wang Y. Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations. J Ethnopharmacol 2009;126:5-17. 5. Akhter MH, Sabir M, Bhide NK. Anti-inflammatory effect of berberine in rats injected locally with cholera toxin. Indian J Med Res 1977;65:133-41. 6. Li SY, Ling LH, Teh BS, Seow WK, Thong YH. Anti-inflammatory and immunosuppressive properties of the bis-benzylisoquinolines: in vitro comparisons of tetrandrine and berbamine. Int J Immunopharmacol 1989;11:395-441. 7. Zhou HY, Mineshita S. The effect of berberine chloride on experimental colitis in rats in vivo and in vitro. J Pharmacol Exp Ther 2000;294:822-9. 8. Iwasa K, Lee DU, Kang SI, Wiegrebe W. Antimicrobial activity of 8-alkyl- and 8-phenyl-substituted berberines and their 12bromo-derivatives. J Natl Proc 1998;61:1150-3. 9. Fukuda K, Hibiya Y, Mutoh M, Koshiji M, Akao S, Fujiwara H. Inhibition by berberine of cyclooxygenase-2 transcriptional activity in human colon cancer cells. J Ethnopharmacol 1999; 66:227-33. 10. Küpeli E, Kos¸ar M, Yes¸ilada E, Hüsnü K, Bas¸er C. A comparative study on the anti-inflammatory, antinociceptive and anti-
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pyretic effects of isoquinoline alkaloids from the roots of Turkish Berberis species. Life Sci 2002;72:645-57. Femiano F, Lanza A, Buonaiuto C, Gombos F, Nunziata M, Piccolo S, Cirillo N. Guidelines for diagnosis and management of aphthous stomatitis. Pediatr Infect Dis J 2007;26: 728-32. Liu J, Zeng X, Chen Q, Cai Y, Chen F, Wang Y, et al. An evaluation on the efficacy and safety of amlexanox oral adhesive tablets in the treatment of recurrent minor aphthous ulceration in a Chinese cohort: a randomized, double-blind, vehicle-controlled, unparallel multicenter clinical trial. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102: 475-81. Babaee N, Mansourian A, Momen-Heravi F, Moghadamnia A, Momen-Beitollahi J. The efficacy of a paste containing Myrtus communis (myrtle) in the management of recurrent aphthous stomatitis: a randomized controlled trial. Clin Oral Investig 2010;14:65-70. Jiang XW, Zhang Y, Song GD, Li FF, Peng HY, Yang SK, et al. Clinical evaluation of allicin oral adhesive tablets in the treatment of recurrent aphthous ulceration. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2012;113:500-4. Wagner H. Natural products chemistry and phytomedicine research in the new millennium: new developments and challenges. ARKIVOC 2000;27:277-84. Scully C, Gorsky M, Lozada-Nur F. The diagnosis and management of recurrent aphthous stomatitis: a consensus approach. J Am Dent Assoc 2003;134:200-6. Porter SR, Scully C, Pedersen A. Recurrent aphthous stomatitis. Crit Rev Oral Biol Med 1998;9:306-21. Greenberg MS, Pinto A. Etiology and management of recurrent aphthous stomatitis. Curr Infect Dis Rep 2003;5:194-8. Shashy RG, Ridley MB. Aphthous ulcers: a difficult clinical entity. Am J Otolaryngol 2000;21:389-93. Marchini L, Campos MS, Silva AM. Bacterial diversity in aphthous ulcer. Oral Microbiol Immunol 2007;22:225-31. Li HM, Wang YY, Wang HD, Cao WJ, Yu XH, Lu DX, et al. Berberine protects against lipopolysaccharide-induced intestinal injury in mice via alpha 2 adrenoceptor-independent mechanisms. Acta Pharmacol Sin 2011;32:1364-72. Cui HS, Hayasaka S, Zheng LS, Hayasaka Y, Zhang XY, Chi ZL. Effect of berberine on monocyte chemotactic protein-1 and cytokine-induced neutrophil chemoattractant-1 expression in rat lipopolysaccharide-induced uveitis. Ophthalmic Res 2007;39: 32-9.
