INTRODUCTION:

Dental caries is a microbiological infection that causes the calcified tissues of teeth to dissolve and be destroyed locally. If left untreated, the illness may cause discomfort, infection, loss of teeth, and in extreme circumstances, even death. Caries is still one of the most serious illness in the world today. The bacteria that cause tooth cavities and plaque development is called staphylococcus aureus. When fermentable carbs like sucrose, fructose, and glucose are present, the acid-producing by this bacteria dissolves tooth structures and causes damage. Food particles, saliva, acid, and bacteria come together in the mouth to create "plaque," which is a sticky material that sticks to teeth.

Even with the commercial availability of numerous anti-caries agents, the hunt for a potent natural agent is still ongoing. Organic items have proven to be an effective substitute for artificial chemical compounds in the prevention of dental cavities.¹

Herbal Toothpaste:

Herbal products used to take care of the teeth and mouth are referred to as oral care products. An essential component of your everyday oral hygiene regimen is toothpaste. It aids in cleaning plaque and food particles off of your teeth and gums.

Polyherbal toothpaste formulation is the composition of Azardirachtan indica leaves, Trigonella foenum -graecum seeds, Punica granatum peel are used and for the evaluation test staphylococcus and candida albicans were used and screened with methanol extractions. Once the antibacterial property of polyherbal formulation extracts is screened under in vitro condition against oral pathogens, evaluation parameters can be carried out for the prepared polyherbal formulation which will treat dental caries.²

MATERIALS AND METHOD:

Plant Material and Extraction Procedure:

Different plants were collected from the garden of Gokaraju Rangaraju College of pharmacy.

Polyherbal toothpaste formulation is the composition of Azardirachtan indica Leaves, Trigonella foenum -graecum seeds, Punica granatum peel, Aloe barbadens is Leaves, are used.

Extraction of Polyherbal Herbal Extract:

The plant parts were powdered and shade-dried before being used in the extraction process. Ten grams of various powdered herbal powder was placed in a conical flask, and 100 milliliters of methanol and water (1:1) ratio was added. The mixture was then kept for maceration for 48 hrs, after that the extracts were filtered through Whatman filter paper.^3-5

Figure 1: Fenugreek extract

Figure 2: Filtration of pomegranate peel extract

Figure 3: Extraction of neem

Table 1: Ingredients for polyherbal toothpaste formulation: (Active ingredients)

S. NO	Ingredients	Quantity (gm)	Uses
1	Fenugreek powder	2.5	Anti-inflammatory
2	Clove oil	2 to 3 drops	Dental Analgesic
3	Neem powder	0.05	Anti-microbial
4	Aloe Vera gel	6	Anti-fungal, Anti-viral
5	Trikatu powder	0.03	Anti-caries, Anti-microbial
6	Pomegranate peel	1.6	Anti-inflammatory

Table 2: Ingredients for polyherbal toothpaste formulation: (base preparation)

S. NO	Ingredients	Quantity (mg)	Uses
1	Calcium Carbonate	41gm	whitening Abrasive,
2	Sodium Fluoride	0.9gm	Fluoride
3	Sorbitol	44gm	Humectant
4	Sodium lauryl Sulphate	1.5gm	Detergent and Foaming agent
5	Sodium CMC	1.8gm	Binding Agent
6	Methyl paraben	0.2gm	Preservative
7	Sodium benzoate	0.1gm	Preservative
8	Sodium saccharine	0.2gm	Sweetening Agent
9	Peppermint Oil	2 to 3 drops	Flavoring Agent
10	Glycerin	Q.s	Humectant

Preparation Method:

Dry Gum Method:

1. The solid ingredients calcium carbonate, sodium fluoride, SLS, sodium CMC, methyl paraben, sodium benzoate, sodium saccharine were weighed accurately as mentioned in the formula and sieved with sieve no.80 so as to maintain the particle size.

2. Further, these chemicals were subjected to mixing in mortar and pestle and triturated with glycerine until semisolid mass formed.

