1.1 Cosmetics

As per Drug and Cosmetics Act 1940 and Rules 1945 “Cosmetics is defined as any article intended to be rubbed, poured, sprinkled or sprayed on or introduced into, or otherwise applied to the human body or any part therefore cleansing, beautifying, promoting attractiveness or altering the appearance. [1].

Anatomy of the oral cavity includes the lips, hard palate (the bony front portion of the roof of the mouth), soft palate (the muscular back portion of the roof of the mouth), retromolar trigone (the area behind the wisdom teeth), front two-thirds of the tongue, gingiva (gums), buccal mucosa (the inner lining of the lips and cheeks), and floor of the mouth under the tongue.^[2]

• Lips
• Buccal mucosa
• Tongue
• Teeth

1.2.2 Teeth

The teeth are the strongest and most rigid substances in the human body. A normal adult has 32 teeth and is divided into incisor, canine, molars and premolars. There is a third molar, which is called the wisdom teeth, which appear in a person’s late teens or early twenties.Each tooth consists of a crown with one or more tips, a neck, and a root. The Pulp cavity is the centre part of the tooth, filled with blood vessels, nerves and connective tissues and is surrounded by an enamel, which protects the tooth against scratch, cut and the invasion of bacteria present in the mouth.

1.2.3 Types of teeth

We have different types of teeth, and each type serves an important purpose. There are four types of permanent teeth in humans.

• Incisors
• Canines
• Premolars
• Molars

A. Incisors

Your incisors are the most visible teeth in your mouth. Most people have four incisors on the upper jaw and four on the lower. These include your front two teeth and the teeth on either side of them. Each incisor has a single narrow edge, which helps cut into food when you bite.

B. Canines

Canine teeth get their name because they resemble a dog’s fangs. They’re pointier than other types of teeth. Most people have four canine teeth — one in each quadrant (upper right, upper left, lower right, lower left). Canine teeth help you tear into foods like meat and crunchy vegetables. Sometimes, people call canines “eye teeth” because of their position directly under your eyes.

 C. Premolars

Also called bicuspids, premolars sit between your canines and your molars (the teeth in the back of your mouth). Premolar teeth have features of both canines and molars. They help you tear, crush and grind food into smaller pieces.

D. Molars

Your molar teeth are in the very back of your mouth. Most of your chewing, about 90% takes place here. Most adults have 12 molar teeth, three in each quadrant. Molar teeth include wisdom teeth (third molars). So, if you’ve had your wisdom teeth removed, or if you were born without them, then you probably have eight molars altogether. ^[3]

1. Tooth decay
2. Periodental disease
3. Trench mouth
4. Tooth erosion
5. Stain teeth
6. Dental cavities

A. Tooth decay

Tooth decay also known as dental caries or cavities. It is a breakdown of teeth due to acids made by bacteria. The most common bacteria associated with dental cavities are Streptococcus mutan and Streptococcus sobrinus and Lactobacillus.

Periodental disease is a serious gum infection that damage the soft tissue and destroy the bone that support the teeth. Periodental disease can cause teeth to loosen or lead to tooth loss. It is caused by Actnomyces.

They are of two types:

• Gingivitis
• Periodontitis

C. Trench mouth

It is a severe gum infection caused by build up of bacteria. It is caused by Prevatella intermedia. It generally occurs in HIV infected patients.

D. Tooth erosion

It is defined as the irreversible loss of tooth structure due to chemical dissolution by acids not of bacterial origin. The most common cause of erosion is by acidic foods and drinks of pH below 5-5.7. It is caused due to accumulation of food particles, accumulation of bacteria in gelatinous mass called as plaque, release of lactic acid and anaerobic fermentation of sugars in saliva.

Teeth can become discoloured by stains on the surface or by changes inside the tooth.

1. Extrinsic: This occurs outside the layers of teeth
2. Intrinsic
3. Age related factors

Permanently damaged areas in teeth that develops into tiny holes. Causes include bacteria, snacking, sipping sugary drinks and poor teeth cleaning. ^[4]

1.4   Herbal Formulations

Herbal formulations means a dosage form consisting of one or more herbs or processed herbs in specified quantities to provide specific nutritional, cosmetic benefits meant for use to diagnose, treat, mitigate diseases of human beings or animals, alter the structure or physiology of human beings or animals. Herbal formulation contain an active substance or herbal substance in combination with one or more herbal preparations. Herbal formulation are obtained by subjecting herbal substances to treatments such as extraction, distillation expression, fractionation, purification, concentration or fermentation include comminuted or powdered. ^[5]

• 1. 1. ADVANTAGES
• These medicines are used to treat the cause of the issue you are having and not just mask the symptom, because herbal medicines contain vitamins, antibodies  and other health promoting agents.

