Antiinflammatory effect of Proanthocyanidins in experimental Periodontitis in rats

 

Jayamathi Govindaraj¹*, Keerthidaa Govindaraj², Vidyarekha U ³, Kesavaram Padmavathy⁴

¹Professor, Department of Biochemistry, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Pallikaranai, Chennai, 600100

²MDS Department of Periodontics, Thai Mookambihai Dental College and Hospital, MGR University, Maduravoyal, Chennai.

³Senior Lecturer in Public Health Dentistry, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Pallikaranai, Chennai, 600100

⁴Associate Professor, Department of Microbiology, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Pallikaranai, Chennai, 600100

 

ABSTRACT:

Background: Periodontal disease is one of the most prevalent oral diseases affecting the supporting tissues of the teeth, gingiva, periodontal ligament, cementum, and alveolar bone.  Oxidative Stress plays an important role in the pathogenesis of periodontitis. Dietary supplementation of proanthocyanidin enhanced the host resistance as well as the inhibition of the biological and mechanical irritants involved in the onset of gingivitis and the progression of periodontal disease. Aim: The aim of the present study is to assess whether proanthocyanidins could exert protective as well as therapeutic effect on endotoxin induced experimental periodontitis in rats. Protective action of  proanthocyanidins is investigated by assaying the free radicals,  acute phase proteins, nonenzymatic antioxidants,  in comparison with a standard antimicrobial agent ‘tinidazole’. Materials and Methods: Experimental periodontitis was induced by injecting E.coli endotoxin. On the 10th day of endotoxin induction, the animals were divided into group,3and4 groups of 6 animals receiving 30mg  proanthocyanidins (sc-sub cutaneously)/kg body weight for 30 days; and  20mg tinidazole (administered orally)/kg body weight for 30 days. After 30 days of treatment procedure, the animals were sacrificed blood was collected for Biochemical assay. Rat maxillae was dissected out for histo pathological evaluation. Results: Levels of free radicals, acute phase proteins and Nonenzymatic antioxidants were significantly increased in  experimental periodontitis rats as compared to PC and tinidazole treated groups   showed significant inhibition of reactive oxygen species and lipid peroxides (P< 0.001). Conclusion: Proanthocyanidin, has been shown to provide a significant anti-inflammatory  effect on endotoxin (Escherichia coli) induced experimental periodontitis in rats and is a promising drug for treating periodontitis.

 

 

INTRODUCTION:

Plaque-induced periodontitis is mixed microbial infections which cause inflammation in the tissues around the teeth, influencing at least 10% of people worldwide^[1]. It has moderate association with several systemic diseases, e.g., cardiovascular disease, diabetes, and adverse pregnancy outcomes The oxidative stress is a phenomenon that occurs within the periodontal disease and has been linked with both onset of periodontal tissue destruction and systemic inflammation. Tissue destruction is mediated by reactive oxygen species (ROS) which are formed during the process of phagocytosis by polymorphonuclear leukocytes^[2].

 

The acute phase reaction are nonspecific and occur in response to many inflammatory conditions^[3]. The pro inflammatory cytokines originating at the diseased site activates the liver cells to produce acute phase proteins as a part of non specific response. Acute phase proteins viz., fibrinogen, C-reactive protein (CRP), α-1-acid glycoprotein, α-2-macroglobulin, α-1-antitrypsin and α-1- antichymotrypsin serve important functions in restoring homeostasis after infection or inflammation^[4].

 

The human system contains a number of protective antioxidant mechanisms, whose specific role is to remove harmful oxidants as they form, or to repair damage caused by ROS in vivo^[5]. There are, however, several nonenzymatic antioxidants to contribute and for the inactivation of free radical reactions.

 

Clinical parameters in the treatment of periodontal diseases are the reduction of the bacterial load or enhancement of the host tissues and ability to defend load or repair itself. Since periodontitis as a manifestation of systemic diseases, adjunctive chemotherapeutic agents may be necessary to control the disease process^[6]. Though chemotherapy has been proven effective in the resolution of diseases, there is always a risk that the disease may reemerge after the treatment has ended. 

