Mouthwash with Tagetes lucida Cav. for Control of Chronic Periodontitis in Older Adults

: Background: Tagetes lucida Cav ( T. lucida ) is an herbaceous plant from the family Asteraceae , native to Mexico, Guatemala, and other Mesoamerican countries. Their common names are: (i) Spanish names: “hierban í s”, “jeric ó n”, “peric ó n vomol”, “Santa Mar í a”, “cuchrucum í n”, “falso hiperic ó n”, “hierba añil”, “peric ó n”, “periquillo”; (ii) indigenous names: “ichka”, “cuahuyauhtli”, “yahuhtli”, “nan á uarhi”, and (iii) English names: “sweet-scented marigold”, “sweet mace”, and “sweet marigold”., Its chemical composition of coumarins, estragole, quercetin, and ﬂavonoids has antioxidant, anti-inﬂammatory, and antimicrobial effects. This study aimed to evaluate the effect of a mouthwash made with T. lucida for control of chronic periodontitis (CP) in older adults. Methods: We carried out a quasi-experimental study with a convenience sample of 60 older adults with CP. The sample was divided into a placebo group (PG) n = 30, administered with a mouthwash made with alcohol diluted with puriﬁed water (10%), and the experimental group (EG) n = 30 treated with a mouthwash made with T. lucida extract in 10% alcohol; both groups were administered three times a day for three months. Results: We found a signiﬁcant improvement in the EG group compared with the PG group in: probing pocket depth (baseline, 5.2 ± 0.81 vs. post-treatment, 3.1 ± 0.43, p < 0.05), the clinical attachment loss (baseline, 6.6 ± 1.9 vs. post-treatment, 2.1 ± 1.5, p < 0.05), lipoperoxides (baseline, 0.056 ± 0.03 vs. post-treatment, 0.034 ± 0.02 µ mol/L, p < 0.05), and chronic inﬂammation markers (IL-1, IL-8, and TNF- α ) ( p < 0.05). Conclusions: Our ﬁndings suggest that mouthwash made with T. lucida extract, administered three times a day, clinically improved chronic periodontitis in older adults, associated to a signiﬁcant decrease in lipoperoxides and proinﬂammatory markers.


Introduction
Chronic periodontitis (CP) is a multifactorial disease, which is associated with the activation of polymorphonuclear leukocytes. Consequently, it generates a high concentration of reactive oxygen species (ROS) and a decrease in the antioxidant system, which causes oxidative stress in the periodontal tissue [1][2][3][4]. CP has a high prevalence and incidence in the world; in Mexico it occurs in more than 70% of the older adult population (≥60 years) [5,6].
If this biochemical disorder is maintained, the epithelial adhesion is destroyed and the alveolar crest loses its height, which translates clinically into dental mobility and formation of periodontal pockets, causing the accumulation of bacteria that increases the inflammation, destroying the periodontal ligament, and consequently, the alveolar bone atrophies, and the tooth is lost. This disease is associated with the activation of polymorphonuclear leukocytes, which in turn can generate a high concentration of reactive oxygen species (ROS) and a decrease in the antioxidant system, which causes oxidative stress in the tissue periodontal [1][2][3][4]. It has been observed that CP has a high prevalence and incidence in the world; in Mexico it occurs in more than 70% of the population of older adults [5,6].
To avoid the damage related to CP, conventional treatment focuses on stopping the progression of periodontal tissue destruction by eliminating local factors through oral hygiene instructions combined with scaling and root smoothing, to maintain and/or restore the function and aesthetics [5]. However, even when this treatment is carried out rigorously, the results are not entirely satisfactory. For this reason, various ethnobotanical therapies have been developed in the treatment of CP, with favorable effects [7][8][9].
In this context, we propose that Tagetes lucida could be useful as a therapeutic option for CP, considering its chemical composition of coumarins, estragole, and quercetin, with antioxidant, anti-inflammatory and antimicrobial effects [10,11].
Experimentally, it has been shown that T. lucida has antifungal and antibacterial properties. Additionally, it has tranquillizing, antidepressant activity, as well as antiinflammatory and antioxidant activities [14][15][16][17][18][19]. Therefore, this study aimed to evaluate the impact of T. lucida on oxidative stress and chronic periodontitis in older adults.

