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A report submitted in fulfillment of the requirements for the degree of Bachelor of Applied Science (Natural Resources Science) with Honours

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(1)by. NUR HADIRAH BINTI KAMARUDIN. A report submitted in fulfillment of the requirements for the degree of Bachelor of Applied Science (Natural Resources Science) with Honours. FACULTY OF EARTH SCIENCE UNIVERSITI MALAYSIA KELANTAN. 2018. FYP FSB. DETERMINATION OF TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF EDIBLE AROIDS, COLOCASIA ESCULENTA (L.) SCHOTT.

(2) I declare that this thesis entitled “Determination of Total Phenolic Content and Antioxidant Activity of Edible Aroid, Colocasia esculenta (L.) Schott” is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.. Signature. : ___________________________. Name. : ___________________________. Date. :___________________________. i. FYP FSB. DECLARATION.

(3) “I/ We hereby declare that I/ we have read this thesis and in our opinion this thesis is sufficient in terms of scope and quality for the award of the degree of Bachelor of Applied Science (Geoscience) with Honors”. Signature. : ………………………….......... Name of Supervisor I. :. Date. : ………………………………... Signature. : ………………………….......... Name of Supervisor II. :. Date. : ………………………………... ii. FYP FSB. APPROVAL.

(4) It is a great pleasure to address people who helped me throughout this project to enhance my knowledge and practical skills especially in this research area which is related to laboratory and chemistry from University Malaysia Kelantan and Prince Songkla University. This work would not have been possible without the help and guide from my Supervisor, Dr. Tule Sirikitputtisak and Dr. Nazahatul Anis Binti Amaludin. My gratitude also been extended to my co-supervisor, Asst. Prof. Dr. Saranyoo Klaiklay and Asst. Prof. Dr. Parinuch Chumkaew who have been actively helping throughout my project timeline. I am grateful to have my fellow undergraduate students which also should be recognised for their support. Finally, I am thankful to all of those with whom I have had the pleasure to work with during this and other related projects. Nobody has been more important to me in the pursuit of this project than my family. I would like to thank my parents, whose love and guidance are with me in whatever I pursue. Most importantly, I wish to thank my friends who also doing their final year project in Prince Songkla University for their support and inspiration given to me.. iii. FYP FSB. ACKNOWLEDGEMENT.

(5) ABSTRACT Colocasia esculenta (L.) Schott is an edible aroid from the family of Araceae. It is a tuber crop which is known for its variety of cooking preparation. In this study, the petioles of Colocasia esculenta (L.) Schott was used as the samples with three types of different solvents based on their polarity in the extraction process. The aims of this studies are to determine the total phenolic content and antioxidant activity by using the Folin-Ciocalteu method and DPPH scavenging assay. Methanol shows the highest absorbance for total phenolic content test whereas the extract obtained by using 95% ethanol showed the highest inhibition for DPPH scavenging assay and IC50 values. These results indicate that Colocasia esculenta (L.) Schott has antioxidant activity including phenolic and can be consumed as food supplements to increase health benefits and lower the risk of diseases.. iv. FYP FSB. Determination of Total Phenolic Content and Antioxidant Activity of Edible Aroid, Colocasia esculenta (L.) Schott.

(6) ABSTRAK Colocasia esculenta (L.) Schott adalah sejenis tumbuhan yang boleh dimakan dari keluarga Araceae. Ia adalah tanaman umbi yang terkenal untuk penyediaan pelbagai masakan. Dalam kajian ini, batang Colocasia esculenta (L.) Schott digunakan sebagai sampel dengan tiga jenis pelarut berdasarkan kekutuban yang berbeza dalam proses pengekstrakan. Tujuan kajian ini adalah menentukan jumlah kandungan fenolik dan aktiviti antioksidan dengan menggunakan kaedah Folin-Ciocalteu dan aktiviti radikal DPPH. Metanol menunjukkan penyerapan tertinggi bagi ujian kandungan jumlah fenolik manakala ekstrak yang diperoleh dengan menggunakan 95% etanol menunjukkan perencatan tertinggi untuk ujian aktiviti radikal DPPH dan nilai IC50. Keputusan ini menunjukkan bahawa Colocasia esculenta (L.) Schott mempunyai aktiviti antioksidan termasuk fenolik dan boleh dimakan sebagai makanan tambahan untuk meningkatkan manfaat kesihatan dan mengurangkan risiko penyakit.. v. FYP FSB. Penentuan Jumlah Kandungan Fenolik dan Aktiviti Antioksidan dari Aroid Boleh Dimakan, Colocasia esculenta (L.) Schott.

(7) PAGE DECLARATION. i. APRROVAL. ii. ACKNOWLEDGEMENT. iii. ABSTRACT. iv. ABSTRAK. v. TABLE OF CONTENTS. vi. LIST OF TABLES. viii. LIST OF FIGURES. ix. LIST OF ABBREVIATIONS. x. LIST OF SYMBOLS. xi. CHAPTER 1 INTRODUCTION 1.1. Background of Study. 1. 1.2. Problem Statement. 4. 1.3. Objectives. 5. 1.4. Scope of Study. 5. 1.5. Significance of Study. 6. CHAPTER 2 LITERATURE REVIEW 2.1. General of Araceae. 7. 2.2. Morphology of Colocasia esculenta (L.) Schott. 8. 2.3. Edible Aroid. 10. 2.4. Extraction of Aroid. 11. 2.5. Total Phenolic Content. 12. 2.5.1. 13. 2.6. Method For Total Phenolic Content Test. Antioxidant Activity. 14. 2.6.1. 15. Method For Antioxidant Activity Assay. CHAPTER 3 MATERIALS AND METHODS 3.1. Materials. 17. vi. FYP FSB. TABLE OF CONTENTS.

(8) Materials for Extraction. 18. 3.1.2. Materials for Antioxidant Activity and Total Phenolic. 18. Content Tests 3.2. 3.3. Methods. 19. 3.2.1. Pre-Extraction Method. 19. 3.2.2. Extraction. 19. 3.2.3. Yield of Extraction. 20. 3.2.4. Dilution of Plant Extract. 21. 3.2.5. Antioxidant Activity DPPH Assay. 22. 3.2.6. Total Phenolic Content. 22. Data Analysis. 23. 3.3.1 Total Phenolic Content and Antioxidant Activity. 23. CHAPTER 4 RESULTS AND DISCUSSIONS 4.1. Total Yield of Extraction. 24. 4.2. Total Phenolic Content. 26. 4.3. Antioxidant Activity Assay. 29. 4.3.1. 33. IC50. CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 5.1. Conclusion. 35. 5.2. Recommendations. 36. REFERENCES. 38. APPENDIX A. 42. APPENDIX B. 43. APPENDIX C. 45. vii. FYP FSB. 3.1.1.

(9) No.. TITLE. PAGE. 4.1. Physicochemical properties of some solvents used in plant extraction. 25. 4.2. Total Phenolic Content for plant extracts. 27. 4.3. DPPH Radical Scavenging Assay for Colocasia esculenta (L.) Schott. 31. viii. FYP FSB. LIST OF TABLES.

(10) No.. TITLE. PAGE. 1.1. Taro, Colocasia esculenta (L.) Schott trees.. 4. 3.1. Colocasia esculenta (L.) Schott collected at Jeli, Kelantan.. 17. 4.1. The total yield extraction for Colocasia esculenta (L.) Schott’s. 25. petioles using three different solvents. 4.2. Standard curve of gallic acid at different concentrations. 27. 4.3. Regression curve of ascorbic acid by DPPH assay.. 29. 4.4. Graph of DPPH assay for plant extracts with different. 30. concentrations. 4.5. Bar chart showing the details on solvents used and the concentrations (µg/ml).. ix. 32. FYP FSB. LIST OF FIGURES.

