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,,, PE PUSTAKAAN UMP
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EFFECT OF DRYING T CAPACITY OF Cosmos
SHAZWANI BINTI IBRAFIIM
Thesis submitted in fulfillment of the requirements for the award of the degree of
Bachelor of Chemical Engineering (Biotechnology)
Faculty of Chemical and Natural Resources Engineering
UNIVERSITI MALAYSIA PAHANG
FEBRUARY 2013
EFFECT OF DRYING METHOD ON THE ANTIOXIDANT
CAPACITY OF Cosmos caudalus EXTRACT
ABSTRACT
Cosmos caudatus have high an ioxidant capacity and able to contribute to the capability
to scavenge free radical ions and reduce oxidative stress. However, the antioxidants may
decrease during the drying process which further reduces the functionality of the herb.
The purpose of this study was to investigate the effect of drying methods on the
antioxidant capacity of C. caudatus extract. Three different types of drying treatment
were set; freeze dryer (-40°C, 72 hours), spray dryer (150°C, 20 rpm of pump setting, 50
rpm of fan setting) and oven dryer (40°C, 72 hours). In this study, Soxhiet extraction
method was used and distilled water and 70% acetone (v/v) as the extraction solvent.
The determination of antioxidant and ascorbic acid content were tested by DPPH assay
and HPLC analysis respectively. Freeze-dried sample of 70% acetone (v/v) exhibits
highest concentration of antioxidant (59.00 mgAAE/g) followed by spray-dried and
oven-dried sample (37.00 mgAAE/g and 36.00 mgAAE/g) respectively. HPLC analysis
shows significant results of ascorbic acid content where freeze-dried sample of 70%
acetone (v/v) exhibits highest ascorbic acid concentration followed by spray-dried and
oven-dried samples (1.09 mgAAE/g, 0.69 mgAAE/g and 0.48 mgAAE/g) respectively.
Freeze dryer was the efficient technique for the preservation of antioxidant compounds
followed by spray dryer and oven dryer. Further study is proposed by differentiating pre-
drying temperature and drying periods used for each dryer to discover the effect of pre-
drying temperature and drying period on the antioxidant compounds preserved.
A
KESAN KAEDAII PENGERINGAN TERHADAP KAPASITI ANTIOKSIDAN
DARIPADA PENGEKSTRAKAN Cosmos caudalus
ABSTRAK
Cosmos caudatus mempunyai kapasiti antioksidan yang tinggi dan mampu menyumbang
kepada keupayaan menghapuskan ion radikal bebas dan mengurangkan tekanan
oksidatif. Akan tetapi, nilai antioksidan akan berkurangan semasa proses pengeringan
sekaligus mengurangkan fungsi herba tersebut. Tujuan kajian adalah untuk mengkaji
kesan kaedah pengeringan terhadap kapasiti antioksidan daripada pengekstrakan C.
caudatus. Tiga kaedah digunakan untuk pengeringan sampel ekstrak iaitu pengering
beku (-40°C, 72 jam), pengering semburan (150°C, penetapan pam sebanyak 20 putaran
seminit, penetapan kipas sebanyak 50 putaran seminit) dan pengering ketuhar (40°C, 72
jam). Kajian mi menggunakan kaedah pengekstrakan Soxh1et dan air suling serta 70%
larutan aseton digunakan sebagai pelarut. Aktiviti antioksidan dan kandungan asid
askorbik telah diuji dengan cerakin DPPH dan analisis kromatografi cecair berprestasi
tinggi. Sampel pengering beku daripada 70% larutan aseton mempamerkan kepekatan
tertinggi sebatian antioksidan (59.00 mgAAE/g) diikuti sampel semburan kering dan
pengering ketuhar (37.00 mgAAE/g dan 36 mgAAE/g). Analisis kromatografi cecair
berprestasi tinggi menunjukkan keputusan dimana sampel pengering beku daripada 70%
larutan aseton mempamerkan kepekatan kandungan asid askorbik tertinggi diikuti
sampel semburan kering dan pengering ketuhar (1.09 mgAAE/g, 0.69 mgAAE/g dan
0.48 mgAAE/g). Teknik pengeringan beku adalah cam yang paling berkesan dalam
pemeliharasn sebatian antioksidan diikuti oleh pengering semburan dan pengering
ketuhar. Kajian lanjut boleh dilakukan dengan membezakan suhu pra-pengeringan dan
tempoh masa pengeringan ekstrak bagi mengetahui kesan suhu pra-pengeringan dan
tempoh pengeringan terhadap pemeliharaan sebatian antioksidan.
