A power point demonstrating the VIVA done on curry leaves

1. THE EFFECT OF DIFFERENT EXTRACTION METHODS ON THE
ANTIOXIDANT PROPERTIES OF CURRY LEAVES
(Murraya koenigii )
SCHOOL OF FOOD SCIENCE AND TECHNOLOGY
STM 4998 FINAL YEAR PROJECT 1
PROPOSAL PRESENTATION
NAME : PARITHYI A/P MURALI THARAN
MATRIC NUMBER : S43811

2. CHAPTER 1: INTRODUCTION
1.1 Background of study
1.2 Problem statement
1.3 Significance of study
1.4 Objectives

3. 1.1 Background of Study
Curry
Leaves
(Murraya
koenigii )
Also fondly known
as”daun kari or “curry
patta”(Verma, 2012).
Recent studies seem
to indicate that
Phytochemicals
largely contribute to
antioxidant
properties
(Salas,P.G.et al.,2010)
Extraction
techniques have
been used to extract
bioactive compounds
from various plant
materials (Gao &
Liu,2005).

4. The information about the secondary metabolite
content and the antioxidant and anticancer activity
of Malaysian curry leaf is still scarce. (Ghasemzadeh
et al.,2014)
In the nutraceutical industry, the extraction process is the
important step for the isolation of phytochemicals from herbs
and spices (Bak et al., 2012).
1.2 Problem Statement

5. Significance of
study
To further conduct studies to improve
antioxidant yield from the curry leaves
using different types of extraction
methods.
To further determine or measure
the maximum antioxidant capacity
of curry leaves .
1.3 Significance of study

6. To determine the effects of the different
extraction methods on the antioxidant properties
of curry leaves
To measure the Total Phenolic Compound (TPC)
and Total Flavonoid Compound (TFC) and
individual flavonoid present in curry leaves.
1.4 Objectives

7. CHAPTER 2: Literature Review
• 2.1 Curry Leaves
• 2.2 Oxidation
• 2.3 Antioxidants
• 2.4 Extraction Methods
• 2.5 Antioxidant Activity Assay
• 2.6 Identification and Characterization technique of antioxidants
• 2.7 Previous study

8. 2.1 Curry leaves
2.1.1 Taxonomy
2.1.2 Botany
2.1.3 Nutraceutical properties
2.1.4 Nutritional composition
2.2 Oxidation
2.2.1 Reaction mechanism of oxidation
2.2.2 Types of lipid oxidation
2.2.1 Auto-oxidation
2.2.2 Photo-oxidation
2.2.3 Effects of oxidation
2.3 Antioxidants
2.3.1 Natural antioxidants
2.3.1.1 Reaction mechanism of natural
antioxidants
2.3.2 Synthetic antioxidants
2.2.2.1 Reaction mechanism of
synthetic antioxidants
2.3.3 Characterization of antioxidants
2.4 Extraction methods
2.4.1 Soxhlet extraction
2.4.2 Ultrasonic Assisted Extraction (UAE)
2.4.3 Microwave Assisted Extraction (MAE)
2.4.4 Supercritical Fluid Extraction (SFE)
2.4.5 Accelerated solvent extraction (ASE)
2.4.6 Subcritical Water Extraction (SWE)
2.4.7 Ultrasonic/Microwave Assisted Extraction (UMAE)
2.4.8 Comparison Of Extraction Methods With Its
Respective Limitations And Strengths
2.5 Antioxidant Activity Assay
2.5.1 Thiobarbituric Acid (TBA) Assay.
2.5.2 Conjugated Diene Assay
2.5.3 𝛽-Carotene Bleaching Assay
2.5.4 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay
2.5.5 Ferric Reducing/Antioxidant Power (FRAP) Assay
2.5.6 Ferric Thiocyanate (FTC) Assay
2.5.7Comparison Of Antioxidant Activity Assay With Its
Respective Limitations And Strengths
2.6 Identification and Characterization technique of antioxidants
2.7 Previous study
Detailed Outline Of Literature Reviews

9. 2.1.1
Taxonomy
2.1.2
Botany
2.1.3
Nutraceutical
properties
2.1.4 Nutritional
composition
2.1 Curry leaf plant ( Murraya koenigii (L.) Spreng.
Curry
leaves

10. Kingdom Plantae– Plants
Subkingdom Tracheobionta– Vascular plants
Superdivision Spermatophyta– Seed plants
Division Magnoliophyta– Flowering plants
Class Magnoliopsida– Dicotyledons
Subclass Rosidae
Order Sapindales
Family Rutaceae– Rue family
Genus Murraya J. Koenig ex L.– murraya
Species Murraya koenigii (L.) Spreng.– curry leaf tree
2.1.1 Taxonomy of curry leaves Murraya koenigii (L.) Spreng.
(USDA, Classification for Kingdom Plantae Down to Species Murraya koenigii (L.) Spreng.)
Table 1 : Taxonomy of curry leaves Murraya koenigii .

11. 2.1.2 Botany
• A small spreading shrub, about 2.5 metres high; the main
stem, dark green to brownish.(Singh,2014)
• Flowers, white, funnel-shaped, sweetly scented, stalked.
(Singh,2014)
• Leaves, exstipulate, bipinnately
compound, 30 cm long, each bearing 24
leaflets, having reticulate venation
(Singh,2014)

12. 2.1.3 Nutraceutical uses
• Aqueous and ethanolic extracts of M. koenigii were evaluated for the anti
candidal activity against the 30 candida albicans, in that no extract exhibited
any anticandidal activity . (Ajay. S et al.,2011)
2.1.3.2 Antifungal properties
2.1.3.1 Antibacterial properties
• The essential oil from Murraya koenigii leaves showed antibacterial effect
against B. subtilis, S.aureus, C. pyogenes, P. vulgaris and Pasteurella multocida.
(Saini et al.,2015)

