Comparative Effectiveness of a Commercial Mouthwash and an Herbal Infusion in Oral Health Care

: Mouthrinse solutions represent a group of products used for maintaining oral hygiene after tooth brushing. Substances contained by plants bring beneﬁts for the whole mouth health. The purpose of this study was to comparatively evaluate the effectiveness of a commercial mouthwash and of an herbal infusion on dental plaque formation and gingival inﬂammation. The participants in the study (90 patients) were divided into two groups, the CM group, which rinsed with a commercial ﬂuoride-containing mouthwash and the IM group, which used an herbal infusion. The Silness–Loe plaque index (PI) and the Loe–Silness gingival index (GI) were assessed at baseline and in three following monitoring sessions. Both mouthwashes used had good results in reducing plaque index and gingival index values. In all monitoring sessions, the average PI value calculated in the CM group was relatively lower than in the IM group, probably because of the ﬂuoride contained in the commercial mouthwash. The average GI value calculated in the CM group was slightly higher in showing gingival inﬂammation than that of the IM group, therefore the IM group had a better average result than the CM group in GI value. Herbal mouthwashes are adequate to induce proper oral prevention through the preservation of good oral health.


Introduction
The formation of the dental biofilm is initially influenced by factors such as the surface free energy, which affects the wettability and the surface tension, the surface roughness, and the adhesion of the salivary proteins [1,2]. It is known that the initial attachment of bacterial biofilm to the dental surface is a result of hydrophobic and electrostatic interactions, respectively, in bacterial attachment to the dental surface [3]. Dental plaque-induced gingival inflammation is modified by various systemic and oral factors.
The prevention of the formation of dental biofilm, respectively, the occurrence of dental cavities and gingival inflammation, is done by several means. The most common are the use of toothpaste with anti-cavity and anti-inflammatory effects and through additional Ethical statement: All subjects provided their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Faculty of Medicine and Pharmacy, University of Oradea, Bihor County, Romania, with the number 21/23.07.2018.

Plant Materials
Polygonum aviculare L. (Polygonaceae family) and Glechoma hederacea L. (Lamiaceae family) plant material comes from the unpolluted spontaneous flora of Oradea area. In the case of Matricaria chamomilla L. (Asteraceae family) and Mentha piperita Huds. (Lamiaceae family), the plant material comes from the cultivated flora of Oradea area. Plant materials were dried at room temperature. The extracts were obtained by infusion.

Physicochemical Characterization of the Mixture of Extracts
In previous papers, we performed the evaluation of the polyphenol and flavonoid content of the Polygonum aviculare L. and Glechoma hederacea L. extracts [11].
Polyphenolic compounds and flavonoids with known beneficial effects act as antioxidants in a biological system under oxidative stress conditions. In order to select the optimal combination ratio of the four extracts, several Mentha piperita Huds. For this study, we evaluated the content of bioactive compounds based on their antioxidant properties demonstrated by in vitro methods.

Determination of the Content in Polyphenolic Compounds
We used the Folin-Ciocâlteu method to determine the content in polyphenolic compounds, and the results are expressed in gallic acid equivalents (GAE) (mg/mL). In order to achieve this determination, we mixed 1750 µL distilled water, 200 µL of Folin-Ciocâlteu reagent (diluted 1:10 v/v), and 1000 µL of 15% Na 2 CO 3 solution with 100 µL of fluid extract and then kept it at room temperature for two hours, away from light. Then, we measured the absorbance using a UV-Vis spectrophotometer at a wavelength of 765 nm. The calibration curve was linear for the concentration range of 0.1-0.5 mg/mL for gallic acid. The content of the total polyphenols in the extracts is expressed as mg equivalent of gallic acid (GAE)/g dry weight extract (DW) [5,[15][16][17]].

