2. MATERIALS AND METHOD
2.1. Plant materials:
Rubus occidentalis is
a deciduous shrub growing to 2–3 m tall,
with prickly shoots. The leaves are pinnate,
with five leaflets on leaves strong-growing
stems in their first year, and three leaflets on
leaves on flowering branchlets. The flowers are
distinct in having long, slendersepals 6–8 mm
long, more than twice as long as the petals. The
round-shaped fruit is a 12–15 mm diameter
aggregation of drupelets; it is edible, and has a
high content of anthocyanins [15,23]
.
2.2. Extraction and purification of
anthocyanins:
The plant material was frozen with Liquid
Nitrogen and powdered using a Blender
suitable for use under extremely low
temperatures. The use of liquid nitrogen
minimizes anthocyanin degradation by
lowering the temperature and providing a
nitrogen environment. The fine powder
maximizes pigment recoveries due to its high
surface area and favors disruption of cellular
compartments. later on from the crushed
material anthocyanin was extracted by
following Methanol and Acetone-chloroform
extraction method4,16].Later on the extract from
respective methods was subjected to column
chromatography and the absorbance was
checked for fraction exhibiting pinkish
colour(checked for all the fractions). TLC was
also done for that fraction using particular
mobile phase.[12]
2.3. pH indicator application
Anthocyanin exhibit various colour at different
pH. So it can be use as natural source for pH
indication [11,14,17]. The Anthocyanin extract was
accurately measured as 1ml and was taken in
different, clean test tubes. The pH of solution in
each test tube was adjusted by 1N HCL and 1N
NaoH sequentially from 1 to 12 using the acid
and base and pH meter for accurate pH adjustments. The contents in each tube were
properly mixed and the colour development
was observed and recorded [