• It helps to strengthen the overall body not just combat illness.
• Less toxic or having less side effects in contrast to synthetic drugs.
• Effective, specific, stable, and potent.

1.4.2 DISADVANTAGES

• They may cause other problems if you’re taking other medicines.
• You may experience a bad reaction or side effects after taking a herbal medicine.
• Not all herbal medicines are regulated.
• Evidence for the effectiveness of herbal medicine is generally very limited.

1.5 Herbal Cosmectics

Herbal cosmetics are formulated using different cosmetic ingredients to form the base in which one or more herbal ingredients are used to cure various skin aliments. Plants are highly used for the development of new drug products for cosmaceuticals and pharmaceutical applications. The demand of herbal medicine is increasing rapidly due to their skin friendliness and lack of side effects. The best thing of the herbal cosmetics is that it is purely made by the herbs and shrubs and thus is side effect free. The natural content in the herbs does not have any side effects on the human body; instead provide the body nutrients^[6].

Tooth paste is a paste or gel dentifrice used with a toothbrush as an accessory to clean and maintain the aesthetics and health of our teeth promoting oral hygiene. Nowadays most of the toothpaste contains chemicals that produce harmful effect to the teeth and gums , hence herbal toothpaste is a wise and healthier choice for those who would like to minimize the amount of chemicals that could potentially endanger general health. Herbal ingredients have been present in oral care products, more commonly produced in south Asian countries.^[7]

1.6.1 Benefits

• Natural toothpastes can eliminate the bacteria in your mouth naturally with out using harmful chemicals.
• Natural toothpastes are free of artificial flavoring and dyes.
• The herbal toothpaste formulated which can satisfy all the required condition to keep the mouth fresh and prevent tooth decay by bacteria.
• Toothpaste have anti-cavity properties

2. Review Of Literature

• Karandikar S M, Pandit V A et al (1997)^[5] Have reported that polyherbal formulations are products from medicinal plants. Pharmacological effects of many plants have been studied in various laboratories in India. However. there are many limitations regarding safety and efficacy of these preparations. Knowledge about active principles of herbal preparations are not well defined, information on toxicity and adverse effects of these formulations are lacking. They also explained about selection of plant materials based on quality which will increase the acceptability of herbal preparation as therapeutic agents.
• Shah  C.S et al (1985-86)^[6] Had undertaken a study on the natural herbs and their products used for aromatic value in cosmetic preparations. The increased demand for the natural product has created new avenues in cosmeceuticals market.
• Mangilal  T  and  Ravikumar  M et al(2016)^[7] Had undertaken a study of on herbal toothpaste containing  natural ingredients  are more acceptable  in  public  belief  than  chemical  based  synthetic  formulations  due  to  their  safety  and  efficacy  in reducing dental caries .They also demonstrate that the extract of natural ingredients posses many activities like anti-cancer, anti-ulcer, anti-caries, anti-bacterial etc.
• Silva  GO,  Abeysundara  AT et al (2017)^[26] Showed that preliminary phytochemical analysis and screening of extracts of plants. Phytoconstituents individually  or in  the  combination,  determine the  therapeutic  value  of a  medicinal  plant.  Alkaloids, flavonoids,  phenolics,  tannins, saponins,  steroids,  glycosides,  terpenes etc are some of the important phyto chemicals with diverse biological activities .It states that  the  pharmacological  activity  of  a  plant  can  be predicted  by  the  identification  of  the  phytochemicals.
• Marongiu, Bruno et al (2005)^[29] Reported in detail the extraction of essential oil and identification of clove oil by clevenger apparatus. Eugenol is the main volatile compound extracted oil from clove bud ( Eugenia caryophyllus), and used in traditional medicine, as a bactericide, fungicide, anesthetic, and others. Its extraction was performed using hydro distillation (CLEVENGER) which is the most common extraction technique.
• Dange VN, Magdum C.S et al (2008)^[33] Showed that  herbal toothpaste containing  natural ingredients  are more acceptable  in  public  belief  than  chemical  based  synthetic  formulations  due  to  their  safety  and  efficacy  in reducing dental caries, and preventing other dental issues.
• Nishad U, Ali M et al (2020)^[35] Have reported that this formulated herbal toothpaste complies with all the required condition and can prevent tooth decay by bacteria. They also demonstrated the various evaluation methods of formulated herbal toothpaste includes determination of pH, spreadability, extrudability, foamability, viscosity, abrasiveness, shape retention etc.