3. Accurately weighed herbal extract were added to the base along with Aloe Vera gel and clove oil.

4. Peppermint oil was added as a flavoring at the end. ⁶

Evaluation and comparison of Polyherbal Toothpaste:

Physical Examination (Colour, odour, taste, smoothness, relative density): Formulated herbal toothpaste was evaluated for its colour, colour was checked visually. Odour was found by smelling the product. Taste was checked manually by tasting the formulation. The smoothness was tested by rubbing the paste formulation between the fingers. ^7-9

Inertness of tube: Deterioration of the container should not be seen after storing the toothpaste in normal storage conditions like heating temperature at 45±2 ℃ for ten days. Inertness of tube was observed by cutting the internal surface of the container and checking whether any sign of deterioration or chemical reactions occurred in the container.

pH: 1gm of the toothpaste was dispensed in a 50 ml beaker and 10ml of freshly boiled and cooled water was added to make aqueous suspension. Stir well to make a thorough suspension an measure the pH using pH meter.

Homogeneity: The toothpaste shall extrude a homogeneous mass from the tube or any suitable container on applying normal force.

Determination of sharp and edge abrasive particles: Toothpaste was scratched on the butter paper for 15-20 cm long to check for the presence of any sharp or abrasive particles. Repeated the same process for at ten times.

Foamability: 2g of toothpaste was taken with 5ml water in a measuring cylinder initial volume was noted and then shaken for 10 times. Final volume of foam was noted.

Determination of moisture and volatile matter: Moisture and volatile matter was determined by using 5gm of herbal toothpaste was placed in a porcelain dish of about 6-8cm in diameter and 2-4cm in depth. Dried in an oven at 105℃.

Calculations:

% by mass = 100Ml / M

Ml -Loss of mass (g) on drying

M- Mass (g) of the material taken for the test.

Determination of Spreadability: About 1-2g of herbal toothpaste was weighed and placed between two glass slides (10 x 10cm) one over each other (sliding, shall not take place), and the slides were pulled in opposite direction. Measure the spreading (in cm) of the toothpaste after 3 minutes. Repeat the experiment and note the average value of three readings.

Antimicrobial activity:

Table 3: Formulation table of agar medium (300ml)

Composition	300ml
Beef extract	3gm
Peptone	3gm
Nacl	1.5gm
Agar	4.5gm
Distilled water	300ml

Procedure:

1. Weigh all ingredients accordingly by given table.

2. Now, add into a conical flask containing 300 ml of distilled water.

3. Boil it for 10mins with continuous stirring until completely dissolved in water.

4. After cooling sterilize in autoclave at 121°C for 15mins.

5. After autoclaving, allow it to cool and add streptococcus species.

6. Pour nutrient agar into each petri dish plate and leave plates on the sterile surface until agar has solidified.

7. Take borer and make wells place the toothpaste in the wells and place it for incubation.

8. The zone of inhibition is measured using a ruler.

Anti fungal activity:

Table 4: Formulation of fungal medium

Composition	Quantity
Glucose	5g
Peptone	2.5g
Agar	3.75g
Distilled water	250ml
pH	5.4

Procedure:

1. Weigh all the ingredients separately by using physical balance.

2. Now, add into a conical flask.

3. Boil it for 10mins with continuous stirring until completely dissolved.

4. After cooling sterilize in autoclave at 121°C for 15mins.

5. After autoclaving, allow it to cool and add candida species.

6. Pour nutrient agar into each petri dish plate and leave plates on the sterile surface until agar has solidified.

7. Take borer and make wells, place the toothpaste in the wells and place it for incubation.

8. The zone of inhibition is measured using a ruler.¹⁰

Protein Denaturation:

Protein denaturation assay was done by taking the reaction mixture (5 ml) consisted of 0.2 ml of 1% bovine albumin, 4.78 ml of phosphate buffered saline (PBS, pH 6.4), and 0.02 ml of extract, and the mixture was mixed, and was incubated in a water bath (37 °C) for 15 min, and then the reaction mixture was heated at 70 °C for 5 min. After cooling, the turbidity was measured at 660 nm using a UV/VIS spectrometer. Phosphate buffer solution was used as the control.¹²

The percentage inhibition of protein denaturation was calculated by using the following formula:

% Inhibition of denaturation = 100 × (1 − A2/A1),

Where A1 = absorption of the control sample, and A2 = absorption of the test sample.