 

Proanthocyanidins (PC):

Potential medicinal plants are developed to treat periodontal pathologies in order to improve the success rate in periodontitis, a novel herbal drug has been developed for the enhancement of host resistance and the inhibition of the pathological and mechanical irritants involved in the progression of periodontal disease. Grape seed extract (GSE), the richest source of PC, has potent antioxidants and exhibits numerous pharmacological activities. PC, one of the most abundant flavonoid in the plant kingdom, are extracted generally from grape seeds and they have antioxidant^[7], free radical scavenging^[8] and anticarcinogenic^[9] property. PC, also known as condensed tannins, are widely distributed in the plant kingdom and it represents a ubiquitous group of plant phenolics which take the form of oligomers or polymers of polyhydroxy flavan-3-ol units such as (+)-catechin, (-)-epicatechin and (-)-epicatechin-3-gallate. Proanthocyanidins consist of phenolic flavan-3-ol nucleus and the flavanol units linked by two C-4 and C-8 interflavan bonds^[10].

 

The present study is intended to assess the ameliorative effects of proanthocyanidins on endotoxin induced experimental periodontitis in rats. Protective and therapeutic effects of proanthocyanidins is investigated by assaying the free radicals, acute phase proteins, nonenzymatic antioxidants, in comparison with a standard antimicrobial agent tinidazole’.

 

MATERIALS AND METHODS:

Proanthocyanidins obtained from M/s Sigma Chemical Co., USA. All other chemicals used were of analytical grade.

 

Male Wistar rats were housed in animal quarters under strict veterinary supervision and maintained in control rooms with 12 h light/dark cycle. This study was conducted with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) and conformed to the guiding principles of Institutional Animal Ethical Committee (IAEC), for the care and use of laboratory animals.

 

Experimental and treatment protocol:

Induction of experimental periodontitis (EP):

EP was extracted induced by injecting E.coli endotoxin as described by Ramamurthy et al (2002)^[11]. On the 10th day of endotoxin induction, the animals were divided into group 3 and 4 of 6 animals each.

It consisted of

Group 1: Control;

Group 2: experimental periodontitis (EP).

Animals of group 2 were further divided into following 2 groups. and received proanthocyanidins (sc), as follows:

Group 3: 30mg proanthocyanidins (sc)/kg body weight for 30 days;

Group 4: 20mg tinidazole (administered orally)/kg body weight for 30 days^[12]

 

After the treatment procedure, the animals were sacrificed, blood was collected to perform Biochemical assay .

 

Biochemical assays:

Assay of Hydrogen peroxide(H₂O_2). H₂O₂ was assayed according to Pick and Mizel (1981)^[13]

 

Assay of Superoxide anion (O₂^•−) Superoxide anion was measured according to Market et al.,(1984)^[14]

 

Myeloperoxidase assay (MPO). MPO activity was assessed by the method of Henson and Johnston^[15].

 

Lipid peroxides. Lipid peroxides in serum was determined by the method of Yagi (1984)^[16]

 

Fibroinogen Fibrinogen assay is based on a method described by Clauss (1957)^[17].

 

Assay of high sensitivity C- reactive protein (CRP) in serum:

Latex- enhanced immunoturbidimetric assay for the quantitative in vitro determination of CRP in serum was carried out according to Kuller et al (1996)^[18] and Thompson et al (1995)^[19].

 

Ascorbic acid:

Ascorbic acid in plasma was estimated by the method of Omaye et al (1979)^[20]

 

α- tocopherol:

α- tocopherol in serum was estimated by the method of Quaife and Dju (1951)^[21]

 

Ceruloplasmin:

Ceruloplasmin is a copper oxidase, which catalyses the oxidation of some polyamines and its action on p-phenylene diamine is used as a measure of the amount present in serum and the method of Ravin (1961)^[22]

 

Reduced glutathione:

Reduced glutathione in serum was measured by the method of Moran et al (1961)^[23]

Protein was determined according to the method of Lowry et al (1951)^[24]

 

Histopathological studies:

Rat maxillary halves were dissected out, and they were analysed by light microscopy.

 

 

 

Statistical methods:

One way analysis of variance was applied to evaluate any significant difference in the mean values. All values used in analysis represented as mean ± SE of 6 rats. POST HOC Tukey’s test was applied to find out the statistically significant groups and Independent t test was applied to compare only group-4 with group 2.

 

RESULTS:

Levels of ROS such as H₂O₂, O₂^•−, myeloperoxidase and the levels of lipid peroxides were significantly increased in endotoxin induced experimental periodontitis rats as compared to PC and tinidazole treated groups showed significant inhibition of reactive oxygen species and lipid peroxides (P< 0.001) (Table 1).

