INTRODUCTION:

Medicinal plants have been used to treat various diseases since ancient times¹. Medicinal plants have been a major source of both curative and preventive medical therapy preparations for human being². However, rural and remote areas where immediate medical facilities are not available, people are dependant mainly on herbal products or traditional medicines which are available easily in their vicinity³. Over the past decade, awareness of medicinal plant usage and their products has markedly at rise. Reports have been observed that conventional medicines cause more adverse effects than herbs derived from plants.

Worldwide, herbal therapies are used to treat oral infections. However, Streptococuus mutans (S. mutans) is an important etiologic agent in dental caries, because of its natural habitat in the human oral cavity^4,5. It is one of the most effective cariogenic bacteria in the initial formation of caries. This micro-organism resides specifically in the dental plaque and forms a multispecies biofilm on hard surfaces of the tooth⁶.

Tooth products provide protection against caries and destroy bacteria to maintain oral hygiene. Today, the dentifrices in the market are made from a variety of compounds, which may not be beneficial for the parts of the teeth. Herbs are considered as safe as these do not affect the human body as synthetic drugs do. Many herbal drugs such as stem and husk are used for oral hygiene since prehistoric time⁷. These plants are used in the raw form and very few researchers have tried to utilize herbal drugs to prepare tooth gel. Therefore, an attempt was made to prepare effective herbal tooth gel using herbal extracts for oral hygiene.

Amlaki (Emblica officinalis) is one of the best rasayanas which has an excellent healing property. It has anti-oxidant, anti-aging properties, immunomodulatory, anti-pyretic, analgesic, cytoprotective, anti-tussive, gastroprotective and antimicrobial activity⁸. It is an effective antiplaque agent due to its antioxidant properties, thus effectively inhibit the biofilm formation⁹ Haritaki (Terminalia chebula) is extensively used in Ayurveda. Terminalia chebula contains tannin such as chebulic acid, chebulagic acid, corilagin, and gallic acid¹⁰. It has anti-bacterial, anti-fungal, anti-viral, antioxidant¹¹ activity, adaptogenic immunomodulatory, hypolipidemic and anti-spasmodic activity¹². Bahera (Terminalia bellerica) is also extensively used in Ayurveda. It is used as antipyretic, anti-diarrheal, anti-inflammatory¹³, antimicrobial, astringent, antioxidant, antispasmodic and antimicrobial¹⁴. It contains gallic acid, ellagic acid and belleric acid.¹⁵Ajowan (Trachycpermum ammi) is used as bronchodilator, anti-microbial, anti-inflammatory, anti-fungal, anti-spasmodic and analgesic. It contains thymol as an important constituent.¹⁶Vidang (Embelia ribes) is used as antibacterial, analgesic, anti-inflammatory, antioxidant¹⁷, anti-hyperlipidemic and antifungal.¹⁸Clove (Syzygium aromaticum) is used as dental analgesic. It has anti-oxidant, anti-microbial activity¹⁹

MATERIAL AND METHODS:

Plant material:

Powders of Emblica officinalis (Amla), Terminalia bellirica (Baheda), Terminalia chebula (Hirda), Trachycpermum ammi (Ajwain) and Embelia ribes (Vidang) were collected from reputed vendor Pune and authenticated correctly by the pharmacognosy department of Dr. Vishwanath Karad MIT World Peace University School of Pharmacy (MITWPU/Pcog/05).

Extraction:

Amla²⁰, Baheda, Hirda, Ajwain²¹ and Vidang powder, each of weight (50g), were weighed and subjected to the process of extraction, separately. Briefly, powders were triturated separately using mortar pestle and subjected to separate thimble for each powder. The thimbles were arranged to Soxhlet apparatus. Then, extraction was continued using ethanol as a solvent until the solution turned colourless. Thereafter, the extract was collected from the round bottom flask which was then subjected to distillation further to remove the alcohol. The process was continued for each extract. The pure extracts were stored in air tight containers till further use.

Phytochemical analysis of plant extracts:

The obtained extracts were subjected for preliminary phytochemical screening to identify the presence of constituents like alkaloids, tannins, flavonoids and carbohydrates²².

