Potential Biotechnological Applications of

UV Absorbing pH Sensitive Pigments in

Kokum (Garcinia indica Choicy)

Nandkumar M. Kamat
Department of Botany, Goa University, Taleigao,
Goa.
Garcinia indica Choisy
 INTRODUCTION
•Plant Anthocyanins are pigments serving as UV
screen and may be present in entire fruit, juice or
confined only to skin (Goodwin, 1976).

•Garcinia indica Choisy or Kokum is a tropical

evergreen, slender ornamental tree having
commercial importance with variety of culinary and
medicinal uses.

•Oval, ripe, dark red Kokum fruits contain

anthocyanin pigments like cyanidin-3-glucoside and
cyanidin-3-sambubioside
(http://www.hvfl.com/kokum.htm).
♦ Strawberry anthocyanins have been found to be
extremely sensitive to fluctuations in pH and at
higher pH colour is lost (Goodwin, 1976).
♦ The pH buffering capacity of Kokum is well
known (http://buyersguide.ift.org/cms).
♦ However, there is relatively poor knowledge of the
effect of changing pH on the colour of the kokum
juice.
♦ This work was aimed to assess the effect of the
increasing pH on the colour and the UV-visible
spectroscopic absorption profile of the Kokum juice
in view of the possibility of the application of the
findings in biotechnology.
Habit of Kokum Different stages of fruit maturation

Ripe red fruits Dry Kokum rind

 OBJECTIVES
•To assess the impact of pH change
on the colour of the fresh Kokum
juice
•To assess any corresponding
change in UV-Visible spectroscopic
profile
•To identify distinct UV-Visible
spectral absorption peaks associated
with the pH and /or colour change
•To evaluate the potential of the
results for appropriate commercial
and biotechnological applications
 MATERIALS AND METHODS
Freshly harvested ripe Kokum fruits were washed thoroughly under tap water

Juice squeezed mechanically with muslin cloth

Centrifuged at 5000 rpm for 20 minutes

Five ml juice aliquot diluted to 25 ml with double distilled water

Original pH (Control Dynamics digital pH meter APX 175) and colour (Camel shade
card) recorded

Titrated against 0.5 N NaOH till a distinct colour change appeared

pH, spectral profile for each colour change noted

Spectral profile was recorded using Shimadzu double beam UV spectrophotometer

(Shimadzu) in range of 190-750 nm, in matched Quartz cuvettes with dist. water as ref.
A sample of freshly extracted Kokum juice
 RESULTS
Table 1: pH and colour change
Sr No. pH Colour change Colour code
1 1.87 Alizarin crimson 002
2 1.99 Vermilion hue 449
3 2.85 Cadmium red 041
4 4.70 Gold ochre transparent 172
5 5.25 Raw sienna 366
6 6.16 Raw sienna 366
7 7.46 Venetian red 447
8 8.47 Venetian red 447
9 10.56 Venetian red 447
10 13.00 Cadmium orange 039
♦ It was observed that the original red colour of the juice changed to
cadmium orange within a pH range of 1.87-13.00

♦ pH 5.25 and 6.16 showed raw sienna with a slight variation in colour.
Similarly for pH 7.46, 8.47 and 10.56.

♦ After pH 13.00 the colour did not change further.

Flasks exhibiting some colours produced by change in the
juice pH. Generally at lower pH the shades are closer to
pink and with alkaline conditions these shift to Orange
 RESULTS
Table 2: pH and spectral profile
Sr. No. pH Spectral peaks
Wavelength Absorbance
(nm)
1 1.87 265.50 1.108
208.50 3.972
2 1.99 264.00 0.249
193.00 2.488
3 2.85 262.00 0.215
193.00 2.408
4 4.70 193.50 2.568
5 5.25 194.00 2.540
6 6.16 194.00 2.619
7 7.46 194.00 2.607
8 8.47 194.00 2.655
9 10.56 194.50 2.640
10 13.00 203.50 3.280
 RESULTS
♦Two peaks are observed at pH 1.87, 1.99 and 2.85, each
with one major peak in the range of 193.00 to 209.00 nm.

