Galls in generally are useful in many industries but sometimes due to pollution they grown in plants, found in road dividers.

1. University of Rajasthan
Submitted to :-
Dr Ranjana Agarwal
(HOD)
Submitted by ;-
Shaifali Bhargava,
B.Sc. Biotechnology
3rd year
(2016-2017)
Kanoria PG Mahila Mahavidyalaya, Jaipur
TRAINING
ON
COMPARATIVE ANTIOXIDANT STUDY OF GALL
INDUCED PLANT LEAVES

2. An Antioxidant
is a molecule that inhibits the oxidation of other molecules.
Oxidation is a chemical reaction that can produce free radicals,
leading to chain reaction that may damage cells.
INTRODUCTION
Galls or cecidia are a kind of swelling growth on the
external tissues of plants or animals. Plant galls are
abnormal outgrowths of plant tissues, similar to
benign tumors or warts in animals.
1) Terminalia arjuna 2) Alstonia Scholaris (L)

3. The leaf samples were collected in clean dry sterile containers from
different environments (favourable and unfavourable). Two plants namely:-
1) Alsotnia scholaris (L)
2) Terminalia arjuna
was obtained from the road dividers of JLN Marg, Jaipur.
Materials-
STUDY SITE AND SAMPLE COLLECTION
REAGENTS PREPARATION
1)1mM of DPPH reagent
Procedure-
The flask was covered with aluminium foil and kept in dark room before use.
0.0019g of DPPH reagent was dissolved in 100ml of distilled water
DPPH reagent

4. Methods:-
AIM: - COMPARATIVE ANTIOXIDANT STUDY OF GALL INDUCED
PLANT LEAVES
 Extraction
 DPPH Assay
 Free radical scavenging percentage (%)
 Inhibition concentration

5. 1) Extraction
Leaves of Alstonia scholaris (L) and Terminalia arjuna has been taken with 2 normal and 2
gall induced.
Procedure-
DPPH assay is performed
10mg (0.01g) of dried sample has been taken and dissolved in 10ml of methanol.
Extract the liquid by filtration and kept for drying.
(Grind till powder)
5gm from each sample is taken and kept in water bath till it get dissolves
Leaves were dried at 45ºC for 3-5 days

6. For the determination of free radical scavenging activity of plant extracts.
1) DPPH Assay
Principle: - DPPH (1, 1-Diphenyl-2-picrylhydrazyl) is a stable free radial with red
color (absorbed at 517nm). If free radicals have been scavenged, DPPH will
generated its color to yellow.
Aim: -
Procedure:-
The absorbance (O.D) was measured at 517nm by spectrophotometer.
Now 1ml of 1mM DPPH reagent is added and kept for 30 min in dark room
From the solution, different concentration of plant extract (100-500µg) has been taken
in tubes and methanol is added (100-900µl) accordingly to make up the solution to
1ml.
10mg of dried plant extract is mixed in 10ml of methanol

7. Observation:-
Fig shows the change in color of DPPH from violet to yellow which shows the
neutralization reaction, now the number of free radicals are counted with the help of
spectrophotometer.

8. Result: - Control DPPH reading – 2.8663nm
Test:- 1) Plant I Alstonia scholaris (L) (normal) and (Gall)
S.no Concentration
of plant
extracts
Concentratio
n of
methanol
Concentratio
n of 1mM
DPPH assay
O.D at 517nm from
spectrophotometer
(normal)
O.D at 517nm from
spectrophotometer
(Gall)
1 100µg 900µl 1ml 1.2776 1.5282
2 200µg 800µl 1ml 1.2535 0.8224
3 300µg 700µl 1ml 1.2346 0.5303
4 400µg 600µl 1ml 1.1471 0.5032
5 500µg 500µl 1ml 1.0810 0.1785

