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Research & Reviews: Journal of Herbal Science Volume 2, Issue 1, ISSN: 2278-2257 __________________________________________________________________________________________ Development of Alcohol-free Herbal Mouthwash Having Anticancer Property Banani Ray Chowdhury*, Souptik Bhattacharya, Madhuparna Deb, Arnav Garai Department of Biotechnology, Bengal Institute of Technology, West Bengal University of Technology, India Abstract Mouthwash is a liquid accessory to clean and maintain the health of our teeth for oral hygiene. Nowadays, we use commercial mouthwash which contains many chemical compounds like sodium lauryl sulfate, thymol, methyl salicylate, benzalkonium chloride, hydrogen peroxide, alcohol which are harmful to our buccal cavity. We have developed a mouthwash with some common food materials and herbs and which can replace costly chemicals like alcohol, coloring agents and preservatives making our mouthwash economically more viable than commercial mouthwash. A herbal mouthwash preparation is developed using the extracts of peppermint oil, clove oil, betel leaf extract, ajwain, ginger, basil, etc., in sterilized conditions having antibacterial, anti-cancer, anti-fungal activity. Basil leaves are known to reduce malodor and possess antibacterial property. Ajwain helps in digestion and removal of malodour. The sensory, physiochemical and microbial analysis of the mouthwash preparation was carried out on the day of preparation and on the seventh day after sample preparation to determine its physiochemical changes, shelf life and its antibacterial property (on the Gram-positive and Gram-negative bacteria) when the sample prepared was kept under two different temperature conditions – ambient temperature and refrigerated temperature. Keywords: Oral hygiene, economically viable, antibacterial, malodour, shelf life *Author for Correspondence E-mail: [email protected] INTRODUCTION Various kinds of mouthwashes have evolved following oral hygienic problems. But apart from this, mouthwashes also serve to refresh breath. Moreover, mouthwash also contains some ingredients that serve as digestive aids. Mouthwashes can be chemical or herbal in nature. Cetylpyridinium chloride (CPC) is a cationic quaternary ammonium compound in some types of mouthwashes, toothpastes, lozenges, throat sprays, anti-sore throat sprays, breath sprays, and nasal sprays. It is thus a type of chemical mouthwash. It is an antiseptic that kills bacteria and other microorganisms. It has been shown to be effective in preventing dental plaque and reducing gingivitis [1]. But it has certain disadvantages. It is combustible. Concentrated solutions are destructive to mucous membranes. It is toxic when swallowed and very toxic when inhaled. Another chemical alcohol free mouthwash consists of sodium chloride, sodium bicarbonate, a flavoring agent and as a solubilizing agent here for, polysorbate 20, being a mixture of partial lauric esters of sorbitol and its mono- and di-anhydrides copolymerized with approximately 20 mol of ethylene oxide for each mol of sorbitol and its anhydrides; and water, said sodium chloride and sodium bicarbonate being dissolved in said water and present in a suitable quantity to produce a solution substantially isotonic with human oral mucosa. A herbal guava mouthwash has been prepared using guava leaf extract, sodium lauryl sulphate, peppermint emulsion and chloroform water. Formulations with highest quantity of guava leaf extract have highest antimicrobial activity due to the presence of bioactive substances. It is more potent against Streptococcus mutans and Escherichia coli. RRJoHS(2013) 7-12 © STM Journals 2013. All Rights Reserved Page 7 Alcohol-free Herbal Mouthwash Having Anticancer Property Chowdhury et al. __________________________________________________________________________________________ So, our present project work is based on the development of new type of herbal mouthwash from guava leaves, peppermint oil, clove oil, betel leaves and ajwain and to study briefly its antibacterial properties and shelf life period. MATERIALS AND METHODS Test Organisms: A freshly prepared slant culture of Streptococcus mutans MTCC-890 and E. Coli. Chemicals/Reagents Required: Culture media used were nutrient agar and nutrient broth. Sterile distilled water, alcohol (95%), anthromycin, and edible apple green food color. medium is now liquefied by keeping in water bath. Sterilized petridishes are kept in hot air oven for 5 min. Now, inside the laminar, 15 mL of liquefied agar is poured in petridishes. The agar-filled petridishes are then allowed to cool; 0.1 mL of mouthwash sample is added to the pertidish maintaining sterilized conditions. The mouthwash sample is then spread with a spreader. Then the petridishes are labeled and kept in the incubator at 37 °C and then checked for appearance of colonies after 24 h and after 7 days in the colony counter. MICROBIAL ANALYSIS Determination of Antibacterial Property of Mouthwash Twenty-four hour freshly prepared cultures of Streptococcus mutans and E. coli are used that are prepared from slant cultures of Streptococcus mutans and E. coli. This is done by: nutrient broth is prepared, pH is tested and sterilized. Using a sterilized loop, some inoculum is transferred from old culture to the newly prepared nutrient broth in the laminar. These subcultures are now placed in incubators at 37 °C for 48 h. Petridishes are filled with 15 mL agar media as above and allowed to get cooled. Using a marker, each petridish is divided into two halves. 