Efficacy of an herbal antioxidant as an adjunct to nonsurgical periodontal therapy on procalcitonin levels in smokers with chronic periodontitis.

CONTEXT: Smokers are at a higher risk of contracting periodontitis. This association is established and reported in many studies. Periodontitis is a polymicrobial disease, wherein smoking is an environmental factor which aggravates the damage caused to the tissues. Procalcitonin (ProCT) is a well-known marker for inflammatory and other infectious diseases and has been assessed in this study. It has been observed that the release of reactive oxygen species (ROS) is exaggerated in smokers. Antioxidants are administered to combat the harmful effects caused by the ROS, and Oxitard™ is a powerful herbal antioxidant that can counteract the action of free radicals. AIMS: As smokers are more prone to tissue injury due to excessive release of ROS, this study aimed to understand the benefits of systemic administration of Oxitard after scaling and root planing (SRP) on serum ProCT levels in smokers with chronic periodontitis (CP). SETTINGS AND
DESIGN: This was a randomized interventional study, wherein forty patients participated.
MATERIALS AND METHODS: Forty smokers with CP aged, between 35 and 60 years, were equally divided into Group A (SRP + Oxitard) and Group B (SRP only), selected from an outpatient ward of a referral care hospital in Hyderabad. A written informed consent was obtained from all the patients, and the study was approved by the institutional ethical committee (DN0026-15). STATISTICAL ANALYSIS USED: Data were analyzed by GraphPad Prism software version 6.01 (GraphPad software incorporation, California, USA). Comparison within the groups was made by paired t-test and between the groups by unpaired t-test. P < 0.05 was considered of statistical significance.
RESULTS: Intragroup assessment showed an improvement in all the variables from baseline to 3 months, which was statistically significant (P < 0.0001). A comparison between the groups, however, yielded better results in Group A (Oxitard + SRP) over Group B.
CONCLUSIONS: Oxitard administered systemically for 3 months after SRP was beneficial in improving both clinical and biochemical parameters.

INTRODUCTION

Periodontitis is caused due to plaque. Plaque, a biofilm teeming with microorganisms, is the perpetuator of the disease. Along with the plaque-laden biofilm, other etiological factors causing periodontitis include an impaired or exaggerated host response and environmental and genetic risk factors. Among the environmental risk factors, smoking plays an indelible role in the etiology of periodontitis. Due to the compromised immune response of the host and the toxic products such as nicotine and tar which are incorporated in cigarettes, smokers are easily affected by lung diseases and cardiovascular diseases, to name a few. Procalcitonin (ProCT) is a peptide precursor of the hormone calcitonin, and its levels are undetectable in health. In inflammatory conditions due to bacterial infections, the levels of ProCT are studied to rise exponentially, and thus it is often assessed as a marker. Oxidative stress often occurs in smokers, and antioxidant therapy helps improve the treatment outcomes in them.

Smokers are four times more prone to periodontitis when compared to nonsmokers. Although nonsurgical periodontal therapy (NSPT) improves the condition of the tissues, optimal results are not obtained as seen in nonsmokers. Antioxidants could be administered after NSPT to improve the results in smokers.

MATERIALS AND METHODS

This was a randomized, interventional, single-blinded study, wherein forty smokers with chronic periodontitis (CP) participated. The study was conducted from August 2016 to May 2017, and approval was accorded by the institutional ethical committee. Written consent was taken from all the patients.

To achieve a mean difference of 0.46 between the test and control groups, with the level of significance being 5% and the power being 80%, it was calculated that twenty patients per group would suffice.

The samples were equally divided into two groups: Group A (twenty patients receiving Oxitard™, The Himalaya Drug Company, Makali, Bengaluru, Karnataka, India – two capsules twice a day for 3 months after scaling and root planing [SRP]). Each capsule contains extracts and powder along with oil.

The extracts comprise Amra (Mangifera indica)* API-Ayurvedic Proprietary Ingredient 94 mg, Ashwagandha (Withania somnifera)* 71 mg, Gajara (Daucus carota)* 47 mg, Yashtimadhu (Glycyrrhiza glabra)* 29 mg, and Draksha (Vitis vinifera)* 12 mg. The powder comprise Amalaki (Emblica officinalis)* 141 mg, Lavanga (Syzygium aromaticum)* 29 mg, Yashada bhashma* 2.5 mg, and the oil is made up of Godhuma (Triticum aestivum)* 6.5 mg [Figure 1]. Group B included twenty patients undergoing SRP only.

