Comparative Evaluation of Antimicrobial Effects of Herbal Extracts Incorporated in Dentifrices among Adults: An In-vitro Study and a Double Blinded Randomized Control Trial

Introduction: A positive correlation has been observed between Streptococcus mutans, Candida albicans and periodontal diseases. Several substances including synthetic chemicals and natural agents have shown promising antimicrobial activity against the S. mutansC. albicans complex. Extracts of Aloe Vera, Triphala, Tulsi, Pomegranate, and Acacia catechu have established therapeutic effects in medicine. The aim of the study was to evaluate the antimicrobial activity and the Minimum Inhibitory Concentration (MIC) of five herbal extracts in-vitro and to compare the antibacterial effects of 5 dentifrices containing the herbal extracts and a commercially available dentifrice on the plaque sample counts of S. mutans and C. albicans in-vivo Methodology: Extracts were obtained using a standardized method. Minimum inhibitory Original Research Article Kalra; JPRI, 34(18A): 63-77, 2022; Article no.JPRI.81106 64 concentration was calculated for the test microorganisms using modified agar-well diffusion and macro broth dilution technique and the inhibition zone diameters were calculated. Dentifrices containing the extracts were formulated and the in vivo antimicrobial activity was evaluated by studying the CFU count from plaque samples of 420 participants. Results: Highest antimicrobial activity against S Mutans and C albicans was shown by Acacia catechu and Tulsi respectively and least was by pomegranate. Mean reduction in microbial counts (CFU/ml) by inoculating nutrient broth from baseline to 60 days was highest in Acacia catechu and Triphala for S Mutans and C albicans respectively. All herbal extracts performed better than the commercially available toothpaste. Conclusion: Dentifrices with herbal extracts of Aloe Vera, Triphala, Tulsi, Pomegranate, and Acacia catechu could serve as an alternative to commercially available dentifrices for treating oral diseases whose causative organisms are S. mutans and C. albicans.


INTRODUCTION
Global burden of diseases (2016) reported periodontal diseases as the eleventh most common disease in the world affecting almost 20-50% of the population of the world [1,2]. Recent studies have suggested that when periodontitis goes untreated, the recovery rate of S. mutans is high from surfaces such as the dorsum of the tongue, buccal mucosa, supra gingival and sub gingival plaque and saliva [3][4][5] demonstrating a positive correlation with chronic periodontitis [6]. Candida albicans is one of the most predominant fungal species and is most often seen in association with S. mutans. Both synthetic chemicals and natural agents have shown promising antimicrobial activity against the S. mutans-C. albicans complex [7]. However, the chemical agents used in the form of mouth rinses and dentifrices are associated with some concerns regarding their untoward effects [8].
The history of herbal medicine can be rooted from ancient civilization which has well documented evidence of their use as traditional medicine. Herbal extracts of Aloe vera, Triphala, Ocimum sanctum (Tulsi), Punica granatum Linn (Pomegranate) and Acacia catechu (AC) have therapeutic properties which have been well demonstrated in herbal medicine. Their antimicrobial properties have been studied invitro [9][10][11][12] and clinically in the form of extracts, mouthwashes [13], gels [14] and dentifrices [15] in randomised controlled trials which have been proven to be effective against both S. mutans [11,[16][17][18][19] and C. albicans [10,19,20]. However, there is paucity of literature in the comparative analysis of these agents together. The rationale of the study was to evaluate the antimicrobial activity and the Minimum Inhibitory Concentration (MIC) of five herbal extracts invitro and to compare the antibacterial, antiplaque and antigingivitis effects of 5 dentifrices containing the herbal extracts and a commercially available dentifrice on the plaque sample counts of S. mutans and C. albicans in-vivo.

Study Design
The study was divided into two phases.
Phase I: In-vitro study to assess the zone of inhibition and minimum inhibitory concentration (MIC) of the five herbal extracts. Phase II-Six group parallel arm double blinded randomised controlled trial

Study Groups
Group I Aloe Vera based dentifrices, Group II-Triphala based dentifrice Group III-Ocimum sanctum (Tulsi) based dentifrice, Group IV-Punica granatum Linn (Pomegranate) based dentifrice Group V-Acacia catechu (AC) based dentifrice Group VI-A commercially available dentifrice

Procurement of the Material
Aloe Vera leaves, Punica granatum fruits and Tulsi leaves were obtained from the botanical garden of Ayurvedic College of YMT group of institutions. Triphala which is a herbal formula composed of the powder of three fruits: amalaki, bibhitaki and haritaki, was made as a powder. Acacia catechu (AC) was obtained from the botanical garden of Ayurvedic College of YMT group of institutions. All plant specimens were identified by a botanist for their authenticity at the Agarkar Research Institute, Pune (Gov. of Maharashtra, letter no. 1-6/485/2019) and a voucher specimen was deposited in the departmental herbarium.