Determination of total tannin content:

Folin-Ciocalteu was used to determine the tannins. The extract (0.1ml) was mixed with 7.5ml distilled water, 0.5 ml Folin-Ciocalteu phenol reagent, 1ml 35 percent sodium carbonate solution, and diluted to 10ml with distilled water in a volumetric flask (10ml). The liquid was thoroughly mixed before being stored at room temperature for 30 minutes. The same procedure was used to make a set of tannic acid reference standard solutions (20, 40, 60, 80, and 100µg/ml). After that, a UV/ Visible spectrophotometer was used to measure the absorbance of the test and standard solutions against a blank at 700nm. The tannin concentration was calculated in mg of tannic acid equivalents/g of dried material in triplicate.²³

Design of Experiment:

The preliminary study of gel was performed by performing trial and error of batches varying the concentrations of sodium carboxymethyl cellulose (S-CMC) (1 to 3%), concentration of Gantrex (0 to 2%) and concentration of sodium lauryl sulphate (SLS) (1 to 3%). Using the programme Design Expert® (version 12.0.4), a 2³ factorial design was conducted to optimise the gel composition). The following three factors each at two levels: concentration of sodium carboxymethyl cellulose (X₁) (2 and 3%); concentration of Gantrex (X₂) (0 and 1%); and concentration of sodium lauryl sulphate (X₃) (1.5 and 2.5%) were selected as independent variables (Table 1). Response variables (dependent variables) were foaming capacity in ml (Y₁) and zone of inhibition (Y₂). Total eight formulation batches were suggested by factorial design (Table 1).

Preparation of herbal tooth gel:

Formulation of herbal tooth gel was prepared as follows. Accurately weighed quantities of sodium benzoate and sodium lauryl sulphate were triturated in a mortar properly. Then, glycerin was added in the mortar and mixed them properly. Thereafter, the extract was dissolved separately in ethanol (2ml) and then added in the mortar. The solution was mixed properly followed by adding liquid sorbitol. In another mortar, weighed amount of sodium carboxymethyl cellulose was added in water. This solution was then added to the earlier solution and mixed properly. Lastly, colour and flavour were added (Table 1).

Table 1: Formulation of tooth gel

Ingredients	Formulation code
Ingredients	F1	F2	F3	F4	F5	F6	F7	F8
Emblica officinalis extract (g)	0.6	0.6	0.6	0.6	0.6	0.6	0.6	0.6
Terminalia bellirica extract (g)	0.6	0.6	0.6	0.6	0.6	0.6	0.6	0.6
Terminalia chebula extract (g)	0.6	0.6	0.6	0.6	0.6	0.6	0.6	0.6
Trachycpermum ammi extract (g)	0.6	0.6	0.6	0.6	0.6	0.6	0.6	0.6
Embelia ribes extract (g)	0.6	0.6	0.6	0.6	0.6	0.6	0.6	0.6
Sodium Carboxymethyl cellulose (g) (X₁)	3	3	3	3	2	2	2	2
Gantrex (g) (X₂)	-	-	1	1	-	-	1	1
Sodium lauryl sulphate (g) (X₃)	2.5	1.5	2.5	1.5	2.5	1.5	2.5	1.5
Sorbitol (g)	73	73	73	73	73	73	73	73
Sodium benzoate (g)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Ethanol (ml)	10	10	10	10	10	10	10	10
Colour (g)	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
Flavour (ml) (clove oil)	0.8	0.8	0.8	0.8	0.8	0.8	0.8	0.8

EVALUATION OF HERBAL TOOTH GEL:

Preliminary Evaluation:

Organoleptic evaluation (colour, odor and taste) was done by sensory and visual inspection. Toothgel (1g) was dispersed in 100ml purified water. The calibration of pH meter was performed using standard buffer solution at 4.0, 7.0 and 9.0. The pH was determined using pH meter. Brookfield viscometer was used to assess the viscosity of the tooth gel¹⁶. The spindle was attached to the viscometer on the lower shaft and it was held firmly while screwing. The trapping of the bubbles was avoided and it was slowly dragged in the tooth gel. About 100g of tooth gel was placed in small sample adapter of the Brookfield viscometer²⁴. Due to high viscosity of the tooth gel, spindle no. 7 was selected and the spindle was lowered in the tooth gel until the "meniscus" of the gel is at the centre of the immersion groove on the spindle’s shaft²³. The viscosity was recorded at angular velocity gradually of 20rpm by using spindle no. 7 for about 5min. After the viscosity measurement was completed, the reading was jot down and calculated the viscosity. Sharp and edge abrasive particles studies were carried out to check the presence of abrasive particles. The content was extruded on a butter paper about 15-20cm long on and the same process was repeated for ten collapsible tubes. The toothgel was checked by figure tip for the presence of particles²⁵.