Figure 1

Oxitard capsules

Randomization procedure

An investigator (KRR) randomly distributed the study samples and was also responsible for administering Oxitard to the test group on a monthly basis for 3 months. The NSPT was performed by another investigator (MVNS) who was blinded to the randomization procedure.

Inclusion criteria

Current smokers having a minimum of twenty permanent teeth with probing depths (PDs) >4 mm and clinical attachment loss >2 mm participated in the study.

Exclusion criteria

Former smokers who have quit the habit, patients with any other systemic diseases, having undergone periodontal therapy within 6 months, and those on antibiotics 3 months prior were excluded from the study.

Clinical parameters

All patients were subjected to clinical examination using William's periodontal probe. The indices assessed were Gingival Index (GI), probing pocket depth (PPD), and clinical attachment level (CAL).

Biochemical variables

Collection of blood sample and serum separation – Blood samples were taken from the antecubital vein. Two milliliters of blood was collected and was subjected to centrifugation. The supernatant was assessed for the levels of ProCT

Assessment of serum ProCT – ProCT assessment was done at baseline and 3 months after SRP using the quantitative sandwich immunoassay technique by Ray Biotech, a bio-techne brand, RayBiotech Life 3607 Parkway Lane, Suite 200 Norcross, GA 30092 USA [Figures 2 and 3]

Figure 2

Frontal view of Human procalcitonin enzyme-linked immunosorbent assay kit

Figure 3

Rear view of Human procalcitonin enzyme-linked immunosorbent assay kit

Reagents used – ProCT microplate, wash buffer concentrate (20X), standard protein detection antibody, horseradish peroxidase-streptavidin concentrate, tetramethylbenzidine one-step substrate reagent, stop solution, and assay diluent [Figure 4]

Figure 4

Reagents used in enzyme-linked immunosorbent assay

Outcome measures – The primary outcome measure assessed included the serum ProCT levels and the secondary outcome measures were the clinical parameters, GI, PPD, and CAL.

RESULTS

Intragroup comparison – There was a statistically significant improvement in all the variables assessed in both the groups (P < 0.001) [Tables 1 and 2].

Table 1

Intragroup comparison of clinical parameters in Group A and Group B

Parameters	Groups	n	Range	Mean±SD	P
GI	Group A at baseline	20	2.2-3.0	2.6±0.3	<0.0001
	Group A after 3 months	20	0.6-1.3	1.0±0.2
	Group B at baseline	20	2.2-3.0	2.6±0.2	<0.0001
	Group B after 3 months	20	0.8-1.9	1.3±0.3
PPD	Group A at baseline	20	7-9	8.1±0.6	<0.0001*
	Group A after 3 months	20	3-4	3.8±0.4
	Group B at baseline	20	6-10	8.0±1.3	<0.0001*
	Group B after 3 months	20	3-6	4.6±0.8
CAL	Group A at baseline	20	5-7	6.1±0.6	<0.0001*
	Group A after 3 months	20	1-2	1.7±0.5
	Group B at baseline	20	4-8	6.0±1.2	<0.0001*
	Group B after 3 months	20	1-4	2.6±0.8

*P<0.05 is statistically significant. GI – Gingival Index; PPD – Probing pocket depth; CAL – Clinical attachment level; n – Number of patients; SD – Standard deviation; P – Probability value

Table 2

Intragroup comparison of procalcitonin levels between Group A and Group B

Parameter	Groups	n	Range	Mean±SD	P
Serum procalcitonin (ng/ml)	Group A at baseline	20	2.01-3.45	2.61±0.47	<0.0001*
	Group A after 3 months	20	0.23-1.82	0.77±0.43
	Group B at baseline	20	1.69-3.65	2.53±0.57	<0.0001*
	Group B after 3 months	20	0.92-2.85	1.74±0.55

*P<0.05 is statistically significant. ng/ml – Nanograms/milliliter; n – Number of patients; SD – Standard deviation; P – Probability value

Intergroup comparison – When a comparison was made between the groups, there was a marked improvement in both the clinical and biochemical parameters in Group A when compared to Group B (P < 0.001) [Tables 3 and 4]. Pertaining to the primary outcome measure, the mean serum ProCT levels dropped from 2.61 at baseline to 0.77 at 3 months in Group A, whereas it was 2.53 at baseline and reduced to 1.74 in Group B. However, the improvement in serum ProCT was more in Group A when compared to Group B (P < 0.0001).

