Effect of Curcumin on Periodontis

Effect of Curcumin on Periodontis INTRODUCTION Periodontitis is a chronic inflammatory disease characterized by destruction of the soft and hard tissues supporting the tooth. This tissue destruction appears to result from complex interaction between the bacteria and the host’s immune and inflammatory system. The host immune response identifies these invading pathogens and tries to neutralize or kill these microorganisms. During this process the host response elaborates a variety of mediators such as pro-inflammatory mediators, cytokines and proteolytic enzymes such as matrix metalloproteinases (MMPs). It is now believed that the major component of the soft and hard tissue destruction seen in periodontitis occurs as a result of activation of these mediators.1 Thus this host response which is essentially protective by intent paradoxically can also result in tissue damage. Hence recent therapeutic efforts are now focusing on altering or modulating this host response. This lead to emergence of a new comprehensive treatment str ategy for periodontitis which involves Host modulatory therapy (HMT) which can be combined with traditional periodontal therapies.2 To date, there is one FDA approved, systemic therapy that is prescribed as a host response modifier in the treatment of periodontal disease and that is adjunctive subantimicrobial dose doxycycline (SDD), which down-regulates the activity of MMPs.3 These MMPs are considered to be primary proteinases involved in periodontal tissue destruction synthesized by a variety of infiltrating inflammatory cells (i.e. neutrophils and macrophages), resident cells and some bacteria. Numerous studies have substantiated the relationship between MMPs and periodontal diseases, with findings of significantly higher MMP-9 (neutrophil derived MMPs) levels in patients with periodontitis compared to healthy controls.4, 5 Doxycycline is the most potent tetracycline for inhibition of these MMPs.6 However long term tetracycline therapy has certain disadvantages, the most important being the development of microbial resistance to this drug.7 At present a health care system exists in India where different types of diseases are being treated by Ayurvedic method. Since ancient times Ayurvedic drugs are being used for the treatment of various diseases, including periodontal and other oral diseases.8 They are widely acclaimed for their minimal side-effects and cost-effectiveness. Use of herbal extracts in treating periodontal diseases does not produce side effects of tetracycline compounds as well as other synthetic drugs. Curcuma longa (Turmeric) is a widely used herb which is distributed throughout tropical and subtropical regions of the world and widely cultivated in India, China and Indonesia.9 It has also long been used in both Ayurvedic medicine and Chinese medicine as an anti-inflammatory agent.9 The active constituent in turmeric is Curcumin.9, 10 Curcumin is known to have an anti-inflammatory effect. Studies show that Curcumin limits the activity of two enzymes, lipoxygenase and cyclooxygenase-2 that are involved in promoting and maintaining inflammation.11, 12 Studies have also shown that Curcumin down-regulates the MMP-9 activity.13, 14 Thus it may have a potential role in the treatment of inflammatory periodontal disease. Its anti-inflammatory activity in periodontal diseases has not been extensively investigated. With this background, the study was conducted to evaluate the anti-inflammatory effect of Curcumin by its inhibitory effect on MMP-9 activity and compare the same with Doxycycline, which is known to have an anticollagenase activity. MATERIALS AND METHODS Subjects: A total of 30 sites from 30 subjects (males and females), above 35 yrs of age, diagnosed clinically as having Chronic Periodontitis with pocket depths ranging from 5-8mm were selected for the study. The patients had no history of pervious dental treatment, antibiotic or anti-inflammatory drug therapy for the past 6 months. Patients with any history of systemic diseases / conditions, pregnant patients and smokers were excluded from the study. An informed consent was obtained from the patients prior to their enrollment into the study, and the study has been independently reviewed and approved by the institutional ethical board. Post collection of samples, the patients who participated in this study received the standard treatment protocol for Chronic Periodontitis. Method of Collection of Samples: Gingival tissue samples were obtained under aseptic conditions after administration of local anesthesia from patients who were diagnosed with Chronic Periodontitis. Gingival tissue samples were