13. 2.1.3.3 Antiprotozoal properties
• Ethanolic extracts (55 %) of Murraya koenigii whole plant excluding roots
showed antiprotozoal action against Ent. Histolytica, antispasmodic effect on
isolated guinea pig ileum.
(Saini et al.,2015)
2.1.3.4 Antimutagenic properties
• The antimutagenicity of M. koenigii benzene fraction has also been
demonstrated against indirect acting mutagens benzo(a)pyrene (BP) and 2-
aminoflourene (2-AF) that infers their mutagenicity by microsomal activation
(Zahin, M. et al.,2013)

14. 2.1.4 Nutritional composition
Nutritional
composition
Phytochemicals
Vitamins
Minerals

15. Table 2: The phytochemical composition of Murraya koenigii leaf
Table 3: The vitamin content of Murraya koenigii leaf
Igara, C.E.et al.,2016
Igara, C.E.et al.,2016

16. Table 4 :Mineral elements of Murraya koenigii leaf
Igara, C.E.et al.,2016

17. 2.2. Oxidation

18. 2.2.1 Mechanism of oxidation
Initiation:
•RH + initiator → R
•ROOH + initiator → ROO•
Propagation:
R + O2 → ROO
ROO + RH → ROOH + R•
Termination:
R + R → R-R
ROO• + R → ROOH
Altemimi et al.,2017

19. 2.2.2 Types of oxidation
Oxidation
Auto-
oxidation
Photo-
oxidation

20. 2.2.2.1 Auto-oxidation (Free radical mediated oxidation)
In senescent phototrophic organisms, the mechanism of initiation of free-
radical oxidation seems to be the homolytic cleavage (catalyzed by some metal
ions) of photochemically produced hydroperoxides.(Rontani, J .F . et al.,2012)
(Shahidi, F., & Zhong, Y.,2010)
Figure 1 : The mechanism of auto-oxidation

21. 2.2.2.2 Photo-oxidation
• Oxidative stress caused by UVA irradiation initiates a series of cellular
responses that can result in cell death either viaapoptosis or necrosis
with modest UV doses.
(Laethem, A. V. et al.,2005)
(Jacobsen,c 2010)
Figure 2 : The mechanism of Photo-oxidation

22. 2.2.3 Effects of oxidative stress
Oxidative stress is a phenomenon caused by an imbalance
between production and accumulation of oxygen reactive
species (ROS) in cells and tissues and the ability of a
biological system to detoxify these reactive products.
When ROS production increases, they start showing
harmful effects on important cellular structures like
proteins, lipids, and nucleic acids
Cells deploy an antioxidant defensive system based mainly
on enzymatic components, such as superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase (GPx),
ROS :
• they are generated
as metabolic by-
products by
biological systems.
• Superoxide radicals
(O2
•), hydrogen
peroxide (H2O2),
hydroxyl radicals
(•OH), and singlet
oxygen (O2)
Pizzino,G.,2017

23. 2.3Antioxidants
• Any substance that directly scavenges or indirectly acts to up-regulate antioxidant
defences or inhibit Reactive Oxygen Species production . (Khlebnikov et al.,2007)
2.3.1 Natural Antioxidants
• Natural antioxidants are found in numerous plant materials and commonly include an
aromatic ring as part of the molecular structure. Example: derivatives or isomers of
flavones, isoflavones, flavonols, catechins, eugenol, coumarin, tocopherols, cinnamic
acid, phosphatides and polyfunctional organic acids (Simic,1981)
2.3.2 Synthetic Antioxidants
• Antioxidants that have synthetically tailored to meet specific requirements. Example:
BHA,BHT,TBHQ (Simic,1981)

24. Pietta (2000), Ratnam et al. (2006) and Godman et al. (2011)
2.3.1 Natural Antioxidants
Figure 3 : The classification of natural antioxidants

25. 2.3.1.1 Reaction mechanism of Natural Antioxidants
Nimse & Pal,2015
Figure 4 : The Reaction mechanism of natural antioxidants

26. 2.3.2 Synthetic Antioxidants
(Carocho et al.,2012)
Figure 5 : The classification of synthetic antioxidants

27. 2.3.2.1 Reaction mechanism of Synthetic Antioxidants
Hameed et al.,2015
Figure 6 : The Reaction mechanism of synthetic antioxidants

28. Characterization
of antioxidants
Based
on their
activity
Based on
their
solubility
Based
on their
size
Non-
enzymatic
enzymatic
Water
soluble
Lipid
soluble
Small
molecule
antioxidants
Big
molecule
antioxidants
(Nimse & pal,2015)
2.3.3 Chracterization of antioxidants

29. Extraction
methods
Soxhlet
extraction Microwave
Assisted
extraction
Ultrasonic
Assisted
extraction
Super-
crtitical fluid
extraction
Accelerated
solvent
extraction
Ultrasonic/
Microwave
Assisted
extraction
Subcritical
water
extraction
2.4 Extraction methods

30. 2.4.1 Soxhlet Extraction
Soxhlet extraction is a very useful tool for preparative purposes in which the analyte is
concentrated from the matrix as a whole or separated from particular interfering
substances. (Encyclopedia of Separation Science, 2000)
Strength
• Good recoveries, inexpensive system, easy to handle (Halfadji.A et al.,2013)
Limitations
• Soxhlet extraction allows use of large amount of sample(Avila V.L et al.,2000)
Shamsuddin et al.,2015
Figure 7 : The soxhlet extractor

31. 2.4.2 Microwave Assisted Extraction (MAE)
MAE utilizes microwave energy to facilitate partition of analytes from the sample matrix into
the solvent Trusheva B.et al.,(2007)
Strength
• Shorter extraction times , shorter cooling times and less use of solvent. (Colvin. D.et
al.,2018)
Limitations
• However, this method is limited to small molecule phenolic compounds (Colvin. D.et
al.,2018)
(Kusuma.H and Mahfud.M,2016)
Figure 8 : The Microwave Assisted Extraction (MAE) Instrument