Table 3

Intergroup comparison of clinical parameters between Group A and Group B

Parameters	Groups	n	Range	Mean±SD	P
GI	Group A after 3 months	20	0.6-1.3	1.0±0.2	0.002*
	Group B after 3 months	20	0.8-1.9	1.3±0.3
PPD	Group A after 3 months	20	3-4	3.8±0.4	0.0005*
	Group B after 3 months	20	3-6	4.6±0.8
CAL	Group A after 3 months	20	1-2	1.7±0.5	0.0001*
	Group B after 3 months	20	1-4	2.6±0.8

*P<0.05 is statistically significant. GI – Gingival Index; PPD – Probing pocket depth; CAL – Clinical attachment level; n – Number of patients; SD – Standard deviation; P – Probability value

Table 4

Intergroup comparison of procalcitonin levels between Group A and Group B

Parameter	Groups	n	Range	Mean±SD	P
Serum procalcitonin (ng/ml)	Group A after 3 months	20	0.23-1.82	0.77±0.43	<0.0001*
	Group B after 3 months	20	0.92-2.85	1.74±0.55

*P<0.05 is statistically significant. ng/ml – Nanograms/milliliter; n – Number of patients; SD – Standard deviation; P – Probability value

In addition, the mean GI was 2.6 which dropped to 1 in Group A, and it was 2.6 which reduced at 3 months to 1.3 in Group B. The mean PPD was 8.1 at baseline and improved to 3.8 after 3 months in Group A, whereas it was 8.0 at baseline and got reduced to 4.6 after 3 months in Group B. The mean CAL was 6.1 at baseline and improved to 1.7 after 3 months in Group A, whereas it was 6.0 at baseline and got reduced to 2.6 after 3 months in Group B.

Thus, when an intergroup comparison was made pertaining to the secondary outcome measures, all the parameters showed better results in Group A when compared to Group B.

DISCUSSION

Periodontitis is highly prevalent worldwide and predominantly, the Gram-negative bacteria residing in the plaque biofilm as well as on tooth surfaces are one of the initiators of the disease. The subgingival microbiota thrives as a polymicrobial community in the biofilm and is difficult to eradicate.[1] It has been observed from various studies that smokers are more prone to have periodontitis when compared to nonsmokers. It has also been studied that smokers have a poor response to therapy whether nonsurgical or surgical.[2]

Neutrophils are the primary inflammatory cells which protect the host, and a quantitative or qualitative reduction in them will cause localized or systemic infections to occur. It is well established by in vitro studies that, in smokers, the neutrophils are deficient both in chemotaxis and phagocytosis.[3]

Smokers have been observed to have elevated levels of tumor necrosis factor-α, prostaglandin, neutrophil elastase, and matrixmetalloproteinase-8.[4]

ProCT has emerged as a promising aid for the diagnosis of infections as higher levels of ProCT have been detected in severe bacterial infections compared to viral and nonspecific inflammatory diseases. Therefore, the clinical application of ProCT has increased.[5]

A rapid rise in serum ProCT concentration can be seen in the case of a poor prognosis or in an active inflammatory condition. ProCT is a useful indicator to determine the degree of infection, predict the prognosis, and monitor the response to treatment. It can be observed at various concentrations in different body fluids, and it has been stored in the extracellular space in tissues.[6]

Some researchers studied the relationship between salivary ProCT and periodontitis in diabetic patients. The other indices assessed were bleeding on probing (BOP) and hemoglobin A1c (HbA1c). The salivary ProCT levels were observed to correlate positively with the BOP and glycated Hb levels, thus making it very clear that salivary ProCT levels can be included as a good marker to assess the disease activity in both the diseases.[7]