obtained from sites with pocket depths ranging from 5-8mm. Immediately following excision, these tissue specimens were briefly washed under cold distilled water and blotted dry. Then the specimens were transferred into sterile plastic vials which contained Phosphate Buffer Solution (PBS), pH 7.2, that acted as the transport medium. Specimens were then immediately carried to the laboratory where they were stored at -80à ¯Ã¢â‚¬Å¡Ã‚ °C until use. IN-VITRO STUDY Chemicals Used: Pure extract of Curcumin in powder form (Samy Labs, Bangalore, India) and Doxycycline i.e. pure doxycycline hyclate in powder form (Aristo Pharmaceuticals Limited, Bangalore, India) was used for the purpose of the study. The Curcumin solution was prepared by dissolving 15mg of Curcumin in 10ml of distilled water (1500 ÃŽ ¼g/ml). Preparation of Doxycycline solution was by dissolving 3mg of Doxycycline in 10ml of distilled water (300 ÃŽ ¼g/ml). Extraction of MMP-9: The frozen gingival tissue samples were allowed to thaw to room temperature. Then each tissue sample was homogenized with 2.5% Triton X-100. This was followed by centrifugation of the homogenized samples for 30 mins at 6000 rpm, at 4à ¯Ã¢â‚¬Å¡Ã‚ °C. The resulting supernatant was separated and used for analysis. Detecting the Optimal Curcumin and Doxycycline Concentration: To determine the optimal inhibition of MMP-9 by Curcumin, different concentrations of Curcumin i.e. 300, 500, 1000, 1500 ÃŽ ¼g/ml were added to 50ÃŽ ¼l of gingival tissue extract and incubated at room temperature for 60mins.The addition of increasing concentrations of Curcumin to the gingival tissue extract resulted in decreased MMP-9 activity as shown in Figure 1. Assessing the MMP-9 activity by Densitometric Analysis demonstrated that the concentration of Curcumin required to inhibit more than 50% of MMP-9 activity was 1500 ÃŽ ¼g/ml. Since Curcumin showed more than 50% inhibition of MMP-9 activity at a concentration of 1500 ÃŽ ¼g/ml as compared to the control (without drug), this concentration was used to pretreat the gingival tissue extract in the study. The Curcumin solution with a concentration of 1500 ÃŽ ¼g/ml was freshly prepared prior to processing of each sample of gingival tissue extract. Similarly increasing concentrations of Doxycycline ie 50, 100, 200, 300ÃŽ ¼g/ml were added to 50ÃŽ ¼l of gingival tissue extract and incubated at room temperature for 60mins. Assessing the MMP-9 activity by Densitometric Analysis demonstrated that the concentration of Doxycycline required to inhibit more than 50% of MMP-9 activity was 300ÃŽ ¼g/ml and thus this concentration was used to pretreat the gingival tissue extract in the study. The Doxycycline solution with a concentration of 300ÃŽ ¼g/ml was freshly prepared prior to processing of each sample of gingival tissue extract. Pretreatment of Gingival Tissue Extract with Curcumin and Doxycycline: To compare the inhibition of MMP-9 activity, 50ÃŽ ¼l of gingival tissue extract was preincubated with freshly prepared solution of 50ÃŽ ¼l of Curcumin (1500ÃŽ ¼l/ml) and 50ÃŽ ¼l of Doxycycline (300ÃŽ ¼l/ml) solution for 60mins at room temperature in separate vials. In addition, 50ÃŽ ¼l of extract was also incubated with 50ÃŽ ¼l of distilled water which was used as the control. Assay for MMP-9 Activity: The presence of MMP-9 activity in collected samples was studied by gelatin zymography. Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) on 10% polyacrylamide containing 10% SDS copolymerized with 1g/l gelatin was used to pretreated tissue extracts under non reducing conditions without prior boiling. After electrophoresis, in order to regain the enzyme activity, the gels were rinsed with 2.5% Triton X-100 for 1hr to remove SDS thus allowing the protein to denature. The gels were then immersed in a proteolysis buffer containing Tris- HCl 50 m M/l (pH 7.6) and CaCl2 20mM/l and incubated at 37  ºC for 16 hours. The gels were subsequently stained Coomassie Blue (0.25% Coomassie Brilliant Blue R250, 40% methanol, 10% acetic acid). Gels were destained (30% methanol, 10% acetic acid and 60% water) until white bands appeared clearly from the blue background. These bands of gelatin lysis detected against the blue background as seen in Figure 2, represented enzymatic act ivities. RESULTS The presence of MMP-9 was studied. The enzymatic activities were detected as unstained bands on gelatin gel by zymography technique. In order to measure the relative MMP-9 levels, multi-image gel documentation systems were used to scan the clear zones and the percentage of inhibition was analyzed. Significant differences were found in the MMP activity in treated groups compared to the control. Figure 3 