32. 2.4.3 Ultrasonic Assisted Extraction (UAE)
UAE involves the use of ultrasound ranging from 20 kHz to 2000 kHz (Handa S.S.et al.,2008)
Strength
• benefits of UAE is mainly due reduction in extraction time and solvent
consumption (Azwanida.N.N 2015)
Limitations
• May destroy the integrity of phytochemicals in high frequencies
(Kaufmann.B and Christen.P ,2002 ), (Handa S.S.et al.,2008)
Molina R.R et al.,2016
Figure 9 : The Ultrasonic Assisted Extraction (UAE) Instrument

33. 2.4.4 Supercritical fluid extraction (SFE)
Separating one component (the extractant) from another (the matrix) using
supercritical fluids as the extracting solvent. (Journal of Chromatography A, 2009)
Strength
• Aspects such as improved selectivity, higher extraction yields, better fractionation
capabilities, and lower environmental impacts (Sánchez A.P et al.,2014)
Limitations
• It complicates system thermodynamics and increases capital costs (Abbas, K.A.,2008)
(Sökmen,M et al.,2018)
Figure 10 : The Supercritical fluid extraction (SFE) Instrument

34. 2.4.5 Accelerated Solvent extraction (ASE)
An automated rapid extraction technique that utilizes common solvents at elevated
temperature and pressure. (Mottaleb, M.A. & Sarker, S. D.,2012)
Strength
• ASE is faster, use less extraction fluids than the “classic” extraction techniques, and
can be readily be automated. (Możajska, H. G., et al., 2001)
Limitations
• high cost (Możajska,H.G., et al.,2001)
Sonya,2012
Figure 11 : The Accelerated Solvent extraction (ASE) Instrument

35. 2.4.6 Subcritical Water Extraction (SWE)
SWE is a new and powerful technique at temperatures between 100 and 374oC and
pressure high enough to maintain the liquid state(Asl ,A.H. et al.,2013)
Strength
• Low price, safety and green character of water, good yields of target compounds
(Nastić,N.et al.,2018)
Limitations
• Applying higher water flow rates is increasing the extract volume (Asl ,A.H. et al.,2013)
(Machmudah,S et al.,2014)
Figure 11 : The Subcritical Water Extraction (SWE) Instrument.

36. 2.4.7 Ultrasonic Microwave Assisted Extraction (UMAE)
A hybrid of both MAE with UAE to ultrasonic-microwave assisted extraction (UMAE), is
a complementary extraction technique with both advantages of MAE and UAE (Wang, Y
et al.,2018)
Strength
• Shorter extraction time, less volume of the solvents needed and higher yield (Lianfu,
Z., & Zelong, L.,2008)
Li,C et al.,2018
Figure 12 : The Ultrasonic Microwave Assisted Extraction (UMAE) Instrument.

37. 2.4.8 Strengths and limitations of extraction methods
Name of extraction methods Strengths Limitations
Soxhlet Extraction Good recoveries, inexpensive system, easy to
handle
large amount of sample
Microwave Assisted Extraction shorter extraction times , shorter cooling times
and less use of solvent
limited to small molecule phenolic
compounds
Ultrasonic Assisted Extraction reduction in extraction time and solvent
consumption
May destroy the integrity of
phytochemicals in high frequencies
Supercritical Fluid Extraction improved selectivity, higher extraction yields,
better fractionation capabilities, and lower
environmental impacts
complicates system thermodynamics
and increases capital costs
Accelerated Solvent Extraction ASE is faster, use less extraction fluids than the
“classic” extraction techniques, and can be
readily be automated
high cost
Subcritical Water Extraction Low price, safety and green character of water,
good yields of target compounds and reduced
energy consumption
applying higher water flow rates is
increasing the extract volume and
consequently, lower concentration
of the final extracts
Ultrasonic Microwave Assisted
Extraction (UMAE)
Shorter extraction time, less volume of the
solvents , high yield
-
Table 5 : Strengths and limitations of extraction methods.

38. Associated with
lipid
peroxidation
• Thiobarbituric Acid (TBA)
Assay.
• Conjugated Diene Assay
• 𝛽-Carotene Bleaching Assay
Associated with
electron and
radical
scavenging
• 2,2-Diphenyl-1-picrylhydrazyl
(DPPH) Assay
• Ferric Reducing/Antioxidant
Power (FRAP) Assay
• Ferric Thiocyanate (FTC)
Assay
Antioxidant
Activity Assay
2.5 Antioxidant activity Assay
Moon & Shibamoto,2009

39. 2.5.1 Thiobarbituric Acid (TBA) Assay.
Strength
• Instrumentation is readily available, inexpensive, simple
(Ghani, M. A. et al.,2017)
Limitations
• lack acceptable reproducibility, and long reaction times
(Buenger et al.,2006)
Yahyavi, H. et al.,2016
Figure 13 : The Thiobarbituric Acid (TBA) reaction.

40. 2.5.2 Conjugated Diene Assay.
Strength
• allows greater sensitivity, easily measurable
(Palmieri & Sblendorio, 2007)
Limitations
• difficulties in measuring conjugated dienes in biological materials
because many of the other substances present
(Palmieri & Sblendorio, 2007)
Goiris,K. et al.,2012
Figure 14 : The Conjugated Diene Assay.