A study was performed to analyze the role of ProCT in CP patients in comparison to healthy controls. The saliva samples of thirty CP patients and thirty healthy controls were examined for the levels of ProCT by enzyme-linked immunosorbent assay (ELISA). The results showed that there was no significant difference in the ProCT levels between the test and control groups, thus negating the value of utilizing ProCT as a diagnostic marker.[8]

Another study assessed the role of ProCT in patients with CP and chronic migraine (CM). One hundred and thirty-eight individuals were divided into four groups based on their migraine and periodontitis history. Thirty-seven patients belonged to the systemically and periodontally healthy group (H), 19 to the systemically healthy and periodontitis group (CP), 34 to the CM and periodontally healthy group (CM), and 48 patients had both CM and CP (CM + CP). The ProCT and high-sensitive C-reactive protein (hs-CRP) serum levels were determined from blood samples taken during migraine interictal period. The results showed that CP + CM group (0.056 ± 0.006 ng/mL) had significantly higher serum ProCT levels in comparison with the systemically and periodontally healthy group (0.029 ± 0.01 9 ng/mL), CM group (0.041 ± 0.002 ng/mL), or CP group (0.034 ± 0.014 ng/mL) (P < 0.001). There were no significant differences in hs-CRP between groups (P = 0.081). Multiple linear regression analysis showed that CP was associated with increased ProCT levels in CM patients (R2 = 0.293, P < 0.001). Thus, it was concluded that CP contributes independently to raised ProCT levels in CM patients.[9]

In this study, the ProCT levels in both the groups were elevated at baseline, the values being 2.61 ng/ml in Group A and 2.53 ng/ml in Group B which could be related to the patients being current smokers with periodontitis.

Reactive oxygen species (ROS) production is a favorable mechanism of the host against the bacterial pathogens. In moderation, the ROS produced is protective; however, when it is excessively released, it causes the oxidation of DNA, lipids, and proteins, which in turn damages the host tissue.[10]

Some researchers have evaluated the association of intensity of smoking on the antioxidant superoxide dismutase levels (SODs) in patients with CP. The SOD levels were evaluated using spectrophotometry in healthy controls (nonsmokers), light smokers (≤10 cigarettes/day), and heavy smokers (≥10 cigarettes/day). The study found that there was a gradual reduction in the SOD levels with an increase in the intensity of smoking.[11]

One hundred and thirty-four male patients with CP and 64 healthy male controls were assessed in another study. The GI, Plaque Index, Papillary Bleeding Index, and CAL were measured using University of North Carolina 15 probe. Total antioxidant capacity (TAOC), red blood cell-SOD, glutathione peroxidase (GSH-Px), Vitamin C, malondialdehyde, and CRP were the biochemical markers assessed. This study showed that smokers with periodontitis had higher periodontal parameters and systemic oxidative stress when compared to the healthy controls.[12]

The TAOC was assessed in smokers and nonsmokers with severe periodontitis in yet another study. Sixty-four patients with severe periodontitis, including 27 smokers, formed the test group and 37 nonsmokers formed the control group. Stimulated saliva samples were taken from all the patients, and the TAOC was estimated using the ferric-reducing antioxidant power technique. It was observed that the Total antioxidant capacity (TAOC) was much lower in smokers in the test group when compared to that of the control group.[13]

Thereby, the literature claims smoking to cause severe oxidative stress that disturbs the balance between periodontal health and disease. It is well known that NSPT remains a gold standard treatment for CP.[12] However, it has been observed in many studies that, when a comparison was made between smokers and nonsmokers who had undergone NSPT, the improvement in all the periodontal parameters was much better in the nonsmokers.[141516]

Another study evaluated the efficacy of NSPT on the oxidant and antioxidant status in smokers with periodontitis. Twenty-nine patients with CP (15 smokers and 14 nonsmokers) and twenty periodontally healthy controls (of whom ten were smokers and ten nonsmokers), totaling 49 individuals, were included in this study. The clinical parameters examined were the PI, GI, BOP, PPD, and CAL. Blood was drawn from each participant to assess both TAOC and antioxidant status. The clinical parameters as well as the oxidant and antioxidant status were assessed at baseline and 6 weeks after SRP. The study concluded that periodontal treatment improves the clinical parameters in both smokers and nonsmokers. These results confirm that NSPT can reduce oxidative stress.[17]

