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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

preserve the antioxidant capacity by selecting drying method which gives the best

results.

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