OOOO Volume 115, Number 2 23. Jeong HW, Hsu KC, Lee JW, Ham M, Huh JY, Shin HJ, et al. Berberine suppresses proinflammatory responses through AMPK activation in macrophages. Am J Physiol Endocrinol Metab 2009;296:E955-64. 24. Li F, Wang HD, Lu DX, Wang YP, Qi RB, Fu YM, Li CJ. Neutral sulfate berberine modulates cytokine secretion and increases survival in endotoxemic mice. Acta Pharmacol Sin 2006;27:1199-205. 25. Bensky D, Gamble A, Kapchuk T. Chinese herbal medicine: materia medica, rev. ed. Seattle: Eastland Press; 1993. p 78. 26. Arayne MS, Sultana N, Bahadur SS. The Berberis story: Berberis vulgaris in therapeutics. Pak J Pharm Sci 2007;20:83-92. 27. Qin X, Guo BT, Wan B, Fang L, Lu L, Wu L, et al. Regulation of Th1 and Th17 cell differentiation and amelioration of exper-
ORIGINAL ARTICLE Jiang et al. 217 imental autoimmune encephalomyelitis by natural product compound berberine. J Immunol 2010;185:1855-63. 28. Kondo Y, Imai Y, Hojo H, Hashimoto Y, Nozoe S. Selective inhibition of T-cell-dependent immune responses by bisbenzylisoquinoline alkaloids in vivo. Int J Immunopharmacol 1992;14: 1181-6. Reprint requests: Xiao-Wen Jiang, DDS, PhD Department of Stomatology The First People’s Hospital of Chenzhou 102 Luojiajing Road, Beihu District Chenzhou 423000, Hunan Province, China [email protected]
Effects of berberine gelatin on recurrent aphthous stomatitis: a randomized, placebo-controlled, double-blind trial in a Chinese cohort Xiao-Wen Jiang, PhD, DDS,a,b Yi Zhang, MD,c Ya-Li Zhu, MD,b Hao Zhang, MD,b Kun Lu, MD,b Fen-Fang Li, MD,b and Hai-Yan Peng, MDb The First People’s Hospital of Chenzhou and University of South China, Hunan, China Objective. Recurrent aphthous stomatitis (RAS) is a common oral mucosal disease, yet effective therapeutic approaches are lacking. This study aimed to determine the effects of application of berberine gelatin in the treatment of minor RAS (MiRAS). Methods. A randomized, double-blind, placebo-controlled, clinical trial was performed. The gelatin containing berberine (5 mg/g) or vehicle only was applied 4 times per day for 5 days. Clinical evaluation included pain level, size, erythema, and exudation of certain ulcers on days 1, 2, 4, and 6. Results. A total of 84 subjects fulfilled the study without obvious side effects. Berberine gelatin treatment reduced the ulcer pain score compared with placebo gelatin (P ⬍ 0.05). Ulcer size was significantly reduced (P ⬍ 0.05) and lower erythema (P ⬍ 0.05) and exudation (P ⬍ 0.05) levels were associated with berberine treatment. Conclusions. Berberine gelatin may be a safe and effective treatment for MiRAS. (Oral Surg Oral Med Oral Pathol Oral Radiol 2013;115:212-217)
Recurrent aphthous stomatitis (RAS), which clinically manifests as distinct and shallow ulceration with a necrotic center covered by a pseudomembrane and surrounded by an erythematous halo, is among the most prevalent oral mucosal lesions affecting the general population.1 Minor RAS (MiRAS) is the most common form, consisting of 70% to 87% RAS.2,3 Although RAS is an episodic and self-limited affection, the ulceration usually severely interferes with eating, speaking, and swallowing. Because no definitive etiology has been identified, there is no uniformly effective and reliable therapy in the termination of this potentially debilitating disorder.3 Available systemic or topical treatments currently are aimed at alleviating pain, decreasing functional disability, and promoting ulcer healing. Some systemic medications, although effective, have side effects that limit their extensive or long-term application. Therefore, topical agents (including glucocorticoids, antibiotics, analgesics, astringents, and laser therapy) remain the mainstay of therapy.3 Competing interests. The authors state that they have no conflict of interest. a Department of Stomatology, the First People’s Hospital of Chenzhou, Chenzhou 423000, Hunan, China. b Institute of Translation Medicine on University of South China, Chenzhou 423000, Hunan, China. c Department of Neurology, the First People’s Hospital of Chenzhou, Chenzhou 423000, Hunan, China. Received for publication July 11, 2012; returned for revision Sept 2, 2012; accepted for publication Sept 9, 2012. © 2013 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter http://dx.doi.org/10.1016/j.oooo.2012.09.009
