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Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment
Complementary Therapies in Medicine (2008) 16, 262—267
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/ctim
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment夽 Supanee Rassameemasmaung a,∗, Anongporn Sirikulsathean a, Cholticha Amornchat b, Pawinee Maungmingsook c, Pleumchitt Rojanapanthu d, Wandee Gritsanaphan e a
Department of Oral Medicine, Faculty of Dentistry, Mahidol University, Ratchathewi, Bangkok 10400, Thailand Department of Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand c Nakornpathom Hospital, Nakornpathom, Thailand d Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand e Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand Available online 25 April 2008 b
KEYWORDS Garcinia mangostana L.; Local drug delivery; Periodontal treatment
Summary Objective: To evaluate the effects of gel containing Garcinia mangostana L. pericarp extract (GM gel) applied topically as an adjunct to periodontal treatment. Design: Subjects who had periodontal pockets on their single-rooted teeth were randomized into the test or control group. Subjects in the test group received periodontal treatment consisting of scaling, root planing and subgingival application of GM gel while those in the control group received scaling and root planing without GM gel application. Setting: Mahidol University, Faculty of Dentistry, Thailand. Main outcome measures: Clinical parameters included probing pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), Gingival Index (GI) and Plaque Index (PI). Microbiological parameter included subgingival microbial composition as examined by phase contrast microscopy. Results: Clinical improvement compared to baseline was found in both groups (P < 0.05). The test group exhibited significantly higher reduction in mean PPD, GI and BOP than the control group at the 3rd month after treatment (P < 0.05). Subgingival microbial composition changed from diseased state to that compatible with health after treatment in both groups. However, significant differences between groups were found only in the mean percentage of cocci at the 1st and 3rd month after treatment (P < 0.05). Conclusions: GM gel could enhance the clinical effects of periodontal treatment. © 2008 Elsevier Ltd. All rights reserved.
Introduction 夽 This work was financially supported by Mahidol University Research Grant. ∗ Corresponding author. Tel.: +66 22036500; fax: +66 23548510. E-mail address: [email protected] (S. Rassameemasmaung).
Periodontitis is the common oral disease affecting many people around the world. It is defined as an inflammation and progressive destruction of the tooth-supporting structures (periodontium). This disease results from interaction
0965-2299/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctim.2007.12.004
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment between specific host defense mechanisms and dental plaque biofilms that colonize on the tooth surfaces at or below the gingival margin. The progression of periodontitis can be arrested by mechanical debridement consisting of scaling, root planing and proper oral hygiene control.1 These treatment modalities aim to remove dental plaque and plaque-retentive factors. However, pathogenic bacteria may not be eliminated in the deep periodontal pockets due to poor access for mechanical debridement, root anatomical complexity2,3 and the ability of the bacteria to invade and reside in the periodontal tissues4 or dentinal tubules.5 The recognition of bacterial plaque as the main etiologic factor has led to increasingly antimicrobial agent intervention. Antimicrobial agents may gain access into the periodontal pockets through both systemic and local route of delivery. Systemic antimicrobial agents can be used to treat multiple sites simultaneously and may target invasive organisms and affect reservoirs of bacteria in the oral cavity.6 However, systemic antimicrobial agents may lead to potential side effects such as development of resistant bacteria7 and gastrointestinal intolerance.8 These drawbacks would be markedly reduced if antimicrobial agents applied locally could be used. For the local antimicrobial agent to be useful, it must be successfully delivered to the base of the periodontal pockets at an efficacious concentration and retain in the pockets for an adequate length of time.9 To achieve these, the sustain-released delivery drugs such as minocycline ointment10 or metronidazole gel11 had been used. It was found that mechanical debridement plus local delivery of these agents produced more favorable outcomes, for example, the reduction of periodontal pocket depth and the number of inflamed gingival sites, than those without local drug delivery.10,11 The use of herbal medicine as an alternative approach has gained much interest nowadays. Garcinia mangostana L., known as the mangosteen tree, contains various compounds such as chrysanthemin, garcinone A, B and C, sesquiterpenoids, gartanin, fructose, sucrose, tannins, xanthones and their derivatives in its pericarp.12 Mangostin could inhibit penicillin-resistant strain of Staphylococcus aureus with an MIC of 3.125 g/ml.13 ␥-Mangostin also inhibited growth of Helicobacter pyroli with the MIC of 1.56 g/ml.14 The antimicrobial screening test performed at our laboratory demonstrated that the 80% ethanolic extract from the pericarp of mangosteen inhibited growth of Porphyromonas gingivalis W50, the main periodontopathic bacteria, at the MIC of 3.91 mg/ml. Other than the antimicrobial activity, mangostin, 1-isomangostin and mangostin triacetate exhibited an anti-inflammatory activity.15 ␥-Mangostin also showed a potent inhibitory activity against prostaglandin E2 released.16 Due to its antimicrobial and anti-inflammatory activity, we hypothesized that topical application of gel containing G. mangostana L. pericarp extract (GM gel) could inhibit bacterial growth and facilitate wound healing after periodontal treatment. Thus, this study aimed to evaluate the clinical and microbiological effects of the gel when used as an adjunct to periodontal treatment.
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Methods Subject selection Before experiment, the study protocol was approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand. This study was performed at the Faculty of Dentistry, Mahidol University. Subjects, aged between 35 and 60 years, were the outpatients of the Department of Oral Medicine, Faculty of Dentistry, Mahidol University. They were systemically healthy and were diagnosed as chronic periodontitis. They had at least two sites with probing pocket depth (PPD) of 5—6 mm and other two sites with PPD of 7—9 mm on their single-rooted teeth. The selected teeth had no endodontic complication. The selected sites must have a radiographic evidence of alveolar bone loss and had bleeding upon probing or suppuration. Subjects were excluded from the study if they were pregnant, lactated, smoker, used antibiotic within the previous 3 months, and received periodontitis treatment within the previous 6 months. Subjects who met the above criteria were explained about the objectives and the details of the study. Those who were willing to participate were asked to sign the informed consent forms before entering the study. After screening, subjects were randomized into the test or control group. Subjects in the test group received mechanical debridement consisting of scaling, root planing and subgingival application of GM gel while those in the control group received mechanical debridement without GM gel application.