(11) ABTS. 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid. Acontrol. Absorbance of DPPH without extract. Asample. Absorbance of the sample with DPPH solution. DPPH. 2,2-diphenyl-2-picryl-hydrazyl. FCR. Folin-Ciocalteu Reagent. FRAP. Ferric Reducing Antioxidant Power. GAE. Gallic Acid Equivalent. M1. Molarity of the stock solution. M2. Molarity of new diluted solution. m. Metre. mg. Milligram. mL. Millilitre. mM. Millimole. nm. Nanometre. V1. Volume of stock solution. V2. Volume of new diluted solution. µg. Microgram. µL. Microlitre. x. FYP FSB. LIST OF ABBREVIATIONS.

(12) >. Greater than. %. Percentage. ⁰C. Temperature (degree Celsius). ±. Plus-minus. xi. FYP FSB. LIST OF SYMBOLS.

(13) INTRODUCTION. 1.1. Background of the Study Tuber crops such as taro, cassava or known as tapioca, sweet potato as well as. potato are widely known as the most important tuber crops. These tubers crops have a notable importance for the agricultural, food sources, and medicinal purposes (Rao et al., 2010). According to Boyce (2012), the production of edible aroids is known in Malaysia for its specialty in cuisine. Variety of dishes can be made from edible aroid especially taro which comes from the Araceae family. Araceae is the fourth largest family of monocotyledons behind sedges, grasses, and orchids. They are one of the best influential mesophytic plant families in tropical Asia. There are several centres of diversity in the Araceae family, especially Sumatera, New Guinea, and Borneo. Boyce stated that the Araceae is a strong monophyletic family comprising about 118 genera and nearly 3,500 published species (Boyce, 2012). The most common type of aroid plant which is edible or ornamental in Malaysia is Colocasia esculenta (L.) Schott, also known as taro. They are produced for commercial purposes and are noted by their common names such as Keladi Sarawak, Keladi Pinang, Keladi Minyak, Keladi Cina, and Keladi Mawar. This taro or aroid is a plant from the arum family (Araceae) which is edible (Temesgen, 2015). 1. FYP FSB. CHAPTER 1.

(14) Oceania. Meanwhile, the bigger producers are in Asia are China, Japan, Philippines and Thailand. In Oceania, the production is dominated by Papua New Guinea, Samoa, Solomon Islands, Tonga and Fiji (FAO, 1999). In this study, Colocasia esculenta (L.) Schott is used as the samples. The plant is tall and tuberous. The plant has flower and leaf together in the middle of the branches. The leaves are simple, with a stout petiole, and lamina peltate. Furthermore, the plant has spadix which is shorter than the petiole and much shorter than the spathe and the appendix is much shorter than the inflorescence (FAO, 1999). The petioles are upright, up to 1-2 metres long with a triangular sinus cut one-third to halfway to petiole, with a dull, not polished surface above. Usually, the peduncle is shorter than the petiole, while the spathe is in pale yellow with length ranging from 15 to 35 cm (Prajapati et al.,2011). It is known that taro has several benefits which are good for human health. The fibre intake in human diet especially in the modern lifestyle that favours fast food or processed food which is high in sugars. It is estimated that 100 gram serving of taro contains about 27% of recommended daily requirement of fibre. In addition, fibre helps in digestive processes as it eases the passage of the digestive system thus resulting in less constipation, bloating, cramping and indigestion. It is important to know the nutritional value of a food source as stated by the consumers where the information on the nutritional contents of the root crops like taro is needed (Huang, 1992).. 2. FYP FSB. The largest quantity of world yield of taro is in Africa, followed by Asia and then.

(15) minerals, vitamins, lipids and others such as total lipid or fat, energy, carbohydrate, total dietary, protein, sugars, riboflavin, zinc, phosphorus, potassium, manganese and iron. In fact, they also have a higher content of starch than any other tropical root crops available (Kay, 1987). High source of starch makes the taro plant staple food for a few countries. It is important to know the nutritional values or chemical compound in the taro tuber via extraction for further studies. As stated by Yadav et al., (2016), the change in the environmental conditions now are increasing the wide range of free radicals in which the plant need to deal for its survival. Free radicals influenced the development of many disorders including inflammation, cancer, and neurodegeneration which leads to the studies of antioxidants for disease prevention and treatment. Antioxidants are natural substances or artificial that may delay or prevent some types of cell damage. Antioxidants are found in many foods, not to mention vegetables and fruits. It is a fact that oxidation reactions are important in life processes, but they can also bring damages. For the last few decades, traditional herbal medicines and dietary foods were the main source of antioxidant that protected the ancient people from the damage caused by free radicals (Baba & Malik, 2015). Thus, antioxidants are widely used in dietary supplements and have been researched for medicinal purposes. Antioxidants such as flavonoids, phenolic, proanthocyanidins, and tannins in plants give protection from many diseases. For instance, the consumption of natural antioxidants has been oppositely related to diseases. Thus, some of the plants are being studied for their antioxidant potential (Baba & Malik, 2015). There are different classes of phytochemicals from natural products, especially from plants sources. Both flavonoids 3. FYP FSB. According to Anon (2016), the taro plant has many nutrients content which are.

(16) fruits, vegetables, cereals and beverages (Genwali et al., 2013).. Figure 1.1: Taro, Colocasia esculenta (L.) Schott trees. Source:(Kamarudin, 2018). 1.2. Problem Statement The purpose of this project is to study the total phenolic content and antioxidant. activity of Colocasia esculenta (L.) Schott or taro which is one of the edible aroids in Malaysia. It is a crucial study as it may contribute to a big scale of taro production in the future. In addition, lack of awareness on the antioxidant activities and nutritional benefits of the taro that should be highlighted in order to promote its advantages in terms of nutritional values and medicinal potential. Some people might not be able to consume taro due to its irritability although there are many health benefits of taro such as controlling 4. FYP FSB. and phenolic acids are the most common polyphenols in our diet and distributed widely in.

(17) and others (Mayo et al., 1997).. Besides, in this study, the taro’s petioles will be used as samples instead of the corm as studies for the petioles are scarce. Moreover, we aim to introduce taro as one of the staple food sources due to its beneficial values contain in the tuber as well as the petioles. The author aims to study the importance of increasing the taro’s commercialization, study the antioxidant activities of the taro, and to introduce taro as one the staple food sources.. 1.3 1.. Objectives To determine the total phenolic content of Colocasia esculenta (L.) Schott’s petioles with three types of solvents (ethanol, ethyl acetate, and methanol).. 2.. To determine the antioxidant activity of Colocasia esculenta (L.) Schott’s petioles with three types of solvents (ethanol, ethyl acetate, and methanol).. 1.4. Scope of Study In this study, the aroid or taro used as the samples are the Colocasia esculenta (L.). Schott. The part of the samples used are the petioles or stems. The main steps in extraction are the reflux extraction method which uses a reflux condenser with a hot plate and a rotary evaporator. For the phenolic content test, Folin–Ciocalteu method is used and the standard reference used is gallic acid. The total phenolic content is calculated from the calibration 5. FYP FSB. blood sugar, anti-cancer properties, improve blood circulation, boosts the immune system.

(18) Meanwhile, for the antioxidant test, the method used is DPPH assay while ascorbic acid act as the positive control.. 1.5. Significance of Study It is important nowadays to know the value of the natural resources availability. and appreciate their benefits. In this research, it is highlighted that the commercial value for aroids such as taro must be continued in order for the younger or future generation to sustain these natural resources. Moreover, these aroids are having various benefits in terms of its nutritional potential. At the same time, the community can also gain income from producing the taro plantation and simultaneously helping the economy of the country. It is necessary to bring natural resources such as aroids to the bio industry sector in our country to increase the awareness of natural resources such as aroids which is beneficial for the industry. Taro plants are well known for a variety of taro chips and icecream made by locals in Malaysia. This kind of food production is in high demand from customers due to it well exposed to the world. The importance of this study is to ensure the sustainability of aroids in Malaysia as one of the natural resources. There are various ways in which the aroids can be introduced more to the community in terms of food sources or as medicinal. It is known that aroids have many beneficial values due to its properties thus ensuring the continuing production of these aroids is important.. 6. FYP FSB. curve, and the results are expressed as mg of gallic acid equivalent per g dry weight..

