International Journal of Research and Development in Pharmacy & Life Sciences
Open Access

Herbal; Extraction; Micro-organisms; Mouthwash

Introduction

The mouth washes are concentrated aqueous anti-bacterial solutions that are used against oral microbes to counter oral infection, cleansing, to get rid of bad breath refreshing, antiseptic. The mouthwash plays an prominent role in the oral hygiene of an individual, it helps to relieve symptoms of inflamed gums gingivitis. And also it reliably used to destruct the pathogenic germs. The mouth washes are used by most of the dental patients to overcome sour mouth i.e. xerostomia, ulcerated throat and sensitive teeth. Dentists always use mouthwash as an antimicrobial agent before oral surgery of the patients, because they help to sterilize the surface of the inflamed gums and teeth, thereby the contamination of any other microorganisms can be avoided. Ancient Egyptians are known to be responsible for the first artistic drawings that emphasize the importance of beauty and hygiene. An unclean body was thought to be impure. PedaniusDioscorides, a Greek physician and surgeon (40–90 AD) whose writings served as a medical textbook, suggested for treatment of bad breath a mouthwash mixture of the following: a decoct of the leaves of the olivetree, milk, the juice of pickled olives, gum myrrh with wine and oil, pomegranate peelings, nutgalls, and vinegar. The ancient Romans included teeth cleaning as part of their [1-9] religious ceremonies. The patriarchy required their slaves to clean their teeth. The Romans included a secret ingredient in their mouthwash: human urine. They imported urine from Portuguese people because they thought it had more strength. Until the 18th century, urine continued to be an active ingredient in toothpaste and mouthwash because of the ammonia’s cleansing abilities.

1. Mouthwashes can be broadly classified as

a. Chemical mouthwashes

b. Herbal mouthwashes

a. Chemical mouthwashes

Usually contains antimicrobial agents, such aschlorhexidinegluconate which is very potent chemoprophylacticagent, it has abroad spectrum action especially against Mutans Streptococci group. But it has many side effectslike staining of teeth, altering the test of the mouth and desquamation of oral mucosa (Figure 1).

Figure 1: Plan of work.

a. Herbal mouthwashes

The importance of herbs is highly considered as effective in contrast to chemical products. Medicinal plants, plays vital role in curing diseases due to their antimicrobial and antifungal activity against human pathogens through decades. Herbs are being widely explored to discover alternatives to syntheticantibacterial agents. Herbal Mouthwash act on oral pathogens and relives pain instantly and are also less side-effective.

a. Uses of mouthwash

1. Antiseptic/As antibacterial

2. Astringent

3. Cooling and refreshing action

b. Aim and objectives

• To prepare Herbal Mouthwash.

• To evaluate prepared Herbal Mouthwash.

• To compare the prepared Herbal Mouthwash with marketed

Chemical Mouthwash.

Details of Selected Herbal Plant

Material and Method

Collection of herbal plant materials

Herbal plant materials like Guavawas collected locally from farm at Solapur city and remaining herbs like Naie, Dikmali, (Table 1) Pomegranate and Henna were purchased from local Market of AyurvedicStore at Solapur, Maharashtra, India in the month of 16thJanuary 2018 (Table 1-9).

Naie
	Synonym	Brilliant Gardenia, Cambi resin tree, Gardenia lucida
	Botanical name	Gardenia gummifera
	Chemical constituents	It contains 89.9%of resins, 0.1%volatile oils and a coloring agent gardenin. Besides these it contains certain alkaloids and minerals.
	Uses	It is used as carminative, stimulant, vermin-fuge, wound healer, painkiller, insect repellent and majorly used in gums and tooth problems, anti septic and anti bacterial.
	Part used	Gum from bark part

Table 1: Pharmacognostic scheme of Dikmali.

Naie
	Synonym	Indian whitehead,Bahuguni,mamejaw
	Botanical name	Enicostemmaaxillare
	Chemical constituents	A fruit mainly contains gum resin and numbers of flavonoids are also present.Whereas it includes gardenin and also woogninderalong with alkaloids, tannins.
	Uses	It is mainly used in diabetes, arthritis, skindiseases, hernia, and swelling. Antibacterial, anti inflammatory and anti oxidant.
	Part used	Leaves

Table 2: Pharmacognostic scheme of Naie.