Subjects and Design
After informed consent, a quasi-experimental, double-blind study was conducted in 60 older adults, 17 (28%) men and 43 (72%) women with an average age of 68 ± 6.52, who attended the odontology service at Zaragoza Clinic, UNAM. They had controlled chronic non-communicable diseases, without consumption of antioxidant supplements in the last 6 months, all with clinical diagnosis of periodontal disease. The patients were not under periodontal treatment or used a supplement for oral cleaning, no change was made in their hygienic and dietary habits. The Ethics Committee of the Universidad Nacional Autónoma de México (UNAM), Zaragoza Campus approved the research protocol (FESZ/DEPI/325/16).
Subsequently, a clinical evaluation for the diagnosis of CP was achieved by the following parameters: the depth and presence of bleeding on probing, the level of clinical insertion, and the presence of tooth mobility.
For the study, a convenience sample of 60 subjects who met the inclusion criteria were randomly divided into 2 groups, the placebo group (PG, n = 30) was administered mouthwash of absolute alcohol (10%) in purified water and the experimental group (EG, n = 30) was treated with a mouthwash treatment based on the extract of T. lucida (10%), 3 times a day for 3 months (5 mL every 8 h) (Figure 1). Saliva samples were taken before and after treatment. Lipoperoxides were quantified by the TBARS (Thiobarbituric acid reactive substance) technique, total antioxidant capacity by the ABTS ® method. Superoxide dismutase enzyme (SOD) activity by the xanthine and xanthine oxidase method; inflammation markers (interleukin-1 (IL-1), IL-8, and tumor necrosis factor-alpha (TNF-α)) were quantified by flow cytometry.

Sample Size
The sample size was calculated based on the formula for the difference of means by the lipoperoxide marker.

Extraction and Preparation of T. lucida
The plant specimen was collected full flowering in October, in Xochimilco, a rural area of Mexico City. It was kept on brown paper to preserve temperature and humidity. It was weighed thoroughly (flowers, leaves, and stems) and cut into proportions of approximately 10 cm.
The alcoholic extraction of active ingredients of T. lucida was performed under the NOM-O72 standard of the General Health Law of herbal supplements using absolute  Saliva samples were taken before and after treatment. Lipoperoxides were quantified by the TBARS (Thiobarbituric acid reactive substance) technique, total antioxidant capacity by the ABTS ® method. Superoxide dismutase enzyme (SOD) activity by the xanthine and xanthine oxidase method; inflammation markers (interleukin-1 (IL-1), IL-8, and tumor necrosis factor-alpha (TNF-α)) were quantified by flow cytometry.

Sample Size
The sample size was calculated based on the formula for the difference of means by the lipoperoxide marker.

Extraction and Preparation of T. lucida
The plant specimen was collected full flowering in October, in Xochimilco, a rural area of Mexico City. It was kept on brown paper to preserve temperature and humidity. It was weighed thoroughly (flowers, leaves, and stems) and cut into proportions of approximately 10 cm.
The alcoholic extraction of active ingredients of T. lucida was performed under the NOM-O72 standard of the General Health Law of herbal supplements using absolute ethyl alcohol. Later, in a 250 mL amber glass crystal, 10% of the mother stain (extract) of T. lucida was diluted with 225 mL of purified water [18].