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Berberine, an isoquinoline alkaloid originally isolated from medicinal herbs, such as Hydrastis canadensis, Coptidis rhizoma, and Berberis vulgaris, is an antimicrobial drug routinely prescribed in traditional Eastern medicine.4 Recently, several investigations have suggested that berberine possesses a wide range of pharmacologic and biological activities, including antiinflammatory,5-7 antimicrobial,8 antitumor,9 and antipyretic10 properties. We hypothesized that berberine may be an effective and favorable treatment candidate for MiRAS because of its multiple bioactivities. The objective of this randomized, double-blind, placebocontrolled clinical trial was to investigate the efficacy and safety of berberine gelatin in the treatment of MiRAS.
MATERIAL AND METHODS Materials Berberine gelatin (10 g) containing 50 mg berberine (98% HPLC, Biopurify Science and Technology Development Co. Ltd., Chengdu, China), hydroxypropyl methyl cellulose, polyethylene glycol 400, glycerine, and flavoring additives was used. The placebo agent (10 g) contained the above-mentioned excipients with
Statement of Clinical Relevance In this placebo-controlled, double-blind trial, berberine gelatin was effective in alleviating pain intensity and promoting oral aphthous healing without notable side effects.
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the exception of berberine and had a similar shape, color, and flavor. All gelatin, packed in soft tubes, was produced by the pharmaceutical department of the First People’s Hospital of Chenzhou, University of China South. Subjects and study design This randomized, double-blind, placebo-controlled clinical trial was conducted under the approval of the Institutional Ethics Committee/Institutional Review Board of the First People’s Hospital of Chenzhou, University of South China. The clinical diagnosis was based on the patients’ medical history and clinical appearance.11 To exclude potentially confounding systemic diseases, all patients underwent careful examination by dermatologists, gastroenterologists, ophthalmologists, and hematologists before enrollment. All patients were selected according to inclusion and exclusion criteria. The inclusion criteria were as follows: (1) male or female individuals aged 18 to 50 years, who could exactly follow the study protocol and sign the informed consent form voluntarily; (2) a history of RAS for a minimum of 6 months with an average of 1 to 2 outbreaks per month and 1 to 5 minor aphthae per outbreak; (3) agreement not to use analgesics or other drugs curing oral ulcer during the study; (4) 1 to 3 aphthous ulcerations with a size ⬎5 and ⬍10 mm in diameter; for more than 1 ulceration, a distance between ulcerations of more than 1 cm; (5) time of ulceration onset within 48 hours; and (6) ulcers in locations that are easily accessible for evaluation and treatment. Exclusion criteria were as follows: (1) history of allergies to berberine; (2) pregnancy or lactation; (3) ulcers as a manifestation of a systemic disease process such as Behçet’s disease, ulcerative colitis, Crohn’s disease, or acquired immune deficiency syndrome; (4) taking systemic nonsteroidal anti-inflammatory drugs, immunomodulatory agents, or systemic antibiotics within 2 weeks; (5) treatment with any oral topical medication or invasive dental procedures within 2 weeks; (6) normal resolution of aphthous ulcers expected in ⬍5 days without any treatment; (7) use of toothpaste containing anti-inflammatory drugs; (8) ulceration secondary to hematinic deficiency, vitamin deficiency, or nutritional deficiency; and (9) ulceration caused by drugs such as nicorandil or methotrexate. Patients were then assigned to a berberine group or a control group using a computer-generated random numbers list. Both the investigators and the participants were blind to the group assignment until study completion. In this trial, only 1 ulcer was selected for analysis. For patients with more than 1 ulcer, the most recent ulcer was selected. The pain level was evaluated by the
