In vivo study on the Subside Effect of Gingivitis by Use of Dentifrice with Centella and Magnolia Components

Transcription

1 Original Article Int J Clin Prev Dent 2016;12(3): ㆍ ISSN (Print) ㆍ ISSN (Online) In vivo study on the Subside Effect of Gingivitis by Use of Dentifrice with Centella and Magnolia Components Jin-Young Lee 1, Seung Chul Shin 1, Geum Sun Lee 2 1 Department of Oral Health, Dankook University College of Dentistry, Cheonan, 2 Department of Dental Hygiene, Suwon Women's University, Suwon, Korea Objective: Centella extract is known for tits anti-inflammatory effect and the synthesis of collagen, and Magnolia officinalis bark extract is known for its anti-microbial effect. For this reason, such dentifrice with these two components was developed and produced, in order to use for treating early gingivitis dental patients. Methods: Ninety adults, volunteers were enrolled for this clinical experiment to compare the subsidal effect of dentifrice with Centella and Magnolia, with the control dentifrice without these components. The study was carried out over 4 weeks of trial, by dividing the subjects into 3 groups; experimental group 1 used the dentifrice on the toothbrush, experimental group 2 rubbed the dentifrice on the gingival tissue before brushing teeth with the dentifrice, and the control group brushed their teeth with no specific anti-septic component. Results: There was no difference on patient hygiene performance index, papillary, marginal, and attached gingival index (PMA) and oral malodor before use and 3 days after use. However, the conditions subsided 2 to 4 weeks later in both the experimental groups 1 and 2, in terms of PMA and oral malodor. Better oral hygiene was reported in experimental group 2 compared to experimental group 1 or the control group (p<0.05). Conclusion: These two components were effective in controlling gingivitis and oral malodor as reported by the clinical study. Keywords: gingivitis, oral hygiene, dentifrices, oral health Introduction Periodontal disease has been known for its pandemic nature. Corresponding author Geum Sun Lee Department of Dental Hygiene, Suwon Women s University, 72 Onjeong-ro, Gwonseon-gu, Suwon, Korea. Tel: , Fax: , bluesky-ljy@daum.net Received August 18, 2016, Revised September 13, 2016, Accepted September 19, 2016 It is widely prevalent among people of middle and advanced ages, with chronic progress and gradually accumulating gingiva in early stages but irreversible after progress [1,2]. Dental plaque has been demonstrated as one of the main causatives for periodontal disease and proper and thorough elimination of dental plaque has been recommended as the best way to control periodontal disease, as apart from performing periodic scaling and dental examination [3,4]. The prevalence rate for periodontal disease has increased to more than 70% in people of middle and senior ages, and prevalence is seen to increase with advance in age [5-7]. Several methods have been introduced for prevention of ca- Copyright c Korean Academy of Preventive Dentistry. All rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 169

2 International Journal of Clinical Preventive Dentistry ries and periodontal disease, and the easy, simple and effective way might be to brush teeth with proper devices. It is also beneficial to use antibacterial and plaque formation inhibitory substances including fluorides and medicinal herbs for the purpose of cleaning tooth surface and maintaining oral health [8-10]. Some of the most effective substances for control of gingivitis in early stage would be Centella and Magnolia, which may be included in the dentifrice. These components are already used individually in some dentifrices and proved to be effective for control of early stage gingivitis. However, there has not been evidence on the effect of using them together as dentifrice components. Hence in this study, these components were used together in the dentifrice and a somewhat different applying method was used, in order to assess whether joint use of these components was more effective in controlling gingivitis. Materials and Methods 1. Materials Different types of dentifrice were prepared: the control group dentifrice with the basic components for dentifrice and experimental group dentifrice with Centlla and Magnolia components together and different applying method for experimental groups 1 and 2, as shown in Table Subjects Ninety-three volunteers of middle and senior ages who had early stage of gingivitis or periodontitis participated in this study. Volunteers were divided into 3 groups with 31 persons in each group. Three subjects were later eliminated because they were absent for oral examinations. Hence the study finally analyzed 90 subjects charts. The age and gender distribution is shown in Table 2, and the