GM gel The crude extract from the pericarp of mangosteen was formulated as a local delivery drug (G. mangostana gel or GM gel) by the Department of Pharmaceutical Technology, Faculty of Pharmacy, Mahidol University, Thailand. The concentration of GM gel was adjusted according to the antimicrobial screening test performed at our laboratory. The gel was tested for short term toxicity by the National Laboratory Animal Center, Mahidol University. It was suggested that the gel could be safely used according to the research regimen. GM gel was kept at 4 ◦ C throughout the study period.
Clinical parameters Probing pocket depth (PPD) and clinical attachment level (CAL) were measured from the selected sites (Fig. 1) using a standard periodontal probe (PCPUNC 15, Hu FriedyTM , IL, USA) and recorded to the nearest millimeter. Bleeding on probing (BOP) was recorded as the presence or absence of bleeding upon probing.17 Gingival Index (GI)18 was measured as follows: 0 = normal gingiva; 1 = mild inflammation, slight change in color; 2 = moderate inflammation, redness, edema and glazing; 3 = severe inflammation, marked redness and edema, ulcerations. Plaque Index (PI)19 was then measured using the following criteria: 0 = no plaque in the gingival area; 1 = a film of plaque adhering to the free gingival margin and adjacent area of the tooth. The plaque may be rec-
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Figure 2 GM gel was delivered into the periodontal pocket by a blunted-tip needle.
Figure 1 Representative illustration showing the placement of a standard periodontal probe into the periodontal pocket and the reference points for the measurement of probing pocket depth and clinical attachment level. A = cemento-enamel junction, B = gingival margin, C = base of the periodontal pocket, A—C = clinical attachment level, B—C = probing pocket depth.
ognized only by running a probe across the tooth surface; 2 = moderate accumulation of soft deposits within the gingival pocket and on the gingival margin and/or adjacent tooth surface that can be seen by the naked eye; 3 = abundance of soft matter within the gingival pocket and/or on the gingival margin and adjacent tooth surface.
Microbiological procedure The experimental teeth were isolated with cotton roll. Tooth surface above the gingival margin was cleaned and dried with an air stream. Subgingival plaque sample from each selected site was collected with three consecutive sterile paper points. The paper points were inserted into the pocket until resistance was met and kept in place for 20 s. After removing from the pocket, the paper points were immediately pooled into a sterile tube containing 1 ml of pre-reduced transport fluid. The sample was then shaken for 10 s. Bacterial suspension was examined under phase contrast microscopy. At least 200 bacteria from a random field were observed at a magnification of ×400 and classified into one of the following groups, i.e., cocci, motile rods, spirochetes and other bacteria.20 Baseline clinical and microbiological examination was performed on the experimental teeth by a blinded examiner who had been calibrated until the intra-examiner error was less than 10%. Full mouth scaling and root planing was performed in all subjects until the operator felt that the root surfaces were smooth. In the test group, GM gel was delivered into the periodontal pockets of the selected teeth
by a 23-gauge, blunted-tip needle until the gel was detected at the gingival margin (Fig. 2). GM gel was repeatedly delivered at 1 week later. Subjects were informed not to drink, eat or rinse within 1 h after gel delivery. Subjects in both groups performed their usual oral hygiene procedures but were not allowed to use any chemotherapeutic mouthrinse or oral irrigator. Clinical and microbiological parameters were re-evaluated at the 1st and 3rd month after the 1st gel application. Clinical adverse experiences were recorded at each follow-up visit.
Statistical analysis Data analysis was achieved by statistical software SPSS for Windows. An individual site was a unit of analysis. For mean PPD, CAL, GI and PI, Wilcoxon match-pairs signed rank test was used for within group comparison and Mann—Whitney U-test was used for between group comparison. Microbiological data were analyzed by paired t-test for within group comparison and by independent t-test for between group comparison. BOP was analyzed by McNemar’s test for within group comparison and by Chi-square test for between group comparison. The level of significance was considered at P < 0.05.
Results Thirty-one subjects completed the study. At baseline, no significant difference between groups was observed for any
Table 1
Subject characteristics at baseline
Characteristics
Test
Control
Numbers of subjects (male, female) Age (year) Mean age (year) Number of sites with initial PPD 5—6 mm Number of sites with initial PPD 7—9 mm
16 (8, 8)
15 (6, 9)
37—60 47.31 32
36—57 45.8 30
32
30
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment Table 2
265
Mean ± standard deviation of probing pocket depth (mm) and clinical attachment level (mm)
Site
Parameter
Interval
Group Test
Sites with initial PPD 5—6 mm
Sites with initial PPD 7—9 mm
Control
PPD
Baseline 1st month 3rd month
5.59 ± 0.50 4.56 ± 0.72* 3.91 ± 0.86*,†
5.77 ± 0.43 4.77 ± 0.57* 4.63 ± 0.61*
CAL
Baseline 1st month 3rd month
9.06 ± 0.72 8.22 ± 0.66* 8.06 ± 0.76*
8.77 ± 1.33 8.00 ± 1.34* 7.90 ± 1.27*
PPD
Baseline 1st month 3rd month
7.69 ± 0.82 6.09 ± 0.89*,† 5.41 ± 0.67*,†
8.00 ± 0.79 6.70 ± 0.92* 6.10 ± 0.80*
CAL
Baseline 1st month 3rd month
11.13 ± 1.43 10.09 ± 1.42* 9.47 ± 1.29*
10.57 ± 1.01 9.70 ± 1.06* 9.03 ± 1.03*
PPD = probing pocket depth, CAL = clinical attachment level. * Significantly different compared to baseline (P < 0.05). † Significantly different between groups (P < 0.05).
subject characteristics (Table 1). For subjects in the test group, neither complaint nor adverse tissue reaction was found after gel application.