Prashant GM, Chandu GN et al (2007)^[49] He demonstrates The antibacterial activity against Staphylococcus aureus reveals that formulated herbal toothpaste exhibited notable activity with zone of inhibition. They undertaken a study about agar well diffusion method by using streptococcus mutans as culture micro oraganism.

3.AIM And Objective

3.1 Aim of the study

Oral diseases like caries and periodontitis affect people all over the world and cause a lot of stress. Toothpaste seems to be the most important chemical product used to keep teeth clean. Toothpaste with chemical agents are mostly used to keep the teeth clean, however the fluoride content in most of the chemical toothpaste have negative side effects, such as altered taste and discoloured teeth, and their deleterious effects on endocrine function including fertility are uncertain. In order to overcome these problems while using toothpaste containing chemical agents herbal toothpaste was used. Herbal preparations are more effective compared to conventional formulations. The present work is to develop toothpaste containing herbal ingredients to be applied in the treatment of tooth decay and bad breath. The main aim of study is to make herbal toothpaste by using herbal plants such as Mangifera indica, Syzygium aromaticum and Ocimum tenuiflorum. All of these plants have effective antibacterial activity.

3.2 Objectives of the study

1. Collection and authentication of the plants Mangifera indica, Syzygium aromaticum and Ocimum tenuiflorum.

2. Study of physiochemical parameters of selected plant materials.

a. Ash values(Total ash ,Acid insoluble ash, water soluble ash).

b. Extractive values (Water soluble extractive value ,alcohol soluble extractive value).

c. Moisture content.

3. Extraction of plant material .

4. Qualitative analysis of the extract (Phytochemical screening) for the presence of various                phytoconstituents.

5.Formulation of herbal toothpaste

6. Evaluations of prepared herbal toothpaste

4.MATERIALS AND METHODS

4.1 List Of Materials

Table no:1 Plant material used for the development of formulation.

4.2 List of chemicals:

Table no:2 Chemical ingredients used for the development of formulation

4.3 List of Equipments

Table no 3: Equipments used for the formulation.

4.4 Plant collection

The fresh leaves of Mangifera indica, Syzygium aromaticum and Ocimum tenuiflorum were collected from Kasargod and Kannur district, Kerala (India) in the month of March 2023. The plant material were identified and authenticated by Dr. Harikrishnan. E, Assistant Professor Department of Botany, Payyanur Collage, Payyanur, Kannur, Kerala. Mangifera indica, Syzygium aromaticum and Ocimum tenuiflorum were dried under shade.

4.4.1 Plant profile

a. Mangifera Indica

       
           
       
    Figure 4.1 Mangiferra indica

Scientific classification of Mangifera indica

Kingdom Plantae – Plants

Subkingdom Tracheobionta – Vascular plants

Superdivisionm Spermatophyta – Seed plants

Division Magnoliophyta – Flowering plants

Class      Magnoliopsida – Dicotyledons

Subclass Rosidae

Order     Sapindales

Family    Anacardiaceae – Sumac family

Genus     Mangifera – mango

Species   Mangifera indica L. – mango

Vernacular names

Sanskrit Ambrah, Madhuulii, Madhuula, Madhuulaka;

English   Mango

Hindi     Aam

French   mangot, mangue, manguier;

Portuguese manga, mangueira

Dutch manja

Tamil Ambiram, Mambazham, Mambalam, Mangai

Punjabi Amb, Wawashi

Gujarati Ambo, Keri, Marvo (unripe)

Kashmiri Amb

Malayalam  Amram, Choothaphalam, Manga, Manpalam, Mavu

Marathi  Amchur, Amba

Synonyms

Mangifera amba Forssk, Mangifera anisodora Blanco, Mangifera austroyunnanensis Hu, Mangifera balba Crevost & Lemarié, Mangifera cambodiana (Pierre) Anon.^[8]

Morphological characteristics

MI is a large evergreen tree in the anacardiaceae family that grows to a height of 10-45 m, dome shaped with dense foliage, typically heavy branched from a stout trunk. The leaves are spirally arranged on branches, linear-oblong, lanceolate – elliptical, pointed at both ends, the leaf blades mostly about 25-cm long and 8-cm wide, sometimes much larger, reddish and thinly flaccid when first formed and release an aromatic odour when crushed. The inflorescence occurs in panicles consisting of about 3000 tiny whitish-red or yellowish – green flowers. The fruit is a well known large drupe, but shows a great variation in shape and size. It contains a thick yellow pulp, single seed and thick yellowish – red skin when ripe. The seed is solitary, ovoid or oblong, encased in a hard, compressed fibrous endocarp.