subject distribution for each group is shown in Table Method 1) In vitro experiment Mouse macrophage and human gingiva fibroblast human gingiva fibroblst (HGF)-1, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) were used for incubation of the micro-organisms. RPMI 1640 (Gibco; Bridgewater, NJ, USA) was used with the addition of 10% of bovine serum (Gibco) and penicillin (Sigma-Aldrich, St. Louis, MO, USA) for incubating raw cells. HGF-1 cell was incubated by use of Dulbecco s modified eagle s medium Table 1. The comparison of the composition of each dentifrice Components Dentifrice group Control (%) Experimental 1 (%) Experimental 2 (%) Dental type Silica a Sodium fluoride b Centella titrated extract c Titrated extract of Zea Mays L unsaponifiable fraction d Magnolia bark CO 2 supercritical dry extract e Applying method Usual brushing of teeth Usual brushing of teeth Topical applying on the gingiva and then brushing as usual a Standardization by American Food & Drug Agency (NF). b Standardization by Korean Food & Drug Agency (KP). c Standardization by Korean Food & Drug Medical Herb regulations. d Standardization by Korean Food & Drug Medical Herb regulation. e Standardization by LG Chemical, Magnolul and Honokiol 40 mg/100 g of dentifrice. Table 2. Age and gender distribution of the subjects Age (yr) Gender (n) Male Female Total Over Total Average age (mean±standard deviation): 62.44±18.25 years. Table 3. Subjects distribution for each group Gender (n) Male Female Total Experimental Experimental Control Total Vol. 12, No. 3, September 2016

3 Jin-Young Lee, et al:gingival Subside Effect by Use of the Dentifrice with Centella and Magnolia Components (DMEM; ATCC) included with 10% fetal bovine serum (FBS, Gibco) and penicillin-streptomycin (Sigma-Aldrich) under the environment of 37 o C, 5% of CO 2 conditions. (1) Inflammation related Cytokine interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-n gene inhibitory efficiency: Incubated raw cell in DMEM media included with 10% FBS and inoculated it with cell/ml concentration. It was then kept in storage under 37 o C, 5% of CO 2 condition for 24 hours. 1 g/ml of B-sitosterol was used as the active component of the titrated extract of Zea Mays L., distributed according to the concentration. The next stage was synthesis of cdna with cdna synthesis kit (PKT, Seoul, Korea) by use of RNeasy mini kit (Qiagen, Hilden, Germany) through the extraction of total RNA. The existence of the inflammatory related cytokine gene was checked by Taqman probe and real time polymerase chain reaction method (Applied Biosystems, Foster City, CA, USA) and house-keeping gene was checked by mouse glyceraldehyde 3-phosphate dehydrogenase. (2) Measurement of prostaglandin E2 (PGE2): PGE2 inhibitory ability was checked on raw cells by use of ipopolysaccharide (LPS) 1 g/ml as inflammatory induction agent and Magnolia bark CO 2 supercritical dry extract by treating according to the concentration. The concentration of PGE2 was measured by use of ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA). (3) Evaluation of the synthesis of collagen: HGF-1 cell was incubated in the 10% FBS contained DMEM media which was inoculated as cells/well concentration and kept under storage at 37 o C, 5% CO 2 condition for 24 hours. The synthesis ability was evaluated through Centella extract treated according to concentration and incubated for an additional 48 hours and used as the upper liquid. ELISA kit (Takara, Shiga, Japan) was used for the measurement of collagen. (4) The matrix metallo-protein 1 (MMP-1) inhibitory ability: MMP-1 inhibitory ability was evaluated through measuring activity changes of the gene promoter of MMP-1. Reporter system was produced through the promoter of MMP-1 gene cloning into PGL3 vector. And then NH-3T3 fibroblast cell was incubated to on the 24-well plate. 1 g of DNA was transfected using Invitrogen lipofectamne, and Centella titrated extract was then added according to the concentration. Each media was changed and subjected to additional incubation for 18 hours and then the cell lysis was carried out through the Promega (Seoul, Korea) reporter lysis buffer. Further, luciferase activity in the lysate was analyzed using luciferase assay of Promega and micro plate analyser (victor analyser). 