(19) LITERATURE REVIEW. 2.1. General of Araceae Boyce & Wong, (2012) stated that Araceae is a family of herbaceous monocots. that are under 125 genera with 3750 species. Most of the genera are in the New World tropics. The family of Araceae is greatly diverse in terms of life expectancy, the leaf morphological, and typically unique. Their existing range from a swamp to terrestrial, and to plant that grows upon another plant. The Araceae leaves are mostly plain and large in size which is easy to characterize based on its uniqueness appearance, unisexual spadix which is a spike with thick axis and peduncle. The internodes between spathe and preceding leaf that is connected by a solitary spathe, a simple bract-like foliar organ associated with spadix. As stated by Mayo et al., (1997) the Araceae are diverse in hemiepiphytes, epiphytes, lithophytes, geophytes, rheophytes, helophytes, submerged or free-floating aquatics which mostly concentrated in trophic Southeast Asia, Malay Archipelago especially Borneo, tropic southern Central America, and West Africa. The Araceae is a plant that is evergreen, lives more than two years and low maintenance. Besides, the Araceae families have two sexual orientations which is unisexual and bisexual in which unisexual Araceae is known as Aroideae which comprises half of the genera. For bisexual,. 7. FYP FSB. CHAPTER 2.

(20) Orontioideae and Pothoideae (Mayo et al., 1997). According to Mayo et al., (1997), roots in Araceae are a dimorphic root which frequently begins in climbing hemiepiphytes, for instance, Monstera deliciosa, and Philodendron bipinnatifidum. Those with bisexual flowers are common in the more primitive groups of climbing genera. Geophytes, which is the plant that have an underground storage organ are found throughout the family Aroideae.. 2.2. Morphology of Colocasia esculenta (L.) Schott In a researched done by Boyce & Wong, (2012) taro, Colocasia esculenta (L.). Schott is grown mostly as a vegetable in India. It is known that taro can adapt in moist perennial as well as others wet areas. In secondary forests, the characteristics of taro plant by the roadsides and areas near the drains are bare, mainly diploid, herbaceous, perennial plant, with a large stem at the bottom. Besides, the corms of the taro plant vary in size. Often, the corms are enclosed by a small number of secondary corms called cormels. It is one of the few crops that can adapt well to changing conditions such as soil types, rainfall, water and temperatures (Devi et al., 2013). According to Mayo et al., (1997) the stem of the plant will continue to grow by the means of a continuation shoot in the foliage of the leaf located below the spathe. Commonly, to form floral sympodium, the production of more than one must take place by the development of a prophyll, spathe and spadix. The first unit arises in the axil of the leaf immediately below the spathe and succeeding ones in the axils of the prophylls.. 8. FYP FSB. they are known as Calloideae, Lasioideae, Gymnostachydoideae, Monsteroideae,.

(21) petiole sheath except for Gymnostachys and some Biarum species which is usually tightly grabbing the subtended internode and has round insertion. Leaf blade shape and size is exceptionally diverse. From smaller size such as Ambrosina to bigger size like Xanthosoma, Alocasia, Anchomanes, Cyrtosperma, Amorphophallus. Meanwhile, shape varies through elliptic, ovate, cordate, sagittate, hastate, trifid or trisect. Taro plant can be described morphologically by qualitative and quantitative characteristics for the production of crops (IPGRI, 1999). Meanwhile, Mulualem, (2013) mentioned in his studies that there are 17 qualitative and 13 quantitative traits used in the morphologies of taro. The qualitative traits used includes leaf position, leaf blade margin, leaf blade margin colour, leaf blade colour, leaf blade colour variegation, leaf vein pattern, leaf vein main colour, petiole junction colour, petiole junction pattern, petiole colour, petiole basal-ring colour, corm skin surface, corm shape, corm cortex colour, corm flesh colour, root colour and corm blanching. For the quantitative traits used are the leaf length, leaf width, number of active leaves, length of petioles, basal ring length, maximum horizontal distance, plant height, number of sucker plant, number of tubers, length of tuber, diameter of tuber, tuber fresh and dry weight (Mulualem, 2013).. 9. FYP FSB. The Araceae genera of the leaf are distinct into an expanded blade, petiole and.

(22) Edible aroid Mulualem, (2013) has stated that the most important food aroids are from the. family of Colocasieae and Caladieae or from the species of Colocasia and Xanthosoma. Edible aroids must be cooked or prepared before can be used as food due to most of the Araceae are harmful and poisonous when it is raw. Colocasia esculenta (L.) Schott which are known as taro and Xanthosoma sagittifolium which is the other name is tannia or new cocoyam are the most important species of edible aroids. According to Hu, (2005), the part of taro plants such as caudices, corms, and cormels are consumed by boiling in China. Traditional ripen food namely Sapal is made by mixing cooked, grated the taro corm with coconut milk and allowed it to ferment at the environment temperature for the country such as Papua New Guinea (Gubag et al., 1996). As stated by Mayo et al., (1997) Colocasia esculenta is an important root crop in the Caribbean, Africa, Madagascar, Asia, and the Pacific Islands due to its humidity. In addition, the tuberous stem is a rich starch source and the leaves of certain cultivars are widely eaten like spinach. It is also known that taro tubers are richer in protein than any other major starch crops and provide a very nutritious food. All parts of the plant are toxic and must be cooked, roasted or heated before consumption. The fresh parts of the uncooked taro plant contain an irritant toxin which can rash the skin. Temesgen (2015) mentioned that it is important to know the nutritional values in our diet. Some consumers considered the beneficial value of food is prominence, thus important needs for the nutritive input for the food source. High in the starch amount in most root crops are good energy source but offer a low protein content. Temesgen also stated that a variety of minerals and some other trace elements, namely potassium and magnesium as well as iron and calcium. These are good sources of vitamins for instance 10. FYP FSB. 2.3.

(23) component which is about 73-80% of taro. Taro content varies depending on the types of soil, growing condition, variety, humidity and the fertilizer used, the point maturity when harvesting, management after the post-harvest and depository or the storage management. Commonly, the content of fat and protein at the root or corm are low but higher in fibre, minerals and carbohydrates. Despite the roots or tubers are the important parts of the plants, the leaves are also eaten by some of the countries. The leaves are can be boiled or prepared in various ways in Asia. The taro can be fried, baked, steam, or roasted in combination with coconut preparations. Taro flour can also be used in pastry filling, sausage doughnut and as a thickening agent in food. According to Lim, (2015) crushing taro corms and cormels in a mortar can produce a thick porridge which is called Achu thus making it as a well-known dish in Cameroon. In Africa, the corms are eaten roasted, boiled, or fried as an additional dish. It is also used for making Fufu, an African food, turning the corm into taro chips as a snack. Meanwhile, in Indonesia, fried corm is eaten with sugar syrup. Slicing the corm into thin slices and deep-fried to make kerepek. In addition, taro is also useful in producing taro bread, infant food, and chips.. 2.4. Extraction of Aroid Azwanida, (2015) reported that the first step in studying the nutritional compound. in the aroid is the preparation of the plant samples or the specimen itself. This step is important in order to ensure that the biomolecules in the plant parts are protected before extraction. Some parts of the plant that can be extracted either from fresh or dried parts which are barks, leaves, roots or tubers, fruits, and flowers. Grinding and drying are also 11. FYP FSB. sweet potato which has a large amount of b-carotene. Starch is the most important.