Aloe Vera [21]
Fresh Aloe Vera leaf gel was dried at 80 degree for 48 hours and then powdered. Twenty grams of powder was soaked in 200ml of methanol and ethanol for 24 hours. This was then filtered and dried. This dried extract was further powdered and then dissolved in distilled water. Acetone extract was prepared in a similar manner except that the extracted powder was dissolved in 0.15N NaOH and was further neutralized with 0.15N HCl.

Triphala [22]
The ingredients used in the Triphala Churna were Amlaki (Embellica officinalis), Bibhitaka (Terminalia bellerica), Haritaki (Terminalia chebula). They were cleaned, dried, sieved, powdered and mixed in the ratio of 1:1:1. A weighed quantity of powder (500 gm.) was passed into sieve no. 40 and subjected to aqueous and Ethanolic extraction (maceration) with distilled water and 90% ethanol and kept at room temperature for 7 days with occasional stirring. Ethanolic and aqueous extracts were concentrated in a water bath (kokate; sofawora 1993). Extracts were dried under reduced pressure using Rotary Vacuum Evaporator (Equitron® Roteva) and were preserved in the desiccator until further use.

Tulsi
The whole plant was weighed, cleaned, air-dried and weighed again. This was then crushed and 300 grams of finely powdered tulsi and mixed with 100% ethanol for the process of maceration. Filtration was done to separate the filtrate and residue which were further processed and subjected to evaporation at 70°C. Extracts were then dried and a dark green residue was obtained. These were refrigerated until further use. Dried extracts were also subjected to phytochemical screening. From 300 gms of powder that was dissolved in 1 liter of ethanol, 18 grams of extract was obtained, thus the yield was 6% w/v.

Pomegranate
Pomegranate fruits were washed with distilled water and cut manually to separate the arils and peel. The juice was manually extracted by pressing the arils. The pomegranate seeds thus obtained were washed with distilled water to remove the adherent flesh and subjected to drying at 60± 5ºC for 6 hrs or till its moisture content reached ~5-6 %. Five grams of pomegranate seed powder was separately blended for 2 mibutes with 300 ml of distilled water or 80% methanol followed by filtration and centrifugation at 3500 rpm for 10mins to obtain extracts which were kept at -20°C prior to analysis.

Acacia Catechu [23]
The heartwood of Acacia Catechu was pulverized after drying at room temperature for four weeks. Heartwood chips were boiled with 10% hydro-alcoholic solution to obtain katha. The specimen was processed for pharmacognostic standardisation.

Preparation of Microbial Strains
Typed pure strains of S. mutan (ATCC 890) and C. albican (ATCC) were obtained from MTCC Chandigarh. Clinical Laboratory, Department of Microbiology. All test strains were re-isolated three successive times on Mueller Hinton agar (MHA) for purification and the identity was confirmed by standard biochemical and staining methods [24].

Preparation of Dentifrices
Laboratory preparation of toothpaste was done by trituration method [25]. A liquid base was prepared first with humectants, preservatives and water. To this base a binder was added, triturated well and kept aside for 15 min to allow the binding agent to swell. Next, powder ingredients except detergent were sifted together and were added gradually to the aqueous mucilaginous mixture with slow but continuous stirring. After addition of all powders, a flavoring agent was added. Surface active agent was added at the end and mixed slowly and thoroughly to prevent aeration or foaming. Mixing was continued till all constituents were evenly distributed. The finished product thus obtained was allowed to stand for 24 h. The paste was finally filled into collapsible tubes, stored and used for further studies to determine hard and sharp edged abrasive particles, the paste was extruded about 15 to 20 cm length from the collapsible tube of each sample on a butter paper. Then all the samples were tested by pressing it along its entire length by a finger for the presence of hard and sharp edged abrasive particles. The five different formulations of herbal toothpastes were prepared by using fine powder of calcium carbonate.
All the formulations differed only with respect to the presence of the active ingredient. MIC% values for both S. mutans and C. albicans were measured for each extract and the higher of the two concentrations (i.e. MIC% S. mutans or C. albicans) for a particular extract was added in the dentifrice, in order to have its effect on both S. mutans and C. albicans. The prepared dentifrices were then transferred to laminate tubes.