vii
TABLE OF CONTENTS
PAGE SUPERVISORS' DECLARATION
STUDENT'S DECLARATION
ACKNOWLEDGEMENT v
ABSTRACT vi
ABSTRAK
TABLE OF CONTENTS viii
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF EQUATIONS xiv
LIST OF ABBREVIATIONS xv
LIST OF SYMBOLS xvi
CHAPTER 1 INTRODUCTION
1.1 Background of the Study i
1.2 Problem Statement 2
1.3 Research Objective 3
1.4 Scope of the Study 4 1.5 Significance of the Study 4
CHAPTER 2 LITERATURE REVIEW
2.1 Cosmos caudatus 6 2.2 Antioxidant components in C. taudatus 7 2.3 Ascorbic Acid
2.4 The Drying Methods ii 2.4.1 Freeze Drying ii 2.4.2 Spray Drying 13 2.4.3 Oven Drying 14
viii
2.4.4 Significance Different of Drying Methods on Herbs 15
2.5 Soxhlet Extraction 20
2.6 Rotary Evaporator 22
2.7 Types of Solvent Used in Extraction 24 2.7.1 Factors Affecting Solvent Selection 24 2.7.2 Significance of Different Solvent Types on Extraction of
Antioxidant Compounds 31
CHAPTER 3 RESEARCH METHODOLOGY
3.1 Research Design 37 3.2 Sample Preparation of C. caudatus Leaves 38 3.3 C. caudatus Leaves Extraction by Using Soxhiet Method 43 3.4 Drying Process of C. caudatus Extract Using Different Types of
Dryer• 44 3.5 Determination of Antioxidant Activity of C. caudatus Extract 48 3.6 Identification of Ascorbic Acid Content from C. caudatus Extract
Using HPLC 50 3.6.1 HPLC Mobile Phase Preparation 51 3.6.2 HPLC Standard Curve Preparation 51 3.6.3 HPLC Sample Preparation 52
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Standard Curve of Ascorbic 54
4.2 Determination of Extraction Yields From C.caudatus 56
4.3 Effect of Drying Methods on the Antioxidant Compounds of
C. caudatus Extracts 61
4.4 Effects of Drying Methods on the DPPH Radical Scavenging Activity
Of C. caudatus Extracts 65
4.5 High Performance Liquid Chromatography (HPLC) Analysis 67
ix
4.6 Identification of Ascorbic Acid Content from C. caudatus
Extracts 69
CHAPTER 5 CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion 72 5.2 Recommendations 73
REFERENCES 75
APPENDIX
Appendix A 81 Appendix B 84 Appendix C 86
x
LIST OF TABLES
PAGE
Table 2.1 Comparison of Various Drying Methods 17
Table 2.2 Coniparison of Different Solvent Used for Extraction
Process 34
Table 3.1 Drying Conditions for Different Drying Techniques 47
Table 4.1 Different Absorbency on the Effect of Ascorbic Acid's
Concentration 55
Table 4.2 Extraction Yields of C. caudatus Leaves Before and
After Drying Process 57
Table 4.3 Concentrations of Antioxidant Compounds 61
Table 4.4 Effect of Drying Methods on the Antioxidant Activities 50
Preserved of C. caudatus Extracts 65
Table 4.5 Concentration of Ascorbic Acid Content of C. caudatus 54
Extracts by Using HPLC Analysis 69
Table A.1 Properties of Acetone 81
Table A.2 Properties of DPPH 81
Table A.3 Properties of Water 82
Table A.4 Properties of Ascorbic Acid 83
Table A.5 Properties of Methanol 83
xi
LIST OF FIGURES
PAGE
Figure 2.1 Cosmos caudatus (Ulam raja) 7
Figure 2.2 The Structure of Ascorbic Acid Molecule 10
Figure 2.3 The Structure of Water Molecule 26
Figure 2.4 The Structure of Ethanol Molecule 28
Figure 2.5 The Structure of Methanol Molecule 29
Figure 2.6 The Structure of Acetone Molecule 29