(24) of phytochemicals in the final extraction. The samples which are fresh or dried are both accepted in medicinal and nutritional plant studies. According to Seidel, (2012), there are many methods used in chemical extraction which includes maceration, ultrasound-assisted solvent extraction, percolation, Soxhlet extraction, pressurized solvent extraction, steam distillation, extraction under reflux and acid or base extraction. Biesaga, (2011) noted many different extraction techniques such as heating reflux, maceration or more innovative techniques such as microwave assisted extraction which is known for their good efficiency, less time, and solvent. Besides, Ghasemzadeh & Jaafar, (2014) reported that the extraction established by Soxhlet or reflux which requires a long period of time at high temperature may degrade phenolic acid and some others compounds. However, according to Do et al., (2014) extraction yield and antioxidant activity not only depend on the extraction method but also on the solvent used for extraction. The presence of various antioxidant compounds with different chemical characteristics and polarities may or may not be soluble in a particular solvent.. 2.5. Total Phenolic Content According to Balasundram et al., (2006) phenolic compound is an essential part of. the human diet and are of considerable interest due to their antioxidant properties towards the colour and sensory characteristics of fruits and vegetables. The phenolic compound also could be a major determinant of antioxidant potentials of foods and natural source of antioxidants. In a work done by Blainski et al., (2013) phenolic include simple phenols, phenolic acids, coumarins, flavonoids, stilbenes, hydrolyzable and condensed tannins, 12. FYP FSB. considered as another pre-preparation of the plant materials which effects the preservation.

(25) metabolites in the plant kingdom, acting mainly as phytoalexins, contributors to plant pigmentation, attractants for pollinators, and protective agents against UV light. Genwali et al., (2013) stated that natural products, notably plants are the sources of different classes of phytochemicals. Phenolic are aromatic compounds bearing one or more hydroxyl substituents. Phenolic acids are divided into derivatives of benzoic acid such as gallic acid and derivative of caeffic acid such as coumaric, caeffic and ferulic acids. Both flavonoids and phenolic acids are the most common polyphenols in our diet and distributed widely in fruits, vegetables, cereals and beverages. Do et al., (2014) also stated that phenolic compounds are the most important for dietary applications and the most widely researched. Phenolic compounds include phenolic acids, polyphenols, and flavonoids. These compounds have been used as antioxidants by humans. Alam et al., (2013) mentioned that generally, in vitro antioxidant tests using free radical traps are relatively straightforward to perform. Among free radical scavenging methods, DPPH method is furthermore rapid, simple (not involved with many steps and reagents) and inexpensive in comparison to other test models.. 2.5.1. Method for Total Phenolic Content Test As for the total phenolic content, the method that are used in this research is from. Baba & Malik, (2015). This is due to the suitability of the sample and the materials available. Folin–Ciocalteu Reagent is used to determine the phenolic content. Briefly, 0.2 mL of crude extract (1 mg/mL) were made up to 3 mL with distilled water, mixed thoroughly with 0.5 ml of Folin–Ciocalteu reagent for 3 min, followed by the addition of 2 mL of 20% (w/v) sodium carbonate. The mixtures are then left to stand for 1 hour before 13. FYP FSB. lignans, and lignins. These compounds are among the most widely occurring secondary.

(26) method to use is from Baba & Malik, (2015). According to Kamath et al., (2015), the amount of total phenolic in extracts are determined according to the Folin-Ciocalteu procedure. The samples should be prepared in triplicates. Based on studies done by Dhawan & Gupta, (2016) gallic acid was used as the standard for the test. The stock solution of the gallic acid was made by dissolving 10 mg in 100 ml of distilled water. Five different concentrations of the gallic acid were used to make a standard curve. Others than that, Sheikh et al., (2016) revealed that in his studies for this method that gallic acid is dissolved in methanol and its different concentrations are prepared prepared in methanol. The solutions are subjected to incubate at room temperature for 30 minutes with intermittent shaking. The absorbance of all the solutions was noticed at 765 nm and methanol are kept as blank.. 2.6. Antioxidant Activity Proestos et al., (2013) defined antioxidants as compounds that present in low. concentration in relation to the oxidant which prevents or delay the oxidation of the substrate. Antioxidants are important in maintaining health and from coronary heart disease and cancer. Besides, Kaneria et al., (2012) mentioned that the importance of the antioxidant constituents raised the interest among food manufacturers, scientists, and consumers as the trend of the future are moving toward functional food with specific health effects. This is due to antioxidants are known as molecules which capable in inhibiting oxidation process in the body and prevent the formation of free radicals.. 14. FYP FSB. being measured at the absorbance of 765 nm. Hence, in this studies, the most suitable.

(27) cardiac, fibrosis, kidney and liver diseases, atherosclerosis, arthritis, neurodegenerative disorders and ageing. It has been demonstrated that medicinal plants, fruits, and vegetables contain various phytochemicals with antioxidant activity, which are responsible for their beneficial health effects. The discovery of new and safe antioxidants from natural sources is of great interest for applications in natural antioxidants, functional foods, and nutraceuticals. According to Do et al., (2014) most of the plants, particularly medicinal plants, have been broadly studied for their antioxidant activity in recent years. An increased intake of food rich in natural antioxidants is associated with lower risks of degenerative diseases. Antioxidants from aromatic, spicy, medicinal, and other plants were studied to develop natural antioxidant formulations for food, cosmetic, and other applications. Additionally, according to Sheikh et al., (2016) medicinal plants are rich in antioxidants which are used to treat many human diseases. Antioxidants are the chemical substances which give their own electron to the free radicals thus check their damagecausing potential in the body.. 2.6.1. Methods for Antioxidant Activity Assay As mentioned by Dhawan & Gupta, (2016) in their studies revealed that 0.2 Mm. of DPPH solution was made using methanol. Ascorbic acid is taken as standard and five different concentrations (200,400,600,800 and 1 ml) are used to make a standard curve. The test samples are made by taking 10μg of each extract and dissolving in 2 ml of the mother solvent. Then, about 1 ml of prepared DPPH are added to all the tubes before being incubated for 60 min. The absorbance for all of the samples is taken at 517 nm. 15. FYP FSB. Moreover, free radicals cause many human diseases like cancer, Alzheimer’s,.

(28) of DPPH in 100 ml of the methanol in order to get the 0.1 mM DPPH solution. Different concentrations of the test samples are prepared in methanol and 1 ml of DPPH solution are added to the 2 ml of test samples. The mixtures are incubated for 10 minutes at room temperature and the absorbance are taken at 515 nm. The methanol is used as a blank. Meanwhile, there are studies done by Baba & Malik, (2015) in which the antioxidant activity of the extract is determined by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay with some modifications. Concisely, 200μL of each extract (100– 500μg/mL) is mixed with 3.8 mL DPPH solution and incubated in the dark at room temperature for 1 h. The absorbance of the mixture was then measured at 517 nm in which ascorbic acid is used as a positive control. Do et al., (2014) stated his method for the antioxidant activity assay of the extract are measured with the DPPH method with slight modifications. A solution of DPPH was freshly prepared by dissolving 6 mg DPPH in 50 mL methanol. 2.5 ml of the extract with different concentrations (60-220 mg/mL) are made. Then, 2.5 ml of DPPH solution are mixed together in a test tube. The test tubes are then incubated in the dark for 20 minutes at room temperature. The absorbance of the mixture was measured at 517. In this studies, the method used is as stated by Do et al., (2014) due to its method suitability with the amount of plant sample and DPPH available.. 16. FYP FSB. Besides, according to Sheikh et al., (2016) the method used is by dissolving 4 mg.

(29) MATERIALS AND METHODS. 3.1. Materials In this studies, the samples used were petioles of Colocasia esculenta L. (Schott).. The samples were collected from a village at Ayer Lanas, Jeli, Kelantan.. Figure 3.1: Colocasia esculenta (L.) Schott collected at Jeli, Kelantan. Source: (Kamarudin, 2018). 17. FYP FSB. CHAPTER 3.

