This document summarizes a student's research on the anti-oxidative effects of green tea extract and tocopherol on the autoxidation of perilla oil. The student purified perilla oil to remove tocopherol and measured tocopherol content using HPLC. Green tea extract was prepared from leaves and powder and analyzed for total phenolic content and DPPH radical scavenging activity. Peroxide value was measured to evaluate the oxidative stability of perilla oil emulsions with different combinations of tocopherol and green tea extract over time. Results showed green tea extract was effective in inhibiting perilla oil autoxidation but was less effective than tocopherol in emulsions, possibly due to their

1. MINISTRY OF EDUCATION AND TRAINING
NONG LAM UNIVERSITY – HO CHI MINH CITY
Faculty of Food Science and Technology
ANTI-OXIDATIVE EFFECTS OF GREEN
TEA EXTRACT AND TOCOPHEROL ON
AUTOXIDATION OF PERILLA OIL
Student: Nguyen Tran Bao Chau
Supervisors: Assoc. Prof. Yukihiro Yamamoto
Dr. Nguyen Minh Xuan Hong
-March 2018- 1

2. OUTLINE
2
1
2
3
4

3. Introduction
Materials and methods
Results and discussion
Conclusion and recommendation
1
2
3
4
3

4. INTRODUCTION
Allergy
4
Perilla oil is a good source of omega 3 fatty acids so it
is often used as a fish oil replacement in diet
Perilla oil
Fish oil

5. INTRODUCTION
One of highest proportion of omega 3
fatty acid (54-64%)
Has the present of omega 6 and omega
9 fatty acid and high calorie content
(900kcal per 100g)
High unsaturated acid content (~90%)
and very low in saturated fat (~10%)
Health benefit of perilla oil
5

6. INTRODUCTION
Mechanism of lipid oxidation in perilla oil
6

7. Anti-oxidation of oxidation
7
INTRODUCTION

8. INTRODUCTION
Perilla oil is a rich
source of
Tocopherol (Toc)
compare to other
seed oil groups
(linseed, sesame,
mustard,...)

9. INTRODUCTION
✓Nowadays, the using of natural antioxidants is being suggested.
The majority of natural antioxidants are phenolic compounds.
✓Green tea (derived from is a good source of polyphenols, mainly flavonoids.
Green tea contains mainly catechins of epigallocatechin gallate (EGCG),
epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC)
• Green tea leaves: In this study, Japanese green
tea leaves known as sencha (煎茶) were used
• Green tea powder: is known as Matcha (抹茶) is
finely ground powder of processed green tea
leaves.
Green tea leaves Green tea powder

10. Introduction
Materials and methods
Results and discussion
Conclusion and recommendation
1
2
3
4
10

11. MATERIALS AND METHODS
Purification of perilla oil to remove Toc
Oil
(170g)
Activated carbon
85g
Hexane 850 mL
Mix 15 mins
Filtering
Evaporation
Oil
without
Toc

12. MATERIALS AND METHODS
Identifying the content of Toc in perilla oil by HPLC
Sample (normal oil and purified oil) 0.05g
HPLC
PMC 0.01 mg/mL 1mL
(standard substance)

13. 200mL
water
80°C
Swirling
for 15
minutes
Filter
paper
Preparation of green tea extract
MATERIALS AND METHODS
20g
(tea leaves/ tea powder)
Green tea extract
(leaves/ powder)

14. 0.5 mL sample
(leaves/powder)
Vortex for 15
seconds
Absorbance
measurement
(765 nm)
4.3 mL distilled
water
0.2 mL Folin
Ciocaltu reagent
(2- fold dilution)
0.5 mL of 20%
of sodium
carbonate
Wait for 30
minutes
MATERIALS AND METHODS
Determination of Total Phenolic Content (TPC)
Ethyl gallate
was used as a
standard for
the calibration
curve

15. Determination of DPPH Radical Scavenging Activity
1 mL of 0.5 mM
DPPH ethanol
solution
2 mL of 0.1M
acetate buffer
(pH5.5)
2 mL of GTE
(100- fold dilution)
DPPH radical
scavenging
activity of the
antioxidants was
evaluated and
converted into the
Toc equivalent
Vortex for 15 sec.
Then stand in dark for
30 min
Absorbance
measurement at 517 nm
Toc was used
as a standard
for the
calibration
curve
MATERIALS AND METHODS

16. MATERIALS AND METHODS
Preparation of oil/water emulsion
Polyphenol added to the substrate was adjusted to 44 ppm as follows, based on the content determined
by radical scavenging activity of polyphenol content t: 266 ppm for Toc, 44 ppm for GTE
Sample 1 Sample 2 Sample 3 Sample 4
Total Toc
(ppm) 0 266 0 133
GTE (ppm
as
polyphenol)
0 0 44 22

17. MATERIALS AND METHODS
Preparation of oil/water emulsion
15 g of perilla
oil (with or
without Toc)
were
prepared for
each sample.
Tween 20
aqueous
solution (5
wt%) was
added to the
same weight
of oil.
The
antioxidants
(GTE) were
added into
sample 3 and
4.
Incubation under constant
stirring at 1000 rpm and
30°C in the dark condition.
Emulsification with a
disperser at 3000 rpm
for 3 minutes.