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Table I. Classification of erythema and exudation levels Classification
Erythema
Exudation
0 1 2 3
No erythema Light red/pink Red but not dark in color Very red, dark in color
No exudation Light exudation Moderate exudation Heavy exudation, with pseudomembrane
subjects following instructions by the investigators, and the size of the ulcers and the erythema and exudation were measured and recorded by the investigator before the first administration and at each evaluation thereafter. To evaluate pain, a visual analog scale consisting of a 100-mm horizontal line between the poles of “no pain” to “unbearable pain” was used. Subjects were instructed to mark the line with a vertical line at the point that best represented the present pain level of the appointed ulcer. Then, the length between the vertical line and the pole representing no pain was measured and recorded to represent the ulcer pain score. To measure the size of the ulcers, the investigators measured the distance between 2 opposite outside edges of the ulcer border with millimeter markings on a calibrated dental probe. The 2 measurements approximately 90 degrees from each other were obtained 3 times, and the largest distance was used as 1 of the measurements. The 2 measurements were then multiplied to represent the cross-sectional area of the ulcer. The degree of erythema and exudation was evaluated and recorded by the investigators on a 4-point scale (Table I) ranging from 0 to 3 based on the methods of Liu et al.12 and Babaee et al.13 All subjects were instructed to dry the ulcer by patting it with clean gauze, squeeze 1 cm of the drug onto a sterile cotton swab, and dab the gelatin onto the ulcer. They were observed for 30 minutes for any possible signs of acute hypersensitivity reactions or other adverse events at the first administration and advised to avoid drinking or eating in the 30 minutes after each administration. The subjects were requested to apply the drug 4 times a day (after meals and before bedtime) for 5 days. The baseline parameters were taken and recorded at the first visit (day 1). The evaluations were made at the same time on the morning of days 2, 4, and 6 before gelatin administration. Participants were requested to bring the soft tube containing the gelatin to every visit to ensure the drugs were used properly. Further, periodic telephone interviews were made to supervise the administration on schedule. Side effects after gelatin administration were recorded, and the oral mucosa was inspected by the investigator at each visit. Subjects were also informed that possible side effects may occur, at which time they should
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terminate use of the gelatin and inform the investigator immediately. To compare the therapeutic effect statistically at different time points, the data were calculated using the following formula [(visit 1–visit 2, visit 4 or visit 6)/visit1] ⫻100% (visitx referring to the values measured at day x) according to previous studies.14 Statistical methods Background and demographic data were summarized with descriptive statistics. Group differences between the berberine group and the control group were evaluated. Comparisons of ulcer history and baseline values of ulcers were performed with the Mann–Whitney test. To evaluate efficacy, the 2 test and the Mann–Whitney test were used. All data were analyzed by SPSS software (SPSS 13.0 for Windows; SPSS, Chicago, IL) and the level of significance was established at a P value less than 0.05.
RESULTS A total of 87 subjects were recruited in this study. Only 2 subjects in the berberine group and 1 subject in the control group dropped out because of a conflicting schedule and not following the instructions. We evaluated demographic and efficacy data without those subjects, which did not influence the interpretation or conclusions. Therefore, 42 subjects in the berberine group and 42 subjects in the control group fulfilled the study.