Figure 2.7 The Structure of Hexane Molecule 30
Figure 3.1 Fresh C. caudatus (Ulam raja) Leaves 39
Figure 3.2 Cleaning Process of C. caudatus Leaves 39
Figure 3.3 Determining the C. caudatus Initial Weight 40
Figure 3.4 Spreading of Leaves on Trays for Pre-drying Process 40
Figure 3.5 Drying Process Using Oven Dryer 41
Figure 3.6 Dried Leaves of C. caudatus After 24 Hours 41
Figure 3.7 Grinding of Dried Leaves of C. caudatus 42
Figure 3.8 Soxhlet Extraction Process of C. caudatus Leaves 43
Figure 3.9 Separation of Solvent from Extraction Product 44
Figure 3.10 Freeze Dryer 45
Figure 3.11 Spray Dryer 46
Figure 3.12 Oven Dryer 46
Figure 3.13 UV-Visible Spectrophotometer 49
Figure 3.14 HPLC Analyzer 53
Standard Curve on the Absorbance Against the Figure 4.1
Concentration of Ascorbic Acid 55
Figure 4.2Total Percentage Yield of C. caudatus Extract from
Different Types of Dryer 59
xii
LIST OF FIGURES
PAGE
Figure 4.3 The Concentration of Antioxidant Compounds from 62
Different Drying Treatment
Figure 4.4 The-Percentage of DPPH Radical Scavenging Activity 66
from Different Drying Treatment
Figure 4.5 Standard Curve on the Identification of Ascorbic Acid 68
Content of C. caudatus by Using HPLC Analysis
Figure 4.6 The Difference of Ascorbic Acid Concentration from 70
Different Drying Treatment
Figure B.1 Extract sample of C. caudatus Using Distilled Water 84
Figure B.2 Extract Sample of C. caudatus Using 70% Acetone (vlv) 84
Figure B.3 Samples of Water Extract from Three Different Dryer 85
Figure B.4 Samples of 70% Acetone Extract from Three Different 85
Dryer
xl"
LIST OF EQUATIONS
PAGE
Equation 3.1 Percentage of Moisture Lost 42
Equation 3.2 PeFcentage of Yields 47
Equation 3.3 DPPH Radical Scavenging Activity 50
Equation 3.4 Linear Equation 52
xiv
LIST OF ABBREVIATIONS
AAA E - Ascorbic acid equivalent
C. caudatus - Cosmos caudatus
DPPH - '2;24liphenyl- 1 -picryihydrazyl
EtOH - Ethanol
hr - Hour
KH2PO4 - Potassium dihydrogen phosphate
MeOH - Methanol
mm - Minute
OD - Optical Density
rpm - Rotation per minute
T - Temperature
UV - Ultra-violet
UV-Vis - Ultra-violet Visible
xv
LIST OF SYMBOLS
% - Percentage
tL - Micro-Liter
g 'Gi'ath
g/mL - Gram per milli-Liter
L - Liter
mAU.s - milliabsorbance units
mg/g - milli-gram per gram
mg/L - milli-gram per Liter
mglmL - milli-gram per milli-Liter
mgAAE/g - milli-gram Ascorbic Acid Equivalent per gram of extract sample
mgAAE/mL - milli-gram Ascorbic Acid per milli-Liter
ML - Moisture lost
mL - milli-liter
nm - nanometer
°C - Degree Celsius
OF - Degree Fahrenheit
v/v - Ratio of solvent to water
w/w - Mass of sample per mass of dry extract
xvi
CHAPTER 1
INTRODUCTION
1.1 Background of Study
Herbs are very significance with their advantages to improve healthiness and
prevent diseases. One of herb that is rich in nutrients is Cosmos caudatus. C. caudatus
belongs to the family of asteraceae, an edible plant having about 20 to 26 species
worldwide (Md Rasdi et al., 2010). It is an annual and short-lived aromatic herb with
purple, pink or white ray florets, grows up from one to eight feet tall, hairless or sparsely
hairy, and the leaves are finely dissected, 10 to 20 cm long (Sliui, Lai & Shih, 2005).