Probing pocket depth (PPD) and clinical attachment level (CAL) At baseline, PPD and CAL were not significantly different between groups (Table 2). At all follow-up visits, PPD and CAL were significantly different when compared to baseline in both groups. For the sites with an initial PPD of 5—6 mm, significant differences in PPD between groups were found at the 3rd month. For the sites with an initial PPD of 7—9 mm, significant differences in PPD between groups were found at the 1st and 3rd month. Regarding CAL, no significant difference was found between groups at the follow-up visits.
Gingival Index (GI) At baseline, all selected sites exhibited moderate gingival inflammation (GI of 2; Table 4). After treatment, the improved gingival status was observed. The percentage of sites with GI of 2 was significantly lower while those with GI of 0 and 1 were significantly higher when compared to baseline in both groups. When compared between groups, significant differences were found at the 3rd month.
Plaque Index (PI) At baseline, the majority of sites exhibited PI of 1 and 2 (Table 5). At the follow-up visits, the improved oral hygiene status was observed in both groups. When compared to baseline, significant differences were found at all follow-up visits in both groups. However, when compared between groups, no significant difference was found.
Bleeding on probing (BOP) At baseline, all sites were bled upon probing (Table 3). As study progressed, percentage of bleeding sites was significantly reduced in both groups. When compared between groups, significant difference was found at the 3rd month.
Table 3 Interval
Baseline 1st month 3rd month * †
Table 4 Number of sites and percentage distribution of Gingival Index (GI) GI
Test (n = 64)
Control (n = 60)
64 (100.00%) 45 (70.30%)* 30 (46.90%)*,†
60 (100.00%) 49 (81.70%)* 39 (65.00%)*
Significantly different compared to baseline (P < 0.05). Significantly different between groups (P < 0.05).
Group Test (n = 64)
Control (n = 60)
0
Baseline 1st month 3rd month
0 (0%) 6 (9.40%)* 25 (39.10%)*,†
0 (0%) 5 (8.30%)* 13 (21.73%)*
1
Baseline 1st month 3rd month
0 (0%) 14 (21.90%)* 9 (14.10%)*,†
0 (0%) 8 (13.30%)* 6 (10.00%)*
2
Baseline 1st month 3rd month
64 (100.00%) 44 (68.80%)* 30 (46.90%)*,†
60 (100.00%) 47 (78.30%)* 41 (68.30%)*
Number and percentage of bleeding sites Group
Interval
* †
Significantly different compared to baseline (P < 0.05). Significantly different between groups (P < 0.05).
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Table 5 Number of sites and percentage distribution of Plaque Index (PI) PI
Interval
Group Test (n = 64)
Control (n = 60)
0
Baseline 1st month 3rd month
4 (6.30%) 15 (23.40%)* 18 (28.10%)*
10 (16.70%) 12 (20.00%)* 22 (36.70%)*
1
Baseline 1st month 3rd month
39 (60.90%) 40 (62.50%)* 38 (59.40%)*
31 (51.70%) 39 (65.00%)* 33 (55.00%)*
2
Baseline 1st month 3rd month
21 (32.80%) 9 (14.10%)* 8 (12.50%)*
19 (31.70%) 9 (15.00%)* 5 (8.30%)*
*
Significantly different compared to baseline (P < 0.05).
Microbiological evaluation After treatment, mean percentage of cocci was significantly increased while those of motile rods, spirochetes and other bacteria were significantly decreased when compared to baseline in both groups (Table 6). When compared between groups, significant differences were found only in the mean percentage of cocci at the 1st and 3rd month.
Discussion The local delivery of antimicrobial agent has gained an increasing interest in the field of periodontal treatment. The aim of the present study was to compare the clinical and microbiological effects of mechanical debridement plus local delivery of GM gel to that of mechanical debridement alone. In this study, blinding was reduced because of no placebo gel in the control group. However, the use of placebo gel may introduce confounding influences of a placebo design into the study.10
Table 6 Interval
From the clinical results, the test group showed an impressive effect on the reduction of pocket depth and gingival inflammation but less pronounced effect on CAL gain. Regarding the pocket depth, PPD reduction was higher in the sites with deep pockets (initial PPD of 7—9 mm) than those with shallow pockets (initial PPD of 5—6 mm). Significant PPD reduction was detected in the test group at the 1st and 3rd month in the sites with deep pockets, and only at the 3rd month in the sites with shallow pockets. This indicated the benefit of scaling, root planing and GM gel application in the reduction of pocket depth over scaling and root planing alone especially in the sites with deep periodontal pockets. Significant gain in CAL as compared to baseline were found in both groups. However, no significant differences of the CAL were found between groups. CAL gain may be due to the decreased gingival inflammation which lead to an increased gingival tissue adaptation and thus a reduction of probe penetration.21 Regarding the GI and BOP, the test group showed a significant improvement in GI and BOP when compared to the control group at the 3rd month. Our result showed that the adjunctive treatment with GM gel could reduce 53% of bleeding sites at the 3rd month and this was comparable to those obtained from other studies.22,23 In those studies, the reduction of bleeding sites was 68% and 53% after adjunctive treatment with minocycline ointment22 and metronidazole gel,23 respectively. PI was also improved after treatment in both groups. However, no significant differences were found when compared between groups. This demonstrated that GM gel provided a more beneficial effect on the reduction of gingival inflammation but not the reduction of plaque. Due to an anti-inflammatory activity, recent investigation reported that the mangostin and its derivatives had an effect on prostaglandin synthesis.16 When prostaglandin level was reduced, the improvement of periodontal-associated parameters could be occurred.24,25 It is possible that GM gel could modulate host inflammatory response. Tannins from the pericarp of mangosteen might also attribute to the PPD reduction due to their astringent property which is usu-
Percentage distribution of bacteria in each group Bacterial group
Group Test (n = 64)
22.61 39.30 13.97 24.13
± ± ± ±
4.61 4.07 6.20 4.09
46.05 ± 4.50*,† 27.62 ± 3.56* 6.21 ± 3.34* 20.18 ± 3.54*
43.68 28.68 7.20 20.39
± ± ± ±
5.88* 3.89* 4.70* 3.06*
48.24 ± 4.56*,† 27.99 ± 3.85* 4.60 ± 2.37* 19.32 ± 2.92*
45.92 28.87 5.11 20.30
± ± ± ±
4.54* 2.90* 2.70* 2.92*
Baseline
Cocci Motile rods Spirochetes Other
24.12 39.85 13.58 22.86
1st month
Cocci Motile rods Spirochetes Other
3rd month
Cocci Motile rods Spirochetes Other
* †
Significantly different compared to baseline (P < 0.05). Significantly different between groups (P < 0.05).