The effect of NSPT on the oxidative stress markers in the gingival crevicular fluid, serum, and saliva in smokers and nonsmokers with and without periodontitis was evaluated in another study. Forty-seven patients with periodontitis (24 smokers and 23 nonsmokers) and 46 periodontally healthy controls (23 smokers and 23 nonsmokers), totaling 93 participants, were included in that study. 8-hydroxydeoxyguanosine, 4-hydroxynonenal, and GSH-Px were analyzed with ELISA. It was observed that NSPT was beneficial in reducing the oxidative markers in patients with periodontitis.[18]

To understand if the administration of antioxidants as adjuvants after NSPT can benefit smokers with periodontitis, in this study, the test group (Group A) was given an herbal antioxidant – Oxitard. There were no side effects reported after the use of these capsules.

A study was done to compare the benefits of administering Oxitard versus Aloe vera gel in patients with submucous fibrosis. The study included 120 individuals with oral submucous fibrosis (OSMF), divided into two groups: group A receiving Oxitard four capsules daily for 3 months and Group B received 5 mg A. vera gel, applied topically thrice daily for 3 months. After 3 months, there was a significant improvement in mouth opening as well as the clinical symptoms in the Oxitard group when compared to the A. vera group.[19]

Another study evaluated the role of Oxitard capsules as monotherapy in OSMF. The study comprised 48 individuals having chronic OSMF. Oxitard was given at a dose of two capsules twice a day, for 3 months. All the patients were evaluated for parameters such as difficulty in opening mouth, hyperkeratosis, pain, and lesion size. Parameters such as difficulty in opening mouth, hyperkeratosis, and pain significantly reduced. The lesion size also reduced to a small extent. It was observed that Oxitard was efficient in treating OSMF and was well tolerated.[20]

In this study also, NSPT was effective in controlling the infection as there was an improvement in all the variables 3 months after therapy. However, when an intergroup comparison was made, the ProCT levels reduced more in Group A (SRP + Oxitard) (2.61 ng/ml at baseline to 0.77 ng/ml after 3 months) when compared to Group B (SRP only) (2.53 ng/ml at baseline to 1.74 ng/ml after 3 months). ProCT values obtained in Group A were statistically significant (P < 0.0001).

Pertaining to the clinical parameters such as GI, PPD, and CAL also, there was better outcome in Group A when compared to Group B. All the clinical parameters have shown improvement from baseline to 3 months, which was statistically significant. Mean GI was 2.6 at baseline and 1.0 after 3 months in Group A and 2.6 at baseline and 1.3 after 3 months in Group B (P < 0.002). Mean PPD was 8.1 at baseline and 3.8 after 3 months in Group A and 8.0 at baseline and 4.6 after 3 months in Group B (P < 0.0005). Mean CAL was 6.1 at baseline and 1.7 after 3 months in Group A and 6.0 at baseline and 2.6 after 3 months in Group B (P < 0.0001).

These superior results in relation to the clinical parameters PD and CAL are rather baffling, as the response to NSPT in nonsmokers showed improvements of 1–2 mm only in CAL. Perhaps, these results could be due to the Hawthorn effect among the patients who might have stopped smoking during the study period.

Thus, it can be inferred that patients in Group A who were administered the herbal antioxidant as an adjuvant to NSPT showed better results than patients in Group B, emphasizing that herbal oxidants can be administered safely as adjuvants in smokers with periodontitis.

Limitations

Patients with periodontitis only did not participate in the study. Only the current smokers were assessed, and also TAOC was not evaluated in both the groups. Moreover, smoking history was not assessed and serum cotinine levels were not measured.

CONCLUSIONS

Although ample evidence is there to point out that treatment outcome would be suboptimal in smokers when compared to nonsmokers, this study was carried out to understand if herbal antioxidants could be safely administered as adjuvants in smokers with periodontitis. Further studies should be done in patients who quit smoking, and Oxitard should be administered at least for 6 months after NSPT to prove its true benefits.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.