Table II. Sociodemographic and basic clinical characteristics of the population
Age (years) Mean ⫾ SD Range Gender Male Female Education Primary school High school College graduate Tobacco Nonsmoker Smoker Allergy history Yes No Ulcer location Cheek Tongue Lip Number of ulcers 1 2 3
Berberine group (n ⫽ 42) (n, %)
Control group (n ⫽ 42) (n, %)
30.86 ⫾ 7.53 19-51
31.19 ⫾ 6.00 20-45
0.638
14 (33.33) 28 (66.67)
10 (23.81) 32 (76.19)
0.337
1 (2.38) 24 (57.14) 17 (40.48)
2 (4.76) 28 (66.67) 12 (28.57)
0.226
4 (9.52) 38 (90.48)
3 (7.14) 39 (92.86)
0.695
0(0) 42 (100)
0(0) 42 (100)
1
18 (42.86) 8 (19.04) 16 (38.10)
24 (57.14) 4 (9.52) 14 (33.34)
0.316
30 (71.43) 10 (23.81) 2 (4.76)
27 (88.10) 8 (9.52) 7 (2.38)
0.328
P
Demographics The demographics of the berberine group and the control group were similar, including age, gender, known allergies, and baseline values of ulcer location and number (Table II). The mean age of the subjects was approximately 31 years. Of the subjects, 71.4% were female and 28.6% were male. Moderation of pain The mean ulcer pain scores of the 2 groups matched well (P ⬎ 0.05) at trial entry, but later significant relief was observed in the berberine group (Figure 1). On day 2, the ulcer pain score of the berberine group decreased by 16.1%, which was greater than that (6.56%) in the control group (P ⬍ 0.05). On day 4, the reduction in ulcer pain score of the berberine group was much greater than that of the control group (44.57% vs 22.60% P ⬍ 0.02). On day 6, pain moderation in the berberine group was 76.65%, which was greater than that (60.87%) in the control group (P ⬍ 0.03). Reduction in ulcer size There was no significant difference between the mean ulcer size of the 2 groups at study entry (P ⬎ 0.05).
Fig. 1. Comparison of ulcer pain score between the berberine and control groups. *Ulcer pain score improvement in the control group was significantly less than in the berberine group, P ⬍ 0.05. #Ulcer pain score in the berberine group did not differ from that in the control group, P ⬎ 0.05.
However, significant group differences appeared at subsequent visits (Figure 2). On day 2, the ulcer size in the berberine group decreased by 35.63%, which was greater than the decrease (14.14%) in the control group (P ⬍ 0.01). On day 4, the ulcer size in the berberine group decreased by 54.07%, which was significantly different from the control group (37.66%, P ⬍ 0.03). On day 6, the berberine group maintained a significantly greater decrease in ulcer
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Fig. 2. Comparison of mean ulcer size between the berberine and control groups. *Ulcer size reduction in the control group was significantly less than in the berberine group, P ⬍ 0.05; # Ulcer size in the berberine group did not differ from that in the control group, P ⬎ 0.05.
size than the control group (70.56% vs 54.35%; P ⬍ 0.005). Reduction in erythema and exudation Both the erythema and the exudation levels improved in the 2 groups (Table III). The group difference demonstrated no statistical significance on day 1 and 2 visits (P ⬎ 0.05, P ⬎ 0.05, respectively); however, the berberine group exhibited significantly lower erythema (P ⬍ 0.01, P ⬍ 0.01, respectively) and exudation (P ⬍ 0.04, P ⬍ 0.05, respectively) levels than the control group on day 4 and 6 visits. Safety evaluation No obviously adverse side effect such as hypersensitivity, infection, or taste sense malfunction occurred. All patients tolerated the gelatin well with a good rating.