Phytochemical Characterization
High performance liquid chromatography (HPLC) of T. lucida analysis was performed using an Agilent Technologies 1260 Infinity Chromatograph coupled to a DAD G1315C diode array detector and a 1290 Infinity G1316C oven; A Luna Omega 1.6 µm Polar C18 100 2.1 × 50 mm Phenomenex column. Chromatograms were recorded at wavelengths of 230 and 320 nm, processed using an Open Lab software program. Chromatographic conditions were developed using 0.1% formic acid (A) and acetonitrile (B) as a mobile phase. For the sample preparation, 11 mg of the dry extract were dissolved in 2 mL of a mixture of methanol: acetonitrile in a 1: 1 ratio. The injection volume was 1 µL, the temperature was kept constant at 35 • C, and the flow was 0.25 mL/min.
The chromatographic profile showed five main compounds compared with the literature through the retention times obtained, and using the UV spectrum at 230 and 320 nm it can be considered that the peaks obtained in the retention times (Rt) of 10.9, 13.7, 14.3, and 22.4 min possess the characteristic spectra of phenolic compounds with antioxidant activity. Additionally, the 7.6 min peak corresponds to the peak of some coumarins. Therefore, it would have anti-inflammatory activity [16,[18][19][20] (Figure 2). Sustainability 2020, 12, x FOR PEER REVIEW 4 of 10 ethyl alcohol. Later, in a 250 mL amber glass crystal, 10% of the mother stain (extract) of T. lucida was diluted with 225 mL of purified water [18].

Phytochemical Characterization
High performance liquid chromatography (HPLC) of T. lucida analysis was performed using an Agilent Technologies 1260 Infinity Chromatograph coupled to a DAD G1315C diode array detector and a 1290 Infinity G1316C oven; A Luna Omega 1.6 μm Polar C18 100 2.1 × 50 mm Phenomenex column. Chromatograms were recorded at wavelengths of 230 and 320nm, processed using an Open Lab software program. Chromatographic conditions were developed using 0.1% formic acid (A) and acetonitrile (B) as a mobile phase. For the sample preparation, 11 mg of the dry extract were dissolved in 2 mL of a mixture of methanol: acetonitrile in a 1: 1 ratio. The injection volume was 1 µ L, the temperature was kept constant at 35 °C, and the flow was 0.25 mL/min.
The chromatographic profile showed five main compounds compared with the literature through the retention times obtained, and using the UV spectrum at 230 and 320 nm it can be considered that the peaks obtained in the retention times (Rt) of 10.9, 13.7, 14.3, and 22.4 min possess the characteristic spectra of phenolic compounds with antioxidant activity. Additionally, the 7.6 min peak corresponds to the peak of some coumarins. Therefore, it would have anti-inflammatory activity [16,[18][19][20] (Figure 2).

Clinical Evaluation
The probing pocket depth (PPD) of all dental organs in each individual was evaluated, using a graduated periodontal probe between teeth and gums at a standard force to measure pocket depth. The clinical attachment loss (CAL) was determined measuring the distance between the pouch base and the cement-enamel junction. To evaluate the bleeding on probing, the probe was inserted in the pocket bottom; this procedure was repeated after 30 s [21].
Dental mobility tests were performed with vestibular-palatine, vestibular-lingual, and mesiodistal movements, considering physiological mobility.

Saliva Samples and Biochemical Techniques
All participants were asked not to eat, drink, or brush their teeth 1 h prior to the trial, and not to consume any alcoholic beverages 24 h prior to sample collection. To stimulate saliva production, participants chewed a piece of paraffin wax for 7 min. Saliva produced during the first 2 min was discarded. Then, the patients chewed the paraffin during the time of saliva collection until 5 mL [22]. Saliva samples were stabilized and conserved after

Clinical Evaluation
The probing pocket depth (PPD) of all dental organs in each individual was evaluated, using a graduated periodontal probe between teeth and gums at a standard force to measure pocket depth. The clinical attachment loss (CAL) was determined measuring the distance between the pouch base and the cement-enamel junction. To evaluate the bleeding on probing, the probe was inserted in the pocket bottom; this procedure was repeated after 30 s [21].
Dental mobility tests were performed with vestibular-palatine, vestibular-lingual, and mesiodistal movements, considering physiological mobility.