41. 2.5.3 𝛽-Carotene Bleaching Assay.
Moon & Shibamoto,2009
Strength
• possesses pronounced radical scavenging properties.
(Stutz,H. et al.,2015)
Limitations
• Limited number of commercial standards imposes analytical
constraint, carcinogenic (Paliakov,E.M., et al.,2009)
Figure 14 : 𝛽-Carotene reaction with antioxidant

42. 2.5.4 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay
Strength
• Rapid, simple and inexpensive method
Limitations
• Time consuming and lengthy procedure (Shalaby & Shanab, 2013)
(Teixeira, J.et al.,2013)
Figure 15 : The reaction of DPPH

43. 2.5.5 Ferric Reducing/Antioxidant Power (FRAP) Assay
Strength
• It is simple, speedy, inexpensive, and robust does not required specialized
equipment. It can be performed using automated, semi-automated, or manual
methods.(Shalaby & Shanab, 2013)
Limitations
• FRAP cannot detect species that act by radical quenching (H transfer),
particularly SH group containing antioxidants like thiols, such as glutathione and
proteins.(Shalaby & Shanab, 2013)
(Danilewicz, J. C.,2015)
Figure 16 : The Ferric Reducing Activity

44. 2.5.6 Ferric Thiocyanate (FTC) Assay
Strength
• Sensitive, rapid, high reproducibility and simple.
(Mihaljevi, B.et al.,1996)
Limitations
• When UV absorption around 500 nm is present, the results are overestimated
or not reliable
(Moon & Shibamoto, 2009)
(Verma,2013)
Figure 17 : The Ferric Thiocyanate Count Assay

45. Extraction Methods Strengths Limitations
Thiobarbituric Acid (TBA) Assay. Instrumentation is readily
available, inexpensive, simple
lack acceptable reproducibility, and
long reaction times.
Conjugated Diene Assay allows greater sensitivity, easily
measurable
difficulties in measuring
conjugated dienes in biological
materials because many of the
other substances present
𝛽-Carotene Bleaching Assay possesses pronounced radical
scavenging properties.
Limited number of commercial
standards imposes analytical
constraint, carcinogenic
2,2-Diphenyl-1-picrylhydrazyl
(DPPH) Assay
Rapid, simple and inexpensive
method
Time consuming and lengthy
procedure
Ferric Reducing/Antioxidant Power
(FRAP) Assay
simple, speedy, inexpensive, FRAP cannot detect species that
act by radical quenching
Ferric Thiocyanate (FTC) Assay Sensitive, rapid, high
reproducibility and simple.
The results are overestimated or
not reliable, when UV absorption
around 500 nm is present
2.5.7 Comparison of antioxidant Activity Assay with its respective strength and limitations
Table 6 : Comparison of antioxidant Activity Assay with its respective strength and limitations.

46. 2.6 Assays for Separation and Quantification of phenolics and flavonoids
Assays for
Separation
and
Quantification
of phenolics
and
flavonoids
Gas
Chromatography
(GC)
Three layer
chromatography
(TLC) High Pressure
Liquid
Chromatography
(HPLC)

47. Assays Advantages Disadvantages
Gas Chromatography (GC) • useful,
• Accurate
• suitable
• Only applicable for volatile
compounds
Three layer chromatography
(TLC)
• Simple
• Applicable for non volatile
or less volatile compounds.
• Limited length of separation
High Pressure Liquid
Chromatography (HPLC)
• Applicable for non volatile
or less volatile compounds.
• preferred technique for
both separation and
quantification of phenolic
compounds
• Expensive
Khoddami, A. et al.,2013
Table 7 : Comparison of Assays for Separation and Quantification of phenolics and flavonoids

48. Previous studies References
Curry leaves
• Azwanida, N.N.
et al.,2015
• Sain, S.C. et
al.,2015
• Medicinal plants are currently in considerable significance view due to their special attributes as a
large source of therapeutic phytochemicals that may lead to the development of novel drugs.
• This plant has been reported to have anti-oxidative, cytotoxic, antimicrobial, antibacterial, anti
ulcer, positive inotropic and cholesterol reducing activities.
Extraction
• Sasidharan, S. et
al.,2011
• Ghasemzadeh,
A. et al.,2014
• Sasidharan, I. et
al.,2011
• Different solvent systems are available to extract the bioactive compound from natural products.
• The application of ultrasound helps develop interesting and novel methodologies in food
processing; these methodologies are often complementary to classical methods.
• The present work was carried out to study the effect of temperature and different solvents on the
antioxidant property of curry leaves
Antioxidants.
• Khoddami,A. et
al.,2013
• Ghasemzadeh,
A. et al.,2014
• Plant foods are rich sources of phenolics and flavonoids , which are molecules that can act as
antioxidants to prevent heart disease reduce inflammation lower the incidence of cancers and
diabetes as well as reduce rates of mutagenesis in human cells .
• To the best of our knowledge, there have been no studies to optimize the flavonoid extraction
from the curry leaf and following that, improvement of the anticancer and antioxidant activities.
2.7 Previous Studies
Table 8 : Previous studies

49. Chapter 3.0 Materials and Methods

50. 3.1 Materials
• Curry leaves will be collected from Pasar Payang ,Kuala
Terengganu.
• Curry leaves will be freeze dried and then finely grinded.

51. 3.2 Methods

52. 3.2.1 Overall View Of Experiment
Curry leaves
Freeze dried, Finely grind, secured in a bottle.
Ultrasonic Assisted
Extraction Microwave Assisted
EXtraction
Soxhlet
Extraction
Determination of Antioxidant activity
Antioxidant quantity
• Determination of Phenolic content
• Determination of flavonoid content
• Determination of individual flavonoid content
using HPLC
•
Antioxidant property
• (DPPH) method
• Ferric Thiocyanate (FTC) method
• Thiobarbituric Acid (TBA) method

53. 3.2.3 Sample preparation
The samples will be obtained
from Pasar Payang , Kuala
Terengganu
The samples will be thoroughly
washed and kept in 4℃.
The samples will be then freeze
and dried and finely grind and
then secured in dark bottles.