18. MATERIALS AND METHODS
3.3 mL of
emulsion
solution
7 mL of chloroform/
methanol mixture
Centrifugation at
3000 rpm in 5 min. Evaporation
Collected oil
Chloroform layer
(lower layer)
Extraction of oil

19. MATERIALS AND METHODS
Measurement of peroxide value (PV, meq/kg)
10 mL mixture of
acetic acid/iso-
octane
Oil sample
0.2 mL of
saturated KI
solution
80 mL water
swirling continuously
for 1 min
Titrator automatically measured and
calculated the value of PV

20. Introduction
Materials and methods
Results and discussion
Conclusion and recommendation
1
2
3
4
20

21. Determination of Toc
Greening Internal of cookies (ziplock)
Time
0 5 10 15 20 25 30
Greening
(-a*)
-20
-15
-10
-5
0
5
10
15
Maple
Sour cream
Buttermilk
Yogurt
Honey
Greening internal of cookies (vacuum)
Time
0 5 10 15 20 25 30
Greening
(-a*)
-20
-15
-10
-5
0
5
10
15
Maple
Sour cream
Buttermilk
Yogurt
Honey
RESULT AND DISCUSSION
-200
0
200
400
600
800
1000
1200
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
mV
Time in minutes
PMC g
b
a
TAG
-200
0
200
400
600
800
1000
1200
1400
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
mV
Time in minutes
TAG
Typical chromatograms of perilla oil (A) and purified perilla oil (B).
TAG: triacylglycerol, PMC: 2,2,5,7,8- pentamethyl- 6- chromanol
(A) (B)
Tocopherol Content (ppm)
Total Toc 265.6
α-Toc 0
bToc 23.4
γ–Toc 230.9
δ - Toc 11.3
Toc composition of perilla oil

22. Determination of the total phenolic content
RESULT AND DISCUSSION
Sample
Total phenolic content
(mg EGE/ gm of dry extract)
Extraction yield vs. dry
extract (wt %)
Leaves 251.1 25.0%
Powder 33.4 3.3%
Amount of total phenolic content in leaves and powder GTE samples

23. Evaluation of the DPPH radical scavenging activity
RESULT AND DISCUSSION
1
6
0
1
2
3
4
5
6
7
Tocopherol Green tea extract
Relative
Activity
Comparison relative DPPH radical activity of GTE and Toc

24. RESULT AND DISCUSSION
Autoxidation test
2.45 5.75
48.43
97.70
486.00
y = 2.3099e0.8193x
0
100
200
300
400
500
600
0 2 4 6 8
PV
(meq/kg)
Time (days)
Sample 1: Purilla Oil without Antioxidant
3.56
13.50
14.00
23.83
51.73
y = 3.4288e0.2196x
0
10
20
30
40
50
60
0 5 10 15
PV
(meq/kg)
Time (days)
Sample 2: Purilla Oil with Toc
2.45 8.22
29.33
55.90
214.00
y = 2.3012e0.5802x
0
50
100
150
200
250
0 2 4 6 8
PV
(meq/kg)
Time (days)
Sample 3: Purilla Oil with GTE
3.56
15.77
25.20
49.93
52.20
y = 3.2827e0.3059x
0
10
20
30
40
50
60
0 2 4 6 8 10
PV
(meq/kg)
Time (Days)
Sample 4: Purilla Oil with Toc and GTE
Effects of GTE and Toc on increasing of PV of perilla oil

25. RESULT AND DISCUSSION
• The following equations were made based on the PV
curve of each sample during storage period:
sample 1, y = 2.3099e0,8193x; sample 2, y = 3.4288e0,2196x;
sample 3, y = 2.3012e0,5802x; sample 4, y = 3.2827e0,3059x
• At PV= 50 meq/kg the order of oxidation stability of the
sample was revealed as: sample 2 (12.2) > sample 4 (8.9)
> sample 3 (5.3) > sample 1 (3.6)
Autoxidation test

26. Autoxidation test
Tryptophan fluorescences
Maple Sourcream Buttermilk Yoghurt Honey
Fluorescence
intensity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Cookies
Dough
RESULT AND DISCUSSION
0
100
200
300
400
500
600
700
0 2 4 6 8 10 12 14
PV
(meq/kg)
Time (Days)
Oxidation Test (PV Measurement)
Sample 1
Sample 2
Sample 3
Sample 4
Effects of antioxidants on perilla oil under in dark condition measured by PV.
Sample 1: PO without antioxidant; Sample 2: PO with Toc, Sample 3: PO with
GTE; Sample 4: PO with GTE and Toc. Data were shown as mean ± standard
deviation

27. Autoxidation test
Schiff bases fluorescences
Maple Sourcream Buttermilk Yoghurt Honey
Fluorescence
intensity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Cookies
Dough
RESULT AND DISCUSSION
486
51.73
214
52.2
0
100
200
300
400
500
600
700
Sample 1 Sample 2 Sample 3 Sample 4
PV
(meq/kg)
Effect of GTE and Toc on perilla oil at the day 6th under
30C in dark condition measured by PV

28. Introduction
Materials and methods
Results and discussion
Conclusion and recommendation
1
2
3
4
28

29. CONCLUSION AND RECOMMENDATION
Conclusion
• Perilla oil has high content of PUFA → unstable oil.
• GTE expressed the effective for antioxidant in perilla oil.
• Radical-scavenging ability test: GTE> Toc
• In perila oil emulsion system: GTE<Toc
Can be explaned by
the position of Toc
and GTE in the
emulsion when
oxidation occurs

30. CONCLUSION AND RECOMMENDATION
Recommendation
Future research:
Controlling the type, location, concentration of emulsifier at the oil-water
interface → concentrating the hydrophilic GTE localize at suitable position
in the oil- water interface.

31. Thank you!