Saliva Samples and Biochemical Techniques
All participants were asked not to eat, drink, or brush their teeth 1 h prior to the trial, and not to consume any alcoholic beverages 24 h prior to sample collection. To stimulate saliva production, participants chewed a piece of paraffin wax for 7 min. Saliva produced during the first 2 min was discarded. Then, the patients chewed the paraffin during the time of saliva collection until 5 mL [22]. Saliva samples were stabilized and conserved after centrifuged at 3000 rpm × 20 min, kept in 400 µL aliquots at −70 • C to preserve them, and then processed. Oxidative stress was quantified using the following techniques:

TBARS
The TBARS assay was performed using the whole saliva according Jentzsch et al. (1996) [23]. In this assay, one molecule of malondialdehyde reacts with two molecules of thiobarbituric acid (TBA), producing a pink pigment with an absorption peak of 535 nm. Amplification of peroxidation during the assay was prevented by adding the chain-breaking antioxidant butylated hydroxytoluene (BHT).

Statistical Analysis
Data were analyzed using descriptive statistics, the mean and standard deviation (SD), frequencies (f), and percentages (%). We performed a chi-square comparison test and repeated measures analysis of variance (repeated measures ANOVA) [27]. A p value of <0.05 was considered statistically significant. p values were determined using SPSS program SPSS, version 16.0.

Clinical Changes Related to Treatment
In the clinical evaluation, after 3 months of treatment, we observed a significant improvement in the EG compared to the PG in PPD (baseline, 5.2 ± 0.81 vs. post-treatment, 3.1 ± 0.43, p < 0.05) and CAL (baseline, 6.6 ± 1.9 vs. post-treatment, 2.1 ± 1.5, p < 0.05) ( Table 1). Regarding bleeding by the dental probe and dental mobility, a significant decrease in both clinical symptoms was observed after treatment in the EG compared with PG (p < 0.001) ( Table 2).

Effect of T. lucida on Oxidative Stress Markers
Concerning oxidative stress markers in saliva, at the end of the three-month treatment with the T. lucida mouthwash, a significant decrease in lipoperoxides levels was observed in the EG group compared with PG (p < 0.05). In contrast, there was an increase in both total antioxidant capacity and superoxide dismutase activity, although these were not statistically significant (p > 0.05) ( Table 3).

Effect of T. lucida on Inflammatory Markers
A significant decrease in interleukin 1β, interleukin IL-8, and TNF-α concentration in saliva was found after three months of treatment with T. lucida compared with PG (p < 0.05) ( Table 4).