(30) Materials for Extraction As for the chemicals used for extraction were 500 mL of methanol (RCL), ethyl. acetate (HoneywellTM) and ethanol (com. g). The apparatus and equipment used were the 50 mL beaker (Pyrex), 10 mL measuring cylinder (Pyrex), Glass Erlenmeyer Flask 250 mL (Pyrex), round bottom flask (Pyrex), glass rod, spatula, boiling chips, test tube, reflux condenser (Pyrex), dropper, aluminium foil, weighing balance, hot plate (IKA), and rotary evaporator (BUCHI).. 3.1.2. Materials for testing method The antioxidant activity test required the crude extract samples, 2,2-diphenyl-2-. picryl-hydrazyl (DPPH) reagent, Loba L- ascorbic acid, and ethanol (com. g). As for the total phenolic content test, the materials used were the crude extract samples, FolinCiocalteu reagent, 20% of sodium carbonate Na2CO3 (Loba), and Gallic acid powder (Sigma), and deionized water. Both tests required ethanol as the solvent to dissolve the crude extract prior to the dilution method. Besides, the tests were carried out by using Genesys 10s Ultraviolet-Visible Spectrophotometer.. 18. FYP FSB. 3.1.1.

(31) Method. 3.2.1. Pre-Extraction Method The beginning stage in this study was the preparation of plant samples to maintain. the bio-molecules in the plants proceeding to extraction. Plant samples, in this case, were petioles or stems that can be extracted either fresh or dried. Extraction is the separation of medicinal effective part of the plant using particular solvents by way of standard procedures (Handa et al., 2008). The reflux extraction method was used in this studies. The step started by cleaning and cutting the petioles into smaller pieces. Next, the petioles were left to dry in the oven with a temperature of 400⁰C for five few days until it was dried. The dried petioles are more brittle and easy to rupture. Then, the dried petioles were grounded and sieved into powder. The total weighed of powder was around 18 g and was divided into three parts, in which each consists of 5 g powdered sample before kept in the refrigerator prior to extraction.. 3.2.2. Extraction In this study, methanol, ethyl acetate, and ethanol were used as solvents. The first. solvent used for the extraction was methanol. Five grams of the powdered sample was added to the round bottom flask with three boiling chips. Then, 50 mL of the methanol was poured into the round bottom flask. Placed the round bottom flask on a hot plate and top it with the reflux condenser, securing it with a clamp which was attached to the condenser. The tubes for water in and out were set up for continuous flow of water around the condenser. The hot plate was turned on and the temperature was adjusted according to. 19. FYP FSB. 3.2.

(32) forming around the condenser. The flowing of the liquid or water vapour in the condenser into the reaction was observed. There was a reflux line in which the methanol was condensed and flowed back down as continuous stream. After six hours, the extraction was stopped. The sample was filtered by using a filter funnel and Whatman No.1 filter paper. The extracted sample was measured and evaporated by using a rotary evaporator. The rotary evaporator is used to rotate the extract in a water bath. The solvent was evaporated while the compound remained. This was the crude extract used for the antioxidant activity and total phenolic test. The same procedures were repeated for ethyl acetate and ethanol sample extractions.. 3.2.3. Yield of Extraction Percentage yield can be defined as the measurement to measure how successful a. reaction in an experiment. In this study, the crude extracts were used to carry out the test. The crude extract from three different solvents (methanol, ethanol and ethyl acetate) were weighed after being evaporated in the rotary evaporator in order to know their percentage of yield based on the formula below: Extraction yield (%) = (weight of the dried extract /weight of the original sample) × 100%. 20. FYP FSB. the boiling point of the methanol. The reaction was heated gradually until there was liquid.

(33) Dilution of Plant Extract The preparation of the stock solution for each extract was the first steps in dilution. procedures. The stock solution was prepared from the dried crude extract samples. In this study, 0.01 gram of the crude extract was dissolved with 10 mL of ethanol to make a concentration of 10 μg/mL of the stock solution. Then, five different concentrations of the plant extract were made from the stock solution. For antioxidant activity DPPH array, the concentration made were 300, 350, 400, 450, and 500 μg/mL based on a researched paper (Sheikh et al., 2016). Meanwhile, the concentrations for the total phenolic test were 20, 40, 60, 80, and 100 μg/mL. The concentrations were made based on the Eq. (3.1) as follow:. M1V1=M2V2. (3.1). M1= molarity of the stock solution V1= volume of the stock solution M2= molarity of the new diluted solution V2= volume of the new diluted solution The concentration of 20 μg/mL was made by pipette out 20 μL of the plant extract in a test tube using a micropipette. Then, 980 μL of the solvent (methanol, ethanol, ethyl acetate) was added making a total volume of 1 mL to make the dilution. The similar method was repeated for the rest of the concentrations.. 21. FYP FSB. 3.2.4.

(34) Antioxidant activity DPPH Array The antioxidant activity of the extract was determined by the 2,2-diphenyl-2-. picryl-hydrazyl (DPPH) assay with some modifications. DPPH solution was prepared by dissolving 0.6 mg of DPPH in 15 mL ethanol with a concentration of 0.1mM. Next, 2.5 mL of the extract with different concentrations (300, 350, 400, 450, 500 μg/ml) was mixed with 2.5 mL of DPPH solution in a test tube making the total solution equal to 5 mL. The test tubes were incubated in the dark for about 20 minutes at room temperature and the absorbance was measured at 517nm by using Ultraviolet-Visible Spectrophotometer. The steps were repeated with the others different solvent extract. The percentage inhibition of radicals was calculated by using the Eq. (3.2):. % of inhibition = (Acontrol-Asample)/Acontrol ×100%. (3.2). Acontrol = absorbance of DPPH without extract Asample = absorbance of the sample with DPPH solution. The half-maximal inhibitory concentration IC50 was used as the amount of antioxidant required to decrease the initial DPPH concentration by 50% (Do et al., 2014).. 3.2.6. Total Phenolic Content For the phenolic content, the method used are from Baba & Malik, (2015). All of. the tests were analyzed by using Ultraviolet-vis spectrometry (Baba & Malik, 2015).. 22. FYP FSB. 3.2.5.

(35) Data Analysis. 3.3.1. Antioxidant activity of Aroids Data analysis for the extraction of antioxidant activity and total phenolic activity. in the taro’s petioles were interpreted by using spreadsheet software. The mean and standard deviation of results were presented in tables with their antioxidant activity and total phenolic content. From the data collected, graphs were drawn to interpret or compare the antioxidant activity properties and the phenolic content in the petioles for the better understanding of the studies.. 23. FYP FSB. 3.3.

(36) RESULTS AND DISCUSSION. 4.1. Total Yield of Extraction The extracts from the dried petioles of the Colocasia esculenta (L.) Schott was. made by using three different solvents ethyl acetate, 95% ethanol and methanol. These plant extracts were extracted for about 6-7 hours in a reflux condenser. The extracted aqueous extracts were put in the rotary evaporator at 400 C to evaporate the solvent until becoming crude extracts. From the result in Figure 4.1, it shows that plant extract with 95% ethanol have the highest mass of material from the petioles of the samples meanwhile ethyl acetate extracted attain the lowest plant sample. The percentage of yield for 95% ethanol was the highest at 3.2% and 2.4% and 1.6% for methanol and ethyl acetate, respectively (*values are expressed as average ± standard deviation of triplicate measurements). Kaneria et al., (2012) stated that the extraction yield depends on solvents, time, and temperature of extraction and the chemical content of the sample itself. The solvent used and the chemical property of the sample are the two most important factors in determining the extraction yield as there are many studies which have been done regarding. 24. FYP FSB. CHAPTER 4.