54. 3.2.4. Extraction Methods

55. 3.2.4.1 Soxhlet Extraction
Finely ground 1g of crude curry leaves are placed
in a porous bag or “thimble” made of strong filter
paper, of the Soxhlet apparatus.
Heat the extracting solvent and condense it in a
condenser.
The condensed extract drips into the thimble
containing the crude drug, and extracts it by
contact.
When the level of liquid in chamber rises to the top
of siphon tube, the liquid contents of chamber
siphon is put into flask.
This process is continuous and is carried out until
a drop of solvent from the siphon tube does not
leave residue when evaporated.
Modified method from
AOAC 1995

56. Mix the Pre-treated and grinded curry leaves with
500 ml of conical flask containing 40 % methanol
Mix the 1g of curry leaves were with 20 ml of
methanol
Submerge the mixture into an ultrasonic cleaner
bath and leave about 6h to extract the samples.
Filter the extracted samples and centrifuge at 700
pm at 4℃ for 10 min.
Remove the methanol from the extract using rotary
evaporator and bottle the resulting extract in the
chiller for next analysis
3.2.4.2 Ultrasonic Assisted Extraction (UAE)
Chemat, F. et al.,2017

57. 3.2.4.3 Microwave Assisted Extraction (MAE)
Make 80%v/v solution of ethanol.
Take 10-40ml of solvent per g curry leaves and
mix with 80%v/v solution of ethanol.
Irradiate the solution for 5-30min at 70– 130 °C
and 200–1000 W microwave power.
Destandau, E.et al.,2013

58. 3.2.5 Antioxidant Activity Assay

59. 3.2.5.1 2,2-dipehenyl -2-picryl-hydrazyl (DPPH) method
Adjust the extracts to 6mg/ml .
Add an aliquot of 3 ml of 0.025 % DDPH radical in
methanol containing 1 ml of sample of extract at 6mg/ml
Vortex the mixture and let it stand in dark room for 1 hour.
Measure the absorbance of the sample at 517 nm
Sample
12 mg sample extracts +
2 ml of methanol =
6mg/ml
1 ml of sample extracts
Negative control
(Without samples)
12 mg sample extract
+ 2 ml BHT= 6mg/ml
1 ml of BHT
12 mg sample extract
+ 2 ml 𝛼 - tocopherol
= 6mg/ml
1 ml of 𝛼 -
tocopherol
12 mg sample
extract
+ 2 ml ascorbic acid=
6mg/ml
1 ml of ascorbic acid
Positive control
Zhang & Hamauzu (2004)

60. Dissolve the samples in 4 ml of absolute ethanol (99.5%),added with 4.1 ml of
2.52 % linolenic acid in absolute ethanol
Mix with 8 ml of 0.05M phosphate buffer (pH7) and 3.9 ml of distilled water
Keep the mixture in crew cap and place it water bath shaker at 40 ℃
Add 0.1ml of samples with 9.7 ml of 75% ethanol and 0.1 ml of 30 %
ammonium thiocyanate.
3.2.5.2 Ferric Thiocynate (FTC) method
Sample
4 mg of BHT 4 mg of 𝛼 –
tocopherol
4 mg of ascorbic acid
Positive control Negative control
(Without samples)
4 mg of sample

61. Add 0.1 ml of 0.02M ferrous chloride in 3.5% Hydrochloric acid into
reaction mixture
After 3 min, measure the absorbance of the resulting red colour at 500
nm every 24 hour until the day the absorbance of the control reached
the maximum value.
Ferric Thiocynate (FTC) method cont’d
Osawa & Namiki (1981)

62. 3.2.5.3 Thiobarbituric Acid (TBA) method
Add 1 ml of sample solution from FTC method with 2 ml
of 20% thrichloroacetric acid and 2 ml of 0.67 % 2-
thiobarbituric acid
Place the mixture in boling water at 100℃ for 10
minutes
Cool the mixture and centrifuge at 300 rpm for 20
minutes
Measure the absorbance of supernatant at 552 nm
Ottolenghi(1985),Kikuzaki & Nakatami
(1993)

63. 3.2.6.1 Determination of phenolic content
Add 1 mg sample with 1 ml of methanol , mix and vortex.
Add the sample with 4.5 ml of deionized distilled water and 5 ml
Follin Cialcalteau reagent.
Let it for 5 minutes in room temperature.
Add 5 ml of sodium carbonate and 2 ml of deionized distilled water.
Let the mixture for 1 h 30 min in the dark room and read the
absorbance at 750 nm
Kim et al.,2003

64. 3.2.6.2 Determination of flavonoid content
Add 50 mg/ml sample methanol with 1.5 ml methanol
Mix with 0.1 ml of 10 % of ammonium chloride
Add 0.1 ml of 1M potassium acetate
Mix with 2.8 ml of distilled water and incubate for 30 min
Read the absorbance at 415 nm
Ebrahimzadeh et al.,2009

65. 3.2.6.3Determination of individual flavonoid content using
(HPLC)
Add 2g of sample into 20 ml solvent ethanol and 6M HCL mixture and then
reflux for 2h at 90 ℃.
Filter the solution through 0.22 𝜇m filter paper and fill into small vials.
Conduct the analysis using HPLC system.
Kumar et al.(2008) , Chen et al.(2009)

66. Experimental unit (EU) Curry leaves
Factor (independent variables) Different types of extraction
Factor level (Treatment) 1. Soxhlet Extraction
2. Ultraviolet Assisted Extraction (UAE)
3. Microwave Assisted Extraction (MAE)
Arrangement One level arrangement
Assignment Completely randomized design ( CRD)
Replication Triplicates
Total of (EU) ( 3 Treatment × 3 replications ) = 9 EU
Response ( dependent variable ) 1. Total Phenolic Compound (TPC) in curry leaves.
2. Total Flavonoid Compound (TFC) in curry leaves.
3. Individual flavonoid present in curry leaves.
Statistical analysis One-way ANOVA
3.3 Experimental design
Table 9 : Experimental Design

67. Statistical analysis will be carried out using
SPSS software at the confidence at α ≤ 0.05.
The result will express as mean ± standard
deviation.
Significant difference at (p< 0.05 ) will
perform by one-way ANOVA and Comparison
of mean will be carried out using Duncan post-
hoc test.
3.4 Statistical Analaysis

68. 4.0 Expected Results.
• The study showed that MAE not only shortened the extraction time, but
also showed better extraction yields compared with conventional thermal
extractions (heat reflux and Soxhlet)
(Mamoori & Janab,2018)
• The extracts contained substantial amounts of effective flavonoid
compounds such as myricetin, epicatechin, and quercetin.
• The curry leaf with the highest TF and TP contents also showed the highest
antioxidant activity as indicated by the FRAP and DPPH assays.
(Ghasemzadeh, A. et al.,2014)

69. 2019 2020
Activities FEB MAR APR MAY JUN JULY AUG SEPT OCT NOV DEC JAN
Preliminary study
Choosing title
Background study
Literature review
Materials & Methods
Proposal presentation
Proposal submission
Sample and Chemical preparation
Extraction
Antioxidant activity assay
Statistical analysis
Thesis writing
Submission Thesis Draft
Final presentation
Thesis Submission
Ghant chart of research activities.