Determination of moisture and volatile matter:

The moisture content²⁶ and volatile matter²⁶ was determined by taking 5g formulation in a porcelain dish. Initially, weight of empty porcelain dish was noted and later weight of the porcelain dish with formulation was taken down. The sample was dried in the oven at 105°C until a constant weight was achieved and the loss of moisture was calculated.

Moisture (%) =100 ML/M

Where, ML-weight (g) after drying, M- Initial weight (g) of the material

Extrudability:

The test was performed by filling tooth gel (20g) into test tube and then sealing it by crimping of the tube. The weight of the tube was along with gel is then recorded. The tube was then placed in between two wooden plates of lab made extrudability apparatus specifically prepared for this purpose. Cap of tube was removed and tube was pierced in order to open the outlet of the tube. 100g weight was placed over the upper wooden plates. The gel extruded through the opening was collected on tared butter paper and quantity of gel extruded was determined. Extrudability using following formula:

Net weight of formulation in tube

Extrudability (%) = –––––––––––––––––––––––––x100

Weight of formulation extruded

Spreadability:

Toothgel (2g) was placed (sandwich like) between two glass slides for 5 min and air was removed to impart uniform film of the gel between slides.²⁷ The extra gel was removed from edges, if any. The top plate was pull off 80g with the help of string attached to the hook and the time (s) required by the top slide and the distance was noted. A short interval indicated better spreadability and was calculated as follows:

Spreadability = Weight in the pan × Length moved by the glass slide/Time

Anti-microbial Activity:

The activity²⁸ was performed by disc diffusion method by using Muller Hinton agar medium against a pathogenic bacterial strain Streptococcus mutans. S. mutans was initially cultured in nutrient broth and incubated at 37ºC for 24h. Thereafter, cultured cells were grown to multiply in the Muller Hinton agar plates. Then the bacterial plates were incubated at 37ºC for the 24 h. The samples of tooth gel were prepared. Further, inert paper discs of uniform size were embedded in this solution. These discs were then placed on the surface of inoculated plates for further inoculation. The diameter of zone of inhibition (ZOI) was measured in millimetres (mm). The samples used were formulated herbal tooth gel (25mg/ml), chlorhexidine gluconate as the positive and ethanol as negative control. The minimum inhibitory concentration (MIC) is the smallest concentration in which the compound displays no visible microbial growth. The test was performed in triplicate.

Foamability:

Modified cylinder shake method was used for determining foaming ability of toothgel²⁹. 1% toothgel solution (1g in 10ml) was introduced in 50ml graduated cylinder and was shaken 10 times. The graduated cylinder was allowed to stand for 15 min and the volume of foam was measured²⁹.

Stability Study:

Stability studies were conducted as per ICH guidelines using triple stability chamber. The collapsible tubes containing formulated gel was stored at different temperature and humidity conditions, 30ºC±2ºC / 65% ± 5% RH, 40ºC±2ºC/75%±5% RH for the period of three months. Color, odor, pH, spredability, viscosity and tannin contents were tested after 0, 15 days, 1month, 2 months and 3 months.

RESULT AND DISCUSSION:

Extraction of phytoconstituents and analysis:

The yield of extract obtained from the crude materials is as follows: Amla (9.7%), Baheda (18.9%), Hirda (21.7 %), Ajwain (16.3%) and Vidang (8.23%). Presence of saponin, alkaloids, glycoside, tannins, carbohydrate and flavonoids in crude drug are as shown in Table 2.