Determination of Total Flavonoids
Using the colorimetric method [17], we determined the total flavonoids. On this line, we mixed 4 mL of distilled water with 1 mL of sample and placed in a 10 mL volumetric flask. Then, we added 3 mL of 5% NaNO 2 solution. After 5 min, we added 0.3 mL of 10% AlCl 3 solution. After 6 min, we added 2 mL of 1 M NaOH. The flask was filled to the mark with distilled water and the absorbance was read at 510 nm. We used the quercetin standard to find the calibration curve. The equation of the calibration curve is y = 56:818571 x _ 0:066498 (R2 = 0:9983), where x represents the absorbance and y represents mg quercetin [18,19].

Evaluation of the Antioxidant Activity of the Mixture of Extracts
A number of analytical methods that are used to determine the antioxidant activity of natural products exist. These are generally based on the reaction between an antioxidant species and a chromogenic compound. We evaluated the antioxidant capacity of the extracts using the following methods: 2,2-diphenyl-1-picryl-hydrazyl (DPPH), 2,2azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric-reducing antioxidant power (FRAP).

DPPH Method
The DPPH method is a spectrophotometric method. It is widely used to test the ability of compounds to remove free radicals or their ability to donate hydrogen. The activity of capturing 2,2-diphenyl-1-picryl-hydrazyl radicals (DPPH) was determined using the method proposed by Pallag et al. [13,20]. Thus, at a volume of 100 mL of plant extract, 2900 mL of DPPH methanolic solution (80 mM) was added. The absorbance of the resulting solution was read at 515 nm after 5 min.
We used the following equation to determine the DPPH inhibitory capacity (%): where A O is the absorption of the blank sample and A S is the sample absorbance at 515 nm [15][16][17].
Cupric Ions (Cu2+) Reducing-Cuprac Assay We used Cuprac assay to determine the cupric ions (Cu2+) reducing antioxidant capacity. We added 0.25 mL CuCl 2 solution (0.01 M), 0.25 mL ethanolic neocuproine solution (7.5 × 10 −3 M), and 0.25 mL CH 3 COONH 4 buffer solution (1 M) to a test tube, then we mixed it with the plant extracts. Afterward, we adjusted the total volume to 2 mL with distilled water and thoroughly mixed it. The tubes were stoppered and kept at room temperature. Absorbance was measured at 450 nm against a reagent blank 30 min later. The increased absorbance of the reaction mixture indicates increased reduction capability. Picryl-hydrazyl-hydrate (DPPH) was determined by the slightly modified method of Brand-Williams et al., 1995 [2,17]. DPPH reacts with an antioxidant compound, which can donate hydrogen and reduce DPPH [15][16][17].

ABTS Method
The ABTS method is often used to evaluate antioxidant activity and uses the method of Arnao et al. [17]. Essentially, ABTS+ was made by reacting the ABTS solution (7 mM) with 2.45 mM potassium persulphate away from light for 16 h at room temperature. At 734 nm, we diluted the stock solution of ABTS to give an absorbance of 0:70 ± 0:02. The mixture was homogenized very well (using vortex) for 30 s after adding 25 L of extract to 2.5 L of diluted ABTS+. At 734 nm, exactly 1 min after homogenization, we measured the interaction between antioxidants and ABTS•+ spectrophotometrically. Trolox was used, and between 0.125 and 2 mmol/L Trolox, a standard linear curve was obtained. The calibration curve equation was y = 1629x + 98:94 (R2 = 0:998), where x represents absorbance and y represents mmol Trolox equivalents [15,16,19].

Ferric-Reducing Antioxidant Power (FRAP) Method
The ferric-reducing antioxidant power (FRAP) method is a simple spectrophotometric procedure for measuring the antioxidant potency of the samples used in the analysis. It is based on the reduction of the ferric tripyridyl-triazine complex (Fe (III)-TPTZ) by a pH-reducing agent [21]. A total of 300 mM acetate buffer, 270 mg FeCl 3 6H 2 O dissolved in 50 mL distilled water, 150 mg TPTZ, and 150 mL HCl dissolved in 50 mL distilled water are among the stock solutions. We mixed together 50 mL acetate buffer solution, 5 mL FeCl 3 6H 2 O solution, and 5 mL TPTZ solution to produce the FRAP solution. We allowed the vegetable extracts (100 mL) to react for 1 h in the dark with 500 mL of FRAP solution and 2 mL of distilled water. We used absorption into the UV-VIS at 595 nm to measure the final colored product (ferric tri-pyridyl-triazine complex). As an antioxidant positive control, we used Trolox, and we obtained a typical linear curve of 50 to 500 mmol/L of Trolox. The FRAP value was calculated using the equation y = 0:0157x + 0:0549 (R2 = 0:9981), where x represents absorbance and y represents mmol of Trolox equivalents [15,17].