Phytochemical constituents

Mangiferin, followed by phenolic acids, benzophenones, and other antioxidants such as flavonoids, ascorbic acid, carotenoids, and tocopherols.

Medicinal uses

Antibacterial, Antioxidant, Immunomodulation, Hypertensive, Antiseptic, Astringent, Anti-asthmatic.^[9]

b. Syzygium Aromaticum

       
           
       
Figure 4.2 Syzygium aromaticum

 Scientific classification of syzygium aromaticum

Kingdom                          Plantae

Sub kingdom                   Tracheobionta

Super division                 Spermatophyta

Division                           Magnoliphyta

Class                                Magnoliopsida

Subclass                           Rosidae

Order                                Myrtales

Family                              Myrtaceae

Genus                               Syzygius

Species                             aromaticum

Vernacular names

Sanskrit Lavanga, Devapuspa, Varala, Bhadrasriya

Hindi     Laung, Lavang

Malayalam    Krayampu, Grampu

Marathi   Luvang

Tamil     Grambu, Kirampu, Kirambu, Lavanga,

Kannada Daevakusuma

Telgu Lavangalu, Devakusumamu

Bengali   Lavanga

Gujarati Lavang

Punjabi Laung

Oriya Labanga

Urudu  Laung, Loung

Morphological characteristics

The Clove tree is a slow growing, evergreen tree that ranges greatly in maximum height, from a relatively small, shrubby treelet at 8m to a medium sized tree of up to 20m. It has a dense conical crown when young, but becomes cylindrical or pyramidal when mature., Propagation, Propagated from softwood cuttings and seeds. Fresh seeds should be sown immediately as viability rapidly declines. Seed germination occurs in 4 to 5 weeks and seedlings can be tranplanted when they reach 25cm in height.

Phytochemical constituents

Quercetin, ursolic acid, ,eugenol  followed by ?-caryophyllene , ?-humulune and eugenyl acetate as the main components. Gallotannic acid, and two crystalline principles; ?- and ?- caryophyllenes, methyl furfural, gum, resin, and fibre.^[10]

Medicinal uses

Antibacterial, Antifungal, insecticidal, antioxidant, Antiseptic in oral infections, Toothache, Dental plaque,  Antiviral.^[11,12]

3. Ocimum Tenuiflorum

       
           
       
Figure 4.3 Ocimum tenuiflorum

Scientific classification of Ocimum tenuif                         

Kingdom                                 Plant

Division                                 Magnoliophyta

Class                                      Magnoliopsida

Order                                      Lamiales

Family                                    Lamiaceae      

Genus                                      Oscimum

Species                                   O. tenuiflorum

Synonym

Holy Basil, Sacred Basil, Tulasi.

Vernacular names

Sanskrit Tulasi, Tulsi,

Surasa, Krishnam, Vishnu-priya

Hindi Kala, Tulasi

English  Holy basil

Bengali Krishna tulasai

Tamil Thulasi  

Morphological characteristics

Tulsi belonging to family Labiatae, is an aromatic herb, is native across the Eastern World tropics and distributed as a cultivated herb and weed. The herb is an erect with many branched sub-shrub.

Height: 30-75 cm.

Stems: Hairy.

Leaves: Green with petiole, ovate or oblong, 5 cm long and slightly toothed, acute with entire or serrate mar…

Phytoconstituents

Isothymusin,ursolic acid, eugenol,  and sinapic acid,methyl cinnamate, camphor and thymol,saponins, flavonoids, triterpenoids, and tannins.^[13,14]

Medicinal uses

Antimicrobial,  Antiviral, Antifungal, Antimalarial, Antiprotozoal, Teeth whitening, Anti asthmatic, Anti stress, Antiemetic.^[15,16]

4.5 Pharmacognostic Study

A. Determination of moisture content

The grams of the leaves were weighed in a tared evaporating dish. It was dried out at 105°C with intermittent weighing. The drying and weighing at 1 hour interval was continued until the difference between two successive weighing was not more than 0.25%. Constant weight was reached when two consecutive weighing after drying for 30 minutes and cooling for 30 minutes in desiccators, showed not more than 0.01 gm difference. The percentage of moisture present in the sample was calculated.^[17,18]

B. Determination of ash value

The ash value is an important parameter for the evaluation of crude drugs, due to the variation of value within fairly wide limits. The ash value of any organic material is composed of inorganic materials like metallic salts and silica.

