7 APPENDICES

3
APPENDIX I
Aluminum Chloride Colorimetric Method:
Total flavonoids in selected plant extracts were determined using the aluminum
chloride colorimetric method (Chang et. al., 2002). Quercetin was used as standard to
make the calibration curve. Ten milligrams of quercetin was dissolved in 80% ethanol
and then diluted to 25, 50 and 100μg/mL. The diluted standard solutions (0.5 mL) were
separately mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% AlCl 3, 0.1 mL of 1M
potassium acetate and 2.8 mL of distilled water. After incubation at room temperature
for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a
Shimadzu UV-1700 Pharma Spec. spectrophotometer. The amount of 10% AlCl3 was
substituted by the same amount of distilled water in blank. Extracted residues (1ml of
1:10 g ml-1) were redissolved in 100 ml ethanol and from that 1 ml is taken for the study.
1mL of each ethanolic extracts was reacted with AlCl3 for determination of flavonoid
content as described above. Total flavonoid content was determined by using the
following formula.

Total Flavonoid content (mg) = QE×V×D×10-6/W

RE - Quercetin equivalent (μg/ml);

V - Total volume of sample (ml);
D - Dilution factor;
W - Sample weight (g)

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 APPENDIX II
Quantification of quercetin aglycones in leaves and cell suspension cultures by
HPLC Analysis (Olszewska, (2007)

Chromatographic equipment and condition

The chromatographic analysis were performed on a 250 mm × 4.6 mm i.d., C18
(ODS), Shimadzu, Japan with 0.5% aqueous solution of Orthophosphoric acid and
Methanol (HPLC Grade) as mobile phase at a flow rate of 1 mL min-1. The HPLC
equipment comprised Hewlett-Packard (HP) 1050 ChemStation Software, an HP model
35900 interface units, an HP 9000 Series 300 computer, and an HP DeskJet 500 Printer.
A Waters 486 tunable absorbance detector was operated at 254 nm; detector sensitivity
was 0.05 AUFS and the column oven temperature was 30°C.

Quercetin Calibration curve

Quercetin was dissolved in HPLC grade Methanol yielding concentrations of 0.1,
1, 5, 10, 25, 50, 100, 250, 500, 1000 µg/ml. the solutions were filtered through a 0.45
µmeter membrane filter. Evaluation of each point was repeated three times and each
calibration curve was fitted by linear regression.

Methodology
Ten µl of acid treated methanolic ethylacetate (60:40) extracts of leaf and cell
suspension cultures this solution was injected for HPLC analysis. Analysis was
performed after three separate extraction of each sample and each extract was diluted
and injected in triplicate. Quercetin in the samples was identified by comparison of their
retention times (Rt) with the standard Quercetin.

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 APPENDIX III
Composition of Murashig and Skoog medium (MS)

Ingredients Composition Stock solution

(mg/L) (W/V) (g)
MS Macro I (10 X) 1000ml
NH4NO3 1650 16.5
KNO3 1900 19
MgSO4.7H2O 370.6 3.7
KH2PO4 170 1.7
100 ml
MS Macro II (10 X) 1000ml
CaCl2.2H2O 439.8 4.398
100 ml
Fe-Na EDTA (1000 X) 100ml
Fe-Na EDTA 36.7 36.7
1 ml
Micro Nutrients (1000 X) 100 ml
NaMoO4.7H2O 0.25 0.025
CuSO4.5H20 0.025 0.0025
CoCl2.2H2O 0.025 0.0025
MnSO4.4 H2O 13.2 1.32
ZnSO4.4H2O 8.6 0.86
H3BO3 6.2 0.62
1 ml
KI (1000 X) 100ml
KI 0.83 0.083
1ml
Myo-Inositol (100 X) 100ml
Myoinositol 100 1
10ml
MS Vitamins (1000 X) 100 ml
Nicotinic Acid 0.5 0.05
Pyridoxine HCl 0.5 0.05
Thiamine HCl 0.1 0.01
Glycine 2 0.2
1 ml

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 APPENDIX IV

(i) Coconut water:

The coconut water is simply drained from dehusked immature coconuts by

drilling holes through two of the micropyles. Extract of water from each fruit separately
was checked properly to ascertain that it is not fermented before addition to the bulk.
Collected water from all the fruits was heated at 80-100 °C for 10 minutes with
continuous stirring to precipitate out the proteins, fats and other materials. The
precipitates were separated by filtration and the filtrate is stored at -20 °C for future use
(George, 1993; Nasib et al., 2008).

(ii) Yeast extract

A carbohydrate fraction isolated from the yeast extract was prepared by Ethanol
precipitation as described by Chen et al. (2001). Briefly, 50 g of the yeast extract was
dissolved in 250 ml of distilled water and Ethanol was added to 80% (v/v). The
precipitate was allowed to settle for 4 days at 6°C and the supernatant was decanted and
discarded. The gummy precipitate was dissolved in 250 ml of distilled water. The
ethanol precipitation was repeated. The second ethanol precipitate was dissolved in
200 ml of distilled water, yielding the crude preparation that was used without further
purification.

(iii) Peptone water

Elicitation was carried out with the peptone water. Stock solutions were prepared
by dissolving peptone in distilled water, and the pH was adjusted at 5.8. The solutions
were sterilized by filtering through a microfilter (0.45 μm). The sterilized peptone
solutions were stored at 4 °C in a refrigerator before use.

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