 

The levels of acute phase proteins such as C-reactive proteins and fibrinogen were increased significantly in plasma was observed in EP rats. PC (Groups 3 ) as well as tinidazole treated groups (Group 4) were observed to have a significant (P < 0.001) decrease in the levels of acute phase proteins (Table 2).

 

Nonenzymatic antioxidants such as ascorbic acid, α-tocopherol, ceruloplasmin and reduced GSH in serum were decreased significantly in EP rats whereas proanthocyanidins (Groups 3) and tinidazole (Group 4) treated groups were found to have significant (P < 0.001) protective effect as reflected by an increase in the levels of antioxidants as shown in Table 3.

 

Table-1: Effect of PC On the levels of free radicals, Mpo And Lipid Peroxides In endotoxin Induced Experimental Periodontitis In Rats

Groups	H2O2	O2•−	Myeloperoxidase	Lipid peroxides
1	2.1± 0.08	4.5 ± 0.15	2.51 ± 0.09	2.9 ± 0.022
2	8.3 ± 0.24a,e	9.2 ± 0.16a,e	4.5 ± 0.09a,e	7.8 ± 0.03a,e
3	3.2 ± 0.08d	5.0± 0.09d	2.8 ±0.05d	3.3 ± 0.10d
4	3.7 ± 0.14d	5.2 ± 0.09d	3.1± 0.03d	3.4 ± 0.13d

All values are mean ± SE from 6 animals in each group.p values: ^a< 0.001 as compared to Group 1 by POST HOC Tukey HSD;

 ^d< 0.001 as compared to Group 2 by POST HOC Tukey HSD;

 ^e< 0.001 as compared to Group 1 by Independent t test.

^f< 0.001 as compared to Group 2 by Independent t test.

H₂O₂ level is expressed as µmol of hydrogen peroxide oxidized / min/mg protein; O₂^•− level is expressed as nmol of superoxide liberated / min/mg protein; Myeloperoxidase level is expressed as unit/min/mg protein.; lipid peroxide level is expressed as nmol/ml

 

Table-2: Effect of PC On acute phase proteins In Endotoxin Induced Experimental Periodontitis in Rats

Groups	Fibrinogen	C-reactive protein
1	265 ± 8.16	0.28 ± 0.022
2	479 ± 9.1a,e	2.6 ± 0.21a,e
3	294 ± 4.6d	0.58 ± 0.07d
4	307 ± 4.9f	0.7 ± 0.02f

All values are mean ± SE from 6 animals in each group. p values: ^a< 0.001 as compared to Group 1 by POST HOC Tukey HSD;

 ^d< 0.001 as compared to Group 2 by POST HOC Tukey HSD; ^e< 0.001 as compared to Group 1 by Independent t test.

 ^f< 0.001 as compared to Group 2 by Independent t test.

Fibrinogen expressed as mg / dl; C-reactive protein expressed as mg / dl of blood.

 

Table-3: Effect of PC On non enzymatic antioxidants In Endotoxin Induced Experimental Periodontitis in Rats

Group	Ascorbic acid	a-tocopherol	Ceruloplasmin	GSH
1	1.30 ± 0.007	4.0 ± 0.06	30 ± 0.28	54 ± 0.28
2	0.61± .007a,e	2.3 ± 0.03a,e	16.7 ±0.2a,e	34.1 ± 0.2a,e
3	1.0 ± 0.07d	3.2 ± 0.04d	28.6 ± 0.16d	47.3 ± 0.67d
4	9.2 ± 0.03f	2.96±0.02f	26.6 ±0.19f	43 ± 0.31f

All values are mean ± SE from 6 animals in each group.p values: ^a< 0.001 as compared to Group 1 by POST HOC Tukey HSD; ^d< 0.001 as compared to Group 2 by POST HOC Tukey HSD;

^e< 0.001 as compared to Group 1 by Independent t test.

^f< 0.001 as compared to Group 2 by Independent t test.