illustrates the reduction in the MMP-9 activity of each sample of gingival tissue extract when incubated with Doxycycline and Curcumin, expressed as percentage of reduction from the control. Table 1 shows the range, mean values and the standard deviation (S.D.) values for Curcumin and Doxycycline. The mean values show 59.58 % reduction in the MMP-9 activity with the addition of Doxycycline and 61.01% reduction in the MMP-9 activity with addition of Curcumin to the gingival tissue extract under identical conditions. Table 2 shows comparison of MMP-9 inhibitory effect between Doxycycline and Curcumin. The results obtained were analysed statistically using students paired t- test. DISCUSSION Periodontal diseases are the result of destruction of the soft and hard connective tissues surrounding and supporting the teeth. Inflammation is the basic cause of initiation of this breakdown. Research in the field of pathogenesis of periodontal disease has shown that various enzymatic activities which are directed towards the destruction of the pathogen also result in destruction of the periodontal connective tissue. Research has shown that, Matrix metelloproteinases (MMP’s) are one group of enzymes which are responsible for this destruction to a large extent. MMPs are involved in a number of physiological events including, tissue remodeling and pathological diseases such as periodontal disease and are the major players in collagen breakdown during periodontal destruction. MMPs are normally tightly regulated and disruption of this regulation leads to the pathologic breakdown of connective tissues. Higher levels of MMPs in the periodontal tissues provoke an imbalance between the production and degradation of collagen, causing tooth attachment loss. Especially, PMN derived MMPs (MMP-8 MMP-9) are the main proteinases related to tissue destruction and remodeling events in periodontal diseases. Numerous studies have substantiated the relationship between MMPs and periodontal diseases, with findings of significantly higher MMP-9 levels in patients with periodontitis as compared to healthy controls.4, 5, 15 With this understanding of the role of host immune response in periodontal destruction the possibility of host modulation so as to reduce the destructive aspects of the host response and hence reduce the damage to the periodontium was investigated. First group of drugs that showed this host modulation activity were Tetracyclines. Studies have demonstrated that tetracycline could significantly inhibit collagenase activity in GCF and gingival tissue, even at lower dosage than traditional antimicrobial dosages ie sub antimicrobial dosage.3 Tetracyclines inhibit collagenases by binding to the Ca2+ or Zn2+ (cations) required for the activation of MMPs such as collagenases and gelatinases.16 It is also possible that tetracyclines can inhibit synthesis of neutrophil-derived oxygen radicals, suppressing neutrophil migration and degranulation.17 However, microbial resistance to tetracyclines has been reported.7 In addition, long term treatment can result in other side- effects such as anorexi a, nausea, epigastric distress and fatty liver. A few recent studies on collagenase inhibition suggested that, like tetracyclines, herbal extracts are also potent inhibitors of pathologically elevated collagenases and hence they may be used as an alternative adjunct in the management of periodontal diseases.18, 19 Ayurvedic drugs like Neem, Triphala, Bakul etc have been used therapeutically since ancient times to treat diseases, including periodontal diseases. With the advent of modern synthetic drugs their convenience of standardized dosage form, dramatic efficacy in acute conditions and most of all simplicity of usage, there was a decline in the use of the plant medicines. But a long term treatment, with these synthetic drugs, has many adverse effects and they are also not cost effective. As Ayurvedic drugs, are widely acclaimed for their minimal side-effects and cost-effectiveness in India, they are now again being used extensively in treatment. Use of herbal extracts in treating periodontal diseases does not produce side effects of tetracycline compounds as well as other synthetic drugs. It has been shown that certain ayurvedic medicines also have the host modulation effect, similar to that of tetracyclines. A few recent studies conducted on collagenase inhibition by herbal extracts suggest that, like tetracyclines, herbal extracts are also potent inhibitors of pathogenically elevated collagenase and hence may be used as an alternative adjunct in the management of periodontal diseases.18, 19, 20 Turmeric which has Curcumin as its active constituent has