2) In vivo experiment Institutional Review Board (IRB) of Dankook University was achieved as (IRB File No. DNK ). Ninety subjects volunteers were divided into 3 groups: 31 participants were added to experimental group 1, 29 to experimental group 2, and 30 to the control group. The volunteers were given education on brushing teeth using the rolling method, after sharing the same kind of toothbrush and 3 kinds of dentifrices with the same shape and color for double blinded test. The subjects were advised to visit the dental clinic and receive oral examinations a total of four times: on the first day, 3 days later, 7 days later, and 14 days later. The volunteers were advised to use the delivered dentifrice 3 times a day for 2 weeks as directed by an examiner. Experimental group 1 was to brush teeth as usual, in addition to applying a small amount of dentifrice on the gingival tissue. They could also include a small amount of dentifrice into the gingival sulcus for 1 to 2 minutes before brushing teeth. Experimental group 2 was advised to brush teeth as usual. Dentifrice with basic components included was used for the control group with usual brushing of teeth. Oral examination was carried for factors such as patient hygiene performance (PHP) index, papillary, marginal, and attached gingival inflammation (PMA) index and oral malodor was checked for 1st, 3rd, 7th, and 14th days. The standardization of each index was introduced as follows. (1) Simplified-PHP index: Indicated 6 teeth as upper and lower 1st molars and upper and lower central incisors and labial or buccal surfaces on the upper molar and central incisors both in upper and lower rows. Lingual surface was checked on lower molar teeth. Plaque deposition was checked and measured by oral examination on the site of the tooth surface after applying the disclosing solution on the pertinent tooth surface, by dividing a surface with 5 sectors as incisal, cervical, mesial, distal and central portions. In case dental plaque was left on the examined portion of the tooth surface, it was given 1 point out of a total score of 5 points. The average score was calculated for 6 teeth as plaque deposit state of 6 surfaces. (2) PMA index: Gingival inflammation state on the anterior portion both in upper and lower rows was checked using PMA index. Labial surfaces of the frontal area both in upper and lower rows were divided into 15 portions as papillary gingiva, marginal gingiva and attached gingiva for upper and lower rows for a total 30 portions, from left canine to right canine. If inflammation symptoms are detected at each portion, it is given 1 point out of a total 30 points. (3) Oral malodor check: Oral malodor was checked for relative amounts of Volatile Sulfur Compound using B&B checker (Plustech, Daejeon, Korea). The tip of the paper is inserted into the mouth and it is breathed onto for 15 seconds after closing the mouth for 3 minutes. If results show values under 50 ppv, 171

4 International Journal of Clinical Preventive Dentistry it would be considered as normal, considering the middle or senior age of the participants; 50 to 100 ppv meant a little malodor, and a value higher than 100 ppv indicated severe malodor requiring treatment. (4) Evaluation of the questionnaire: An interview was performed on the last day of the clinical experiment in order to check the subjects sensory response to reduction of gingivitis and to check the side effects of using the dentifrice. (5) Statistical analysis: All data for PHP index, PMA index and oral malodor were examined and estimated for average of each group and each day, to compare them with each other using the IBM SPSS ver program (IBM Co., Armonk, NY, USA). ANOVA test was used to compare the data between the groups. Results The influence of -sitosterol on inflammatory cytokine IL-1, IL-6 TNF gene expression is shown in Figure 1 as inhibitory activity for gene expression. The inhibitory effect of PGE2 production is shown in Figure 2. Collagen synthesis effect of Centella extract for HGF-1 cell is shown in Figure 3. The influence of Centella extract on MMP-1 expression is shown in Figure 4. S-PHP index changes as time passed. S-PHP changes as time passed is shown in Table 4, showing significantly difference after 14 days. It revealed the lowest of PHP in experimental group 2, 14 days later. PMA index changes as time passed. PMA changes as time passed was shown in Table 5. It shows significantly different values between the two experimental groups and the control Figure 1. The influence of -sitosterol on inflammatory cytokine interleukin (IL)-1, IL-6 TNF gene expression. LPS: lipopolysaccharide, TNF: tumor necrosis factor. Figure 3. Collagen synthesis effect of Centella extract for human gingiva fibroblst-1 cell. Figure 2. The inhibitory effect of prostaglandin E2 (PGE2) production. LPS: lipopolysaccharide. Figure 4. The influence of Centella extract on matrix metalloprotein 1 (MMP-1) expression. 172 Vol. 12, No. 3, September 2016