Discussion
Chronic periodontitis (CP) is a degenerative and inflammatory disease with damage and loss of connective tissue, which in general is associated with the activation of polymorphonuclear leukocytes that generate reactive oxygen species (ROS) [28,29]. It has also been linked with physiopathological mechanisms of numerous aging-related diseases which lead to the production of ROS during inflammatory conditions [30][31][32]. In this sense, medicinal herbal medicine can be a therapeutic alternative in the dental field, since research has been carried out to support the use of various plant species such as T. lucida, which, due to its chemical composition, has anti-inflammatory and antioxidant properties. It could have a positive effect on chronic periodontitis, as an alternative treatment or in parallel with another dental treatment [18,33].
The results showed that older adults with CP treated with the T. lucida mouthwash for 3 months had a significant decrease in concentration of lipoperoxides associated with an improvement in the signs and symptoms of CP compared to untreated older adults. This finding supports the proposal of the antioxidant effect of T. lucida [11]. Additionally, the decrease in ROS and the increase in the efficiency of the antioxidant system agree with results reported by some authors who found an association between decrease of oxidative stress and CP [34][35][36][37][38]. Consequently, the excess of ROS can selectively damage the proteoglycans associated with soft periodontal tissues and alveolar bone, activating nuclear factor kappa B (NF-kB) that triggers signaling cascades that activate osteoclasts, leading to inflammation [35,[39][40][41][42].
On the other hand, an increase in the total antioxidant capacity and in the activity of the SOD enzyme was observed, although not statistically significant, probably due to the sample size. Similarly, there have been studies conducted with different treatments based on plant species, such as Commyphora myrrha, Mentha piperita, Bursa Pastoris, Clematis ternifolia, Timus vulgaris, and Aquillea millefolium, at different times of intervention and in some cases, changing hygienic-dietary habits of the patients [32][33][34][35][36][37]. In our study, we observed that the post-treatment concentration of lipoperoxides decreased significantly (p ≤ 0.05). In this regard, other studies have reported a statistically significant decrease in the concentration of lipoperoxides, when performing periodontal treatments; however, such treatments increase damage to periodontal tissues caused by the scraping and curettage procedure [43].
Likewise, some studies have shown that the salivary system is formed by some enzymes such as SOD. This enzyme is capable of increasing in response to different inflammatory diseases such as pulpitis and periodontitis. SOD is able to protect the oral cavity against the effects of ROS, which could be considered a partial compensatory response when there is oxidative stress in the cavity, and SOD represents the first defense in the antioxidant tissues [44,45].
In this work, the results of oxidative stress markers show a global improvement in the EG by decreasing lipoperoxidation, preserving the activity of SOD, and increasing the total antioxidant activity.
Regarding the levels of pro-inflammatory interleukins, it was observed that they decreased significantly in the treatment group with T. lucida, an effect due to the decrease in the periodontal infectious process. Therefore, the decrease in the secretion of bacterial lipopolysaccharides is caused by a decrease in the secretion of these interleukins of the macrophages, and consequently the decrease in osteoclastic activity and expression of adhesion molecules to the vascular endothelium [46,47].
Accordingly, some animals studies where CP has been induced and treated with some plants rich in curcumin, tannins, and phenolic compounds showed a significant decrease in TNF-α [48].
In this sense, it is essential to consider that extracts of T. lucida have a potential antiseptic effect; this bactericidal activity decreased the microorganisms number leading to anti-inflammatory effects and decreased oxidative stress [11,14,19].
This was clinically observed with the favorable modifications found in the clinical evolution of CP in the group treated with T. lucida, since the CAL and the PPD decreased in this group (p < 0.05); probably due to the decrease in periodontopathogens and leukocyte infiltrate associated with the reduced concentrations of pro-inflammatory cytokines. Additionally, the decrease in periodontal pockets was probably due to the osteoclastic action did not continue because the secretion of IL-1β and TNF-α decreased [49,50]. Furthermore, mobility and gingival bleeding reduced significantly in the EG (p < 0.05) due to the restoration of periodontal tissue integrity and the fibroblastic protective action of T. lucida quercetin [50].
Finally, it is important to point out as limitations of the study: (i) the reduced sample size to measure the effect on all oxidative stress markers; (ii) the groups were not proportional by sex, consequently, the influence of this variable could not be evaluated; (iii) study follow-up was only once at 3 months, so the effectiveness over time cannot be guaranteed; (iv) the plaque index was not measured; and (v) a comparison group with a mouth rinse as the gold standard was not included, such as 0.12% chlorhexidine [51,52]. In this sense, it is advisable to carry out a longitudinal study with subsequent clinical evaluations considering the indicated limitations.

Conclusions
Our findings suggest that T. lucida has an antioxidant and anti-inflammatory effect in older adult subjects with CP, presumably due to the presence of curcumin and quercetin contained in this plant. Funding: The publication of this paper was supported by a grant from the Secretaría de Educación, Ciencia, Tecnología e Innovación de la Ciudad de México CM-SECTEI/200/2020 "Red Colaborativa de Investigación Traslacional para el Envejecimiento Saludable de la Ciudad de México (RECITES)". Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México. Consejo Nacional de Ciencia y Tecnología (Beca de Posgrado CONACYT CVU 70011).

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Ethics Committee of Universidad Nacional Autónoma de México (UNAM), Zaragoza Campus (protocol code FESZ/DEPI/325/16).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are because they are owned by UNAM.