THE USE OF ARTIFICIAL SALIVA RECIPES IN DISCRIMINATING AGAINST THE

INTERFERENCE DISPLAYED TOWARDS ANTIMICROBIAL FORMULATIONS

PRESENTED BY SONDESS AZIZSUPERVISOR: DR. JIM ROCHEBIOTECHNOLOGY YEAR 4

AIMS AND OBJECTIVES To formulate artificial saliva recipes close to natural saliva. To investigate a potential interference of artificial saliva recipes with

antimicrobial action of oral formulations. Microbiological assessment of the AIT lozenge in the presence of artificial

saliva. To evaluate and measure the physical properties of artificial saliva i.e. pH

and viscosity. To evaluate drug release profile of oral formulation in artificial saliva by

dissolution studies. Using artificial saliva recipes, examine for leaching of ethylene oxide from

oral mouthpieces.

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INTRODUCTIONOral dental formulations

Mouth wash Antimicrobial formulations ability is to interact with the oral surfaces and slow

releasing property (substantivity).

Solid dosages of antimicrobial formulations 2,4- dichlorobenzyl alcohol (C7H6Cl2O)

in 1.2 mg Amylmetacresol (0.6 mg) AIT lozenge (AIT candidate molecule)

Oral pathogens Candida albicans Streptococcus pyogenes Streptococcus mutans 3

Strepsils®

LITERATURE REVIEWNatural Saliva

Saliva forms a bolus that allows the mouth to be clear of bacteria and food particles. About 500 to 1500 mL/day secreted with a pH range is +/- 7.4.

Secretion of saliva follows a circadian rhythm, decreasing during sleep hours and increasing during waking hours.

Oral fluid that has diverse functions including, lubricating properties; cleansing action or adherence of oral flora, protection and enzymes i.e. lactoferrins, lysozymes & lactoperoxidase (Silva et al., 2012).

Artificial Saliva Acts as a substitute to natural saliva (Ana et al., 2002). Physical properties such as viscosity, density and pH are essential. Composed of mucilaginous agents i.e. cellulose and xanthan gum or mucin, which increase

the moistening (Silva et al., 2012).

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MATERIALS & METHODS

Chemicals StarchComposition

(g/L)

Saliveze Composition

(g/L)

Xialene 2Composition

(g/L)

Starch 5.0 - -

NaCMC - 10.0 -

Xanthan Gum - - 0.18

KCL 0.62 0.62 1.2

NaCL 0.84 0.84 0.86

MgCL 0.06 0.06 0.04

CaCL 0.17 0.17 0.12

K2HPO4 0.8 0.8 0.12

KH2PO4 0.3 0.3 -

Prototype 1: Formulation of artificial saliva

Figure 1: Oswald viscometer (Sakka, 2005 )

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MATERIALS & METHODSPrototype 2: Density & Volume of polymer mouthpieces

in deionized water

Artificial Saliva Samples

Density of Mouthpieces (g)

Addition of Sterile Water

(mL)

Starch 1.43 50

Xialene 2 1.61 45

Saliveze 1.78 45

Prototype 3: Erosion Evaluation

One Tablet of Strepsils was dissolved

into 100 mL of deionized water. Strepsils® were diluted (1:10), to

achieve the maximum lambda Maximum absorbance detected at

284 nm using UV spectrophotometer.

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Results

Figure 2: Xialene 2 Chromatogram

Prototype 3: Concentration of Ethylene oxide vs. peak area.

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0 50 100 150 200 250 3000

20

40

60

80

100

120

f(x) = 0.442414491863009 x − 2.94611529009004R² = 0.990904064349305

Ethylene oxide Standard curve Concentration vs. Area

peak areaLinear (peak area)

Concentration (µg/mL)

Area

Concentration(ppm or µg/mL)

Peak Area

0 00.5 0.73995 2.416025 0.3840

125 53.1803250 107.9483

Figure 3: Standard curve of Ethylene Oxide standards with unknown concentration of artificial saliva samples.

Xialene 2: 1.23 cP Xialine 2: 1.36 cP Saliveze: 5.76 cP Saliveze: 5.71 cPStarch: 0.98 cPNatural saliva: 2.75 cP

Mean Viscosity values for saliva recipes

(Preetha et al., 2005)

RESULTS

Organism 2 min 4 min 6 min 8 min

MouthwashC. albicans 0 0 0 0S. mutans 0 0 0 0S. pyogenes 0 0 0 0

StrepsilsC. albicans TNTC TNTC TNTC TNTCS. mutans 0 0 0 0S. pyogenes 1714 142 0 0

AIT lozengeC. Albicans 1580 TNTC TNTC TNTCS. Mutans 0 0 0 0S. pyogenes 1078 874 483 217

Prototype 4: Artificial saliva testing for recipe Xialene 2

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Organism

 

2 min

 

4 min

 

6 min

 

8 minMouthwash      C. albicans 0 0 0 0S. mutans 0 0 0 0S. pyogenes 0 0 0 0

         Strepsils        C. albicans 2293 1500 TNTC 2800S. mutans TNTC TNTC TNTC TNTCS. pyogenes 1215 0 0 0

         AIT lozenge        C. albicans TNTC TNTC TNTC TNTCS. mutans TNTC TNTC TNTC TNTCS. pyogenes 1063 131 0 0

Prototype 5: Control for Deionized water.

RESULTS

Figure 4: Drug release profile of Strepsils® against artificial Saliva samples.9

0 1 2 3 4 5 6 7 8 90

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0

0.013

0.0250.026

0.025

0

0.00700000000000001

0.018

0.033 0.033

0

0.014

0.028

0.035

0.024

0

0.0190000000000002

0.0230.024 0.024

Sterile WaterXilene2SalivezeStarch

Time Period (minutes)

Amou

nt o

f dru

g re

leas

e (m

g/m

L)

RESULTS

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Saliva Samples and time intervals (min)

Absorbance at 284 nm

Absorbance Calculated(mg/mL)

Water 0 02.0 0.59 0.0194.0 0.70 0.0236.0 0.74 0.0248.0 0.72 0.024

Xialene 2 0 02.0 0.42 0.0144.0 0.86 0.0286.0 1.05 0.0358.0 1.09 0.024

Saliveze 0 02.0 023 0.0074.0 0.56 0.0186.0 1.00 0.0338.0 1.00 0.033

Starch 0 02.0 0.40 0.0134.0 0.77 0.0256.0 0.80 0.0268.0 0.73 0.025

Prototype 6: Absorbance detected using UV spectrophotometer for saliva samples

CONCLUSION In order for these Saliva substitutes to work effectively it is necessary to bring them close

to natural Saliva and this task is difficult due to the role of saliva being “multifunctional”. In this study Xialene 2 was physically similar to normal Saliva due to its viscosity of 1.23 cP

Saliva interferences: Low but with Xialene 2 it was significantly high against the antimicrobial agent.

Antimicrobial effects: Mouthwash was the most effective and consistent antimicrobials agent tested against studied artificial saliva recipes.

Dissolution & erosion studies: Amount of drug released by Strepsils was significantly high, which increased with time period(2.75 cP). Therefore the viscosity determined was high for AS recipes.

GC analysis: Further research is considered

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