5 Jin-Young Lee, et al:gingival Subside Effect by Use of the Dentifrice with Centella and Magnolia Components Table 4. Simplified patient hygiene performance index changes as time passed Time passed Experimental 1 (n=31) Experimental 2 (n=29) Control (n=30) ANOVA (Scheffe) Base 2.71± ± ±0.52 a a a 3 days after 2.71± ± ±0.64 a a a 7 days after 1.65± ± ±0.65 a a a 14 days after 1.28± ± ±0.39 b a c Values are presented as mean±standard deviation. Statistical significant by ANOVA (p<0.05). a, b, c: The same letter means there is no difference in statistical value. Table 5. Papillary, marginal, and attached gingival inflammation index changes as time passed Time passed Experimental 1 Experimental 2 (n=31) (n=29) Control (n=30) ANOVA (Scheffe) Base 11.06± ± ±2.11 a a a 3 days after 10.55± ± ±2.19 a a a 7 days after 7.55± ± ±2.25 a a b 14 days after 6.55± ± ±2.14 b a c Values are presented as mean±standard deviation. Statistical significant by ANOVA (p<0.05). a, b, c: The same letter means there is no difference in statistical value. group after 7 days and significantly different values between the three groups after 14 days later. The oral malodor level changes as time passed. The relative oral malodor level is shown in Table 6. Values are significantly different after 14 days. Discussion Dentifrice, or what we commonly call tooth paste, was developed as additional material for brushing teeth in order to enhance the cleaning effect in the past. However the concept has evolved and different dentifrices are developed for special purposes such as caries prevention, control of early gingivitis, prevention of periodontal disease, desensitization of hyper-sensitive dentin, oral malodor control or tooth whitening and so on in the present day [1-4]. One of the important roles of toothpaste is to control early state of gingival inflammation, also known as gingivitis [8]. Several of dentifrices were developed focusing on the periodontal tissue as prevention or treatment and many of them were proved as effective for periodontal disease. Many that included fluorides proved effective for dental caries [10]. Table 6. The oral malodor level changes as time passed Time passed Experimental1 (n=31) Experimental 2 (n=29) Control (n=30) ANOVA (Scheffe) Base 70.97± ± ±20.67 a a a 3 days after 70.48± ± ±16.24 a a a 7 days after 59.74± ± ±16.03 a a a 14 days after 53.16± ± ±18.34 a a b Values are presented as mean±standard deviation. Statistical significant by ANOVA (p<0.05). a, b, c: The same letter means there is no difference in statistical value. Cream-type dentifrices that contain various ingredients to prevent dental caries and gingivitis are widely used. In particular, it is a well-known fact that the dentifrice with fluoride is effective in preventing dental caries. In addition to this, dentifrices containing sodium claim to remove biofilm on the teeth and provide effective plaque control [11,12]. Cream-type dentifrices with chitosan and some auxiliary components are used for prevention of periodontal diseases [13]. It was found that teeth discoloration and gingivitis were controlled by using a dentifrice with pine and bamboo salt in conjunction with chitosan powder [14]. As many studies on complex herbal medications have been conducted recently and the safety of herbal medications and the increased effects of the herbal medications when used by mixture have been verified in previous studies [15-19]. Titrated extract of the unsaponifiable fraction of Zea Mays L. is effective against inflammatory diseases and it has been reported that it reduced periodontal pocket depth, tooth mobility and gingival bleeding [20]. From the in vitro experimental part of the study, the inhibitory effect of inflammatory related cytokine IL-1, IL-6, TNF- gene express was proved. -sitosterol, titrated as extract of Zea Mays L. unsaponifiable fraction was shown to influence inflammatory cytokine IL-1, IL-6, and TNF- gene expression on the raw cell. About 21% of the inhibition of the expression in treated IL-1 and IL-6 and 25% of the inhibition in TNF- were revealed to have been treated with -sitosterol 100 mol/ml. A 100% expression was assumed in case of the inflammatory cytokine gene expression, it was treated with -sitosterol according to the concentration, and the relative expression rate was calculated. So it was concluded that the titrated extract of Zea Mays L. unsaponifiable fraction could inhibit inflammatory gene expression, as shown in Figure 1. It was also revealed that Magnolia bark CO 2 supercritical dry extract had the effect of inhibiting the production of PGE