± ± ± ±
Control (n = 60)
4.21 4.17 6.50 3.70
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment ally accompanied by contraction and wrinkling of the tissue as well as their vasoconstrictor effect which reduces local edema, exudation and inflammation. As tissue shrinkage naturally occurs after scaling and root planing, the additional PPD reduction observed in the test group might likely to be facilitated by the anti-inflammatory effect of mangostin and its derivatives as well as the astringent property of tannins. In terms of the microbiological observation, baseline subgingival bacterial counts revealed low proportion of cocci but high proportions of motile rods, spirochetes, and other bacteria in both groups. After treatment, the compositions of subgingival microflora were compatible to that generally observed in healthy periodontal condition.26 In our study, a continuous increase in the percentage of cocci was observed, while the percentages of motile rods, spirochetes, and other bacteria decreased over the 3-month period. At all follow-up visits, the test group showed a statistically higher mean percentage of cocci. The results from this study did not obviously show a difference between scaling and root planing with and without GM gel in changing composition of subgingival microflora. It is possible that the phase-contrast microscopy used in our study might not be sensitive enough to detect any apparent microbial changes after treatment. It is also possible that scaling and root planing could not disrupt dental plaque biofilms because of some limitations, and thus the concentration of GM gel might not effectively cope with the periodontopathic bacteria. In conclusion, this randomized, controlled, single-blinded study suggested superior clinical benefits of GM gel when used as an adjunct to scaling and root planing in periodontitis subjects. The greater reduction in probing depth, gingival inflammation and bleeding on probing observed in the test group might be facilitated by an anti-inflammatory activity of the gel.
Conflict of interest The authors had no potential conflict of interest.
References 1. Isidor F, Karring T. Long-term effect of surgical and non-surgical periodontal treatment. A 5-year clinical study. J Periodont Res 1986;21:462—72. 2. Caffesse RG, Sweeney PL, Smith BA. Scaling and root planing with and without periodontal flap surgery. J Clin Periodontol 1986;13:205—10. 3. Nordland P, Garrett S, Kiger R, Vanooteghem R, Hutchens LH, Egelberg J. The effect of plaque control and root debridement in molar teeth. J Clin Periodontol 1987;14:231—6. 4. Sandros J, Papapanou P, Dahlen G. Porphyromonas gingivalis invades oral epithelial cells in vitro. J Periodont Res 1993;28:219—26. 5. Adriaens PA, Edwards CA, De Boever JA, Loesche WJ. Ultrastructural observations on bacterial invasion in cementum and radicular dentin of periodontally diseased human teeth. J Periodontol 1988;59:493—503. 6. Christersson LA, Zambon JJ. Suppression of subgingival Actinobacillus actinomycetemcomitans in localized juvenile periodontitis by systemic tetracycline. J Clin Periodontol 1993;20:395—401.