C. caudatus is originated from tropical Central of America and already
widespread in almost part of tropicl regions including Malaysia, Thailand and other
South-East Asian countries, Mexico, and South America. C. caudatus usually consumed
freshly as salad or cooked. They are also used as an appetizer and natural food seasoning
due to their unique taste and aroma. In Malaysia, C. caudatus is well-known as ulam raja
(King's salad), in Indonesia, it is known as kenikir or randa midang, and in Thailand, it
1
is called daoruang-phama or khamhae (Global Information Hub on Integrated Medicine,
2010).
The freshness of herbs cannot last longer although been refrigerated for a long
period of time, and may affect the nutrient mainly antioxidants presence. Therefore,
searching of technique that is suitable to be used to preserve the bioactive nutrients is
strongly encouraged although is it not consumed freshly. One of the best methods is by
drying process. There are various drying techniques that can be used in which give
different results based on the method of drying, drying period and temperature used for
the drying process.
1.2 Problem Statement
Drying is the oldest method of preserving food by removing water content inside
foods. Examples of foods that usually been dried are fruits and herbs such as corn, apple
slices and kiwi slices, ginger, peppermint, meats and fishes and others (Troftgruben,
1977). In addition, dried foods can last-longer and no microorganisms are able to grow
because of low humidity of water content. However, drying will not preserve the taste,
texture and appearance of the foods compared to canning and freezing methods. This is
because of the changes of the water content inside the food that may affect the taste
itself, the shrinkage of the dried product and also the change in dried foods colour. There
are lots of methods to dry foods; by air drying, oven drying, spray drying and also freeze
drying.
2
Although drying of foods helps in preserving and ensure long-lasting of foods'
expiry date, there are problems in drying method especially for herbs processing. It is
known that herb is rich in nutrients that can be easily affected by high temperature when
drying process octtfrs. Active chemical compounds such as various types of antioxidants
compounds and phenolic contents may decrease as the drying process occurred thus
reducing the functionality of the herbs. The total antioxidant compounds preserved
during drying process is affected by several factors. The factors include types of drying
method used, temperature applied for drying process and types of solvent used for
extraction process.
Therefore, it is essential to determine which drying methods that may be the best
to be used to preserve the antioxidant capacity of C. caudatus in addition with type of
solvent used as extraction medium so that the beneficial of its nutrients can be
manipulated and used as supplement or food additive to increase nutrients intake.
1.3 Research Objective
The objective of this research is to study the effect of drying method on the
antioxidant capacity of C. caudatus extract.
3
1.3 Scope of Study
The scopes of this study include:
1.4.1 Extracting antioxidant content from C. caudatus by using Soxhiet extraction
method for eight hours for distilled water and 70% acetone solution.
1.4.2 Investigating the effect of freeze-drying, spray-drying and oven-drying method
on antioxidant capacity of C. caudatus extract.
1.4.3 Identifying the ascorbic acid content from C. caudatus extracts by using High
Performance Liquid Chromatography (HPLC).
1.5 Significance of Study
C. caudatus has been used traditionally in various scopes as remedy to cure
diseases. The effectiveness of the remedies is studied so that the usefulness of this
substance can be proving scientifically. Antioxidant contents of C. caudatus leaves were
studied in relation to the processes involved in food and herbal medicine preparation.
(Sukrasno et al., 2011). Thus, the antioxidant capacity of the herb must be preserved so
that the production yields of supp1ernnts or new applications can be perform. The best
drying methods also must be known in search of the best preserved antioxidant capacity
from C. caudatus extract. Fresh C. caudatus is rich in flavonoid and antioxidant
activities. Therefore, it is best to manipulate this content by using drying method to
4
CHAPTER 2
LITERATURE REVIEW
2.1 Cosmos caudatus
Cosmos caudatus (genus: Cosmos, family: Asteraceae) are plants that can be
eaten raw mainly its leaves part as a form of local salad. It is a well-known traditional
salad that got a high demand on market nowadays not only because of its unique aroma
and taste, but also because of its high nutritional values. The main nutrients of C.
caudatus are vitamins and minerals in which ensure their consumers' health with no
need of modern supplements.