Table 2: Phytochemical screening of crude drugs

S. No.	Crude drug	Eo	Tb	Tc	Ta	Er
1	Saponin	-	+	+	+	+
2	Alkaloid	+	+	+	+	+
3	Glycoside	+	-	-	-	-
4	Tannins	+	+	+	+	+
5	Flavonoids	+	+	+	+	-
6	Carbohydrates	+	+	+	+	+

Eo: Emblica officinalis, Tb: Terminalia bellirica,
Tc: Terminalia chebula, Ta:Trachycpermum ammi, Er: Embelia ribes

Determination of total tannin content:

The quantitative analysis of tannin content was performed by Folin-Ciocalteu method. The results revealed highest tannin content in the extract of Terminalia chebula (89.02 ± 0.41 mg/g dried extract) and least in Trachycpermum ammi (1.78 ± 1.98 mg/g dried extract) as shown in Table 3.

Table 3: Estimation of total tannin content

Sr. No	Crude drug	Total tannin content (mg/g dried extract)
1	Emblica officinalis	29.06 ± 1.23
2	Terminalia bellirica	52.09 ± 1.45
3	Terminalia chebula	89.02 ± 0.41
4	Trachycpermum ammi	1.78 ± 1.98
5	Embelia ribes	25.23 ± 0.65

Preparation and optimization of herbal tooth gel:

Eight formulations (F1 to F8) were successfully formulated using varying concentration of sodium carboxymethyl cellulose, Gantrex as gelling agents and Sodium lauryl sulphate as foam producing agent

All these formulations were further subject for optimization and evaluation studies.

Evaluation of herbal tooth gel:

Preliminary Evaluation:

All the formulation were found to be uniform light green in color, they all were having typical clove odour and were sweet to taste. Toothgel were found to have pH in the alkaline range of 7.2 to 7.54. Viscosity is essential parameters in semisolid preparations low viscosity would cause draining of the preparation from the brush while high viscosity would result in poor spreadability on teeth and poor extrudability. Viscosity of the formulated gel (Table 4) was found to be optimum in the range of 28000cps to 70000 cps. Sharp edged abrasive particles will lead to damage of tooth enamel and hence this parameter was undertaken for study. Toothgel were found to be smooth with even and regular surface; they were free from perceptible projections, lumps, or indentations.

Moisture and volatile content:

The percentage of moisture and volatile content in formulated toothgel showed values (Table 4) that matched with the marketed formulation (Colgate gel maxfresh). These results explained that both marketed and lab made formulation had good moisture and volatile content.

Extrudability:

Extrudability values of formulations were found in optimum range of 5.11% to 7.22% were found, implying that the gel would not cause draining of the preparation from the tube as in case of low extrudability or would not result in difficulty of removal of gel from tube in which it is packed as in case of high extrudability. Also, the observation showed that viscosity and extrudability were going hand in hand and are directly proportional as it should be.

Spreadability:

Spreadability values of formulations were found in range of 57 to 73g.cm/s indicating that the gel is easily spreadable by small amount of shear. The toothgel will thus spread easily on the surface of tooth and enamel without applying extra force during brushing. This will in turn protect the teeth and enamel from damaging which might result when one applies extra pressure for uniform spreading. Further the spreadability of formulated gel was found to be higher than marketed formulation

Effect of variables on formation of tooth gel:

Foamability:

Pareto chart was used to screen the effect of factors on foamability and further to avoid the non-significant terms. Pareto chart revealed only the main positive effect of SLS (C) which was considered as significant factor as bar graph was nearer to Bonferroni limit t-value limit (Fig. 1a). Even though, effects of sodium CMC (A) and Gantrex (B) were non-significant with negative effect, both the factors were found to affect the formation of foam. Factors with interactions such as AC, AB, ABC and BC are far below Bonferroni line, therefore were non-significant.

Fig. 1 Effect on foamability showing (a) Pareto chart (b) 3D plot

Herein, foamability of gel was ranged between 32.1±0.2 to 48.2±0.4 ml. Based on the results of ANOVA, the effect of foamability is shown in polynomial equation:

Y₁= + 39.25 - 1.50A - 1.50B + 4.75C + 0.25AB - 0.5AC + 0.25ABC

SLS is a surfactant which is responsible for formation of more amount of foam by reducing surface tension.