Clinical Trial of a Commercial Mouthwash and an Herbal Infusion
The randomized multicenter comparative clinical study was conducted in accordance with the ethical standards, according to good practice and ethical principles. The study comparatively evaluated the effectiveness of a commonly used mouthwash and of an infusion of herbs that was created in the Faculty of Medicine and Pharmacy Oradea, University of Oradea. The clinical trial was conducted in the Faculty of Medicine and Pharmacy of Oradea University, the Dental Medicine Faculty of George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, and the Dental Medicine Faculty of Titu Maiorescu University of Bucharest. The null hypothesis of the clinical trial was started from the premise that the efficacy of the used methods in decreasing plaque index (PI) and gingival index (GI) is not different.
The participants included in this study were randomly assigned to one of the two groups, CM group, which rinsed with the commercial mouthwash, and the IM group, which used the herbal infusion, using an algorithm [22] that generated a random number for every patient (odd for the CM group, even for the IM group). The participants were informed about procedures, agreed to voluntarily participate in the study, and signed their consent forms for participation in the study (Ethics Committee number 21/23.07.2018).
The inclusion criteria were represented by the patients who voluntarily registered in the study program: cooperating patients with good oral hygiene who did not use mouthwashes in the last six months; systemically healthy patients without acute or chronic diseases; patients without dental, periodontal, mucosal lesions of the oral cavity; no antibiotic therapy and no other therapies or professional cleaning at least six months prior to the start of this study; minimum number of teeth 24; and age range between 20-29 years old. The exclusion criteria were represented by patients with dental, mucosal, or/and periodontal diseases/treatments within the period of research; anti-inflammatory and antibiotic therapy within the period of research; adverse effects or allergic reactions after the use of substances contained by the researched mouthwash and/or infusion; history with general chronic diseases; pregnancy; and lactation.
All examinations of this clinical trial were performed by dentists who realized an inter-examiner calibration before the beginning of the study to ensure the coherence of examinations and diagnosis. They used the same bacterial plaque-disclosing agent (erythrosine tablets) and the same examination instruments. The working protocol received by all examiners from the coordinators of the Oradea center included the assessment of the Silness-Loe plaque index (PI) and the Loe-Silness gingival index (GI).
The Silness-Loe plaque index (PI) was imagined by authors in 1964 [8] and estimates the presence of dental plaque deposits on the cervical area of the 4 coronal surfaces of the teeth. The arithmetic mean of the values represents the plaque index that follows the stages of plaque formation at the level of the dento-periodontal junction: 0 = absence of plaque; 1 = plaque that can be evidentiated with the probe; 2 = visible plaque; 3 = abundant plaque deposits. PI = sum of all areas/no. of examined surfaces. From the average of the 4 values (labial/buccal, mesial, oral, and distal), the specific index of each tooth resulted. The plaque indices for the six teeth (1.6, 1.2, 2.4, 3.6, 3.2, 4.4) were added and then divided by six in order to obtain the PI for each patient [8]. The Loe-Silness gingival index (GI) quantifies the state/severity of gingival inflammation, determined at the level of the same four cervical areas of a dental crown as in the PI. The GI was assessed by inserting a periodontal probe in the gingival sulcus or in the gingival recessions. The GI scores are: 0 = normal gum without signs of inflammation and without bleeding; 1 = slight inflammation, slight color changes, no bleeding; 2 = moderate inflammation, erythema, edema, bleeding on probing or pressure; 3 = severe inflammation, pronounced redness, edema, spontaneous bleeding, and sometimes ulceration. The GI calculation is identical to the PI calculation [9,10,23].
After the initial analysis and examination of 150 patients who were initially included in the study, 90 patients continued (42 males = 46.66% and 48 females =53.33%) until the study endpoint, 37 of research center Targu Mures and 53 of research center Oradea.
The 60 participants were eliminated or self-eliminated during the study as follows: 2 women were confirmed pregnant, 14 patients began treatment for acute conditions during the study, 3 patients developed allergic reactions to the commercial mouthwash, 4 patients underwent surgery, 21 patients gave up during the study or did not show up for follow-up, and 13 patients were eliminated because they did not comply with the appropriate oral hygiene rules (brushing and additional mean).
The age range of the 90 remaining patients was a median age of 24.5 years and a mean of 24.5 ± 4.5 years.
The patients in the IM group (45) used an infusion of 10 g Mentha piperita Huds., 10 g Matricaria chamomilla L., 10 g Polygonum aviculare L., and 20 g Glechoma hederacea L. in 1 L of water, an infusion that was made in the pharmaceutical laboratory of the Pharmacy Department, Faculty of Medicine and Pharmacy, University of Oradea.
Patients were instructed to rinse vigorously for 30 s with approximately 20 mL solution after brushing their teeth, without swallowing, for a maximum of 2 times a day. Patients were instructed not to ingest food or liquids for one hour after rinsing the mouth.
The patients were monitored during the period of April 2018-March 2019 in the dental clinics of the faculties for evaluation of PI and GI status. At the first examination (baseline), the determinations of PI and GI were realized.
The monitoring examinations were performed after 3 days (first examination session), 7 days (second examination session), respectively, and 2 weeks (third examination session), registering the findings in the patients' record for each session, thus having the possibility to assess the parameters in evolution.
In order to create the same conditions for each patient included in the study, we offered the participant patients the same type of toothbrush and fluoride-free toothpaste. All subjects were trained to acquire proper oral hygiene: correct brushing technique (manual brushing, Bass technique) and the proper use of the mouthwash.
There were no study sponsorships or other types of third-party funding so that the results of the study would not be influenced.
The working protocol that the researchers from all three centers followed consisted of inter-examination calibration; bacterial plaque highlighting with a disclosing agent (erythrosine tablets) to reduce potential drift in scoring; determination of PI and GI at baseline; professional cleaning; individual oral hygiene training with the toothpaste and toothbrush provided by the researchers; the proper use of mouthwash (CM group) or the herbal infusion (IM group) after the distribution in the research groups; and 3 monitoring sessions of PI and GI.