70. REFERENCES.
 Abbas, K.A., Mohamed, A., Abdulamir, A.S., Abas, H.A. (2008). A Review on Supercritical Fluid
Extraction as New Analytical Method. American Journal of Biochemistry and Biotechnology 4 (4): 345-
353.
 Ajay S, Rahul S, Sumit G, et al. Comprehensive review: Murraya koenigii Linn. Asian Journal of
Pharmacy and Life Sciences. 2011;1(4):417–425.
 Al Mamoori, F., & Al Janabi , R. (2018). Recent Advances In Microwave-assisted Extraction (Mae) Of
Medicinal Plants: A Review. International Research Journal Of Pharmacy, 9(6), 22–29.
 Altemimi, A., Lakhssassi, N., Baharlouei, A., Watson, D., & Lightfoot, D. (2017). Phytochemicals:
Extraction, Isolation, and Identification of Bioactive Compounds from Plant Extracts. Plants, 6(4), 42.
 AOAC. (1995) (16th ed.). Official methods of analysis, Vol. 41. Washington, DC: Association of Official
Analytical Chemists.
 Azwanida, N.N. (2015). A Review on the Extraction Methods Use in Medicinal Plants, Principle,
Strength and Limitation. Medicinal & Aromatic Plants, 04(03).doi:10.4172/2167-0412.1000196
 Bak M-J, Ok S, Jun M, Jeong W-S: 6-shogaol-rich extract from ginger up-regulates the antioxidant
defense systems in cells and mice. Molecules 2012, 17(7):8037–8055.
 Buenger, J., Ackermann, H., Jentzsch, A., Mehling, A., Pfitzner, I., Reiffen, K. A., Wollenweber, U.
(2006). An interlaboratory comparison of methods used to assess antioxidant potentials. Int. J.
Cosmet. Sci. 2006; 28:135–146

71.  Carocho, M., & Ferreira, I. C. F. R. (2013). A review on antioxidants, prooxidants and related
controversy: Natural and synthetic compounds, screening and analysis methodologies and future
perspectives. Food and Chemical Toxicology, 51, 15–25.
 Castro-López,C.,Rojas,R., Ernesto,J.,Alejo,S., Niño-Medina,G., Guillermo, C.G. Ávila., M.(2016).
Phenolic Compound Recovery from Grape Fruit and By- Products: An Overview of Extraction
Methods. Grape and Wine Biotechnology
 Chemat, F., Rombaut, N., Sicaire, A.-G., Meullemiestre, A., Fabiano-Tixier, A.-S., & Abert-Vian, M.
(2017). Ultrasound assisted extraction of food and natural products. Mechanisms, techniques,
combinations, protocols and applications. A review. Ultrasonics Sonochemistry, 34, 540–560.
 Chen ,Q.C., Zhang ,W.Y., Jin, W., Lee, I.S., Min, B.S.,Jung,H.J., et al. Flavonoids and isoflavonoids
from Sophorae Flos improve glucose uptake in vitro. Planta Med. 2010;76:79–81.
 Colvin,D.( 2018). A Review on Comparison of the Extraction Methods Used in Licorice Root: Their
Principle, Strength and Limitation.
 Danilewicz, J. C. (2015). Folin-Ciocalteu, FRAP, and DPPH* Assays for Measuring Polyphenol
Concentration in White Wine. American Journal of Enology and Viticulture, 66(4), 463–471.
 Destandau, E., Michel, T., & Elfakir, C. (n.d.). CHAPTER 4. Microwave‐assisted Extraction. Natural
Product Extraction, 113–156.
 Gao, M. and Liu, C. Z.(2005).“Comparison of techniques for the extraction of flavonoids from cultured
cells of Saussurea medusa Maxim,” World Journal of Microbiology and Biotechnology, vol. 21, no. 8-9,
pp. 1461–1463, 2005

72.  Garcia-Salas, P.; Morales-Soto, A.; Segura-Carretero, A.; Fernandez-Gutierrez, A. Phenolic-compound-
extraction systems for fruit and vegetable samples. Molecules 2010, 15, 8813–8826.
 Ghani, M. A., Barril, C., Bedgood, D. R., & Prenzler, P. D. (2017). Measurement of antioxidant activity with
the thiobarbituric acid reactive substances assay. Food Chemistry, 230, 195–207.
 Ghasemzadeh, A., Jaafar, H. Z. E., Rahmat, A., & Devarajan, T. (2014). Evaluation of Bioactive Compounds,
Pharmaceutical Quality, and Anticancer Activity of Curry Leaf (Murraya koenigiiL.). Evidence-Based
Complementary and Alternative Medicine, 2014, 1–8.
 Giergielewicz-Możajska, H., Dąbrowski, Ł., & Namieśnik, J. (2001). Accelerated Solvent Extraction (ASE) in
the Analysis of Environmental Solid Samples — Some Aspects of Theory and Practice. Critical Reviews in
Analytical Chemistry, 31(3), 149–165.
 Godman, M., Bostick, R.M., Kucuk, O., Jones, D.P., 2011. Clinical trials of antioxidant as cancer prevention
agents: past, present and future. Free Radic. Biol. Med. 51,1068–1084
 Goiris, K., Muylaert, K., Fraeye, I., Foubert, I., De Brabanter, J., & De Cooman, L. (2012). Antioxidant
potential of microalgae in relation to their phenolic and carotenoid content. Journal of Applied Phycology,
24(6), 1477–1486.
 Haghighi, A., & Khajenoori, M. (2013). Subcritical Water Extraction. Mass Transfer - Advances in
Sustainable Energy and Environment Oriented Numerical Modeling. doi:10.5772/54993