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7. Kornman KS, Karl EH. The effect of long-term low-dose tetracycline therapy on the subgingival microflora in refractory adult periodontitis. J Periodontol 1982;53:604—10. 8. Loesche WJ, Grossman N, Giordano J. Metronidazole in periodontitis. IV. The effect of patient compliance on treatment parameters. J Clin Periodontol 1993;20:96—104. 9. Goodson JM. Controlled drug delivery: a new means of treatment of dental diseases. Compend Contin Educ Dent 1985;6:27—32, 35-6. 10. Lu HK, Chei CJ. Efficacy of subgingivally applied minocycline in the treatment of chronic periodontitis. J Periodont Res 2005;40:20—7. 11. Griffiths GS, Smart GJ, Bulman JS, Weiss G, Shrowder J, Newman HN. Comparison of clinical outcomes following treatment of chronic adult periodontitis with subgingival scaling or subgingival scaling plus metronidazole gel. J Clin Periodontol 2000;27:910—7. 12. Fansworth NR, Bunyapraphatsara N. Thai medicinial plants recommended for primary health care system. Bangkok: Prachachon; 1992. 13. Phongpaichit S, Ongsakul M, Nilrat L, Tharavichitkul P, Bunchoo S, Chuaprapaisilp T. Antibacterial activities of extracts from Garcinia mangostana pericarps on methicillin-resistant Staphylococcus aureus and Enterococcus species. Songklanakarin J Sci Technol 1994;16:399—405. 14. Hasegawa H, Sakai S, Aimi N, Takayama H, Koyano T. Helicobacter pylori inhibitors containing xanthones from Garcinia mangostana. Patent: Jpn Kokai Tokkyo Koho JP 08, 231, 396 (96,231,396) (1996), 5 pp. 15. Shankaranarayan D, Gopalakrishnan C, Kameswaran L. Pharmacological profile of mangostin and its derivatives. Arch Int Pharmacodyn Ther 1979;239:257—69. 16. Nakatani K, Nakahata N, Arakawa T, Yasuda H, Ohizumi Y. Inhibition of cyclooxygenase and prostaglandin E2 synthesis by gamma-mangostin, a xanthone derivative in mangosteen, in C6 rat glioma cells. Biochem Pharmacol 2002;63:73—9. 17. Ainamo J, Bay I. Problems and proposals for recording gingivitis and plaque. Int Dent J 1975;25:229—35. 18. L¨ oe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand 1963;21:533—51. 19. Silness J, L¨ oe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:21—35. 20. Listgarten MA, Levin S. Positive correlation between the proportions of subgingival spirochetes and motile bacteria and susceptibility of human subjects to periodontal deterioration. J Clin Periodontol 1981;8:122—38. 21. Jeffcoat MK, Reddy MS, Haigh S, et al. A comparison of topical ketorolac, systemic flurbiprofen, and placebo for the inhibition of bone loss in adult periodontitis. J Periodontol 1995;66:329—38. 22. Graca MA, Watts TL, Wilson RF, Palmer RM. A randomized controlled trial of a 2% minocycline gel as an adjunct to nonsurgical periodontal treatment, using a design with multiple matching criteria. J Clin Periodontol 1997;24:249—53. 23. Stelzel M, Flores-de-Jacoby L. Topical metronidazole application as an adjunct to scaling and root planing. J Clin Periodontol 2000;27:447—52. 24. Li KL, Vogel R, Jeffcoat MK, et al. The effect of ketoprofen creams on periodontal disease in rhesus monkeys. J Periodont Res 1996;31:525—32. 25. Pihlstrom BL. Measurement of attachment level in clinical trials: probing methods. J Periodontol 1992;63(Suppl 12):1072—7. 26. Savitt ED, Socransky SS. Distribution of certain subgingival microbial species in selected periodontal conditions. J Periodont Res 1984;19:111—23.
available at www.sciencedirect.com
journal homepage: www.elsevierhealth.com/journals/ctim
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment夽 Supanee Rassameemasmaung a,∗, Anongporn Sirikulsathean a, Cholticha Amornchat b, Pawinee Maungmingsook c, Pleumchitt Rojanapanthu d, Wandee Gritsanaphan e a
Department of Oral Medicine, Faculty of Dentistry, Mahidol University, Ratchathewi, Bangkok 10400, Thailand Department of Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand c Nakornpathom Hospital, Nakornpathom, Thailand d Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand e Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand Available online 25 April 2008 b
KEYWORDS Garcinia mangostana L.; Local drug delivery; Periodontal treatment
Summary Objective: To evaluate the effects of gel containing Garcinia mangostana L. pericarp extract (GM gel) applied topically as an adjunct to periodontal treatment. Design: Subjects who had periodontal pockets on their single-rooted teeth were randomized into the test or control group. Subjects in the test group received periodontal treatment consisting of scaling, root planing and subgingival application of GM gel while those in the control group received scaling and root planing without GM gel application. Setting: Mahidol University, Faculty of Dentistry, Thailand. Main outcome measures: Clinical parameters included probing pocket depth (PPD), clinical attachment level (CAL), bleeding on probing (BOP), Gingival Index (GI) and Plaque Index (PI). Microbiological parameter included subgingival microbial composition as examined by phase contrast microscopy. Results: Clinical improvement compared to baseline was found in both groups (P < 0.05). The test group exhibited significantly higher reduction in mean PPD, GI and BOP than the control group at the 3rd month after treatment (P < 0.05). Subgingival microbial composition changed from diseased state to that compatible with health after treatment in both groups. However, significant differences between groups were found only in the mean percentage of cocci at the 1st and 3rd month after treatment (P < 0.05). Conclusions: GM gel could enhance the clinical effects of periodontal treatment. © 2008 Elsevier Ltd. All rights reserved.
Introduction 夽 This work was financially supported by Mahidol University Research Grant. ∗ Corresponding author. Tel.: +66 22036500; fax: +66 23548510. E-mail address: [email protected] (S. Rassameemasmaung).
