Saponin 1 and 20 ml of 40% saturated

Saponin content was determined using
Brunner’s (1984) method. One gram of air-dried organ was ground and then mixed
with 100 ml of isobutyl alcohol in a 250 ml beaker. To ensure uniform mixing of
the extract, the mixture was shaken for five hours on a mechanical shaker. Thereafter,
the mix­ture was filtered with a Whatman paper No. 1 and 20 ml of 40% saturated
magnesium carbonate solution was added to it. To obtain a clear colorless
solution, the mixture was again filtered. One ml of colorless solution was
pipetted into 50 ml volu­metric flask and two ml of 5% iron chloride solution
(FeCl3) was added and made up to the mark with distilled water and kept
for 30 min. The standard saponin solutions were prepared and were treated
similarly with 2 ml of 5% Fe­Cl3 solution. The absorbance of samples
and standard solutions were read by a spectrophotometer (SPEKOL 1500, Germany)
at 380 nm wavelength and the regression equation was calculated between the
read numbers from spectrophotometer and concentration of standard solutions and
saponin content of each sample was expressed as mg g-1 dry weight.

2.3.2. Tannin compounds

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Tannin content was measured
according to Van Bardon and Robinson (1969). 500 mg of powdered dry sample and
50 ml of distilled water were poured into a glass container and then shaken for
an hour by mechanical shaker. The solution was filtered into a 50 ml volumetric
flask and made up to that volume by distilled water. Five ml of the filtered
solution was poured into the test tube and mixed with 2 ml of 0.1 M FeCl3
(1.622 g FeCl3 in 100 ml of distilled water), 0.1 N HCl (0.829 ml of
37% HCl with a density of 1.19 in 100 ml of distilled water) and 0.008 M
potassium ferrocyanide (0.2946 g of K4Fe(CN)6.3H2O
in 100 ml distilled water). The absorbance was read at 395 nm within 10 min by
a spectrophotometer (Irakui et al., 2013). Finally, the tannin content of each
sample was calculated using the standard curve of tannic acid and expressed as
mg g-1 dry weight.

2.3.3. Mucilage

Five grams of milled dry plant
sample was added to a glass container and homogenized with 100 ml of distilled
water and were kept in a refrigerator at 4°C for 24 hours. Then, the solution
was filtered using a Buchner funnel and 50 ml of filtered solution was mixed
with 100 mL of 95% ethanol and kept again for 24 hours until sediment of the
gum and mucilage in the solution. Then, the solution was filtered on the pre-weighed
filter paper (w0) and dried in an oven at 75 ºC and again weighted
(w1). The difference of w1 – w0 determined the
mucilage and gum weights together. Then, filter papers containing the gum and
mucilage were placed in the volumetric flask and 100 ml of distilled water was
added to gum to be dissolved in the water and separated from the filter papers
and the mucilage remained on the paper. Then filter papers dried again in an
oven at 75 ºC and weighted (w2). The mucilage weight was calculated
by subtraction of w1 from w2.