73.  Halfadji, A., Touabet, A.,BADJAH HADJAH, A.Y. (2013).Comparison Of Soxhlet Extraction, Microwave-
Assisted Extraction And Ultrasonic Extraction For The Determination Of PCBS Congeners In Spiked Soils
By Transformer Oil International Journal of Advances in Engineering & Technology(ASKAREL), Jan.
2013.©IJAETISSN: 2231-196363Vol. 5, Issue 2, pp. 63-75
 Handa, S.S., Khanuja, S.P.S., Longo, G., Rakesh, D.D. (2008) Extraction Technologies for Medicinal and
Aromatic Plants, (1stedn), no. 66. Italy: United Nations Industrial Development Organization and the
International Centre for Science and High Technology.
 Igara, C.E.,Omoboyowa,D.A., Ahuchaogu, A.A., Orji,N.U. and Ndukwe, M. K.(2016).Phytochemical and
nutritional profile of Murraya Koenigii (Linn) Spreng leaf
 Jacobsen, C. (2010). Challenges When Developing Omega-3 Enriched Foods. Oléagineux, Corps Gras,
Lipides, 17(4), 251–258.
 Kaufmann, B. and Christen, P.(2002).Recent extraction techniques for natural products: microwave-
assisted extraction and pressurized solvent extraction. Phytochemical Analysis.13: 105-113.
 Khlebnikov, A.I., Schepetkin, I.A., Domina, N.G., Kirpotina, L.N., Quinn, M.T., 2007.Improved quantitative
structure–activity relationship models to predictantioxidant activity of flavonoids in chemical, enzymatic,
and cellularsystems. Bioorg. Med. Chem. 15, 1749–1770.
 Khoddami, A., Wilkes, M., & Roberts, T. (2013). Techniques for Analysis of Plant Phenolic Compounds.
Molecules, 18(2), 2328–2375.

74.  Kumar, N., Bhandari, P., Singh, B., Gupta, A. P., & Kaul, V. K. (2008). Reversed phase-HPLC for rapid
determination of polyphenols in flowers of rose species. Journal of Separation Science, 31(2), 262–267
 Kusuma, H. S., & Mahfud, M. (2016). Preliminary study: kinetics of oil extraction from sandalwood by
microwave-assisted hydrodistillation. IOP Conference Series: Materials Science and Engineering, 128,
012009.
 Lee, S. E., Hwang, H. J., Ha, J. S., Jeong, H. S., & Kim, J. H. (2003).Screening of medicinal plant
extracts for antioxidant activity. Life Sciences, 73, 167–179.
 Li, C., Zhang, J., Zhao, C., Yang, L., Zhao, W., Jiang, H., Ren, X., Su,W., Li,Y., Guan, J.
(2018). Separation of the main flavonoids and essential oil from seabuckthorn leaves by
ultrasonic/microwave-assisted simultaneous distillation extraction. Royal Society Open Science, 5(7),
180133.
 Lianfu, Z., & Zelong, L. (2008). Optimization and comparison of ultrasound/microwave assisted
extraction (UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomatoes. Ultrasonics
Sonochemistry, 15(5), 731–737.
 Luque de Castro, M. D., & García Ayuso, L. E. (2000). ENVIRONMENTAL APPLICATIONS | Soxhlet
Extraction. Encyclopedia of Separation Science, 2701–2709.doi:10.1016/b0-12-226770-2/06681-3
 Machmudah, S. (2015). Subcritical Water Extraction of Xanthone from Mangosteen (Garcinia
Mangostana Linn) Pericarp. Journal of Advanced Chemical Engineering, 05(01).
 Mihaljevic, B., B. Katusin-Razem and D. Razem, 1996. The reevaluation of the ferric thiocyanate
assay for lipid hydroperoxides with special considerations of the mechanistic aspects of the
response. Free Radic. Biolo. Med., 21: 53-63.

75.  Moon, J.-K., & Shibamoto, T. (2009). Antioxidant Assays for Plant and Food Components. Journal of
Agricultural and Food Chemistry, 57(5), 1655–1666.
 Mottaleb, M. A., & Sarker, S. D. (2012). Accelerated Solvent Extraction for Natural Products Isolation.
Natural Products Isolation, 75–87.
 Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Hamidinia A, Bekhradnia AR. Determination of antioxidant
activity, phenol and flavonoids content of Parrotia persica Mey.Pharmacologyonline,2008,2,560-567.
 Nastić, N., Švarc-Gajić, J., Delerue-Matos, C., Barroso, M. F., Soares, C., Moreira, M. M., Radojković, M.
(2018). Subcritical water extraction as an environmentally-friendly technique to recover bioactive
compounds from traditional Serbian medicinal plants. Industrial Crops and Products, 111, 579–589.
 Nimse, S. B., & Pal, D. (2015). Free radicals, natural antioxidants, and their reaction mechanisms. RSC
Advances, 5(35), 27986–28006
 Osawa, T. and M. Namiki, 1981. A novel pype of antioxidant isolated from leaf wax
of Eucalyptus leaves. Agric. Biol. Chem., 45: 735-735.
 Palmieri B., Sblendorio V. (2007b). Oxidative stress tests: overview on reliability and use. Part II. Eur.
Rev. Med. Pharmacol. Sci. 11, 383–399.
 Pietta, P. (2000). Flavonoids as antioxidants. J. Nat. Prod. 63, 1035–1042
 Pizzino, G., Irrera, N., Cucinotta, M., Pallio, G., Mannino, F., Arcoraci, V., Squadrito, F., Altavilla, D.,Bitto,
A. (2017). Oxidative Stress: Harms and Benefits for Human Health. Oxidative Medicine and Cellular
Longevity, 2017, 1–13.