Determination of the Minimal Inhibitory Concentration (MIC)
The MIC was determined by two test methods, a modified agar-well diffusion method and the macrobroth dilution technique [26].
In the agar-well diffusion technique, a two-fold serial dilution of each extract in 50% DMSO was prepared to obtain a 0.1-100 mg/ml concentration range. 100-ul of each dilution was introduced in triplicate in pre-inoculated on the MHA plates. All the plates were incubated at 37 °C for 24 hours. The least concentration of each extract or control drug showing a clear zone of inhibition was taken as the MIC.
For the macrobroth dilution antibacterial assay, two fold serial dilutions of the extracts and control drug were prepared in tubes with MHB as diluent. Each dilution was seeded with the test organism to the standard concentration (5×10 5 CFU/ml). The MIC was taken as the last dilution showing no noticeable growth (turbidity).
Using the values of MIC determined for each extract by the agar-well diffusion and macrobroth dilution techniques, the MIC index was calculated for each test organism.

In vitro Determination of Antimicrobial Efficacy
The zone of inhibition was determined using the Kirby Bauer method or disc diffusion. A piece of filter paper impregnated with the extract was placed onto the surface of MH agar impregnated with the inoculum of test microorganism. The extract diffused into agar after which it's concentration radially decreased from the spot at which the antibiotic was applied outwards. If the extract was effective against the bacteria at a particular concentration, the bacteria will not grow when the concentration of the agar at that point is more than the effective concentration. This region of no bacterial growth is called the Zone of Inhibition. It was measured in millimeters as the clear zone surrounding the well containing the extract, using vernier callipers. All the zones of inhibition were measured by two different methods of illumination (illuminated by reflected light and by transmitted light) as follows: (i) Reflected light. The test plates were placed, medium side up, on a black, nonreflecting surface and were illuminated with reflected light from a desk lamp. The test plates were held 2 to 3 inches (ca. 5.1 to 7.6 cm) above the black surface to expedite zone measurement. The zones were then measured by holding a vernier calliper against the back of the petri plate.
(ii) Transmitted light. The test plates were held in front of a desk lamp, and the zones were measured with a vernier calliper held against the back of the petri plate. In either situation, the diameters of the zones of inhibited growth were measured to the nearest whole millimeter, including the diameter of the 0.25-inch (6.35-mm) disk. If there was no inhibition, a zone of 6 mm was recorded. The endpoint was to be taken as the area showing no obvious growth that could be detected with the unaided eye, not including a faint haze of growth or tiny colonies which can be detected only with difficulty at the edge of the zone of inhibited growth. If a few large colonies appeared within an otherwise clear zone of inhibition, contamination was suspected, and the test was repeated after checking for purity.

Microbial
Growth in Salivary Samples to Determine the Antimicrobial Efficacy In-vivo Samples of saliva were taken at baseline, 30 days and 60 days. They were inoculated in the respective nutrient broth and colony forming units (CFU/ml) were counted for each of the six groups.

Sample Size Calculation
Sample size was calculated using the least mean difference as 0. 15

Inclusion and Exclusion Criteria
Subjects with informed consent, subjects having Gingival Bleeding Index (GBI) score >40%, subjects with at least 20 natural teeth present were included in the study. Subjects with medical disorders or probing depth >3 mm, individuals under antimicrobial therapy at least 1 month prior to the study and using mouth rinses or dentifrices containing substances with anti-inflammatory properties, smokers and pregnant women were excluded from the study.

Collection of Plaque Sample
On the day of collection, the participants were instructed not to eat or drink anything for at least one hour before the collection of saliva samples. The collected samples were transferred into 5ml. sterile disposable vials and carried in a vaccine carrier with freezing mixture to the laboratory, where analysis of the samples were done on the same day. Samples were collected at baseline, at the end of 1 month, 2 months after the use of assigned dentifrice at baseline by a single, previously trained & calibrated examiner.
All subjects were asked to Maintain and fill a compliance sheet and get the used toothpaste packs at every visit which was weighed and assessed for compliance. Frequent reminder phone calls as a part of motivation.

Randomization
Participants were randomly allocated 6 study groups with 70 subjects in each group. Allocation sequence was generated using the lottery method by graph pad (https://www.graphpad.com/quickcalcs/randomiz e2/).

Allocation Concealment
Allocation concealment was done using the SNOSE (sequentially numbered, opaque, sealed envelopes) technique which concealed the sequence until interventions were assigned. Allocation ratio was 1:1.