been shown to have anti-inflammatory, anti-oxidant, anti- bacterial, anti- viral activities etc.10 Its anti- inflammatory activity has not been investigated in periodontal diseases. With the above details in mind the present study was conducted to evaluate the anti-inflammatory property of Turmeric. In the present study the anti-collagenase activity of Curcumin was compared with that of Doxycycline which has proven anti-collagenase activity. In the present study we chose Doxycycline because it has been proved that it is a more potent collagenase inhibitor as compared to minocycline and tetracycline.6 Studies have shown that Curcumin down-regulates MMP-9 activity13, 14, 21 and thus, in the present study we chose Curcumin to find out its anti-collagenase activity by checking its effect on MMP-9 activity. Research has shown that the predominant MMPs in inflamed gingival and periodontal tissues are PMN- type MMPs (MMP-8 MMP-9). Elevated activity of gelatinases (MMP-2 and MMP-9) has also been found in inflamed gingival tissues from chronic periodontitis patients.5, 22 The activity of PMN- type MMPs has been found not only to positively correlate to the severity of inflammation and pocket depth but also the amount of these enzymes recovered in an active rather than latent form appears to increase with greater severity of periodontal disease in the gingival tissue.20 Thus, since the above studies have shown that activity of PMN- type MMPs is increased in inflamed gingival tissues, in the present study we decided to use gingival tissue samples that were obtained from the patients diagnosed clinically with chronic periodontitis. The reduction in the MMP-9 activity of each sample of gingival tissue extract when incubated with the drugs .i.e. Doxycycline and Curcumin, was expressed as percentage of reduction from the control (without drug). The mean values showed 59.58% reduction in the MMP-9 activity with the addition of Doxycycline and 61.01 % reduction in the MMP-9 activity with addition of Curcumin to the gingival tissue extract under identical conditions. These results show that Curcumin has a significant inhibitory effect on PMN-type MMP-9, and this inhibitory effect of Curcumin is comparable to that of Doxycycline when the percentage of inhibition as compared to control was analysed. Curcumin also showed a significant reduction in the MMP-9 activity with an average inhibition of about 61% which is also statistically highly significant (p 23 and 12 g per day.24 But the overall results show that Curcumin has the ability to significantly inhibit the MMP-9 activity. The present study suggested that Curcumin could produce significant inhibition of MMPs at 1500 ÃŽ ¼g/ml concentration, which is well under the safe drug profile confirmed by toxicological studies.23, 24 Thus this study shows that Doxycycline and Curcumin possess anticollagenase activity in vitro. The finding of the present study is significant and confirms the use of Curcumin in treating periodontal diseases. A recent in vivo study showed that Curcumin modulates periodontal disease and had potent anti-inflammatory effects when it was systemically-administered in ligature-induced periodontitis in rats.25 Studies have shown that one of main mechanisms for the anti-inflammatory effects of Curcumin may be the inhibition of NF-ÃŽ ºB (nuclear factor kappa B).13, 25 The Curcumin-mediated inhibition of MMP-9 gene expression appears to occur via NF-ÃŽ ºB and AP-1 (Activator Protein-1) because their DNA binding activities were suppressed by Curcumin.14, 21 When compared to tetracycline, Curcumin has better anti-inflammatory effect, is more cost effective and has minimal side effects and thus can be tried as a substitute for Tetracycline as an anticollagenase agent. Curcumin also allows suppression of collagenase activity well within the safe dosage profile confirmed by toxicological studies. The in vivo environment is substantially different, and inherent limitations of an in vitro replication may constrain our understanding of the systemic effects of Curcumin. Further in vivo studies of Curcumin are needed to define its toxicological profile before making it a part of the therapeutic regimen in periodontal treatment. CONCLUSION In the light of observations from the current study it can be concluded that Doxycycline and Curcumin possess anticollagenase activity in vitro. Since Curcumin has better anti-inflammatory effect compared to Tetracycline, Curcumin being more cost effective and with no side effects can be tried as a substitute for Tetracycline as an anticollagenase agent. Thus Curcumin may have therapeutic potential as a host modulation agent in periodontal diseases.