0.05 mL of Streptococcus mutans is added to one petridish and 0.05 mL of E. coli is added to the other. After adding the inoculum, the inoculum is spread by the spreader. The petridishes are kept in the incubator at 37 °C for 5 min. The petridishes are taken out and placed in the laminar. Using the borer, wells are created in the inoculum-applied agar media on each of the halves of the petridishes. This method is known as the bore well method. 0.05 mL of the refrigerated mouthwash sample is applied in one half and 0.05 mL of ambient temperature mouthwash is placed in the other half. This process is repeated respectively for the Streptococcus mutans and E. coli petridishes. The petridishes are now kept in the incubator at 37 °C for 24 h. After 24 h, petridishes are looked for inhibition zones around the wells Procedure for Shelf Life Determination The shelf life of the sample was determined by pour plate method with the diluted sample in water using total plate count method for viable cells. The agar medium is prepared, pH is tested and autoclaved. The sterilized agar Minimum Inhibitory Concentration of the Herbal Extract This is the lowest concentration expressed in mg/L that under given in vitro conditions inhibit the growth of bacteria in a given period Herbal Ingredients: Guava leaves, betel leaves, ajwain (local market), peppermint oil (local market), clove oil (local market). Equipment Used: Dhona balance (160D, Kolkata, India), autoclave (Instind, Kolkata, India) hot air oven (Multispan MDC-1901), 100 °C water bath (ICT, Kolkata, India ), grinder, Whatman filter paper, laminar air flow colony counter, spreader, incubator (Lambda, l/032/01-02-03), refrigerator (LG, Kolkata, India). Preparation of Mouthwash: The distilled water, glass wares are sterilized; the leaves are weighed and washed in tap water. The leaves are dried for 24 h in a hot air oven. The dried leaves are now grinded in a grinder and the powdered form is obtained. The leaf powder is boiled in sterilized water for 15 min by keeping it in the water bath (10 g/100 mL). The liquid thus obtained is filtered using a Whatman paper (40 mL of each extract). Clove oil and peppermint oil (each 2 mL) are added finally to the extract mixture. The edible food color is mixed in it. The sample thus prepared is now kept in two volumetric flasks at two varied temperatures, one at ambient temperature and the other at low temperature. RRJoHS (2013) 7-12 © STM Journals 2013. All Rights Reserved Page 8 Research & Reviews: Journal of Herbal Science Volume 2, Issue 1, ISSN: 2278-2257 __________________________________________________________________________________________ of time. Conventionally, this is determined using a series of dilutions of the herbal sample in the liquid broth. The herbal sample is ten times diluted from the original sample. After inoculation of the test strain in the test tubes, growth is determined by visible turbidity after 24 hr. MIC tests are also done by extended breakpoint sensitivity test and by using commercially available E-Test strips. These methods are technically time consuming and expensive. The original herbal extract is diluted ten times using 1 mL sample and 9 mL of sterile distilled water. The test tubes are filled with broth and the diluted herbal sample in varied proportions. The test tube containing only broth is the positive control while the test tube containing only herbal sample is the negative control. 0.1 mL of freshly prepared Streptococcus mutans culture is added to all the test tubes maintaining sterile conditions throughout. The test tubes are now kept at incubator at 37 °C for 24 h and observed for the presence of visible turbidity. herbal extract with the standard antibiotic. The diameter of the inhibition zone for both, the extract and antibiotic is measured. Hot agar is added to the petridishes in the laminar and allowed to cool. Using the borer wells are made on the petridishes that have been divided into two halves by the marker. The 500 mg antibiotic is diluted to 250 ppm concentration On the S side, 0.1 mL of the extract is added and on the A side 0.1 mL of the antibiotic althromycin (250 ppm) is added. The petridish is kept