Natively, C. caudatus can be found in Northern and Southern Mexico, Southern
America such as Costa Rica, El Salvador, Guatemala, and Panama, Northern South
America such as Venezuela and Western South America like Colombia. Because of its
ability to grow wild especially in tropical region, C. caudatus can be easily found in
mo
I •\ •_U
P -. .-
• .. ;.'r
Iv ... S
.fr 1S :--__
South-East Asian region including Malaysia, Indonesia and Thailand, including Africa
and Asia Pacific Country (GRIN, 2012).
I - '; .,
; -.' -. (Z
..- - . .
r- 1 / - . .'ë• -. t •-'
Figure 2.1 Cosmos caudatus (Ulam raja)
2.2 Antioxidant Components in C. caudatus
Antioxidant molecules are used to neutralize the free radical ions inside body by
supplying another electron to balance it. Free radical molecules which are created from
oxidative process of chemical reaction, exposure to pollution, cigarette smokes and fatty
foods in the body are unstable because of the loss of electron and able to react with other
molecules to form other free radical. Omenn, Goodman and Thomquist (2009) identifies
that massive amount of free radical niolecules inside body may cause damage to the cell,
and have relation to the development of heart and liver disease, cancers, arthritis,
accelerated aging and cataract. Without antioxidant, our body cells are unable to
rejuvenate them and can easily been damage. This may results in improper health for
human.
7
C. caudatus plays an important role to nature to be used as remedy for various
diseases. Based on previous research, it is indicated that the C. caudatus had extremely
high antioxidant capacity especially proanthocyanidins and quercetin which is believed
to have some medicihàl fuiictions (Shih, Lai & Koh, 2005). It contributes to the ability
to scavenge free radicals and reducing oxidative stress (Sukrasno et al., 2011). Analysis
of the extractions of C. caudatus leaves shows highest linoleic acid oxidation, DPPH,
ABTS, and ferric cyanide antioxidant capacities as compared to other vegetables tested
(Andarwulan et al., 2010). This results supported by Shui, Lai & Shih (2005) which
prove that the Cosmos caudatus had the highest DPPH free radical scavenging activity.
It is reported that C. caudatus contains several types of bioactive components for
examples, antimutagen and antifungal compounds. This is supported by Md Rasdi et al.
(2010) that showed antimicrobial activity against microorganisms tested. This result
shows the effectiveness and multiple functions of Cosmos caudatus in which beneficial
to the society. It is necessary to search for new methods of preserving antioxidant
components rather than depending on intake of fresh raw vegetables which are enrich in
vitamins and minerals. Altering the fresh raw of C. caudatus as a new potent in dry
condition and used as supplements or food additives may be useful for obtaining enough
nutrients needed. By preserving the antioxidant contents of C. caudatus in dry state
helps in prolonging the antioxidant compounds durability. Thus, is it important to
determine the best drying method need to be used for this study to obtain highest amount
Of antioxidant compounds preserved from the drying process.
8
2.3 Ascorbic Acid
Ascorbic acid is one of many antioxidants present naturally in fruits and vegetables.
The ascorbic acid with forfnula C 611806 appears in white to yellowish colour in powder
and crystal form with odourless characteristic. Ascorbic acid is a light sensitive
substance but stable under normal temperature (Sigma-Aldrich, 2012; Sciencelab.com
Inc., 2005). Ascorbic acid is a type of formerly known antioxidant presents richly in
fruits and vegetables. Ascorbic acid also known as Vitamin C is water soluble vitamins
which can only be obtained through consumptions of foods enriched in Vitamin C such
as citrus fruits, dark leafy greens, broccoli and herbs. This type of vitamin is easily
excreted from the body. Thus, continuous supply of Vitamin C is needed to ensure
enough continuous supply to fulfill the requirement of body cells instead of depending
on the cell-self production (The George Mateljan Foundation, no date).