Periodontitis is the common oral disease affecting many people around the world. It is defined as an inflammation and progressive destruction of the tooth-supporting structures (periodontium). This disease results from interaction
0965-2299/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctim.2007.12.004
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment between specific host defense mechanisms and dental plaque biofilms that colonize on the tooth surfaces at or below the gingival margin. The progression of periodontitis can be arrested by mechanical debridement consisting of scaling, root planing and proper oral hygiene control.1 These treatment modalities aim to remove dental plaque and plaque-retentive factors. However, pathogenic bacteria may not be eliminated in the deep periodontal pockets due to poor access for mechanical debridement, root anatomical complexity2,3 and the ability of the bacteria to invade and reside in the periodontal tissues4 or dentinal tubules.5 The recognition of bacterial plaque as the main etiologic factor has led to increasingly antimicrobial agent intervention. Antimicrobial agents may gain access into the periodontal pockets through both systemic and local route of delivery. Systemic antimicrobial agents can be used to treat multiple sites simultaneously and may target invasive organisms and affect reservoirs of bacteria in the oral cavity.6 However, systemic antimicrobial agents may lead to potential side effects such as development of resistant bacteria7 and gastrointestinal intolerance.8 These drawbacks would be markedly reduced if antimicrobial agents applied locally could be used. For the local antimicrobial agent to be useful, it must be successfully delivered to the base of the periodontal pockets at an efficacious concentration and retain in the pockets for an adequate length of time.9 To achieve these, the sustain-released delivery drugs such as minocycline ointment10 or metronidazole gel11 had been used. It was found that mechanical debridement plus local delivery of these agents produced more favorable outcomes, for example, the reduction of periodontal pocket depth and the number of inflamed gingival sites, than those without local drug delivery.10,11 The use of herbal medicine as an alternative approach has gained much interest nowadays. Garcinia mangostana L., known as the mangosteen tree, contains various compounds such as chrysanthemin, garcinone A, B and C, sesquiterpenoids, gartanin, fructose, sucrose, tannins, xanthones and their derivatives in its pericarp.12 Mangostin could inhibit penicillin-resistant strain of Staphylococcus aureus with an MIC of 3.125 g/ml.13 ␥-Mangostin also inhibited growth of Helicobacter pyroli with the MIC of 1.56 g/ml.14 The antimicrobial screening test performed at our laboratory demonstrated that the 80% ethanolic extract from the pericarp of mangosteen inhibited growth of Porphyromonas gingivalis W50, the main periodontopathic bacteria, at the MIC of 3.91 mg/ml. Other than the antimicrobial activity, mangostin, 1-isomangostin and mangostin triacetate exhibited an anti-inflammatory activity.15 ␥-Mangostin also showed a potent inhibitory activity against prostaglandin E2 released.16 Due to its antimicrobial and anti-inflammatory activity, we hypothesized that topical application of gel containing G. mangostana L. pericarp extract (GM gel) could inhibit bacterial growth and facilitate wound healing after periodontal treatment. Thus, this study aimed to evaluate the clinical and microbiological effects of the gel when used as an adjunct to periodontal treatment.
263
Methods Subject selection Before experiment, the study protocol was approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand. This study was performed at the Faculty of Dentistry, Mahidol University. Subjects, aged between 35 and 60 years, were the outpatients of the Department of Oral Medicine, Faculty of Dentistry, Mahidol University. They were systemically healthy and were diagnosed as chronic periodontitis. They had at least two sites with probing pocket depth (PPD) of 5—6 mm and other two sites with PPD of 7—9 mm on their single-rooted teeth. The selected teeth had no endodontic complication. The selected sites must have a radiographic evidence of alveolar bone loss and had bleeding upon probing or suppuration. Subjects were excluded from the study if they were pregnant, lactated, smoker, used antibiotic within the previous 3 months, and received periodontitis treatment within the previous 6 months. Subjects who met the above criteria were explained about the objectives and the details of the study. Those who were willing to participate were asked to sign the informed consent forms before entering the study. After screening, subjects were randomized into the test or control group. Subjects in the test group received mechanical debridement consisting of scaling, root planing and subgingival application of GM gel while those in the control group received mechanical debridement without GM gel application.
GM gel The crude extract from the pericarp of mangosteen was formulated as a local delivery drug (G. mangostana gel or GM gel) by the Department of Pharmaceutical Technology, Faculty of Pharmacy, Mahidol University, Thailand. The concentration of GM gel was adjusted according to the antimicrobial screening test performed at our laboratory. The gel was tested for short term toxicity by the National Laboratory Animal Center, Mahidol University. It was suggested that the gel could be safely used according to the research regimen. GM gel was kept at 4 ◦ C throughout the study period.
Clinical parameters Probing pocket depth (PPD) and clinical attachment level (CAL) were measured from the selected sites (Fig. 1) using a standard periodontal probe (PCPUNC 15, Hu FriedyTM , IL, USA) and recorded to the nearest millimeter. Bleeding on probing (BOP) was recorded as the presence or absence of bleeding upon probing.17 Gingival Index (GI)18 was measured as follows: 0 = normal gingiva; 1 = mild inflammation, slight change in color; 2 = moderate inflammation, redness, edema and glazing; 3 = severe inflammation, marked redness and edema, ulcerations. Plaque Index (PI)19 was then measured using the following criteria: 0 = no plaque in the gingival area; 1 = a film of plaque adhering to the free gingival margin and adjacent area of the tooth. The plaque may be rec-
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Figure 2 GM gel was delivered into the periodontal pocket by a blunted-tip needle.
Figure 1 Representative illustration showing the placement of a standard periodontal probe into the periodontal pocket and the reference points for the measurement of probing pocket depth and clinical attachment level. A = cemento-enamel junction, B = gingival margin, C = base of the periodontal pocket, A—C = clinical attachment level, B—C = probing pocket depth.
ognized only by running a probe across the tooth surface; 2 = moderate accumulation of soft deposits within the gingival pocket and on the gingival margin and/or adjacent tooth surface that can be seen by the naked eye; 3 = abundance of soft matter within the gingival pocket and/or on the gingival margin and adjacent tooth surface.
Microbiological procedure The experimental teeth were isolated with cotton roll. Tooth surface above the gingival margin was cleaned and dried with an air stream. Subgingival plaque sample from each selected site was collected with three consecutive sterile paper points. The paper points were inserted into the pocket until resistance was met and kept in place for 20 s. After removing from the pocket, the paper points were immediately pooled into a sterile tube containing 1 ml of pre-reduced transport fluid. The sample was then shaken for 10 s. Bacterial suspension was examined under phase contrast microscopy. At least 200 bacteria from a random field were observed at a magnification of ×400 and classified into one of the following groups, i.e., cocci, motile rods, spirochetes and other bacteria.20 Baseline clinical and microbiological examination was performed on the experimental teeth by a blinded examiner who had been calibrated until the intra-examiner error was less than 10%. Full mouth scaling and root planing was performed in all subjects until the operator felt that the root surfaces were smooth. In the test group, GM gel was delivered into the periodontal pockets of the selected teeth
by a 23-gauge, blunted-tip needle until the gel was detected at the gingival margin (Fig. 2). GM gel was repeatedly delivered at 1 week later. Subjects were informed not to drink, eat or rinse within 1 h after gel delivery. Subjects in both groups performed their usual oral hygiene procedures but were not allowed to use any chemotherapeutic mouthrinse or oral irrigator. Clinical and microbiological parameters were re-evaluated at the 1st and 3rd month after the 1st gel application. Clinical adverse experiences were recorded at each follow-up visit.