in the incubator at 37 °C for 24 h. The diameters of the zone of inhibition are measured. Sensory Analysis The color, flavor and taste of 0-day and 7th day samples were determined using 9-point Hedonic scale method. Physiochemical Test The pH of the of 0-day and 7th day mouthwash samples at ambient and low temperature storage are measured to monitor the changes of acidity of the sample, considering pH < 7 to be suitable for our mouth. Standardization by Agar Diffusion Method This is done to compare the strength of the RESULTS Sample 0 day 0 day 7 day 7 day Table 1: Sensory Characteristics. Temperature Color Flavor Taste Ambient 6 8 9 Refrigerated 6 8 9 Ambient 4 6.5 7 Refrigerated 5 7 8 Texture Clear Clear A little turbid Clear 0-extrmely bad, 3-not bad not good, 5-moderate, 7-good, 9-excellent. Color: The color change observed here is the original color of the mouthwash sample. The color is dull brownish on the 0th day and degrades faster at the ambient temperature. Flavor: The flavor is almost unchanged and has an excellent fragrance of clove and peppermint. Only a week after, the fragrance is somewhat lost when kept at ambient temperature. Taste: The taste is strong and remains almost same over the week except for the ambient temperature sample. Texture: Samples remain clear liquid over a week except the one kept at the ambient temperature which develops turbidity after the 3rd day. RRJoHS(2013) 7-12 © STM Journals 2013. All Rights Reserved Page 9 Alcohol-free Herbal Mouthwash Having Anticancer Property Chowdhury et al. __________________________________________________________________________________________ Table 2: Physiochemical Characteristics. Day of Measurement pH of the Sample 0th day 6.5 7th day 6.5 Thus, no change was found in the acidity of the sample over a week and the sample was observed to be quite stable chemically. Table 3: Microbial Analysis. Storage Life (7 days) Storage temperature sample Ambient Refrigerated of Storage life (log cfu/mL) – 0th day No growth No growth The sample at ambient temperature develops more colonies than the one at refrigerated temperature when observed over a week. Antibacterial Properties: E. coli (Gramnegative bacteria) – Inhibition zone observed Storage life (log cfu/mL) – seventh day 4.90 4.48 (Gram-positive bacteria) Streptococcus mutans – inhibition zone observed. So, antibacterial property is observed against both Streptococcus mutans (Gram-positive) and E. coli (Gram-negative). MIC Method Table 4: MIC 10D Extract 0 0.5 1 1.5 2 2.5 3 3.5 4 5 Broth Dilution 5 0.5 4 3.5 3 2.5 2 1.5 1 0 Positive control 0.1 (10D) 0.2 (10D) 0.3 (10D) 0.4 (10D) 0.5 (10D) 0.6 (10D) 0.7 (10D) 0.8 (10D) Negative control MIC was thus determined to be 3 mg/ml. Agar Diffusion Method The diameter of inhibition zone for anthromycin – 30 + 0.5 mm. The diameter of inhibition zone for original sample – 20 + 0.5 mm. Sample activity unit = 250 ppm anthromycin × (20/30) unit = 166.66 anthromycin unit. DISCUSSION Betel Leaf: An analysis of the betel leaf shows it consists of moisture 85.4%, protein 3.1%, fat 0.8%, minerals 2.3%, fiber 2.3% and carbohydrates 6.1% per 100 g. Its minerals and Result + + + - vitamin contents are calcium, carotene, thiamine, riboflavin, niacin and vitamin C. Its calorific value is 44. Recent studies have shown that betel leaves contain tannins, sugar and diastases and an essential oil. The essential oil is a light yellow liquid of aromatic odor and sharp burning in taste. It contains a phenol called chavicol which has powerful antiseptic properties. The alkaloid arakene in it has properties resembling cocaine in some respects. The betel leaf has analgesic and cooling properties. According to traditional Ayurveda medicine, chewing areca nut and betel leaf is a remedy for bad breath [2]. RRJoHS (2013) 7-12 © STM Journals 2013. All Rights Reserved Page 10 Research & Reviews: Journal of Herbal Science Volume 2, Issue 1, ISSN: 2278-2257 __________________________________________________________________________________________ Peppermint: Peppermint has a high menthol content, and is often used as tea and for flavoring ice cream, confectionery, chewing gum, and toothpaste. The oil also contains menthone and menthyl esters, particularly menthyl acetate [3]. It is the oldest and most popular flavor of mint-flavored confectionery. Peppermint can also be found in some shampoos and soaps, which give the hair a minty scent and produce a cooling sensation on the skin [4]. Peppermint oil is versatile oil: it is analgesic, anti-inflammatory, anti-viral, digestive, anti-septic, an astringent, carminative and anti-spasmodic Peppermint, essential oil is used to treat migraine, bronchitis, sinusitis, indigestion, nausea, irritable bowel syndrome, irregular periods and nervous conditions. It is also very useful in the treatment of cold and flu. Ajwain: Ajwain is used as an antiseptic. It is used for