(37) studies that extraction yield and antioxidant also depend on the solvent used for extraction.. Total extraction yield (%) 3.5. 3.2. Yield Extraction (%). 3 2.4. 2.5 2. 1.6. 1.5 1 0.5 0 Ethanol. Methanol Ethyl Acetate Solvents used in plant extracts. Figure 4.1: The total yield extraction for Colocasia esculenta (L.) Schott’s petioles using three different solvents. (*values are expressed as average ± standard deviation of triplicate measurements).. Table 4.1: Physicochemical properties of some solvents used in plant extraction. Solvent. Polarity. Boiling. Point Viscosity. Index. (°C). (cPoise). water (% w/w). Ethyl acetate. 4.4. 77. 0.45. 8.7. Ethanol. 5.2. 78. 1.20. 100. Methanol. 5.1. 65. 0.60. 100. Water. 9.0. 100. 1.00. 100. Source:(Seidel,2012). 25. Solubility. in. FYP FSB. the extractive yield varied with different solvents. Do et al., (2014) also mentioned in his.

(38) Total Phenolic Content. The total phenolic test was carried out by using the method stated by Baba & Malik, (2015) with a modest modification. In this method, 1 mg of crude extract was dissolved in 1 mL of ethanol. Ethanol (95%) was used in this test due to its high polarity which can dissolve most of the plant extract. The reagent used was Folin-Ciocalteu Reagent (FCR) which is responsible for determining specific protein concentrations. According to studies done by Sinica et al., (2012) FCR works on the mechanism of the oxidation–reduction reaction. The method fully depends on the reduction of the mixture heteropolyphosphotungsates–molybdates by the phenolic compound which results in the formation of the blue coloured chromogen. The phenolic compounds react with FCR only under basic conditions adjusted by sodium carbonate solution. In the total phenolic contents test, the compound used as the standard reference was gallic acid and the results are expressed as mg of gallic acid equivalent per g dry weight. The standard calibration curve was made in order to get the curve equation which is y= 0078x-0.1489, R2=0.9632 where y= absorbance measured at 765nm, x= concentration of total phenolic content in μg/mL of the extract. Figure 4.2 shows the standard calibration curve of TPC in which showing the increase in the concentration of plant samples resulting in the increase of the absorbance. The absorbance showed that as the concentration of the gallic acid is increased, the absorbance also increasing.. 26. FYP FSB. 4.2.

(39) FYP FSB. Standard Curve of Gallic Acid 0.7. y = 0.0078x - 0.1489 R² = 0.9632. 0.6. Absorbance. 0.5 0.4 0.3 0.2 0.1 0 0. 20. 40. 60. 80. 100. 120. Concentration (µg/ml) Figure 4.2: Standard curve of Gallic acid at different concentrations.. Table 4.2: Total Phenolic Content for plant extract. Extract. Total Phenolic Content (mg GAE/g extract). Ethyl Acetate. 25.885±0.001. 95% Ethanol. 30.367±0.006. Methanol. 34.345±0.001. *values are expressed as average ± standard deviation of triplicate measurements.. Table 4.2 shows the results for the total phenolic content for the plant extract with three different solvents. It is shown that the reading with methanol as the solvents was the highest. This is due to the capability of methanol to dilute the plant extract higher than ethyl acetate. 27.

(40) the methanolic extract was the highest compared to another solvent. It is known that phenolic compounds have redox properties as some antioxidants. Additionally, high in phenolic and flavonoid content influences the bioactivity of crude extracts. Moreover, another study from Do et al., (2014) revealed that the ethanol extract exhibited the highest phenolic content (40.5 mg GAE/g of defatted L. aromatic). The presence of various antioxidant compounds may or may not be soluble in a particular solvent. Polar solvents are frequently used for recovering polyphenols from plant matrices. The solvents which are the most used for chemical testing are ethanol, methanol, acetone, and ethyl acetate. Based on studies done by Dhawan & Gupta, (2016) it showed that the maximum concentration of phenol in his plant samples (Datura metel) was observed when ethyl acetate record the highest readings followed by methanol when used as extraction solvents. This may be depending on the bioactive constituents of the samples itself. It is known that ethanol is a good solvent for polyphenol extraction whereas methanol has been generally found to be more efficient in the extraction of lower molecular weight polyphenols. Bhebhe et al., (2016) stated that the highest TPC in his studies for C. carthagenensis was obtained in ethanol (50%) as the solvent is known as has been the most commonly used solvents for extracting polyphenolic compounds from plant materials. The studies also revealed that natural phenols tend to prefer solvents which have an intermediate polarity such as acetone compare to water which is high polarity or ethyl acetate and hexane which have low polarity. Krishna et al., (2013) mentioned in their studies that phenolic content also influences the oxidative stress tolerance on plants which are caused by biotic and abiotic stress conditions that produce reactive oxygen species 28. FYP FSB. Besides, Baba & Malik, (2015) also have stated that the total phenolic content of.

(41) materials that rich in phenolic are highly used in the food industry for their antioxidative properties and health benefits.. 4.3. Antioxidant Activity Assay According to Do et al., (2014) DPPH radical is a stable organic free radical which. loses absorption when accepting an electron and it is shown from the discolouration of the solution from purple to yellow. Besides, it is mentioned that DPPH can accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentrations.. DPPH Radical Scavenging Activity 92. Inhibition (%). 90. 89.008. 88. 86.998. 86.761. 89.243. y = 0.0952x + 80.84 R² = 0.7644. 86 84 82. 80.733. 80 0. 20. 40. 60. 80. 100. Concentration (µg/ml) Figure 4.3: Regression curve of Ascorbic acid by DPPH assay.. 29. 120. FYP FSB. (ROS) in plants. Most of the crude extracts from vegetables, herbs, fruits, and other plant.

(42) scavenging activity assay from their method with a slight modification. The concentration used for the plant extracts was 300, 350,400,450, and 500 μg/ml and the concentration for the ascorbic acid, the concentrations used was 20, 40, 60, 80, and 100 (μg/ml). This was based on the report from Sheikh et al., (2016) in which the sample used was Colocasia esculenta leaves and the concentration used started at 400 μg/ml until 1600 μg/ml.. DPPH Assay for Plant Extracts 80. Inhibition (%). 70 60 50 40 30 20 10 0 300. 350. 400. 450. 500. Concentration (µg/ml) Ethyl Acetate Methanol Ethanol Linear (Ethyl Acetate) Linear (Methanol) Linear (Ethanol) Figure 4.4: Graph of DPPH Assay comparing the inhibition (%) of the plant extracts different concentrations.. From Figure 4.4, it is showed that the inhibition of ethanol (95%) are the greatest as it shows the highest inhibitions in each concentration. The highest DPPH scavenging activity for ethanol was 74.11% at concentration 500 μg/ml, while for methanol 57.33% and ethyl acetate was 40.78%. In comparison, the reading of the control, ascorbic acid was 30. FYP FSB. In this studies, the antioxidant activity test was done by using the DPPH radical.

(43) ethanol was 65.96±6.24% while for ethyl acetate was 33.76±6.80 % inhibition. Studies were done by Do et al., (2014) has stated that the extract obtained by ethanol yielded the highest DPPH radical scavenging activity at concentrations ranging from 60 mg/mL to 180 mg/mL. There is a finding by (Lee et al., 2011) in which revealed that the petioles of Colocasia esculenta showed the highest antioxidant activity compared to root and leaf. It is also mentioned that the antioxidant activity of the root was the lowest in the recent study. This may be due to the different ways of the plant grows whether from cultivation or naturally growing plants.. Table 4.3: DPPH Radical Scavenging Assay for Colocasia esculenta (L.) Schott. Extract. DPPH Radical Scavenging Assay. Ethyl Acetate. 33.76±6.80%. Ethanol. 65.96±6.24%. Methanol. 50.71±5.57%. *values are expressed as average ± standard deviation of triplicate measurements.. 31. FYP FSB. 89.24% at 100 μg/ml. Table 4.3 showed the mean and standard deviation readings for.