Statistical analysis Data analysis was achieved by statistical software SPSS for Windows. An individual site was a unit of analysis. For mean PPD, CAL, GI and PI, Wilcoxon match-pairs signed rank test was used for within group comparison and Mann—Whitney U-test was used for between group comparison. Microbiological data were analyzed by paired t-test for within group comparison and by independent t-test for between group comparison. BOP was analyzed by McNemar’s test for within group comparison and by Chi-square test for between group comparison. The level of significance was considered at P < 0.05.
Results Thirty-one subjects completed the study. At baseline, no significant difference between groups was observed for any
Table 1
Subject characteristics at baseline
Characteristics
Test
Control
Numbers of subjects (male, female) Age (year) Mean age (year) Number of sites with initial PPD 5—6 mm Number of sites with initial PPD 7—9 mm
16 (8, 8)
15 (6, 9)
37—60 47.31 32
36—57 45.8 30
32
30
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment Table 2
265
Mean ± standard deviation of probing pocket depth (mm) and clinical attachment level (mm)
Site
Parameter
Interval
Group Test
Sites with initial PPD 5—6 mm
Sites with initial PPD 7—9 mm
Control
PPD
Baseline 1st month 3rd month
5.59 ± 0.50 4.56 ± 0.72* 3.91 ± 0.86*,†
5.77 ± 0.43 4.77 ± 0.57* 4.63 ± 0.61*
CAL
Baseline 1st month 3rd month
9.06 ± 0.72 8.22 ± 0.66* 8.06 ± 0.76*
8.77 ± 1.33 8.00 ± 1.34* 7.90 ± 1.27*
PPD
Baseline 1st month 3rd month
7.69 ± 0.82 6.09 ± 0.89*,† 5.41 ± 0.67*,†
8.00 ± 0.79 6.70 ± 0.92* 6.10 ± 0.80*
CAL
Baseline 1st month 3rd month
11.13 ± 1.43 10.09 ± 1.42* 9.47 ± 1.29*
10.57 ± 1.01 9.70 ± 1.06* 9.03 ± 1.03*
PPD = probing pocket depth, CAL = clinical attachment level. * Significantly different compared to baseline (P < 0.05). † Significantly different between groups (P < 0.05).
subject characteristics (Table 1). For subjects in the test group, neither complaint nor adverse tissue reaction was found after gel application.
Probing pocket depth (PPD) and clinical attachment level (CAL) At baseline, PPD and CAL were not significantly different between groups (Table 2). At all follow-up visits, PPD and CAL were significantly different when compared to baseline in both groups. For the sites with an initial PPD of 5—6 mm, significant differences in PPD between groups were found at the 3rd month. For the sites with an initial PPD of 7—9 mm, significant differences in PPD between groups were found at the 1st and 3rd month. Regarding CAL, no significant difference was found between groups at the follow-up visits.
Gingival Index (GI) At baseline, all selected sites exhibited moderate gingival inflammation (GI of 2; Table 4). After treatment, the improved gingival status was observed. The percentage of sites with GI of 2 was significantly lower while those with GI of 0 and 1 were significantly higher when compared to baseline in both groups. When compared between groups, significant differences were found at the 3rd month.
Plaque Index (PI) At baseline, the majority of sites exhibited PI of 1 and 2 (Table 5). At the follow-up visits, the improved oral hygiene status was observed in both groups. When compared to baseline, significant differences were found at all follow-up visits in both groups. However, when compared between groups, no significant difference was found.
Bleeding on probing (BOP) At baseline, all sites were bled upon probing (Table 3). As study progressed, percentage of bleeding sites was significantly reduced in both groups. When compared between groups, significant difference was found at the 3rd month.
Table 3 Interval
Baseline 1st month 3rd month * †
Table 4 Number of sites and percentage distribution of Gingival Index (GI) GI
Test (n = 64)
Control (n = 60)
64 (100.00%) 45 (70.30%)* 30 (46.90%)*,†
60 (100.00%) 49 (81.70%)* 39 (65.00%)*
Significantly different compared to baseline (P < 0.05). Significantly different between groups (P < 0.05).
Group Test (n = 64)
Control (n = 60)
0
Baseline 1st month 3rd month
0 (0%) 6 (9.40%)* 25 (39.10%)*,†
0 (0%) 5 (8.30%)* 13 (21.73%)*
1
Baseline 1st month 3rd month
0 (0%) 14 (21.90%)* 9 (14.10%)*,†
0 (0%) 8 (13.30%)* 6 (10.00%)*
2
Baseline 1st month 3rd month
64 (100.00%) 44 (68.80%)* 30 (46.90%)*,†
60 (100.00%) 47 (78.30%)* 41 (68.30%)*
Number and percentage of bleeding sites Group
Interval
* †
Significantly different compared to baseline (P < 0.05). Significantly different between groups (P < 0.05).