RESULTS AND DISCUSSION:

The herbal toothpaste formulation was prepared from neem leaves, pomegranate peel, fenugreek powder and other natural ingredients. Formulated herbal toothpaste evaluation tests were carried out and are compared with different properties of formulated herbal and marketed toothpastes. All the results of evaluating parameters are given in below tables.

Table 5: Evaluation parameters and comparison of polyherbal toothpaste with marketed herbal formulation

S No	Parameters	Polyherbal toothpaste	Dant kanti	Dabur red
1	Color	Light green	Brown	Red
2	Odor	Characteristic	Characteristic	Characteristic
3	Taste	Characteristic	Characteristic	Characteristic
4	Smoothness	Smooth	Smooth	Smooth
5	pH	9.3	7.8	8.6
6	Homogeneity	Good	Good	Good
7	Abrasiveness	Good	Good	Good
8	Foamability	3.5 cm	3 cm	3.3 cm
9	Moisture content	12%	14%	18%
10	Spreadability	6 cm/sec	5 cm/sec	5.5 cm/sec
11	Stability	Stable	Stable	Stable
12	Protein denaturation	82%	--	--
13	Anti-bacterial activity	24.5 mm	23.8 mm	23.5 mm
14	Anti-fungal activity	20.5 mm	19.8 mm	20.3 mm

Along with the commercial formulations, the organoleptic assessment of polyherbal toothpaste was conducted.

The polyherbal toothpaste's smoothness was comparable to that of the other two commercially available formulations.The pH of formulated polyherbal toothpaste was compared with the marketed formulations and it showed pH 9.3 which is within the range that is 7-10.

Figure 4: Digital pH meter

The foamability of polyherbal toothpaste is more when compared with the marketed formulations. It is 3.5 cm which is greater than the other two marketed formulations.

Figure 5: Comparison of Foamability

Spreadability of poly herbal toothpaste was good and it is more when compared with the other two formulations that is 6 cm.

Figure 6: Comparison of Spreadability

The loss on drying of formulated poly herbal toothpaste as compared to other formulations is less and it is more efficient when compared to other two marketed formulations.

Figure 7: Comparison of % Moisture Content

Formulated polyherbal toothpaste has good antibacterial activity. The zone of inhibition was found to be 24.5mm, it showed good anti-bacterial activity against the staphylococcus species when compared with the marketed herbal formulations.

Figure 8: Anti-bacterial activity (zone of inhibition) shown by polyherbal toothpaste.

Formulated polyherbal toothpaste has good anti-fungal activity against candida species. Zone of inhibition was found to be 20.5mm, which is greater when compared with the other two herbal marketed formulations.

Figure 9: Anti-fungal activity (zone of inhibition) shown by polyherbal toothpaste.

Figure 10: pH of formulated herbal toothpaste and marketed herbal toothpaste.

Figure 11: Foamability of formulated herbal toothpaste and marketed herbal toothpaste.

Figure 12: % of moisture content of formulated toothpaste and marketed herbal toothpaste

Figure 13: Comparison of anti-bacterial activity (zone of inhibition) of formulated polyherbal toothpaste and marketed toothpastes.

Figure 14: Comparison of anti-fungal activity (zone of inhibition) formulated polyherbal toothpaste and marketed toothpastes.

CONCLUSION:

The results of the study also support the traditional application of the plant and suggest that plant extracts possess compounds with antimicrobial properties that can be used as antimicrobial agents. And the developed polyherbal toothpaste could be utilized in the treatment of various dental diseases.

ACKNOWLEDGEMENT:

We are thankful to Principal and management of Gokaraju Rangaraju College of Pharmacy for providing the facilities required for the research.

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Received on 12.07.2024 Revised on 22.12.2024

Accepted on 14.04.2025 Published on 18.04.2025

Available online from April 22, 2025

Asian J. Res. Pharm. Sci. 2025; 15(2):129-134.

DOI: 10.52711/2231-5659.2025.00020

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.