(44) FYP FSB. Inhibition (%). DPPH Assay for Plant Exracts 80 70 60 50 40 30 20 10 0 Ethyl Acetate. Methanol Solvents 300. 350. 400. 450. Ethanol 500. Figure 4.5: Bar chart showing the details on solvents used and the concentrations (μg/ml).. Based on Figure 4.5, it shows the comparison between the three solvents used and the inhibition with the concentration of the plant extracts. The pattern for the ethanol’s concentrations as the solvent can be seen as the most efficient solvent in this assay followed by methanol and ethyl acetate. Previous studies were done by Sheikh et al., (2016) has stated that when the odd electron joins together in the presence of a free radical scavenger, the DPPH solution is decolourized from deep violet to pale yellow as the absorption reduces. The degree of reduction in absorbance measurement is indicating the power of the radical activity of the extract. In his studies, ethanolic extract of Colocasia esculenta seemed to be as effective as Vitamin C due to its IC50 value was 18.53 μg/ml.. 32.

(45) IC50 IC50 or half maximal inhibitory concentration of a compound is the amount of. antioxidant needed to decrease 50% of the DPPH concentration. It is inversely related to its antioxidant capacity which can be obtained by interpolation from a linear regression analysis. The IC50 for each plant extracts ethyl acetate, ethanol and methanol were calculated by using the equation calibrated from the Excel spreadsheet which was y= 0.0844x, y=0.1623x and y=0.1251x. Based on the results from Figure 4.6, the concentration for ethanolic plant extract achieved it 50% of inhibition with value 308 µg/ml. Plant extracts with a lower value of IC50 indicate that their antioxidant activity is higher (Do et al., 2014).. IC50 Concentration (µg/ml). 700 600. 592. 500. 400. 400. 308. 300 200 100 0 Ethyl Acetate. Ethanol Plant extracts. Methanol. Figure 4.6: The value of IC50 for the plant extracts calculated from the equation.. 33. FYP FSB. 4.3.1.

(46) recorded the highest IC50 value. It can be interpreted that ethyl acetate has lower antioxidant activity as the higher value of IC50 represented the lower antioxidant activity. As for methanol, the value was in between ethanol and ethyl acetate thus indicating that the antioxidant activity of that solvents was moderate. In a study done by Lee et al., (2011), the inhibition concentration of the Colocasia esculenta stem for ic50 was 0.125 ppt (parts per trillion, 10^-12), as for the leaf extract was 0.28 ppt and the highest value was from the corm or tuber with 4.8 ppt. These studies were done to determine the antioxidant activity as well as the anticancer activity of the species. It was found that at the concentration of 20 and 30 µg/ml, the stem and root of the samples indicated 30% of cancer cell whereas there was no anticancer activity observed in the leaf of the samples.. 34. FYP FSB. Figure 4.6 shows the value of IC50 for the plant extracts in which the ethyl acetate.

(47) CONCLUSION AND RECOMMENDATIONS. 5.1. Conclusion Colocasia esculenta (L.) Schott is known as a tuber crop which has many benefits. as it is edible. This species also has medicinal values which can be consumed by people. In this study, the experiments were conducted with the objectives to determine the total phenolic content and antioxidant activity of the plant extracts with three different types of solvents which are ethanol, ethyl acetate and methanol. The total yield of extraction for this studies revealed that it is important in determining the most suitable solvents with high polarity, extraction method whether conventional or classic, the time taken for extraction and well as the temperature of the extraction. Yield of extraction plays an important role in determining the percentage of sample extracted were left in the extraction for further use. Moreover, it is found out that the extract of Colocasia esculenta (L.) Schott petioles inhibit a fair value of total phenolic content as well as the antioxidant activity. The total phenolic content for the plant extract was carried out by using the FolinCiocalteu reagent (FCR) method with some modification. It resulted in higher total phenolic content for methanol solvent compared to ethanol and ethyl acetate which is the lowest among the other three solvents. 35. FYP FSB. CHAPTER 5.

(48) activity assay in which ascorbic acid was used as the standard reference. The results showed that 95% ethanol of plant extracts inhibit higher antioxidant activity. Some studies done by researchers mentioned that most of the solvents used with high polarity inhibit a higher antioxidant activity of the plant extracts. Besides, there was a study done by (Krishnapriya & Suganthi, (2017) stated that Colocasia esculenta (L.) Schott is acknowledged for its nutritional benefits as some parts of the plant contain 56.8% of moisture, 1.22% of ash, protein, carbohydrate and starch. It has a high nutritional value which is good to be consumed by people. Thus, it is acknowledged that edible aroid such as Colocasia esculenta (L.) Scott has many benefits and can be accepted as a staple food as it has many nutritional potential and values which is needed in daily dietary.. 5.2. Recommendations In future studies, the determination of total phenolic content and antioxidant. activity of this plant extract shall be done with more than one part of the plants. The leaves, fruits, and roots of the plants can be used in the experiment to explore more of the phenolic contents and antioxidant activity. Some parts of the plants are known to have a high antioxidant activity such as leaves and roots. Moreover, collect more samples to be used in the experiment in order to have enough and adequate weight of the samples. This is to prevent the possibility of lack in samples weight in the future experiment.. 36. FYP FSB. The antioxidant activity tests were carried out by using the DPPH scavenging.

(49) tests for certain phenolic such as tannin, alkaloid, flavonoid, steroid and terpenoid. In addition, the tests for antioxidant activity can be done through another method such as ABTS method and FRAP method. The method for plant extraction can also use the Soxhlet method in the plant extract will be extracted continuously with a less time taken compared to percolation or maceration. The extraction solvents used shall also add hexane which has the lowest polarity and water which has the highest polarity index in order to differentiate the different between solvents used. These recommendations shall be taken into account for a better study about this project in the future.. 37. FYP FSB. Besides, add more method in the experiments such as phytochemical screening.

(50) Alam, M. N., Bristi, N. J., & Rafiquzzaman, M. (2013). Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharmaceutical Journal, 21(2), 143–152. Anon. (2016). National Nutrient Database for Standard Reference for Raw Taro. Retrieved from https://ndb.nal.usda.gov/ndb/foods/ Azwanida NN. (2015). A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Medicinal & Aromatic Plants, 04(03), 3–8. Baba, S. A., & Malik, S. A. (2015). Determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of Arisaema jacquemontii Blume. Journal of Taibah University for Science, 9(4), 449–454. Balasundram, N., Sundram, K., & Samman, S. (2006). Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry, 99(1), 191–203. Bhebhe, M., Füller, T. N., Chipurura, B., & Muchuweti, M. (2016). Effect of Solvent Type on Total Phenolic Content and Free Radical Scavenging Activity of Black Tea and Herbal Infusions. Food Analytical Methods, 9(4), 1060–1067. Biesaga, M. (2011). Influence of extraction methods on stability of flavonoids. Journal of ChromatographyA, 1218 (18), 2505–2512. Blainski, A., Lopes, G. C., & De Mello, J. C. P. (2013). Application and analysis of the folin ciocalteu method for the determination of the total phenolic content from limonium brasiliense L. Molecules, 18(6), 6852–6865. Boyce, P. C & Wong, S. Y. (2012). The Araceae of Malesia I: Introduction. Malayan Nature Journal, 64(1), 9–43. Dhawan, D., & Gupta, J. (2016a). Comparison of Different Solvents for Phytochemical Extraction Potential from Datura metel Plant Leaves. International Journal of Biological Chemistry, 11(1), 17–22. Dhawan, D., & Gupta, J. (2016b). International Journal of Biological Chemistry Research Article Comparison of Different Solvents for Phytochemical Extraction Potential from Datura metel Plant Leaves Dixon Dhawan and Jeena Gupta. International Journal of Biological Chemistry, 11(1), 17–22. Do, Q. D., Angkawijaya, A. E., Tran-Nguyen, P. L., Huynh, L. H., Soetaredjo, F. E., Ismadji, S., & Ju, Y. H. (2014). Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica. Journal of Food and Drug Analysis, 22(3), 296–302. FAO. (1999). Taro Cultivation in Asia and the Pacific. Food and Agriculture Organization Of The United Nations. Rome, Italy. Genwali, G. R., Acharya, P. P., & Rajbhandari, M. (2013). Isolation of Gallic Acid and 38. FYP FSB. REFERENCES.