6 International Journal of Clinical Preventive Dentistry About 72% of inhibitory effect of PGE2 production was shown in case of applying 0.2 g/ml of Magnolia bark CO 2 supercritical dry extract, and applying 2 g/ml had 91% of inhibitory effect on the production of PGE2. Here it is assumed that PGE2 production would be induced by LPS, which in turn is influenced by Magnolia bark CO 2 supercritical dry extract, as shown in Figure 2. The influence of HGF-1 cell from Centella extract on the synthesis of collagen is shown in Figure 3. Supposing that 100% of collagen synthesis would be formed in the control group, about 30% increase of collagen synthesis was shown in case of applying 20 g/ml of Centella extract. Therefore it was concluded that Centella extract could increase collagen synthesis. It has been known that gingival cells are weakened by destruction of collagen in case of inflammation response through the production of protease like MMP. Therefore the protective effect of Centella extract on the tissue could be assessed if the inhibitory rate of MMP production was estimated. About 23% of inhibition of MMP-1 production was shown with the application of 20 g/ml of Centella extract, as shown in Figure 4. It was concluded that Centella extract could inhibit the destruction of gingiva tissue through inflammation. In this clinical study, S-PHP showed no significantly different results till 7 days, but the applications showed measurable outcomes in 14 days. In general, PHP index is not easily changed by a new dentifrice within a short period. This is because there are lots of variables such as the frequency and method of brushing teeth, the sort of toothbrush, and the use of the supplementary oral hygiene devices, in addition to the kinds of dentifrice used. From the results measured after 14 days, the best effect for plaque removal was revealed to be the method used by experimental group 2, namely applying dentifrice with these components on the gingiva for a minute before brushing teeth. Topical application combined with brushing teeth might get the double benefits of anti-bacterial and gingival control effects in the long long term. A similar concept was considered for the PMA index in terms of gingival inflammation of the frontal area both in upper and lower.at labial side. PMA index is known to be an easy and simple method to configure out the inflammatory portion from the normal portion at the divided areas by comparing each side as papillary, marginal and attached gingiva, and using a relatively large scale of 30 points of full score. It is easy to interpret the different scores as different results. In the results, significant differences were seen between the two experimental groups and the control group 7 days later, and results were also different between experimental groups 1 and 2 14 days later, with the method used by experimental group 2 appearing to be more effective. In oral malodor check, significantly different results between two experimental groups and the control were revealed only after 14 days. Even though components for anti-plaque formation and gingival subside effect such as Centella titrated extract, titrated extract of Zea Mays L., unsaponifiable fraction and Magnolia bark CO 2 supercritical dry extract were included, it was not easy to get the evidence for malodor control. In this experiment, it was considered that these components together in the toothpaste could be effective to control the volatile sulfur compound gas through controlling gram negative anaerobic bacteria. In conclusion, these components, namely Centella extract and titrated extract of Zea Mays L., unsaponifiable fraction and Magnolia bark CO 2 supercritical dry extract, contained in the dentifrice, have been effective for control of gingivitis well as malodor control in 2 weeks of use. Conclusion Centella extract and the titrated extract of Zea Mays L., unsaponifiable fraction and Magnolia bark CO 2 supercritical dry extract have been known as effective in controlling early stage gingivitis. This study was composed of in vitro and in vivo experiments to prove the effect of gingival control effect for early stage of gingivitis patients. The obtained the results were as followings. 1. In the in-vitro experiment, Centella extract and the titrated extract of Zea Mays L. unsaponifiable fraction were considered for the inhibition of the inflammatory gene expression. 2. In the in-vivo study, dentifrice with Centella extract and the titrated extract of Zea Mays L. unsaponifiable fraction could decrease S-PHP index, PMA index and oral malodor in 14 days. 3. It is recommended to use dentifrice with Centella extract and the titrated extract of Zea Mays L. unsaponifiable fraction in order control early stage gingivitis. It is more effective to apply topically a minute before brushing teeth. References 1. Owens J, Addy M, Faulkner J. An 18-week home-use study comparing the oral hygiene and gingival health benefits of triclosan and fluoride toothpastes. J Clin Periodontol 1997;24: McClanahan SF, Beiswanger BB, Bartizek RD, Lanzalaco AC, Bacca L, White DJ. A comparison of stabilized stannous fluoride dentifrice and triclosan/copolymer dentifrice for efficacy in the reduction of gingivitis and gingival bleeding: six-month clinical results. J Clin Dent 1997;8: Hong SJ, Choi EG, Son JB, Jeong SS. 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