Blinding
All laminate tubes procured were without any labels. Labelling was done by wrapping a paper which had letters A, B, C, D, E, F. Double blinding was done, i.e. primary investigator and subjects were not aware of the intervention.

Statistical Analysis
Data obtained was compiled on a MS Office Excel Sheet (2016). Data was subjected to statistical analysis using the Statistical package for social sciences (SPSS v 23.0, IBM). Descriptive statistics like frequencies and percentage for categorical data, Mean & SD for numerical data have been depicted. Normality of data was checked using Shapiro-Wilk test & Kolmogorov-Smirnov test and it was found that data did not follow a normal curve. Inter group comparison for microbial counts were done using Kruskal-Wallis Analysis of Variance followed by Mann-Whitney U test for group wise comparison. Intra group comparison for microbial counts was done using Friedman's test followed by pair wise comparison using Wilcoxons Signed rank test. For all the statistical tests, P <.05 was considered to be statistically significant, keeping α error at 5% and β error at 20%, thus giving a power to the study as 80%. For MIC, means and standard deviations were calculated.

Antimicrobial Activity of Extracts at 48 Hours
( Fig. 1) The antimicrobial activity of all the extracts at 48 hours was more than the negative control. For S. mutans, antimicrobial activity among groups was in the following order: AC>Triphala>Aloe-Vera>Tulsi>Pomegranate. For C. albicans, it was as follows: Tulsi>Triphala>AC>Aloe-Vera>Pomegranate.

Baseline Demographic Data
A total of 420 subjects were recruited in the study with a distribution of 70 subjects in each group.
The mean age of the subjects was 38.72(±2.62), with female preponderance.

Intergroup (Table 1) and Pair Wise (Table 2) Comparison of Colony Forming Units of S. mutans at Baseline, 1 month and 2 months Follow Up
S Mutans: A significant difference was observed for the values between the groups (P =.008) for S. mutans at baseline. The CFU values were in the following descending order group 2 (Triphala) > group 4 (Pomegranate) > group 6 (Commercially available dentifrice) > group 3 (Tulsi) > 5 (Acacia catechu) > group 1 (Aloe Vera). This difference was significant when comparing group 2 with the other groups, with the values of group 2 being higher. However, the difference in the CFU count was not statistically significant between other pairs of groups.
At 1 month there was a statistically significant difference (P<.001) observed between the CFU counts in the following descending order group 6 > group 4 > group 3 > group 5 > group 2 > group 1 . The difference was significant between all the pairs of groups except when comparing group 1 (Aloe vera) with group 2 (Triphala) and group 1 with group 5 (Acacia catechu) where no significant difference was observed.

Intergroup (Table 3) and Pair Wise (Table 4) Comparison of Colony Forming Units of C. albicans at Baseline, 1 Month and 2 Months Follow Up
At baseline: A significant difference was observed for the values between the groups (P =.010). The CFU counts were in the following descending order group 5 > group 6 > group 4 > group 1 > group 2 > group 3. When performing a pair wise comparison there was a highly significant difference (p<0.01) seen when comparing group 5 (Acacia Catechu) with other groups whereas no significant difference was observed between the pairs of other groups (p>0.05).
At At 2 months: A significant difference (P<.001) was observed for the values between the groups. The CFU counts were in the following descending order group 6 > group 3 > group 5 > group 4 > group 2 > group 1. The difference observed was significant between all the pairs of the groups except between group 1 vs. 2, 1 vs. 4, 1 vs. 5 & 2 vs. 5 where there was no significant difference observed (p>0.05).

Intragroup comparison of CFU at different time interval
S. Mutans-There was a statistically highly significant difference seen for the values between the time intervals with higher CFU counts observed at baseline and the least count observed at 2 months for group 1,2,3 and 4 while there was an increase seen at 2 months for group 5 and 6 ( Table 5).
C. albicans-There was a statistically significant difference seen for the CFU values between the time intervals with higher values at baseline and least at 2 months (Table 6). (Table 7) When comparing the CFU counts of S. mutans at baseline vs. 1 month and baseline vs. 2 months, a significant reduction in the count was observed for groups 1, 2, 3, 4 and 5. For C albicans, when comparing the CFU at baseline vs. 1 month and baseline vs. 2 months, a significant reduction in the count was observed for all the groups except group 3 and 6 where as between 1 month and 2 months only group 4 and 5 showed a significant reduction in the CFU count.