The following three different methods were adopted.

•   Total ash

•    Acid insoluble ash

•    Soluble ash

Ashing involves the oxidation of components of the products and a high ash value indicates the contamination, substitution, adulteration, or carelessness in the preparation of crude drugs for marketing^[19].

i. Total ash

Three grams of ground air dried material was accurately weighed out in a crucible previously ignited for 30 minutes. The material was spread in an even layer and ignited at a temperature of 500-600°C until it was white indicating the absence of carbon; cooled in a desiccator and weighed. The content of total ash per gram of air dried material was calculated.

ii. Acid insoluble ash

To the crucible containing the total ash, 25 ml of 2N hydrochloric acid was added, covered with a watch-glass and boiled gently for 5 minutes. The watch-glass was rinsed with 5 ml of hot water and this liquid was also added into the crucible. The insoluble matter was collected on an ashless filter paper and washed with hot water until the filtrate was neutral. Filter-paper containing the insoluble matter was transferred to the original crucible, dried on a hot plate and ignited to constant weight. The residue was allowed to cool in a desiccator for 30 minutes and then weighed. The content of acid insoluble ash per gram of air-dried material was calculated^[20,21].

iii. Water soluble ash

To the crucible containing the total ash, 25 ml of water was added and boiled for 5 minutes. The insoluble matter was collected in a sintered-glass crucible, washed with hot water and ignited in a crucible for 15 minutes at a temperature not exceeding 450°C. The weight of this residue in milligram was subtracted from the weight of total ash. The content of water soluble ash was calculated per gram of air-dried material. The results are given in Table no. –

       
           
       
   Figure 4.4 Ash                                          

       
           
       
Figure 4.5 Muffle furnace

C. Determination of extractive value

This method determines the amount of active constituents in a given amount of plant material when extracted with a solvent. As mentioned in different official books, the extractive value is used as a means of evaluating crude drugs which are not readily estimated by other means. For example, lowering from the prescribed values indicates the addition of exhausted or unwanted material with the original drug or incorrect processing of the drug.^[22]

i. Alcohol soluble extractive value

Macerated 5 grams of coarsely powdered air dried leaf (Mangifera indica, Eugenia caryophyllata, Ocimum tenuiflorum) separately with 100 ml ethanol in a stoppered flask for 24 hours, with occasional shaking during the first 6 hours and then allowed to stand undisturbed for another 18 hours. Filtered rapidly, by taking precaution against loss of alcohol. Then 25 ml of the filtrate was evaporated to dryness in a tared flat bottomed shallow dish, dried at 105°C and weighed. Calculated the percentage w/w ethanol soluble extractive with reference to air dried material. The results are given in Table no.^[23]

ii. Water soluble extractive value

Macerated 5 grams of coarsely powdered air dried leaf(Mangifera indica, Eugenia caryophyllata, Ocimum tenuiflorum) separately with 100 ml chloroform water in a stoppered flask for 24 hours, with occasional shaking during the first 6 hours and then allowed to stand for another 18 hours. Filtered rapidly, then 25 ml of the filtrate was evaporated to dryness in a tared flat bottomed shallow dish and dried at 105°C and weighed. Calculated the percentage w/w of water soluble extractive with reference to air dried material^[24].

       
           
       
   Figure 4.6 Extraction

4.6 Preparation of plant extracts

The collected leaves are carefully washed under water and was dried at shade for 10-15 days. These shade dried plant material were homogenized to a coarse powder using an electronic blender and then stored at air tight container until further use .The organic solvents such as ethanol and water was used for extraction .10 gm of homogenized coarse leaf powder of Mangifera indica, Syzygium aromaticum, and Ocimum tenuiflorum was soaked in different conical flasks containing 10 ml of ethanol and water .Then it is allowed to stand 30 minutes with occasional shaking, finally each sample extract was filtered through whatmanNO:1 filter paper .This filtrate used to detect various biologically active constituents present in various solvent extract.^[25]

4.7 Phytochemical Screening

4.7.1 Preliminary phytochemical screening of the various extracts^[26,27,28]

A. Chemical test for alkaloids

5. Mayer's test: To the leaf extract add 2 ml of Mayer's reagent shaken, a dull white or creamy precipitate indicates the presence of alkaloids
5. Dragendorff's test: Take 1 ml of extract in a test tube and few drops Dragendroff's reagent appearance of orange or red colour reveals the presence of alkaloids.
5. Hager's test: Add 2 ml of Hager's reagent to the leaf extract, formation of yellow colour precipitate confirms the presence of alkaloids^.