Statistical Analysis
All continuous variables were checked for distribution with Kolmogorov-Smirnov test and represented with average (standard deviation) or median (interquartile range), respectively. Variables with normal distribution were compared using the Student t-test for independent samples, those with skewed distribution using the Mann-Whitney test. Statistical difference was considered when the probability of the null hypothesis (p) was under 0.05. The statistical analysis was performed with the aid of MedCalc ® statistical software version 12.5.0.0 (MedCalc ® Software, Mariakerke, Belgium).

Physicochemical Characterization of the Mixture of Extracts
The total and flavonoid polyphenol contents of the extract mixture are different depending on the ratio of the associated extracts, as shown in Table 1. We observed that the extract combination Mp:Mc:Po:Gh (1:1:1:2) had a well-balanced ratio regarding the bioactive compounds. There was a total of 372.82 mg GAE/g DW content of total polyphenols and 6.85 mg QE/g DW of total flavonoids.   Thus, we evaluated the antioxidant activity of the plant extract combinations using the DPPH, ABTS, and FRAP methods. The obtained results are summarized in Table 2

Study of the Effects of Commercial Mouthwash and an Herbal Infusion on Oral Cavity
No adverse side effects were reported in either group. The PI and GI were determined and marked in the patients' records. All the PI and GI values determined in CM and IM groups, at the first examination (baseline) and in the monitoring sessions, were statistically calculated using the Mann-Whitney methods and t-tests, in order to examine the relationship between the PI and GI, respectively, the sex influence on these values.
In Table 3, the median (interquartile range) of PI and GI are presented at baseline in all participant patients, in Table 4, the median (interquartile range) of PI and GI at baseline in males, and in Table 5, the median (interquartile range) of PI and GI at baseline in females.   Statistical analysis revealed that both CM and IM groups of patients presented homogeneous values of PI and GI (p > 0.5, median between 2.21-2.25 per total) at baseline. The GI had slightly lower values than PI values, probably due to the age of the patients participating in the study (20-29 years).
In Table 6, the average (standard deviation) of PI and GI in the first monitoring session in all patients are presented, in Table 7, the average (standard deviation) of PI and GI in the first monitoring session in male patients are presented, and in Table 8, the average (standard deviation) of PI and GI in the first monitoring session in female patients are presented.   After the first treatment session, in the first monitoring session, significant differences were observed overall in both PI and GI (p < 0.0001). It can also be seen that the significance is even more evident in GI and PI indicators, but with slight differences between men and women (p = 0.0051 men and p = 0.0001 women), and highly significant for GI in both men and women (p < 0.0001), and less significant for PI (p = 0.0051 men, p = 0.0001 women).
In Table 9, the average (standard deviation) of PI and GI in the second monitoring session in all patients is presented, in Table 10, the average (standard deviation) of PI and GI in the second monitoring session in male patients is presented, and in Table 11, the average (standard deviation) of PI and GI in the second monitoring session in female patients is presented.   In the second monitoring session, we did not find any notable differences compared to the first monitoring session.
In Table 12, the average (standard deviation) of PI and GI in the third monitoring session in all patients is presented, in Table 13, the average (standard deviation) of PI and GI in the third monitoring session in male patients is presented, and in Table 14, the average (standard deviation) of PI and GI in the third monitoring session in female patients is presented.    There are highly significant differences overall in both categories (GI and PI) (p < 0.0001), which denotes the clearly different effect in the two groups, IM and CM.
At all monitoring evaluations (first, second, third), there are strongly significant differences (p < 0.0001) for GI, both in total and by sex analysis. In addition, in all evaluation sessions, there are higher values for PI in the IM group and lower for GI in the IM group, which results in IM efficiency, especially on the gingival component, and CM efficiency on the bacterial plaque component.
It is also observed that the differences are more clearly significant as the treatment progresses. Standard deviation (0.06-0.07) also indicates the homogeneity of the obtained results, which indicates that the used substances had the same effects on the whole studied batch.
Both types of mouthwash demonstrated their efficacy in reducing PI and GI values. In all monitoring sessions, the medium value of PI calculated in the CM group was relatively lower than in the IM group, probably because of the fluoride-containing mouthwash. In all monitoring sessions, the GI medium value calculated in the CM group was slightly higher in showing gingival inflammation than those of the IM group; therefore, the IM group had an average better result than the CM group in GI values.
The commercial mouthwash that was used in this research (CM group) was randomly chosen. It was intended to be an alcohol-free mouth rinse solution (knowing that the alcohol is an irritant for the oral mucosa), but it contains fluoride with antiplaque and caries-preventive effects.
The herbal infusion used in the IM group, due to the selected plants with the best anti-inflammatory, antioxidant, and antiseptic properties, had a better response in reducing the gingival inflammatory phenomena, probably due to its content of Mentha piperita Huds., Matricaria chamomilla L., Polygonum aviculare L., and Glechoma hederacea L. extracts, plants which possess beneficial features and substances for the oral cavity.
It has also been taken into account that the herbal infusion had the property of giving a feeling of comfort to the patients by adding plants that perfumed the breathing and removed the oral halitosis. No local or general side effects of the herbal mouthwashes have been reported.

