,,, PE PUSTAKAAN UMP SHAZWANI BINTI IBRAFIIM

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1 EFFECT OF DRYING,,, PE PUSTAKAAN UMP 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

2 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 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 predrying temperature and drying periods used for each dryer to discover the effect of predrying temperature and drying period on the antioxidant compounds preserved. A

3 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

4 TABLE OF CONTENTS SUPERVISORS' DECLARATION STUDENT'S DECLARATION ACKNOWLEDGEMENT ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF EQUATIONS LIST OF ABBREVIATIONS LIST OF SYMBOLS PAGE v vi viii xi xii xiv xv xvi CHAPTER 1 INTRODUCTION 1.1 Background of the Study i 1.2 Problem Statement Research Objective Scope of the Study Significance of the Study 4 CHAPTER 2 LITERATURE REVIEW 2.1 Cosmos caudatus Antioxidant components in C. taudatus Ascorbic Acid 2.4 The Drying Methods ii Freeze Drying ii Spray Drying Oven Drying 14 viii

5 2.4.4 Significance Different of Drying Methods on Herbs Soxhlet Extraction Rotary Evaporator Types of Solvent Used in Extraction Factors Affecting Solvent Selection Significance of Different Solvent Types on Extraction of Antioxidant Compounds 31 CHAPTER 3 RESEARCH METHODOLOGY 3.1 Research Design Sample Preparation of C. caudatus Leaves C. caudatus Leaves Extraction by Using Soxhiet Method Drying Process of C. caudatus Extract Using Different Types of Dryer Determination of Antioxidant Activity of C. caudatus Extract Identification of Ascorbic Acid Content from C. caudatus Extract Using HPLC HPLC Mobile Phase Preparation HPLC Standard Curve Preparation HPLC Sample Preparation 52 CHAPTER 4 RESULTS AND DISCUSSION 4.1 Standard Curve of Ascorbic Determination of Extraction Yields From C.caudatus Effect of Drying Methods on the Antioxidant Compounds of C. caudatus Extracts Effects of Drying Methods on the DPPH Radical Scavenging Activity Of C. caudatus Extracts High Performance Liquid Chromatography (HPLC) Analysis 67 ix

6 4.6 Identification of Ascorbic Acid Content from C. caudatus Extracts 69 CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 5.1 Conclusion Recommendations 73 REFERENCES 75 APPENDIX Appendix A 81 Appendix B 84 Appendix C 86 x

7 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

8 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 Figure 4.1 Figure 4.2 Standard Curve on the Absorbance Against the Concentration of Ascorbic Acid 55 Total Percentage Yield of C. caudatus Extract from Different Types of Dryer 59 xii

9 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"

10 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

11 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

12 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

13 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 fr eshly 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

14 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

15 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

16 1.3 Scope of Study The scopes of this study include: Extracting antioxidant content from C. caudatus by using Soxhiet extraction method for eight hours for distilled water and 70% acetone solution Investigating the effect of freeze-drying, spray-drying and oven-drying method on antioxidant capacity of C. caudatus extract 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

17 preserve the antioxidant capacity by selecting drying method which gives the best results.

18 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

19 South-East Asian region including Malaysia, Indonesia and Thailand, including Africa and Asia Pacific Country (GRIN, 2012). I \ _U P ;.'r I - ';., ; -.' -. (Z Iv... S.fr 1S : 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

20 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

21 2.3 Ascorbic Acid Ascorbic acid is one of many antioxidants present naturally in fruits and vegetables. The ascorbic acid with forfnula C 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).

22 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. me Figure 2.2 The structure of ascorbic acid molecule 10

23 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 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

24 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