76.  Poliakov, E., Gentleman, S., Chander, P., Cunningham, F. X., Grigorenko, B. L., Nemuhin, A. V., &
Redmond, T. M. (2009). Biochemical evidence for the tyrosine involvement in cationic intermediate
stabilization in mouse β-carotene 15, 15’-monooxygenase. BMC Biochemistry, 10(1), 31.
 Ratnam, D.V., Ankola, D.D., Bhardwaj, V., Sahana, D.K., Kumar, N.M.V.R., 2006. Role of antioxidants in
prophylaxis and therapy: a pharmaceutical perspective. J.Control Release 113, 189–207.
 Rontani, J. F. (2012). Photo- and Free Radical-Mediated Oxidation of Lipid Components during the
Senescence of Phototrophic Organisms, Senescence, Dr. Tetsuji Nagata (Ed.), ISBN: 978-953-51-0144-
4, InTech.
 Rostagno, M. A., Villares, A., Guillamón, E., García-Lafuente, A., & Martínez, J. A. (2009). Sample
preparation for the analysis of isoflavones from soybeans and soy foods. Journal of Chromatography A,
1216(1), 2–29.
 Saini, S.C. and Reddy. (2015). A Review on Curry Leaves (Murraya koenigii): Versatile Multi-Potential
Medicinal Plant.
 Sánchez-Camargo, A. P., Mendiola, J. A., Ibáñez, E., & Herrero, M. (2014). Supercritical Fluid Extraction.
Reference Module in Chemistry, Molecular Sciences and Chemical Engineering.
 Sasidharan, I., & Menon, A. N. (2010). Effects of temperature and solvent on antioxidant properties of
curry leaf (Murraya koenigii L.). Journal of Food Science and Technology, 48(3), 366–370.

77.  Sasidharan, S., Chen, Y, Saravanan, D., Sundram, K.M., Latha, Y. (2011). Extraction, Isolation and
Characterization Of Bioactive Compounds From Plants’ Extracts. nstitute for Research in Molecular
Medicine (INFORM), Universiti Sains Malaysia, Minden
 Shahidi, F., & Zhong, Y. (2010). Lipid oxidation and improving the oxidative stability. Chemical Society
Reviews, 39(11), 4067.
 Shalaby, A. (2013). Antioxidant compounds, assays of determination and mode of action. African Journal
of Pharmacy and Pharmacology, 7(10), 528–539.
 Shamsuddin, N. M., Yusup, S., Ibrahim, W. A., Bokhari, A., & Lai Fatt Chuah. (2015). Oil extraction from
Calophyllum inophyllum L. via Soxhlet extraction: Optimization using response surface methodology
(RSM). 2015 10th Asian Control Conference (ASCC).
 Simic, M. G. (1981). Free radical mechanisms in autoxidation processes. Journal of Chemical Education,
58(2), 125.
 Singh, S.,Omreb, P.K., And Mohan, S.M. (2014) Curry Leaves .Murraya Koenigii Linn. Sprengal)- A Mircale
Plant .
 Sökmen, M., Demir, E., & Alomar, S. Y. (2018). Optimization of sequential supercritical fluid extraction
(SFE) of caffeine and catechins from green tea. The Journal of Supercritical Fluids, 133, 171–176.
 Stutz, H., Bresgen, N., & Eckl, P. M. (2015). Analytical tools for the analysis of β-carotene and its
degradation products. Free Radical Research, 49(5), 650–680.

78.  Teixeira, J., Gaspar, A., Garrido, E. M., Garrido, J., & Borges, F. (2013). Hydroxycinnamic Acid
Antioxidants: An Electrochemical Overview. BioMed Research International, 2013, 1–11.
 Trusheva, B., Trunkova, D., Bankova,V. (2007) Different extraction methods of biologically active
components from propolis: a preliminary study. Chem Cent J 13.
 USDA, Classification for Kingdom Plantae Down to Species Murraya koenigii (L.) Spreng
 V. Lopez-Avila and R. Young, “Microwave-assisted extraction of organic compounds from standard
reference soils and sediments.
 Verma, R. S. Padalia R.C Arya V. and Chauhan A. (2012). Aroma Profile of the curry leaf, Murraya
Koenigii (L.) Spreng. Chemotypes Variability in North India during the year. Industrial Crops and
Product, 36:343-348.
 Verschooten L, Claerhout S, Laethem AV, Agostinis P, Garmyn M. New strategies of photoprotection.
Photochem Photobiol. 2006; 82:1016–1023. doi: 10.1562/2006-04-27-IR-884.
 Verschooten, L., Claerhout, S., Laethem A.V., Agostinis, P., Garmyn,M.(2006). New strategies of
photoprotection. Photochem Photobiol.;82:1016–1023.
 Yahyavi, H., Kaykhaii, M., & Hashemi, M. (2016). A rapid spectrofluorimetric method for the
determination of malondialdehyde in human plasma after its derivatization with thiobarbituric acid and
vortex assisted liquid–liquid microextraction. RSC Advances, 6(3), 2361–2367.

79.  Yehye, W. A., Abdul Rahman, N., A. Alhadi, A., Khaledi, H., Ng, S. W., & Ariffin, A. (2012).
Butylated Hydroxytoluene Analogs: Synthesis and Evaluation of Their Multipotent
Antioxidant Activities. Molecules, 17(7), 7645–7665.
 Zahin, M., Aqil, F., Husain, F. M., & Ahmad, I. (2013). Antioxidant Capacity and
Antimutagenic Potential ofMurraya koenigii. BioMed Research International, 2013, 1–10.

80. Thank You