Zone of inhibition:

Pareto chart revealed only the main positive effect of SLS (C) on zone of inhibition which was considered as significant factor as bar graph was far above Bonferroni limit trend line (Fig. 2a). However, effect of sodium CMC (A) was positive while that of Gantrex (B) was negative on zone of inhibition. The effects of factors are depicted in polynomial equation. Zone of inhibition was found to be ranged between 3.4 ± 0.1 to 4.4 ± 0.2 cm. The polynomial equation based on ANOVA is as follows:

Y₂= +1.67+0.4A-0.2B+1.35C-0.3AC

Here, increase in concentration of SLS increased the zone of inhibition due to its surfactant activity, which helped to penetrate more amount of phyto-constituents present in tooth gel in the media.

Fig. 2 Zone of inhibition showing (a) Pareto chart (b) 3D plot

Table 4. Evaluation of Formulations

Parameters	Formulation
Parameters	F1	F2	F3	F4	F5	F6	F7	F8	Marketed
Moisture content %	98.26±0.23	98.98± 0.67	98.20±0.98	98.78±0.78	98.35± 0.88	98.46±0.34	98.59±0.56	98.78± 0.66	98.38± 0.98
Extrudability (%)	5.11± 0.32	6.23 ± 0.45	5.11 ± 0.44	6.20 ± 0.65	6.11 ± 0.55	7.22 ± 0.36	6.20 ± 0.76	6.50 ±0.97	5.73 ± 0.45
Spreadability	62 ± 0.10	66 ± 0.30	62 ±0.30	80 ±0.10	66 ±0.20	80 ±0.20	72.7 ±0.30	80 ± 0.10	57 ± 0.20
pH	7.24	7.13	7.43	7.37	7.24	7.54	7.43	7.33	7.2
Viscosity (cp)	66,000	40,000	66,000	38,000	55000	30000	40000	28000	71,000
Stability	Stable	Stable	Stable	Stable	Stable	Stable	Stable	Stable	Stable
Foamability (ml)	43± 1.00	35 ± 0.70	41 ± 0.40	32 ± 0.10	48 ± 0.30	37 ± 0.40	44 ± 0.90	34 ± 0.50	32 ± 0.40
Zone of inhibition (cm)	4 ± 0.33	3.5 ±0.20	3.8 ±0.23	3.4 ±0.12	4.4 ± 0,20	3.6 ± 0.40	4.1 ±0.35	3.4 ± 0.28	3.7 ± 0.35

Formulations F1 to F8 were green in color and uniform in consistency with relatively good uniformity. However, formulations F5, F6, F7 and F8 did not form the gel which may be due low concentration of gel forming agents sodium carboxymethyl cellulose and Gantrex.

SELECTION OF OPTIMIZED BATCH:

The formulation of tooth gel which attained both maximum foamability and zone of inhibition was considered as optimized batch. F5 showed highest foamability (48.2±0.4ml) and zone of inhibition (4.4±0.2cm) with the desirability value 1 (most desirable response) at design space X₁ = 2%, X₂ = 0%, X₃ = 2.5 %. Therefore, F5 was optimized formulation.

Stability Studies:

Optimized batch F5 was selected for optimization study. Formulation F5 were found stable even after 3 months, no change in colour, pH, viscosity and spreadability of gel was observed. Observations of stability studies are tabulated in Table 5.

Table 5. Stability studies of formulation F5

Colour	Odour	Taste	Spreadability	pH	Viscosity
At 30ºC± 2ºC / 65% ± 5% RH (3^rd month)
Green	Characteristic	Sweet	18.15cm/sec	8.4	66,000cp
At 40ºC± 2ºC / 75% ± 5% RH (3^rd month)
Green	Characteristic	Sweet	18.10cm/ssec	8.3	64,000cp

CONCLUSION:

The formulated tooth gel of herbal extracts of Emblica officinalis, Terminalia bellirica, Terminalia chebula, Trachycpermum ammi, Embelia ribes and Syzygium aromaticum revealed good anti-bacterial activity and foaming capability to prevent tooth decay. The phytoconstituents were successfully extracted and analysed, which showed highest tannin content in the extract of Terminalia chebula and least in Trachycpermum ammi, which helped to control plaque. Therefore, formulated herbal tooth gel can be better alternative to marketed gel. In conclusion, formulated herbal tooth gel was considered as helpful to take care of oral hygiene of human being.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 02.12.2021 Modified on 11.04.2022

Research J. Pharm. and Tech 2023; 16(3):1283-1288.

DOI: 10.52711/0974-360X.2023.00211