DISCUSSION
In the in vivo study, on using the herbal extracts in the form of a dentifrice, a reduction in the microbial count of S. mutans and C. albicans was observed over a period of 2 months which was greater than that of the commercial dentifrice. Triphala and Aloe Vera were observed to be consistently highly effective against S. mutans and C. albicans when compared to other herbal extracts. This is a novel study which has compared the efficacy of all 5 herbal extracts when used in a dentifrice.
In the in vitro studies, Acacia catechu demonstrated highest antimicrobial activity against S. mutans while also proving to be the third most efficacious agent against C. albicans. Acacia catechu formulations in various concentrations when tested along with other herbal products have shown significant antimicrobial effects against both gram positive and negative organisms and similar efficacy as chlorhexidine against gingival and periodontal infections. The results of the present study are in accordance with the observations made by Negi and Dave [17]. It has been extensively used in various ayurvedic formulations for many years including mouthwashes to treat sore throat, various oral diseases including caries and gingival diseases. The antimicrobial properties of AC have been attributed to the active ingredients present in it which include catechins, gallic acid and tannins [27,28]. Although very few studies are available in literature demonstrating its applications in dentistry, it has demonstrated no side effects when used long term for its antiinflammatory benefits [29].
The second highest antimicrobial activity in vitro was that of Triphala which was also the most effective extract against S Mutans in vivo.
Studies of its antimicrobial effects have been performed in various aspects in vitro such as to evaluate its efficacy as an endodontic irrigant, for orthodontic wires [30] and in cultures. When compared with sodium hypochlorite triphala showed promising results [31,32] Active ingredients like tannins, which restrict adhesion; quinines which provide free radicals and cause inactivation of microbial proteins and flavones, flavonoids, and flavonols, which disrupt microbial membrane and cell walls contribute to its antimicrobial its properties [28].
Aloe-vera demonstrated the third highest antimicrobial properties against S. mutans and fourth highest against C. albicans in vitro. Similar effect has been demonstrated in previous In-vitro studies with aloe-vera [33]. Its antimicrobial properties have been attributed to lignin, vitamins, minerals, salicylic acids, amino acids, and saponins. Aloe vera also contains a latex compound that has been found to be bacteriostatic in nature [34]. In clinical studies Aloe-vera Mouthwash has demonstrated significant antimicrobial effects [13]. Upon its incorporation into dentifrices, aloe-vera was as efficacious as other commercially available dentifrices [15,35].
In the In vitro evaluation, Tulsi demonstrated fourth highest efficacy against S. mutans and was the most effective against C. albicans. Previous In-vitro studies have also demonstrated significant antimicrobial effects of Tulsi against S. Mutans [12] as well as C. albicans [11]. Clinical study, involving chewing of Tulsi leaves in children showed significant reduction in S. mutans count [36]. Similarly Mouthwashes have also shown similar results. Tulsi mouthwashes in Candidiasis, demonstrated significant reduction in counts of C. albicans and relief in clinical symptoms [37]. Antimicrobial properties of tulsi can be attributed to the presence of eugenol (1hydroxy-2-methoxy-4-allylbenzene), ursolic acid and carvacrol [34].
Extract to demonstrate least in vitro antimicrobial activity in our study against S. Mutans and C. albicans was Punica granatum linn (pomegranate). However, it was second most efficacious in vivo against C. Albicans but still the least efficacious agent against S. mutans invivo. However a previous in vitro study has demonstrated significant reduction in bacterial adherence of S. Mutans and reduction in counts of C. Albicans with Pomegranate gels [14] A previous Invivo study has reported significant reduction in S Mutans counts when the extract was used as a mouth rinse [38]. Although there were not many studies evaluating its efficacy in the form of a dentifrice reported in literature, one unani study highlighted better significant antimicrobial properties against both gram negative and positive organisms [39]. The antimicrobial activity can be attributed to the presence of flavonoids and tannins [40].

CONCLUSION
Within the limitations of the studies it can be concluded that all herbal extracts when introduced in the form of dentifrices were effective in reducing the microbial load of S. mutans and C. albicans with Triphala and Aloe Vera showing the highest efficacy. Dentifrices consisting of these herbal extracts can be considered as alternatives to commercial toothpaste which target oral diseases caused by S. mutans and C. albicans. However, further studies using standard protocol and concentrations in order to enable commercial incorporation of these extracts need to be conducted.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
Ethical clearance was obtained from the Institutional review board (IRB) and ethics committee of Dr G.D. Pol Foundation's Yerala Medical Trust Dental College and Hospital, Navi Mumbai.