6.

Appendices _________________________________
A. Reagent and media preparation B. Definitions C. Properties of organic solvents D. Cut-off values of different solvents

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Appendix A. Reagent / Media Preparation Reagents 28 mM sodium phosphate It was prepared by dissolving 3.35 g of sodium phosphate (SRL) in 1 L of distilled water.

4 mM ammonium molybdate 4.94 g of ammonium molybdate (S-d fine chemicals) was dissolved in 1 L of distilled water.

0.6 M of sulfuric acid 33.33 mL of concentrated (18 N) sulfuric acid (Rankem) was added to distilled water to make up the final volume of the reagent to 1 L.

2.5 mM gallic acid 0.47 g gallic acid (SRL) was dissolved in 1 L methanol (95%).

0.5 mM ascorbic acid 0.088 g of ascorbic acid (Merck) was dissolved in 1 L of distilled water.

0.05 M phosphate buffer 0.34 g of potassium phosphate monobasic (KH2PO4; SRL) and 0.35 g of anhydrous sodium phosphate dibasic (Na2HPO4; SRL) was dissolved in distilled water to make up the final volume to 100 mL.

DPPH 0.0394 g DPPH (Suyog chemicals) was dissolved in 1000 mL methanol.

10% v/v Folin-Ciocalteu reagent 1 mL 2 N F-C reagent was diluted to 10 mL.

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0.1mg/mL quercetin 0.1mg of quercetin (SRL) was dissolved in 1 mL methanol (99%).

10% w/v aluminum chloride 10 g of aluminum chloride (Merck) was added to 100 mL distilled water.

0.5 McFarland Standard (http://www.ijmm.org/documents/Antimicrobial.doc): 0.5 mL of 0.048 M BaCl2 ( SD Fine Chemicals, Mumbai) was added to 99.5 mL of 0.18 M H2SO4 with constant stirring. OD was ensured to fall within the range of 0.08 -0.1 at 625 nm. It was stored in dark.

Media Nutrient agar 40 g nutrient agar (HiMedia, Mumbai) in 1000 mL distilled water. Mueller Hinton agar 38 g of MH agar ( HiMedia, Mumbai) in 1000 mL distilled water. Mueller Hinton broth 21 g of MH broth ( HiMedia, Mumbai) in 1000 mL distilled water. Aeromonas starch ampicillin agar 52 g of Aeromonas starch ampicillin agar in 1000 mL distilled water. After autoclaving, a pinch of ampicillin was added. All the media were sterilized by autoclaving at 121C, 15 psi for 15 min. All the media contained vegetable peptones.

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Appendix B. Definitions

Broad spectrum: Drugs that act on both gram-positive and gram-negative bacteria are referred to as broad spectrum antibiotics (Romich, 2004).

Cut-off value: The cut-off value is the UV absorbance value of a particular solvent (Schmidt, 2005).

hRf: Rf value multiplied by 100 is referred to as hRf. Rf value is quoted in the form of hRf as a whole number is used to describe qualititative description of thin-layer chromatograms (Deinstrop and Leach, 2007).

IC50 (Inhibitory concentration 50 %): The concentration of a compound needed to reduce population growth of organisms by 50% in vitro (Barile, 2004).

Minimum inhibitory concentration (MIC): The lowest concentration of test agent that inhibits the visible growth of test organism during overnight incubation (Greenwood et al., 2002).

Minimum bactericidal concentration (MBC): The lowest concentration of test agent at which the organism is killed (death of 99.9% of population) (Bannister et al., 2003).

Narrow spectrum: Drugs that act specifically on the gram-positive group or specifically on the gram-negative group of bacteria are referred to as narrow spectrum antibiotics (Romich, 2004).

Retardation factor (Rf): The distance travelled by the solute divided by the distance traveled by the solvent from the origin where sample was loaded (Stahl, 1969).

Synergistic activity: Synergistic activity is the enhanced performance of the mixture of two or more components compared to the simple additive performance of the components at the same concentration (Houghton and Raman, 1998).

Total activity: Total activity is a measure of the amount of material extracted from plant in relation to the MIC of the extract, fraction or isolated compound (Eloff, 2004).

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Appendix C. Properties of organic solvents (Smallwood, 1996)

(D = Density, DC = Dielectric constant, DF = Dissipation Factor) Solubility in water (g/100mL) Miscible Miscible 0.18 100 0.795 0.05 7.5 Miscible 8.7 Miscible 0.014 Miscible 0.05 6.15 20.5(25) 2.28 78 4.81 2.71 4.34 24.6 6(25) 37.7 1.89 32.6(25) 2.38(25) 1570 2500 6400 DC DF (tan)

Solvent

M.W.

B.P. (oC )

M.P. ( oC ) 16.6 -94.3 5.5

D ( g/mL) 1.049 0.786 0.879 1.000

Acetic acid Acetone Benzene Water Chloroform Chloro-benzene Diethyl ether Ethanol Ethyl acetate Ethylene glycol Hexane Methanol Toluene

60.05 58.08 78.11 18 119.38 112.56 74.12 46.07 88.11 62.07 86.18 32.04 92.14

118 56.2 80.1 100 61.7 131.7 34.6 78.5 77 195 69 64.6 110.6

-63.7 -45.6 -116.3 -114.1 -83.6 -13 -95 -98 -93

1.498 1.1066 0.713 0.789 0.895 1.115 0.659 0.791 0.867

Appendix D. Cut-off values of different solvents (Sorenson, 1999)

Solvent Water Hexane Ethanol Chloroform Acetone Methanol Cut off value (nm) 180 195 210 245 330 205

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