B. Chemical test for glycosides

5. Legal's test: The extract was dissolved in pyridine, to this add freshly prepared sodium nitroprusside solution then add sodium hydroxide solution to make this alkaline, Formation of pink red colour indicates the presence of cardiac glycoside.
5. Keller killiani: To the extract add sulphuric acid and glacial acetic acid and ferric chloride solution. Formation of blue colour indicates the presence of glycoside.
5. Borntrager's test: The extract boiled with dilute sulphuric acid filtered and to this add benzene and shake well, the inorganic layer was separated, to this add ammonia solution  slowly. Formation of pink, red or violet colour indicate the presence of free hydroxyl antraquinone^.

C. Chemical test for flavonoids

5. Lead acetate test: To the extract add lead acetate solution. Formation of yellow colour precipitate reveals the presence of flavonoids.
5. Alkaline reagent test: To the extract add sodium hydroxide solution, a yellow colour is formed which becomes colourless on the addition of dilute acetic acid indicate the presence of flavonoids.

D. Chemical test for carbohydrates

5. Molish's test: To the filtrate add Molish's reagent and add slowly concentrated sulphuric acid along the side of the test tube.Formation of violet ring at the junction indicate the presence of carbohydrates.
5. Benedict's test: To the filtrate add Benedict's reagent and heat. Orange red precipitate indicates the presence of reducing sugar.
5. Fehling's test: To the filtrate add dilute HCL, then it is heated with fehling's A and B. Formation of red precipitate indicates the presence of reducing sugar.

E. Chemical test for saponins

5. Foam test: A small quantity of extract diluted with 20 ml of water in a cylinder. The suspension was shaken for 15 min. Formation of foam indicates the presence of saponins.

F. Chemical test for phytosterols

5. Liberman Burchard's test: Extract is treated with chloroform and the solution is filtered. Then boiled the solution with a few drops of acetic anhydride solution and cooled. Then concentrated sulphuric acid was added through the side of the test tube. Formation of a brown ring at the junction indicates the presence of phytosterol
5. Salkowski's test: Extract is mixed with chloroform and filtered. The filtrate was treated with few drops of concentrated sulphuric acid, shaken well and allowed to stand. Formation of yellow our indicates the presence of terpenes.

G. Chemical test for Tannins and phenolic compounds

5. Ferric chloride test: A small quantity of extract is diluted with water, then it is treated with ferric chloride solution and formation of bluish black indicates the positive result.
5. Gelatin test: The extract was dissolved in water and filtered. To this add sodium gelatin containing sodium chloride. Noted for the presence of milky white precipitate.
5. Lead acetate test: The extract dissolved in water and added lead acetate solution. Positive result shown when bulky white precipitate formed.
5.  Decolourisation test: The extract dissolved in water treated with dilute potassium permanganate solution, decolourisation of potassium permanganate shows positive results.

H. Chemical test for proteins and amino acids

5. Million's test: To the extract add a few ml of Million's reagent and observe for the presence of white precipitate which turns to red.
5. Biuret test: To the extract add sodium hydroxide solution and heat. Then add a few drops of copper sulphate solution, forming a purplish violet colour.

Ninhydrin test: To the extract add ninhydrin reagent and heat. Formation of blue colour indicates the presence of amino acid.

       
           
       
Figure 4.7 Preliminary phyto chemical screening

4.8 Extraction Of Plant Material

4.8.1 Extraction of clove flower buds^[29,30]

Extraction of clove using Clevenger Apparatus consist of one round bottom flask of 500 ml which is connected with another two way round flask which holds raw material. The top flask is connected with condenser through the connecter. The separating funnel is used for the separation of essential oil and water. Buds (50 gm)of Syzygium aromaticum then washed the buds with water . After collection and 50 g boiled with 500 ml of distilled water in a Clevenger apparatus until oil distillation ceased after 5-6 hrs. The oil obtained after extraction was collected.