DISCUSSION For the treatment of MiRAS, several topical and systematic agents have been adopted. However, efficacious and safe therapy in termination of the potentially debilitating disorder is limited.3 Therefore, MiRAS remains a challenge for stomatological specialists. Recently, many research-based pharmaceutical companies have been investing time and money in the development of medicinal natural products as a method for addressing the drug resistance and cost-effectiveness of synthetic drugs.15 Berberine is an isoquinoline alkaloid present in some herb materials and has proven to have a range of pharmacologic and biological activities. In this population, by a mechanism that remains unclear, berberine gelatin is associated with relief of pain at-
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tacks and the promotion of healing without obvious side effects. The precise etiology of RAS remains unknown. The inflammatory process plays an important role in RAS, and neutrophils and mononuclear cell infiltration in the lamina propria are histologic characteristics.16,17 Also, focal immune dysfunction exerts a significant role.18,19 Although it is unlikely that oral bacteria play a primary pathogenic role in the development of RAS, the presence of abnormal oral flora was associated with an aberrant immune and inflammation response.18,20 Because of the potential for multiple etiologies and pathophysiologies, the optimal therapeutic agent should posses multiple bioactivities. Berberine was selected as a possible candidate treatment for MiRAS in this study because of its known anti-inflammation, antimicrobe, and immunomodulation properties. The literature is promising with regard to the direct anti-inflammation properties of berberine. Li et al.21 and Cui et al.22 demonstrated that berberine could diminish inflammatory cell infiltration in animal models. Additional studies9,23,24 reported that berberine could repress proinflammatory responses through activated protein kinase activation in macrophages; downregulate the expression of proinflammatory genes (tumor necrosis factor-␣, interleukin [IL]-1, IL-6, and so on); and upregulate IL-10, which exerted the main negative regulatory feedback on Th1 cytokine production. Berberine was also demonstrated to possess significant antimicrobial activity against a wide range of microorganisms, including Staphylococcus, Streptococcus, Candida, and Salmonella.25,26 Furthermore, berberine is an immunosuppressive that demonstrates a safe drug profile.27,28 Although the exact mechanisms of berberine in the treatment of MiRAS remain to be clarified, the multiple bioactivities may be beneficial in accelerating pain relief and ulcer healing. Interestingly, some curative effect for pain relief was also observed in the control group, which may be because the placebo gelatin provides a protective film to isolate the physical and chemical stimuli and promote the healing process. The double-blind experiment protocol may also cause some beneficial psychotherapy effect. The berberine gelatin could adhere to the oral mucosa to release berberine slowly at the site of the ulcer, resulting in higher drug concentrations and a longerlasting release period. Berberine is easily accessible in China at a low cost. The gelatin is also easy for patients to administer and carry. In view of the many drawbacks of synthetic compounds used as topical agents, berberine, which has a plant origin and no obvious side effects, would be a promising candidate in the treatment of MiRAS.
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Table III. Changes in erythema and exudation Berberine group (n ⫽ 42) Erythema Day 1 Day 2 Day 4 Day 6 Exudation Day 1 Day 2 Day 4 Day 6
Control group (n ⫽ 42)
0
1
2
3
0
1
2
3
P*
0 10 26 31
0 10 13 9
31 17 2 2
9 5 1 0
0 6 10 16
3 5 10 15
29 23 15 8
10 8 7 3
⬎0.05 ⬎0.05 ⬍0.01 ⬍0.01
0 9 24 30
3 11 13 9
27 14 3 2
12 8 2 1
0 9 17 22
4 9 10 10
25 15 9 7
13 9 6 3
⬎0.05 ⬎0.05 ⬍0.04 ⬍0.05
*Mann–Whitney test.
In conclusion, the present study findings demonstrate that the topical application of berberine gelatin could decrease pain intensity and promote ulcer healing with minimal safety concerns. Therefore, in clinical practice, berberine gelatin could be a well-tolerated and safe addition to the treatment armamentarium for MiRAS. This study was supported by the Scientific Research Project of Xiangnan University and the First People’s Hospital of Chenzhou, University of South China, and the Science and Technology Bureau Research Project of Chenzhou Municipal. We gratefully acknowledge Drs. Jiali Tan, Dunting Zhang, and Haiwen Zhou as investigators.
11.
12.
13.
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ORIGINAL ARTICLE Jiang et al. 217 imental autoimmune encephalomyelitis by natural product compound berberine. J Immunol 2010;185:1855-63. 28. Kondo Y, Imai Y, Hojo H, Hashimoto Y, Nozoe S. Selective inhibition of T-cell-dependent immune responses by bisbenzylisoquinoline alkaloids in vivo. Int J Immunopharmacol 1992;14: 1181-6. Reprint requests: Xiao-Wen Jiang, DDS, PhD Department of Stomatology The First People’s Hospital of Chenzhou 102 Luojiajing Road, Beihu District Chenzhou 423000, Hunan Province, China [email protected]