9. 2) Plant II Terminalia arjuna (normal) and (Gall)
S.no Concentration
of plant
extracts
Concentratio
n of
methanol
Concentratio
n of
1mMDPPH
assay
O.D at 517nm from
spectrophotometer
(normal)
O.D at 517nm from
spectrophotometer
(Gall)
1 100µg 900µl 1ml 0.5856 0.4774
2 200µg 800µl 1ml 0.5787 0.1665
3 300µg 700µl 1ml 0.5675 0.0825
4 400µg 600µl 1ml 0.3353 -0.0779
5 500µg 500µl 1ml 0.0972 -0.3849

10. Determination of Free radical Scavenging activity
Scavenging % has been calculated from different plant material of different concentration.
Formula-
Scavenging % = Absorbance (O.D) of Control – Absorbance (O.D) of sample × 100
Absorbance (O.D) of control
•Absorbance(O.D) of control =2.8663
1) Plant I Alsotnia scholaris (L) (normal)
a) Concentration of 100µg plant extract
= 2.8663-1.7276 × 100
2.8663
= 39.72%
b) Concentration of 200µg plant extract
= 2.8663-1.2535 × 100
2.8663
= 56.26%

11. Result:-
Sno Concentration of
plant extract
Scavenging %
Plant I
(normal)
Plant I
(Gall)
Plant II
(normal)
Plant II
(Gall)
1 100µg 32.72% 56.10% 79.63% 83.34%
2 200µg 56.26% 71.30% 79.81% 94.19%
3 300µg 56.92% 81.49% 80.20% 97.12%
4 400µg 59.97% 82.44% 88.30% 102%
5 500µg 62.28% 93.77% 96.60% 113%

12. 0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
100µg 200µg 300µg 400µg 500µg
Comparative analysis of
scavenging % pg plant I
Alsotnia scholaris
scavenging % of normal scavenging % of gall
Interpretation
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
100µg 200µg 300µg 400µg 500µg
Comparative analysis of
scavenging % of plant II
Terminalia arjuna
scavenging % of normal scavenging % of gall
Fig shows a rise in scavenging % of gall induced leaves as compare to normal leaves that
depicts higher concentration of free radicals are formed in gall induced plants.

13. Inhibition Concentration
 The IC 50 value of the sample, which is the concentration of sample required to
inhibit 50% of the DPPH free radical.
 It means Absorbance is inversely proportional to inhibition concentration.
To calculate IC 50 value
Formula: - y=120x+8.1
Here, y= scavenging %
x = IC 50 calculated value
1) For plant I Alstonia scholaris (L) (normal)
a) For 100µg concentration-
Scavenging % (y) = 39.72%
y =120x+8.1
39.72 = 120x+8.1
x = 39.72-8.1
120
x = 0.26µg ⁄ ml

14. Interpretation:-
0
0.2
0.4
0.6
0.8
1
100µg 200µg 300µg 400µg 500µg
Comparative Analysis of IC
50 value of plant I Alsotnia
scholis (L)
IC 50 value of normal IC 50 value of gall
0
0.2
0.4
0.6
0.8
1
100µg 200µg 300µg 400µg 500µg
Comparative Analysis of IC
50 value of plant I
Terminaliaarujuna
IC 50 value of normal IC 50 value of gall
Fig shows higher IC 50 value in gall induced Alsotinia scholis
(L) and Terminilia arjuna plant

15. Conclusion
This study determined that Ethanolic extract of gall induced leaves of Alsotina
scholaris (L) and Terminalia arjuna plant species showed better antioxidant potential
by DPPH radical scavenging method when compare to standard ascorbic acid and IC
50 value is higher than absorbance of plant extracts and found to be as 9.3 to 24.8
µg/ml for ascorbic acid and alcoholic extract respectively.
So, we can say this plant is having good antioxidant activity.

16. Applications
 It play a major role in plant defence mechanism.
 Galls are rich in resins and tannic acid and have been used in the
manufacture of permanent inks (such as iron gall ink) and astringent
ointments, in dyeing, and in tanning.
 The larvae in galls are useful for a survival food and fishing bait.
 It possess many medicinal properties.