4.8.2 Extraction of holy basil^[31,32]

The leaves were dried in order to reduce the initial moisture content (90 % of humidity), in an oven with air renewal and circulation. The drying temperature was 50 °C and was kept constant for 5 hours until constant moisture. After drying, the leaf’s were grinded in order to increase the contact surface reducing the resistance to oil extraction. The grinded leaves were placed in sealed plastic bags, protected from light and moisture .The extraction was done in a Clevenger apparatus, coupled to a bottom flask of 500 ml. It was added 30 g of crushed leaves of basil and 300 mL of water into the flask. The extraction time was fixed at 4 hours. The extract oil was diluted in hexane and filtered after separation oil was collected after extraction.

Table no 4: Formula of the composition of herbal toothpaste

Procedure

1. The solid ingredients calcium carbonate, sodium lauryl sulphate, glycerin, sodium benzoate, sodium saccharin were weighed accurately as mentioned in the formula.
2. These ingredients were mixed in a mortar and pestle, then triturated with precisely weighed glycerin until a semisolid substance was created.
3. Accurately weighed herbal ingredients like Tulsi oil, clove oil and mango leaf powder were added to the base and triturated to form a paste.

4.9.2 Evaluation of herbal toothpaste^[35,36,37]

A. Physical Evaluation

Physical parameters such as colour and appearance were evaluated.

B. Determination of pH^[38]

Take 1gm of the toothpaste in a 150ml beaker and add 10ml of freshly boiled and cooled water. Stir well to make a thorough suspension and determine the pH of the suspension using pH meter.

       
           
       
    Figure 4.10 Determination of pH using digital pH meter

C. Foamability ^[39]

2g of toothpaste was measured and mixed with 5ml of water in a measuring cylinder. Shake it for 10 minutes and final volume was taken.

Foamability = Final volume – Initial volume

       
           
       
    Figure 4.11 Determination of foamability using measuring cylinder

D. Determination of spreadability^[40]

Slip and drag characteristics of paste are used to determine the spreadability technique. About 1-2g of herbal toothpaste was weighed and placed between two glass slides (10 x10) that were stacked one on top of the other, and then slides were moved in opposing directions. After 3 minutes measure the amount of toothpaste that has spread. Repeat three times.

S = M x L/T

M – Weight placed

L – Length moved by glass slide

T – Time taken to separate upper slide from lower

       
           
       
Figure 4.12 Determination of spreadability

E. Extrudability ^[41,42]

In this method, the formulated paste was filled in standard capped collapsible aluminum tube and sealed by crimping to the end. The weights of tubes were recorded. The tubes were placed between two glass slides and were clamped. 500g was placed over the slides and then cap was removed. The amount of the extruded paste was collected and weighed. The percent of the extruded paste was calculated.

F.Abrasiveness^[43,44]

Extrude the content 15-20 cm long on through butter paper. Repeat the same process for at least ten collapsible tubes. Press the contents of the entire length with fingertip for the presence of sharp and hard edged abrasive particles.

H.Fragrance test^[45,46]

It was based on individual observation for its acceptability. 5 people were asked for acceptability of fragrance and their opinion was taken and fragrance was evaluated based on the below described criteria.

1. The fragrance was good, as good as the fragrance of reference toothpaste.
2. The fragrance of the toothpaste was not so good but comparable to the reference toothpaste.
3. The fragrance of the toothpaste was poor than reference toothpaste.

I. Shape retention^[47,48]

Toothpaste was squeezed out from the tube and put entirely on a toothbrush and the state of the toothpaste after it was allowed to stand for 10 seconds was evaluated based on the,

1. Shape just after the toothpaste is squeezed out on the toothbrush is maintained.
2. Shape just after the toothpaste is squeezed at on the toothbrush is almost maintained.
3. The toothpaste squeezed from the toothbrush and cannot maintain its shape.

       
           
       
Figure 4.13 Determination of shape retention using toothbrush

4.10 Screening For Antimicrobial Activity^[49,50]

4.10.1 Invitro- antimicrobial activity

Antibacterial activity was determined by agar well diffusion method using amoxicillin as standard. Activity of herbal toothpaste was tested against E.coli and lactobacillus.

5. Media: Nutrient agar medium

The media was prepared by the addition of 2% agar (Bacteriological grade) selected strains were preserved by sub-culturing them periodically on nutrient agar slants and storing them under frozen condition.

