Antioxidant activities and HPLC analysis of phenolic compounds from Saffron (Crococus

sativus)

Introduction:

At least two pages

Objectives:

1. To find out the antioxidant activity of hot water extract of Crococus sativus.

2. To determine the phenolic profile of aqueous extract of Crococus sativus.

Material and methods:

Chemicals

Standards used in HPLC analysis were purchased from Sigma Aldrich. Iron, sodium

nitroprusside, DPPH, ammonium molybdate and 1,10-phenanthroline were purchased from

Biochemicals (Lahore, Pakistan).

Preparation of plant extract

The flowers and leaves of plant will be locally purchased, identified by a botanist and a

voucher specimen will be deposited at the Herbarium of University of Poonch, Department

of Botany.

Finely grounded fruit material of the plant (25g) will be placed for 15 minutes in boiling

water (500 ml)after boiling the extract was cooled and filtered with filter paper. The solvent was

evaporated by rotary evaporator (450C) producing 3 g (12% w/w) extract.

In vitro lipid peroxidation assay

The anti-lipid peroxidative properties of extracts were studied by a method (Khaliq eta al., 2015).

In brief the egg yolk was weighed to 1 gram and diluted to 100 ml with 100 mM Tris-HCl, pH

7.4 and used as homogenate. The homogenate was incubated with Fe (II) or sodium

nitroprussside with or without the extract and colour reaction was carried out by adding 600 µl of

TBA and 600 µl of acetic acid (pH 3.4) for 1 h. The tubes were cooled and 2 ml of n-butanol was

finally added and centrifuged. The absorbance was read at spectrophotometer at 532 nm.
 DPPH radical scavenging activity

The scavenging of the DPPH radical was reported by the method (Hatano et al., 1988).

Scavenging of stable DPPH radical was studied in vitro and the absorbance was measured at

517 nm. Percent inhibition was calculated from the control. Ascorbic acid was used as a

standard compound in DPPH assay.

Metal chelating activity

The iron chelating ability of the extract was studied by the method (Puntel et al., 2005). Briefly

150 µL of freshly formed 2 mM FeSO 4·7H2O was added in a mixture which have 168µL of the

0.1M tris HCl (pH 7.4), (218µL) of saline and (25-200 µL/ml) concentration of plant extracts.

The mixture of sample was incubate for 5 minutes before addition of 13µL of 0.25 percent 1,10-

phenanthroline (w/v). Absorbance was checked at 510 nm in spectrophotometer.

Antioxidant potential assay

The reducing ability of the extract was followed by phosphomolybdenum method (Prieto et

al., 1999). The results were expressed as ascorbic acid equivalent. The assay was based on the

reduction of molybdenum, Mo(VI)–Mo(V) by the extract and subsequent formation of a green

phosphate/ Mo(V) complex at acidic pH. The extract (0.1 mg/ml) was mixed with 3 ml of the

reagent

solution (0.6 M H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes

were incubated at 95 C for 90 min. The mixture was cooled to room temperature and the

absorbance of the solution was measured at 695 nm.

HPLC analysis of phenolics and flavonoids

The dried hot water extract of Crococus sativus will be dissolved in HPLC grade methanol (1
mg/ml), filtered through a 0.45 μm membrane filter and subjected for qualitative and quantitative
analysis by using Shimadzu HPLC system, Prominence Auto-Sampler (SIL-20A), equipped with
Shimadzu LC-20 AT reciprocating pumps connected to the degasser DGU 20A5 with integrator
CBM 20A, UV–VIS detector DAD SPD-M20A and Software LC solution 1.22 SP1. Reverse
phase chromatographic analyses will be carried out under isocratic conditions using C18 column
(4.6 mm × 250 mm) packed with 5 μm diameter particles. For the separation of phenolics the
mobile phase composition methanol:acetonitrile:water (40:15:45) containing 1% acetic acid will
be used. The mobile phase will be filtered through a 0.45 μm millipore membrane filter and then
degassed by an ultrasonic bath prior to use. Standard curves were prepared by using gallic acid
(y = 1.09x104c526,314, r2 = 0.997), rutin (y = 1.92 × 10008.85c-16,949, r2 = 0.999) and
quercetin (y = 3.01 × 10017.6c-235,135, r2 = 0.996). Data was monitored at 257 nm for the
detection of phenolic acids and flavonoids. Quantification was carried out by the integration of
the peak using external standard method. The flow rate was 1 ml/min and the injection volume of
aqueous extract will be10 μl. Results will be obtained by comparison of retention times and
DAD-UV spectra (λmax = 257 nm) with those of the reference standards. All chromatographic
operations will be carried out at ambient temperature and in triplicate.

References

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thiobarbituric acid reactive species (TBARS) production in rat brain in vitro. Neurochem
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