Pomegranate
	Synonym	Apple fruit
	Botanical name	Punicagranatum
	Chemical constituents	Ellagicacid,punicalagin,punicalin.punicicacid,urolithinA with 85%of water and others are ascorbic acid citric acid
	Uses	It is mainly used as rich source of vitamin C,detoxifyingagent,combinant tool against heart diseases and also for dental hygiene, anti oxidant, antimicrobial etc.
	Part used	Peel part

Table 3: Pharmacognostic scheme of Pomegranate.

Henna
	Synonym	Egyptian privet, Lawsonia  Alba, Mehandi
	Botanical name	Lawsoniainermis
	Chemical constituents	The phytochemicals present in Henna are phenols,andglycosides.Lawsoneisb an active constituent while others are gallicacid,resin, sugars,tannins.
	Uses	It is mainly used as antibacterial, treat headache, antifungal, astringent, aromatic, cooling and analgesic
	Part used	Leaves

Table 4: Pharmacognostic scheme of Henna.

Guava
	Synonym	Psidium, guava bush, Peru
	Botanical name	Psidiumguajava
	Chemical constituents	Polyphenol,  Flavonoids, carotenoids, essential oils, 10% tannins, saponins
	Uses	Anti-oxidant, anti-infalmmatory, anti-bacterial
	Part used	Leaves

Table 5: Pharmacognostic scheme of Guava.  

Table 6: Zone of inhibition in Dikmali extracts.

Table 7: Zone of inhibition in Naie extracts.

Table 8: Zone of inhibition in Henna extracts.

Table 9: Zone of inhibition in Pomegranate extracts.

Preparation of extract

Preparation of powder

• Dikmali, Naie and Henna: Readymade powders utilized directly (Table 2).

• Guava leaves were shadow dried at room temperature for 1 week before extraction process.

• Leaves were cut into small pieceswith the help of scissorandgrinded in mortar & pestle to fine texture powder form and stored in air –tight polythene bag (Figure 2).

Figure 2: Set up for the Extraction.

Solvent extraction

Powdered plant material was extracted by Percolation Method (Exhaustive extraction) using ethanol and distilled water (1:4).

1. Procedure Followed For Percolation

• Drug in a suitably powdered form.

• Imbibe the powdered drug with menstruum for a period of one hour.

• Packed evenly into the percolator.

• A piece of filter paper is placed on surface followed by a layer of clean sand so that top layers of drugs are not disturbed.

• Sufficient menstruum is poured over the drug slowly and evenly to saturate it, keeping the tap at bottom open for passing of occluded gas to pass out.

• Sufficient menstruum is also added to maintain a small layer above the drug and allowed to stand for 24 hours (Figure 3).

Figure 3: Collection of extraction after 24 hours.

• 75% of the volume of the finished product is collected.

• Mark is pressed and expressed liquid is added to the percolate giving 80% to 90% of the final volume.

• Volume is adjusted with calculated quantities of fresh menstruum.

• Evaporation and concentration to get finished products by applying suitable techniques and apparatus.

Evaluation Test

1. Color: Yellowish Brown

2. Odour: Aromatic

3. Taste: Sweet, Pleasant

Microbial test

Collection of Micro-organisms

Mouth organisms like Staphylococcus aureus, Streptococcus pyrogens, Escherichia coli, and Candida albicans were procured from AshwiniSahakariHospital& Research Centre, Solapur and maintained and incubated at 360 C for 2 hrs, before performing the activity.

Collection of oral swab

The sterile cotton swab was rubbed on to the gums and periodontal region of both upper and lower jaws of the individuals (totally 6 oral swabs) were collected.

Antibacterial Activity

Disc Diffusion Method

A sterile 500 ml conical flask was used to prepare nutrient agar

(HIMEDIA M001-100G) Lot 0000293649 Expiry: MAR 2022

• Prepared Media and petri dishes were autoclaved at 1210C for 15min.

(Autoclave LabHosp size 12X12)

• Then 15-20 ml of agar was poured in each sterile Petri dish.

• The plates were air dried under UV radiated in the laminar air flow (KlenzaidsBioclean devices Ltd.) for 15 min.

• The bacterial colonies were inoculated into nutrient broth and incubated for 24hrs.

• In broth turbid solution indicated the viable microorganisms ready to use for testing the antibacterial activity.

• From this turbid solution havingmicro-organism inoculated into prepared agar plate.

• The discs were diffused in testing samples.