♦The minor peak with less absorption diminishes from

pH 1.87 to 2.85 from 265.5, 264.00 to 262.00 nm
respectively and completely disappears at higher pH.

♦pH 5.25 to 8.47 shows a common peak at wavelength

194.00 nm with slight variation in absorption.

♦There is sudden increase in wavelength at pH 13.00

from 194.50 nm (pH 10.56) to 203.50
 RESULTS
Table 3. pH change, colour change and spectral profile

Sr. No pH Colour change Spectral shift

change (nm)
1 1.87 Alizarin crimson 208.50
2 1.99 Vermilion hue 193.00
3 2.85 Cadmium red 193.00
4 4.70 Gold ochre transparent 193.50
5 5.25 Raw sienna 194.00
6 6.16 Raw sienna 194.00
7 7.46 Venetian red 194.00
8 8.47 Venetian red 194.00
9 10.56 Venetian red 194.50
10 13.00 Cadmium orange 203.50

♦ With increase in pH there is distinct change in colour and the spectral

characteristics of the juice
 pH 1.87 pH 1.99 pH 2.85

Fig. 1 a Fig 1 b Fig 1 c

Fig 1 a. shows two peaks. Major peak at 208.5 with 3.972 absorbance and minor
peak at 265.5 nm with 1.108
1b. Shows two peaks. Major peak at 193.00 nm with 2.488 absorbance and
second peak at 264.00 nm with very less absorbance of 0.249
1c. Shows two peaks. Major peak at 193.00 nm with 2.408 absorbance and
second peak at 262.00 nm with very less absorbance of 0.215
 pH 4.70 pH 5.25 pH 6.16

Fig. 2 a Fig. 2b Fig. 2c

Fig. 2 a. shows only one prominent peak at 193.5 nm with

absorbance of 2.568.
2 b & c. shows common peak at 194.00 nm with
absorbances of 2.540 and 2.619 respectively.
 pH 7.46 pH 8.47 pH 10.56

Fig. 3 a
Fig. 3 b Fig. 3 c
pH 13.00
Fig. 3. (a-c) display sharp peaks with
high absorbance at a very narrow range
193-194.5 nm and a spectral shift occurs
at high pH (d) at 203 nm with change of
the colour from venetian red to cadmium
orange

Fig. 3d
 DISCUSSION
•Kokum juice at natural pH (1.87) does not show
any absorbance between 600-700 nm. However
there is a weak absorbance over a broad
bandwidth of 400-600 nm which was found to
diminish with higher pH.

•At the original pH there is a distinct absorption at

265 nm which diminishes in intensity with
increasing pH and then disappears at pH 4.7
indicating a distinct chemical change.

•The absorbance of the solution gradually

increases between 400-250 nm between 4.70-13.00
pH
 DISCUSSION
• At 13.00 pH the absorption profile is very
smooth probably indicating presence of
single chemical component with strong
absorption in the UV range at 203.50 nm.
• Initially a slight change in pH shows a UV
spectral shift between 208.50-193.00 nm.
The absorbance peak remains almost
constant from 1.99-10.56 pH.
• Between 10.56-13.00 pH the peak is shifted
again from 194.50 nm to 203.50 nm.
 DISCUSSION
Potential applications of the UV absorbance
capacity of the pigment :-
• The strong absorbance values ( 1.108-3.972) over
193 nm-265.5 nm range shows a UV protective
function of the soluble pH sensitive pigment

• The Nucleic acids (DNA & RNA) are known to

strongly absorb at 260 nm
 DISCUSSION
Potential of UV protective
action: -
• The UV protective action of the Kokum
pigment may be useful in masking the
skin from DNA damaging solar radiation.

• The natural juice could be an important

resource for developing cheap, organic,
eco-friendly, UV-protecting sun-
screens/ointments/lotions and could
have immense potential in cosmetics
industry
 DISCUSSION
• The Chromic shift associated with pH
change could be useful property for
producing either low cost pH
indicator papers (for a pH range of 3-
13) or pH sensitive opto-electronic
biosensors which can be employed
under highly alkaline bioreactor
conditions
• The simple Kokum juice can thus serve
to be a forerunner to new products and
processes in cosmetics and biosensor
industry