9 Minerals determination

For minerals determination the rose petals were shade dried. Then dried petals were grounded to
make fine powder. Then 0.5 gram sample was dissolved in the mixture of nitric acid (10ml) and
perchloric acid (5ml). This dissolved mixture was kept for one night at room temperature. On next
day, digestion of sample was done on hot plate at 180°C. When digestion was completed, the
sample was further cooled, filtered and diluted with pure deionised water upto 50 ml. Rose petals
sample was prepared in triplicate and mean value was used to analyse statistically. Digested
samples were evaluated by flame photometer and atomic absorption spectrophotometer. Sodium,
potassium and calcium were determined by using flame photometer. Lead, manganese, nickel,
copper, iron , cadmium and zinc were determined by atomic absorption spectrophotometer
(Vincevica-Gaile et al., 2011).

Antioxidant properties
3.2.2.1 Preparation of Extract
Extracts rose petals were prepared to estimate their total phenolic content and antioxidant
properties. Rose petals were shade dried and grounded to fine powder. This rose powder was used
to prepare rose extracts. Four different solvents used to prepare extracts including hexane, acetone,
ethanol and water. Ratio of rose petal powder to solvent was kept1:10 for all kind of solvents. Rose
powder was mixed with solvents in four flasks and kept for one night at orbital shaker at 1200
rpm. Next morning extracts were filtered with filter paper (whatman filter paper no. 01). After
filtration all extracts were concentrated using rotary evaporator at temperature half to their boiling
temperatures respectively. Concentrated extracts were used to evaluate total phenolic content and
antioxidant properties (Patturaja et al., 2016).

3.2.2.2 DPPH assay (2,2-diphenyl-1-picrylhydrazyl)

Radical scavenging activity of Rosa gruss-an-teplitz was examined by using 1,1-diphenyl-2-
picrylhydrazyl radical. Four solvents were used to make extracts of rose petals including water,
acetone, ethanol and hexane. DPPH solution of 1.0 mmol/L was prepared. Assay mixture was
consisted of 2 ml of DPPH solution and 1ml of extract. Two different concentrations of extract
were used including 50 and 100 µg/mL. Incubation period for test assay was 20 minutes in dark at
room. Decrease in absorbance for every solution was checked for at 517 nm by UV
spectrophotometer. Ascorbic acid was used as positive control which is an antioxidant. Mixture of
ethanol and DPPH solution was used as blank (Alhakmani et al., 2013). Radical scavenging
percentage was calculated by the following formula.

Antioxidative activity (%) = (Absorbance of control solution-Absorbance of sample solution)/

(Absorbance of control solution) X100

3.2.2.3 Total phenolic content

Folin Ciocalteu method was used to investigate total phenolic content of Rosa gruss-an-teplitz’s
petals. A volume of 2 ml of folin ciocalteu’s regaent (ten time diluted with distilled water) was
mixed with 0.5 ml of rose flower extract. About 4 ml solution of sodium carbonate (7.5%) was
also added to extract. For half an hour, the mixture was kept in dark. The absorbance of extract
was checked at 765 nm after incubation period by using double beam UV spectrophotometer.
Reference standard used for this experiment was gallic acid. Standard curve was constructed. Total
phenolics were calculated by linear equation that obtained from standard curve. Results were
represented as mg/g GAE (gallic acid equivalent) of dried extract (Alhakmani et al., 2013).