• Then these diffused discs were placed on agar plate and kept it in incubator for 48 hrs.at 37oC.

• After 48 hrs the results were identified and recoded the zone of inhibition (Figure 4).

Figure 4: Zone of inhibition for E.coli.

• His zone of inhibition was identified for the resistance of the micro-organisms too (Figure 5-12).

Figure 5: Zone of inhibition for S.aureus.

Figure 6: Zone of inhibition for C. albicans.

Figure 7: Zone of inhibition for S. pyrogen.

Figure 8: Zone of inhibition of formulations for E. coli.

Figure 9: Zone of inhibition of formulations for S.aureus.

Figure 10: Zone of inhibition of formulations for C.albicans.

Figure 11: Zone of inhibition of formulations for S.pyrogens.

Figure 12: Zone of inhibition of random mouth saliva.

Antimicrobial testing by Disc Diffusion method

• Extracts:-(Naie, Dikmali, Henna, Guava & Pomegranate)

• Formulated Herbal Mouthwash:- (Formula-1, Formula-2, Formula-3)

• Standard Mouthwash: (Standard-1 & Standard-2)

Result & Discussion

Solvent extraction of herbal plants

The herbs used in the prepared herbal mouthwash like Dikmali, Naie, Pomegranate, (Table 3) Henna and Guava can be used in oral care formulations like mouthwash. The solvent extracts were prepared as it shows very less side-effects than commercially available chemical mouthwashes.

Collection of Micro-organisms

The test organisms were identified & the results were recorded based on morphological & cultural characters.

Antimicrobial activity

The extracts were taken in different dilutions. The sensitivity & resistance of the organisms against formulated herbal mouthwash were analyzed. The result showed that all the herbslike Dikmali, Naie, Pomegranate, Henna (Table 4) and Guava used in this study have antimicrobial activity (Table 5, Table 9).

Dikmali: shows sensitivity towards Staphylococcus aureus, Strptococcuspyrogens, Candida albicans and shows resistance (Table 6) towards Escherichia coli.

Naie: shows sensitivity (Table 7) towards Staphylococcus aureus, Streptococcus pyrogens, Candida albicans& Escherichia coli.

Guava: shows sensitivity towards Staphylococcus aureus, Candida albicans& Escherichia coli& shows resistance towards Streptococcus pyrogens (Table 10).

Table 10:  Zone of inhibition in Guava extracts.

Pomegranate: shows sensitivity towards Staphylococcus aureus, Streptococcus pyrogens, Candida albicans& Escherichia coli.

Henna: shows sensitivity (Table 8) towards Streptococcus pyrogens, Candida albicans& Escherichia coli & shows resistance towards Staphylococcus aureus.

It was found that the antimicrobial activity of 10% w/v of extract was found to be substantial and at the same time it is tolerable as regards the palatability due to bitterness. So, the formulation prepared by the 10% w/v of extract provides optimal antimicrobial activity and palatability to the formulation. The prepared formulations shows positive results against the randomly taken mouth saliva sample. The formulation of 10% w/v of extract shows significant effect in terms of antimicrobial activity compare with the marketed synthetic drug like chlorhexidine (Table 11-17).

Table 11: Zone of Inhibition in Formulated Herbal Mouthwash.

Table12 :  Zone of Inhibition in Standard Mouthwash.

Table 13: Following table shows total yield obtained after percolation.

Table14 : Formulation:  After percolation all the extracts were dried for a week to get the residual mass. Using this dried mass prepared the herbal mouthwash by using following different formulae for 100 ml.

Table 15: Standard Mouthwash Used for comparison study.

Table 16: pH Test :( Standard Range: 5.1 to 7.3).

Table 17: Viscosity: (Standard Range: 1 to 4 cps).

Conclusion

Inclusion of Naie in mouthwash showed sensitivity and significantly effective against oral micro-organisms. Pomegranate in mouthwash helps in destruction of microbes. Hence the prepared herbal mouthwash shows effectiveness against oral micro-organisms namely Staphylococcus aureus, Streptococcus pyrogens, Candida albicans& Escherichia coli. An attempt has been made to outline some of the commonly available herbs which are readily available can be used as effective mouthwashes. People can use and promote such cost effective measures of maintaining the oral health which are also devoid of many unwantedside effects. Thus the prepared herbal mouthwashes can also be routinely used for improving oral hygiene of children and adults as well as in patients with dental caries and gingivitis.

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