5. Ingredient

Beef extracts -0.3%

Peptone – 0.5%

Agar-1.5%

5. Preparation

The ingredients were dissolved in distilled water with aid of heat and pH was adjusted to 7.2-7.6 using alkali or dilute acid.

5. Sterilization

15-20 ml of nutrient agar was transferred to petri plates and covered. It was then autoclaved at a temperature of 121 degree celcius and pressure of 15 psi for not less than 20 minutes.

5. Sample used : Formulated herbal toothpaste
5. Standard drug: Amoxicillin
5. Culture used:

Lactobacillus

The bacteria lactobacillus is prepared from curd. The curd is filtered and the broth is incubated at 25 degree celcius for 24 hours.

      E. coli

E. coli was cultured by adding 1-2 grams of sugar to 100 ml of clear beef broth and boil for several minutes. E. coli could grow well in any medium that has a little protein and some carbohydrate.

The micro organisms are cultured and stored in our pharmaceutical biotechnology laboratory.

5. Working condition

The entire work has done using horizontal laminar flow hood so as to provide aseptic conditions. Before Commencement of the work air sampling was carried out using a sterile nutrient agar plate and exposing it to the environment inside the hood .After incubation it was checked for the growth of micro organism and absence of growth confirmed aseptic working condition.

5. Method

Agar well diffusion method was used. Agar plates were prepared aseptically to get a thickness of 5-6 mm. The plates were allowed to solidify and inverted to prevent condensate falling on the agar surface. The test micro organisms was spread on the agar plates by means of   sterile cotton swab. The wells were then punched into agar medium and filled with herbal toothpaste and standard drug. The plates were incubated for 24 hours at 37 degree celcius. Observation were made for zones of inhibition  around the formulation and compared with that of standard.

5. RESULT AND DISCUSSION

5.1 Plant collection and drying

Collected plant materials were identified and authenticated by Dr. Harikrishnan. E, Assistant professor, Department of Botany, Payyanur collage, Payyanur, Kannur, Kerala. Mangiferra indica, Syzygum aromaticum and Oscimum tenuiflorum.

5.2 Preparation of plant extracts

The collected plant materials are subjected to extraction using various solvents such as ethanol and water. The extracts obtained after maceration process was the used for phytochemical studies to choose the most suitable solvent for further extraction.

5.3 Physico-chemical parameters

 After the collection of plant materials, they were shade dried and powder costly in an electronic blender and stored in air tight containers until further use. Physico-chemical parameters of plant were tabulated in table no. Parameter such as ash value, extractive values and moisture content were estimated.

Table no 5: Physico-chemical parameters of Mangiferra indica, Syzygum aromaticum and Oscimum tenuiflorum

Table no 6: Phytochemicals present in ethanol and aqueous extracts of  Mangiferra indica, Syzygum aromaticum and Oscimum tenuiflorum

5.4 Formulation of herbal toothpaste

In the present study herbal toothpaste were prepared.

       
           
       
Figure 5.2 Packed herbal toothpaste

5.5 Evaluation of herbal toothpaste

Table no 7: Physical evaluation

The formulated herbal toothpaste were visually inspected for their colour, odour, state, and its constituency was determined .Result are shown in the table 7.

Table no 8: Evaluation result

Table no 9: Extrudability

Table no 10: Fragrance test

5.5.1 Shape retention

Toothpaste was squeezed out from the tube and put entirely on a toothbrush and the state of the toothpaste after it was allowed to stand for 10 seconds was evaluated as, Shape just after the toothpaste is squeezed out on the toothbrush is maintained.

5.6  In-Vitro Anti -Microbial Activity Evaluation

The anti microbial activity of the developed formulation was done by using agar well diffusion method. The anti-bacterial activity was measured in terms of zone of inhibition. The zone of inhibition obtained for herbal toothpaste was compared with the standard Amoxicillin  The developed formulations showed anti-bacterial activity against Lactobacillus and E.coli produced a better zone of inhibition of about 6mm,7mm respectively which was near to the zone of inhibition produced by standard Amoxicillin (10mm,8mm).

           
           
       
    
    Figure 5.3 Preparation of culture media

   
           
              
    Figure 5.4 Zone of inhibition of formulated herbal toothpaste against lactobacillus

Figure 5.5 Zone of inhibition of formulated herbal toothpaste against E.coli

Table no.11 Result showing Anti-bacterial activity of Herbal Toothpaste formulation

Result showing Anti- bacterial activity of Herbal toothpaste formulation