Discussion
The benefits of the used mouthwashes that contain substances extracted from plants are detailed in many articles. Mentha piperita Huds. (Lamiaceae family) has an antiseptic and analgesic action in the oral cavity due to its components. Menthol is extracted from this plant, which, besides the antiseptic and antioxidant action, eliminates bad breath and is used to flavor chewing gums [24][25][26]. Matricaria chamomilla L. (Asteraceae family), also known as chamomile, has an anti-inflammatory and scar-promoting effect, inactivates bacteria (especially Staphylococcus and Streptococcus), and due to its essential oil content, has a strong calming effect and disinfecting action in the oral cavity [27][28][29].
In the case of Glechoma hederacea L. (Lamiaceae family), the studies have shown the presence of large amounts of phenolic compounds [7,11]. Among the phenolic compounds, caffeic, chlorogenic, and rosmarinic acids are the substances with a strong antioxidant effect in the plant [40]. Antimicrobial, anti-inflammatory effects were demonstrated in numerous in vitro studies [7,11,23]. Several studies have shown a strong antioxidant effect and free radical scavenging activity [7,11,41,42].
In the case of Glechoma hederacea L. (Lamiaceae family), the studies have shown the presence of large amounts of phenolic compounds [7,11]. The biologic actions of Glechoma hederacea L. are owed to the existence of metabolites such as caffeic, chlorogenic, and rosmarinic acids, with proven antioxidant effects [40]. The antioxidant effect, free radical scavenging activity, and antimicrobial and anti-inflammatory effects were demonstrated in numerous in vitro studies [7,23,41]. The in vitro research performed by Chou et al. [42] revealed that the components of Glechoma hederacea L. have an antimutagenic and antioxidant effect. Kumarasamy et al. demonstrated the antibacterial and free radical scavenging activity of the aerial parts of Glechoma hederacea L. [41].
The treatment of gingival and periodontal diseases is recommended to be started in the earliest stages in order to be effective. The more advanced the periodontal disease, the harder it will be to approach. Studies show that the use of mouthwashes based on natural extracts has been shown to be effective in preventing and reducing gingival inflammation. Essential oils mouthwashes have an impact on saliva and gingival plaque flora and reduced levels of supragingival and subgingival plaque by a mean of 53% [11]. Cortelli et al. [43] demonstrated that the essential oils group revealed a significant reduction in the occurrence of Porphyromonas gingivalis in saliva, comparing baseline and 45 days control, and this difference still remains at 180 days. The clinical parameters are positively modified after the use of mouth rinse containing essential oils [43,44].
Gingival inflammation is the reaction to the development of microbial biofilms and subsequently of bacterial plaque. It is considered to be a risk factor in odontal and periodontal lesions. Therefore, the control of gingival inflammation by the maintenance of oral health is essential for the primary prevention of oral diseases [45][46][47]. Kulkarni et al. [48] recommended using alcohol-free mouth rinses to avoid side effects, i.e., burning mouth [44], reduced salivary flow, etc. According to Horst et al. [48], fluoride is not sufficient to control dental cavities in high-risk patients. Topical therapies and dietary modifications should be employed, decreasing the transmission of the cariogenic bacteria or the impact capacity of dental plaque organisms (e.g., antimicrobials).
The current knowledge presents practical significance for oral hygiene, including the evolution of oral prevention programs that search for decreasing the incidence of oral diseases and the accurate evaluation of oral health integrating the regional differences [49].

Conclusions
Within the limits of our study, our results demonstrated that the anti-inflammatory effect in the IM group of patients was higher than the antiplaque effect. In conclusion, herbal mouthwashes are adequate to induce proper oral prevention through the preservation of good oral health, but it is important to find the right proportion for the herbal solution to be administered.
Concurrently, commercial mouthwashes, used for a long time, have adverse effects: the disappearance of taste, staining of teeth, and imbalance of oral bacterial flora effects that have not been found in mouthwashes with natural products. In our study, the comparative evaluation time of the effects of rinse solutions was relatively short; that is why long-term researches are required. Informed Consent Statement: All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Faculty of Medicine and Pharmacy, University of Oradea, Bihor County, Romania, with the number 21/23.07.2018.