cleaning wounds and treating skin infections. Thymol in Ajwain seeds is known as a strong germicide, anti-spasmodic and fungicide. Oil of Ajwain is also used in toothpaste and perfumery. Ajwain leaves can be crushed and applied on infections. Ajwain seeds are also used in prevention of bad breath. Thymol from Ajwain seeds is also used in various mouthwashes. Regularly chewing of Ajwain seeds along with fennel seeds prevents bad breath [7]. with other species remaining unstudied. From preliminary medical research in laboratory models, extracts from apple guava leaves or bark are implicated in therapeutic mechanisms against cancer, bacterial infections, inflammation and pain [9–11]. Essential oils from guava leaves display anti-cancer activity in vitro [12]. Guava leaves are used in folk medicine as a remedy for diarrhea [13] and, as well as the bark, for their supposed antimicrobial properties and as an astringent. Guava leaves or bark are used in traditional treatments against diabetes [14–16]. In Trinidad, a tea made from young leaves is used for diarrhea, dysentery and fever [17], and against aging processes in human body. Our skin cells contain fewer naturally occurring antioxidants, as we age. It is helpful in providing photo-protection, treating photodamaged skin and pigmentation disorders in the skin. The antioxidant nature of guava enables it to reduce the harm caused to skin due to its exposure to sunlight. It serves as a mouth freshener. For people suffering from diarrhea and amoebiasis, unripe guava is useful for treating halitosis. It is rich in tannic, malic, oxalic and phosphoric acids as well as calcium, oxalate and manganese. Chewing fresh guava leaves also stops bleeding from gums along with bad breath. Most mouthwashes contain antimicrobial substances as antiseptic ingredients against germs that commonly cause mouth infections. Phytochemical studies indicate the guava leaves bioactive components like tannins, tri terpenes, phenols, flavonoids, essential and fixed oils, sapinins, lectins, carotenoids, etc. However, despite the popular ehnomedical use of guava leaves there is still a dearth of information on the use of guava leaves in the mouthwash formulations. This study was therefore carried out to determine the antimicrobial potency of guava leaves in mouthwash formulations. Along with all the use of individual constituents of the mouthwash, the herbs used are cheap, easy to use and are easily available Guava Leaves: Since the 1950s, guavas – particularly the leaves – have been the subject for diverse research on their constituents, pharmacological properties and history in folk medicine [8]. Most research, however, has been conducted on apple guava (P. guajava), Sample Sterility The herbal extract is prepared maintaining sterile conditions throughout and the sterility is observed at two dilutions 10−1, 10−3. No microbial growth was observed in the sample prepared. Clove: Cloves are used in Indian Ayurveda medicine, Chinese medicine, and Western herbalism and dentistry where the essential oil is used as an anodyne (painkiller) for dental emergencies. Cloves are used as a carminative to increase hydrochloric acid in the stomach and to improve peristalsis. Cloves are also said to be a natural anthelmintic [5]. The essential oil is used in aromatherapy when stimulation and warming are needed, especially for digestive problems. Topical application over the stomach or abdomen are said to warm the digestive tract. Clove oil, applied to a cavity in a decayed tooth, also relieves toothache [6]. RRJoHS(2013) 7-12 © STM Journals 2013. All Rights Reserved Page 11 Alcohol-free Herbal Mouthwash Having Anticancer Property Chowdhury et al. __________________________________________________________________________________________ CONCLUSIONS A sample of herbal mouthwash was developed and tested for 0 and 7th day in ambient and refrigerated temperature and both are accepted in respect to sensory and microbial characteristics. The developed sample has positive effect on both S. mutans (Grampositive) and E. coli (Gram-negative) culture. REFERENCES 1. Asadoorian Joanna. Cetylpyridinium Chloride Mouth Rinse on Gingivitis And Plaque. Journal of Dental Hygiene 2008. 2. Naveen Pattnaik. The Tree of Life. PDR for Herbal Medicines, 4th Edn. Thomson Healthcare: 640p. ISBN 978-1-56363678-3. 3. R. Eccles. Menthol and Related Cooling Compounds. J. Pharm. Pharmacol 1994; 46(8):618–630p. PMID 7529306. 4. Balch Phyllis, Balch James. Prescription for Nutritional Healing, 3rd edn. Avery Publishing: 2000; 94p. 5. USA (2012-05-24). 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Cote ce Cote la: Trinidad & Tobago Dictionary, Arima, Trinidad 1986. 17. HI Abdelrahim, AZ Almagboul, MEA Omer, et al. Antimicrobial Activity of Psidium guava. L. Fitoterapia 2002; 73:713–715p. RRJoHS (2013) 7-12 © STM Journals 2013. All Rights Reserved Page 12