Antioxidant such as Vitamin C is an essential nutrient required to help in reducing
the oxidative stress of free radical ions inside the body. Vitamin C helps to reduce
potency of some damages on the body caused by free radicals by which able to cause
cancer, heart disease and also arthritis. In addition, higher free radicals are also
responsible for the increase rate of aging process (U.S. Natinal Library of Medicine,
2012). Vitamin C also helps to promote effective production of collagen. Collagen is a
type of protein which is important to be used for healing wounds on skins, build up
structure to bones, tendons cartilages, muscles and blood vessels. Furthermore, Vitamin
C also helps for iron absorption inside the body. The United States Adequate Intake (Al)
outlined the intake of Vitamin C is 90 milligrams per day and 75 milligrams per day for
men and women respectively (Mosure, 2004).
me
Despite of lessen the oxidative stress, Vitamin C is also important for maintaining
healthy body from diseases. Some common diseases related to the deficiency of Vitamin
C are scurvy, weak immune functions and lung-infections. Scurvy is a disease where
lacking of Vitamin Cócc1if. Symptoms of scurvy are bleeding of gums, loosened teeth,
loss of appetite, bleeding in eyes and others (Medical News Today, 2009). Other than
that, peoples who lack of Vitamin C may have weak immune systems and easily affected
by colds and other infections. Respiratory tracts in which depends a lot with Vitamin C
for protection may affected by respiratory infection and lung-related conditions in
lacking of Vitamin C (The George Mateljan Foundation, no date).
The ascorbic acid is widely used in food and beverages processing industry for its
nutritional values and also for its ability to preserve product's quality. The ascorbic acid
is added to the processed fruit juices to restore or as fortified nutrient and also helps to
improve the colour and the appearance of products. According to Takeda U.S.A
Incorporation (no date), ascorbic acid may act as an oxidizing agent in surrounding with
low oxygen concentration. Thus, the oxidizing agent reduces the available oxygen
present in environment. This action makes the ascorbic acid as an effective antioxidant.
Figure 2.2 The structure of ascorbic acid molecule
10
2.4 The Drying Methods
One of methods that always been use to preserve foods like herbs is by using
drying method.'. DryiñgiS a process where the water content been removed from foods.
Low of water content and humidity helps to ensure for long-lasting of foods because of
less possibility for germination of microorganisms. There are lots of method to dry
foods, some of well-known drying process including freeze drying, spray drying and
also oven drying. For each drying method, there are pro and cons depending on what
types of foods that we want to dry. Different method results in different quality of foods.
Thus, the best method to be used is depending on the types of foods, water content,
times taken and also preservation of foods' nutrients as well as appearance and colour.
2.4.1 Freeze Drying
Freeze drying process has been widely use in a number of applications especially
in food and pharmaceutical industries. Freeze drying also known as lyophilization is a
dehydration process which preserves perishable material. This process can be done by
freezing material (food) and the reducing the surroundings pressure to allow sublimation
of frozen water. Sublimation proces 's occurs when a frozen liquid directly goes into
gaseous state without turned into liquid phase first (Labconco Corp., 2010). There are
three stages in the process of freeze drying; prefreezing, primary drying and secondary
drying.
11
For the pre-freezing stage, fresh or cooked foods are flash frozen, and then
placed in a vacuum chamber (Emergency Essentials, 2012). The methods of handling
the pre-frozen and the final temperature of the products' frozen can affect the
successfulness offreeze dry process. Next, during primary drying, about 98% of the
foods moisture is evaporated by evaporating process of the ice. This is done at
temperature as low as -50°F (Emergency Essentials, 2012). Conditions must be
established in which ice can be removed from the frozen product. This step requires two
important parameters, which are temperature and pressure (Labconco Corp., 2010). For
secondary drying step, Continued drying is necessary at the warmer temperature to
reduce the residual moisture content to optimum values. Heat or high temperature is
applied to the frozen product to accelerate the sublimation process so that the remaining
moisture can be removed. Next step, low temperature condenser plate remove the
vaporize solvent from the vacuum chamber by converting it back to solid. This complete
the separation process (Bellis, 2012).
Some advantages of using freeze drying method are freeze dried products
maintain the nutrients, shape and size, colour, flavor, and also the texture of the food
products. In addition, the frozen product reconstitute to its original state when placed in
water, no requirement for cold storage and offers highest quality in a dry product.
However, this method have its own disadvantages, including need expensive equipment
for freeze drying process, cost effective for selective products and usually restricted to
delicate, heat sensitive materials of high value (Emergency Essentials, 2012).
12