(51) Ghasemzadeh, A., & Jaafar, H. Z. E. (2014). Optimization of Reflux Conditions for Total Flavonoid and Total Phenolic Extraction and Enhanced Antioxidant Capacity in Pandan (Pandanus amaryllifolius Roxb.) Using Response Surface Methodology. Scientific World Journal, 2014. Gubag, R., Omoloso, D.A. , Owens, J. D. (1996). Sapal: a traditional fermented taro [ Colocasia esculenta (L.) Schott] corm and coconut cream mixture from Papua New Guinea. Int J Food Microbiol, 28(3), 361–367. Handa, S.S., Khanuja, S.P.S., Longo, G., Rakesh, D. D. (2008). Extraction Technologies for Medicinal and Aromatic Plants. United Nations Industrial Development Organization and the International Centre for Science and High Technology. Italy. Hu, S. Y. (2005). Food plants of China. Hong Kong: The Chinese University Press. Huang, A. S. and L. S. T. (1992). Application of anion exchange high-performance liquid chromatography in determining oxalates in taro (Colocasia esculenta (L.) Schott) corms. Journal of Agriculture and Food Chemistry, 40, 2123–2126. IPGRI. (1999). Descriptors for taro (Collocasia spp.). International Institute for Tropical Agriculture, Ibadan. Kamarudin, N. H. (2018). Colocasia esculenta L. (Schott). Kamath, S. D., Arunkumar, D., Avinash, N. G., & Samshuddin, S. (2015). Determination of total phenolic content and total antioxidant activity in locally consumed food stuffs in Moodbidri, Karnataka, India. Advances in Applied Science Research, 6(6), 99– 102. Kaneria, M. J., Bapodara, M. B., & Chanda, S. V. (2012). Effect of Extraction Techniques and Solvents on Antioxidant Activity of Pomegranate (Punica granatum L.) Leaf and Stem. Food Analytical Methods, 5(3), 396–404. Kay, D. E. (1987). Crop and Product Digest (2nd ed.). London: Tropical Development and Research Institute. KrishnaMurthy, A., & Rathinasabapathi, B. (2013). Oxidative stress tolerance in plants: Novel interplay between auxin and reactive oxygen species signaling. Plant Signaling and Behavior, 8(10), 1–5. Krishnapriya, T. V, & Suganthi, A. (2017). Biochemical and phytochemical analysKrishnapriya, T. V, & Suganthi, A. (2017). Biochemical and phytochemical analysis of colocasia esculenta ( L .) Schott tubers, 2(3), 21–25.is of colocasia esculenta ( L .) Schott tubers. International Journal of Research in Pharmacy and Pharmaceutical Sciences, 2(3), 21–25. Lee, S. W., Dqg, F., Urrw, W. K. H., Ulfk, L. V, Do, H. W., Wkdw, F., … Syamsumir, D. F. (2011). Antimicrobial, antioxidant, anticancer property and chemical composition of different parts (corm, stem and leave) of Colocasia esculenta extract. 39. FYP FSB. Estimation of Total Phenolic Content in Some Medicinal Plants and Their Antioxidant Activity. Nepal Journal of Science and Technology, 14(1), 95–102..

(52) Lim, T. . (2015). Edible medicinal and non-medicinal plants. Journal of Chemical Information and Modeling (Vol. 53). https://doi.org/10.1017/CBO9781107415324.004 Mayo, S. J., Mayo, S., Bogner, J. and Boyce, P. C., & Boyce, P. . (1997). The Genea of Araceae. (E. Catherine, Ed.) (illustrate). Royal Botanic Gardens, Kew. Mulualem Beyene, T. (2013). Morpho−Agronomical Characterization of Taro (Colocasia esculenta) Accessions in Ethiopia. Plant, 1(1), 1. https://doi.org/10.11648/j.plant.20130101.11 Prajapati, R., Kalariya, M., Umbarkar, R., Parmar, S., & Sheth, N. (2011). Colocasia esculenta: A potent indigenous plant. International Journal of Nutrition, Pharmacology, Neurological Diseases, 1(2), 90. https://doi.org/10.4103/22310738.84188 Proestos, C., Lytoudi, K., Mavromelanidou, O., Zoumpoulakis, P., & Sinanoglou, V. (2013). Antioxidant Capacity of Selected Plant Extracts and Their Essential Oils. Antioxidants, 2(1), 11–22. https://doi.org/10.3390/antiox2010011 Rao, V.R., Hunter, D., Eyzaguirre, P.B. & Matthews, P. J. (2010). Ethnobotany and global diversity of taro. Boiversity International. Seidel, V. (2012). Natural Products Isolation. In Methods in Molecular Biology (Vol. 864, pp. 27–42). Sheikh, M. A., Tembhre, M., & P, G. N. M. V. H. M. (2016a). Preliminary Phytochemical Screening , in vitro Antioxidant Activity , Total Phenolic and Total Flavonoid Contents of Colocasia esculenta Leaf Extract . Asian Journal of Experiment Sciences, 30(1), 39–43. Sheikh, M. A., Tembhre, M., & P, G. N. M. V. H. M. (2016b). Preliminary Phytochemical Screening , in vitro Antioxidant Activity , Total Phenolic and Total Flavonoid Contents of Colocasia esculenta Leaf Extract ., (1), 39–43. Sinica, D. P., Jadhav, A. P., Kareparamban, J. A., Nikam, P. H., & Kadam, V. J. (2012). Pelagia Research Library Spectrophotometric Estimation of Ferulic Acid from Ferula asafoetida by Folin - Ciocalteu ’ s Reagent. Pelagia Resrarch Library, 3(6), 680– 684. Sultana, B., Anwar, F., & Ashraf, M. (2009). Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules, 14(6), 2167– 2180. Temesgen, M. (2015). Nutritional Potential , Health and Food Security Benefits of Taro Colocasia Esculenta ( L .): A Review. Food Science and Quality Management, 36(June), 23–31. Vinutha, K. B., Devi, A. A., & Sreekumar, J. (2015). Morphological Characterization of above Ground Characters of Taro ( Colocasia esculenta ( L .) Schott .) Accessions 40. FYP FSB. Annales, XXIV(3), 9–16..

(53) Yadav, A., Yadav, A., Kumari, R. & Mishra, J. P. (2016). Antioxidants and its Functions in Human Body- A Review. Res. Environ. Life Sciences, 9(11), 1328–1331.. 41. FYP FSB. from North East India. Journal of Root Crops, Indian Society for Root Crops, 41(1), 3–11..

(54) Raw Material. Description The samples of Colocasia esculenta (L.) Schott petioles.. 42. FYP FSB. APPENDIX A.

(55) Pre-Extraction. Description The petioles were freshly cut and weighed.. The samples were left to dry in the oven (40⁰C) for five days.. 43. FYP FSB. APPENDIX B.

(56) The samples were blended until become powdered.. 44. FYP FSB. The dried samples..

(57) Laboratory Works. Description The extraction process of the samples.. The extracted samples.. 45. FYP FSB. APPENDIX C.

(58) Description TPC test for gallic acid as references.. The DPPH assay for ethyl acetate plant extracts.. The DPPH assay for methanolic plant extracts.. The DPPH assay for 95% ethanol plant extracts.. 46. FYP FSB. Tests.

(59) 47. FYP FSB. The UV-Visible Spectrophotometer used..

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Rujukan

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