Ascorbic acid is expressed as mg/dl serum; a-tocopherol is expressed as mg/dl serum; Ceruloplasmin is expressed as mg/dl serum; GSH is expressed as mg/dl serum;catalase is expressed as μmoles of H₂O₂ decomposed / min/mg protein;

 

Figure -1 Histopathological changes observed in endotoxin induced rats EP treated with proanthocyanidins (PC)

(A)     Group 1 - normal architecture of gingival tissues; (B) Group 2 - dense chronic inflammatory cells; (C) Group 3-few scattered chronic inflammatory cells and blood vessels; (D) Group 4 - occasional inflammatory cells

 

Histopathological evaluation on rat maxillae are shown in Fig. 1 (A-D). Fig. A represented normal structure of rat maxillae on histopathological evaluation. The histopathological evidence of EP in animals indicated that progressive disease was associated with the presence of cellular infiltration of inflammatory cells (Fig. B). Proanthocyanidin treated groups especially 30 mg (Fig. C) and Fig. D for 30 days and tInidazole treated group showed no such abnormalities and scattered, diffused inflammatory cells and blood vessels were observed. This showed that the severity of periodontal inflammation in EP was reduced after treatment with PC and tinidazole.

 

DISCUSSION:

Periodontal disease is a common, complex inflammatory disease characterized by the destruction of periodontal tissues and loss of connective tissue attachment. Neutrophils were implicated to play a destructive role in the periodontal tissue breakdown process due to high levels of lysosomal enzymes and generation of reactive oxygen derivatives^[25]. Reactive oxygen species (ROS) are continuously generated by most tissues as an integral part of normal cellular metabolism. MPO possessed potent antimicrobial activity, and as an indicator of neutrophilic degranulation, it was reported to play a crucial role in tissue injury^[26]. MPO and lactoferrin were considered to reflect the strength of oxidative stress^[27] and the imbalance between the levels of MPO and lactoferrin could result in tissue damage by ROS in periodontitis^[28] and oxygen derived free radicals and their products were known to play an important role in the pathogenesis of chronic inflammatory disorders^[29] found that O₂^•− and/or H₂O₂ were implicated in the destruction of periodontal tissues. The increased levels of ROS suppressed the antioxidant systems, leading to the damage of periodontium. The protective role of PC through its free radical scavenging property both in vitro and in vivo was also demonstrated by Ye et al (1999)^[30] and agree with the study indicated that PC had inhibitory effect on the ROS generation.

 

The acute phase response was a nonspecific process that may occur in the initial host response to injuries, infections, ischemic necrosis or malignancy^[31]. Thus acute-phase response plays an important role in protection against bacterial products such as endotoxin.^[32]. Tissue destruction in periodontitis leads to release of pro-inflammatory cytokines which inturn modulate the systemic acute-phase reaction and the hepatic acute-phase proteins release. Increased level of fibrinogen was also observed in the present study and it was restored to normal in proanthocyanidin treated and tinidazole treated rats.

 

The findings of the present study concurred with an earlier report which showed diminished levels of ascorbic acid in periodontitis and this reduction progressed along with advancement of the disease. In this study, GSH content was significantly reduced in EP rats and this could be explained by the assimilation of GSH by the rapidly generating free radicals. Canakci et al.^[33] detected reduced antioxidant activities in saliva reflecting increased oxygen radical production or activity during periodontal inflammation. However, antioxidant property of PC imparted some degree of GSH salvation as GSH levels were shown to increase significantly in PC treated groups.

 

Proanthocyanidins were suggested to be more potent when compared to flavanols in their antioxidant capacity due to the fact that oxidation of proanthocyanidins predominantly produced semiquinone radicals that coupled to produce oligomeric compounds through nucleophilic addition^[34]. Chemical structure determined relative ease of flavonol or proanthocyanidin oxidation and free-radical scavenging activity although the presence of galloyl groups and the number and position of hydroxyl groups (based on redox potential) was noticed to enhance antioxidant activity^[35]. Identification of histopathological manifestations is a necessary step to assess the pathological changes in EP and also in EP treated with PC. Histopathological studies showed the degree of decreased inflammation after PC treatment.

 

Natural antioxidants strengthened the endogenous antioxidant defense from ROS ravage and it restored the optimal balance by neutralizing ROS. Chemically, the important features of flavonoids, were their remarkable antioxidant properties: the hydrogen donating substituents (hydroxyl groups) attached to the aromatic ring structures of flavonoids, which enabled them to undergo redox reactions scavenging free radicals more easily and the stable delocalization system, consisting of aromatic and heterocyclic rings as well as multiple unsaturated bonds, which helped to delocalize and regulate the free radicals^[36].

 

CONFLICT OF INTEREST:

Nil

 

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Received on 07.04.2019           Modified on 21.05.2019

Accepted on 28.06.2019         © RJPT All right reserved