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Table 5 Number of sites and percentage distribution of Plaque Index (PI) PI
Interval
Group Test (n = 64)
Control (n = 60)
0
Baseline 1st month 3rd month
4 (6.30%) 15 (23.40%)* 18 (28.10%)*
10 (16.70%) 12 (20.00%)* 22 (36.70%)*
1
Baseline 1st month 3rd month
39 (60.90%) 40 (62.50%)* 38 (59.40%)*
31 (51.70%) 39 (65.00%)* 33 (55.00%)*
2
Baseline 1st month 3rd month
21 (32.80%) 9 (14.10%)* 8 (12.50%)*
19 (31.70%) 9 (15.00%)* 5 (8.30%)*
*
Significantly different compared to baseline (P < 0.05).
Microbiological evaluation After treatment, mean percentage of cocci was significantly increased while those of motile rods, spirochetes and other bacteria were significantly decreased when compared to baseline in both groups (Table 6). When compared between groups, significant differences were found only in the mean percentage of cocci at the 1st and 3rd month.
Discussion The local delivery of antimicrobial agent has gained an increasing interest in the field of periodontal treatment. The aim of the present study was to compare the clinical and microbiological effects of mechanical debridement plus local delivery of GM gel to that of mechanical debridement alone. In this study, blinding was reduced because of no placebo gel in the control group. However, the use of placebo gel may introduce confounding influences of a placebo design into the study.10
Table 6 Interval
From the clinical results, the test group showed an impressive effect on the reduction of pocket depth and gingival inflammation but less pronounced effect on CAL gain. Regarding the pocket depth, PPD reduction was higher in the sites with deep pockets (initial PPD of 7—9 mm) than those with shallow pockets (initial PPD of 5—6 mm). Significant PPD reduction was detected in the test group at the 1st and 3rd month in the sites with deep pockets, and only at the 3rd month in the sites with shallow pockets. This indicated the benefit of scaling, root planing and GM gel application in the reduction of pocket depth over scaling and root planing alone especially in the sites with deep periodontal pockets. Significant gain in CAL as compared to baseline were found in both groups. However, no significant differences of the CAL were found between groups. CAL gain may be due to the decreased gingival inflammation which lead to an increased gingival tissue adaptation and thus a reduction of probe penetration.21 Regarding the GI and BOP, the test group showed a significant improvement in GI and BOP when compared to the control group at the 3rd month. Our result showed that the adjunctive treatment with GM gel could reduce 53% of bleeding sites at the 3rd month and this was comparable to those obtained from other studies.22,23 In those studies, the reduction of bleeding sites was 68% and 53% after adjunctive treatment with minocycline ointment22 and metronidazole gel,23 respectively. PI was also improved after treatment in both groups. However, no significant differences were found when compared between groups. This demonstrated that GM gel provided a more beneficial effect on the reduction of gingival inflammation but not the reduction of plaque. Due to an anti-inflammatory activity, recent investigation reported that the mangostin and its derivatives had an effect on prostaglandin synthesis.16 When prostaglandin level was reduced, the improvement of periodontal-associated parameters could be occurred.24,25 It is possible that GM gel could modulate host inflammatory response. Tannins from the pericarp of mangosteen might also attribute to the PPD reduction due to their astringent property which is usu-
Percentage distribution of bacteria in each group Bacterial group
Group Test (n = 64)
22.61 39.30 13.97 24.13
± ± ± ±
4.61 4.07 6.20 4.09
46.05 ± 4.50*,† 27.62 ± 3.56* 6.21 ± 3.34* 20.18 ± 3.54*
43.68 28.68 7.20 20.39
± ± ± ±
5.88* 3.89* 4.70* 3.06*
48.24 ± 4.56*,† 27.99 ± 3.85* 4.60 ± 2.37* 19.32 ± 2.92*
45.92 28.87 5.11 20.30
± ± ± ±
4.54* 2.90* 2.70* 2.92*
Baseline
Cocci Motile rods Spirochetes Other
24.12 39.85 13.58 22.86
1st month
Cocci Motile rods Spirochetes Other
3rd month
Cocci Motile rods Spirochetes Other
* †
Significantly different compared to baseline (P < 0.05). Significantly different between groups (P < 0.05).
± ± ± ±
Control (n = 60)
4.21 4.17 6.50 3.70
Topical application of Garcinia mangostana L. pericarp gel as an adjunct to periodontal treatment ally accompanied by contraction and wrinkling of the tissue as well as their vasoconstrictor effect which reduces local edema, exudation and inflammation. As tissue shrinkage naturally occurs after scaling and root planing, the additional PPD reduction observed in the test group might likely to be facilitated by the anti-inflammatory effect of mangostin and its derivatives as well as the astringent property of tannins. In terms of the microbiological observation, baseline subgingival bacterial counts revealed low proportion of cocci but high proportions of motile rods, spirochetes, and other bacteria in both groups. After treatment, the compositions of subgingival microflora were compatible to that generally observed in healthy periodontal condition.26 In our study, a continuous increase in the percentage of cocci was observed, while the percentages of motile rods, spirochetes, and other bacteria decreased over the 3-month period. At all follow-up visits, the test group showed a statistically higher mean percentage of cocci. The results from this study did not obviously show a difference between scaling and root planing with and without GM gel in changing composition of subgingival microflora. It is possible that the phase-contrast microscopy used in our study might not be sensitive enough to detect any apparent microbial changes after treatment. It is also possible that scaling and root planing could not disrupt dental plaque biofilms because of some limitations, and thus the concentration of GM gel might not effectively cope with the periodontopathic bacteria. In conclusion, this randomized, controlled, single-blinded study suggested superior clinical benefits of GM gel when used as an adjunct to scaling and root planing in periodontitis subjects. The greater reduction in probing depth, gingival inflammation and bleeding on probing observed in the test group might be facilitated by an anti-inflammatory activity of the gel.
Conflict of interest The authors had no potential conflict of interest.
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