(1)FYP FIAT ANTIOXIDANT POTENTIAL AND CHEMICAL ANALYSIS OF FRESH AND DRIED YOUNG LEAVES OF Flemingia Macrophylla (WILLD.) MERRILL (LEGUMINOSAE)

Tekspenuh

(1)FYP FIAT ANTIOXIDANT POTENTIAL AND CHEMICAL ANALYSIS OF FRESH AND DRIED YOUNG LEAVES OF Flemingia Macrophylla (WILLD.) MERRILL (LEGUMINOSAE). By SHAHIRAH BINTI AHAMAD. A report submitted in fulfilment of the requirements for the degree of Bachelor of Applied Science (Technology Product Development) with Honours. Faculty of Agro Based Industry UNIVERSITI MALAYSIA KELANTAN 2018.

(2) I hereby declare that the work embodied in this report is the result of the original research and has not been submitted for a higher degree to any universities or institutions.. ______________________ Student Name. : SHAHIRAH BINTI AHAMAD. Matric no: F15A0204 Date. :. I certify that the report of this final year project entitled “Antioxidant Potential and Chemical Analysis of Fresh and Dried Young Leaves of Flemingia macrophylla (Willd.) Merrill (Leguminosae)” by Shahirah Binti Ahamad, matric number F15A0204 has been examined and all the correction recommended by examiners have been done for the degree of Bachelor of Applied Science (Product Development Technology) with Honours, Faculty of Agro-Based Industry, Universiti Malaysia Kelantan.. Approved by: ___________________ Supervisor Name: Dr Shamsul Bin Muhammad Date:. FYP FIAT. DECLARATION.

(3) In the name of Allah, the Most Graceful and the Most Merciful.. All praise to Allah, for giving me many opportunity and chances in order to assist me completing the final year project thesis in fulfilling the requirement of my Degree of Bachelor of Applied Science (Hons.) Technology Product Development. I finally finished my thesis entitled “Antioxidant Potential and Chemical Analysis of Fresh and Dried Young Leaves of Flemingia Macrophylla (Willd.) Merrill (Leguminosae)” in a given time. Upon the completion of this thesis, it had given me a lot of new knowledge and experience that may be very useful for me in the future.. First and foremost, I would like to dedicate my sincere thanks to Dr Shamsul bin Muhammad and Miss Nurul Amira binti Buslima, my supervisors who gives a lot of useful guidance and advice during my research. I was able to gain a lot of beneficial information and knowledge through their comments and views. Without their modesty in sharing the knowledge, I cannot assure the success of this thesis. I also would like to acknowledge all the laboratory assistant for their dedication and cooperation in providing the chemicals and equipments during conducting the laboratory work.. Moreover, I would like to express my gratefulness to the big family of mine, especially to my parents, my husband Mohd Arif Syafiq bin Muhammad Ridhwan Pang, and my parent-in-law for their endless moral and financial support that are needed upon the completion of this thesis. They had been a source of encouragement and motivation for me in completing this project. Thank you for the endless support and love through years.. Besides, a sincere appreciation to Cik Siti Fatimah, a postgraduate student for her guidance and advices during conducting laboratory work and the contribution of ideas to keep me writing this thesis. Lastly, I would like to express my utmost gratitude to all my lecturers and friends, especially Esther, Syuhada, Hazirah and Izzanis that have involved whether directly or indirectly towards the compilation of this thesis. i. FYP FIAT. ACKNOWLEDGEMENT.

(4) Macrophylla (Willd.) Merrill (Leguminosae). ABSTRACT. Flemingia macrophylla (Willd.) Merrill (Leguminosae) is a medicinal plant that had been used for traditional treatment and sources of remedies. Locally, the F. macrophylla is locally known as ‘pokok seringan jantan’. The decoction F. macrophylla leaves had locally been used as fever and smallpox treatment. However, there are still lack of scientific evidence on phytochemical and functional properties of the leaves part of F. macrophylla. The fresh leaves of F. macrophylla are frequently used in traditional treatment, however for further utilising this plant in industrial application, dried leaves are more preferable by industry. There is also an urge in searching for agro solvent such as glycerol which is safer to be used in extraction. Glycerol solvent has been used due to its natural and safety properties, however, there are lack of study about the effectiveness of glycerol in extraction. In the present study, antioxidant potential of the ethanol and glycerol extracts of the young leaves of F. macrophylla were evaluated by using phytochemical screening, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay, total phenolic content (TPC) and total flavonoid content (TFC) in order compare the effectiveness of the solvent. The extract showed significant activities (p ≤ 0.05) in DPPH and TPC, however, no significant different was observed in TFC assay. Moreover, F. macrophylla extract showed strong reducing power. These results suggest that F. macrophylla may act as a chemo preventative agent, providing antioxidant properties and offering effective protection from free radicals. Apart from that, glycerol solvent showed better antioxidant activity and it is good enough to substitute ethanol solvent, thus support the usage of safer solvent extract. Dried sample also showed better extraction result against fresh sample in quantification assay. As conclusion, the young leaves of F. macrophylla is worthy to further discover its antioxidant compound and other curative compound by using safer solvent.. Keywords: Flemingia Macrophylla (Willd.) Merrill (Leguminosae), antioxidant activities, DPPH radical scavenging, glycerol extraction, phytochemical screening. ii. FYP FIAT. Antioxidant Potential and Chemical Analysis of Fresh and Dried Young Leaves of Flemingia.

(5) ABSTRAK. Flemingia macrophylla (Willd.) Merrill (Leguminosae) adalah sejenis tumbuhan perubatan yang telah digunakan sebagai rawatan tradisional. Di kalangan penduduk tempatan, F. macrophylla dikenali sebagai 'pokok seringan jantan'. Rebusan daun F. macrophylla telah digunakan sebagai rawatan demam dan cacar. Walau bagaimanapun, masih terdapat kekurangan bukti saintifik terhadap sifat fitokimia dan fungsi daun F. macrophylla. Daun segar F. macrophylla sering digunakan dalam rawatan tradisional, namun untuk menggunakan tumbuhan ini dalam aplikasi perindustrian, daun kering lebih sesuai untuk industri. Terdapat juga usaha untuk mengenalpasti pelarut agro seperti gliserol yang lebih selamat digunakan untuk pengekstrakan. Pelarut gliserol telah digunakan kerana sifat semulajadi dan keselamatannya, namun, masih kekurangan kajian tentang keberkesanan gliserol dalam pengekstrakan. Dalam kajian ini, potensi sebagai antioxidan daripada ekstrak etanol dan gliserol daun muda F. macrophylla dinilai dengan menggunakan pemeriksaan fitokimia, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) pengedaman assay, jumlah kandungan fenolik (TPC) dan jumlah kandungan flavonoid (TFC) untuk membandingkan keberkesanan pelarut tersebut. Ekstrak menunjukkan aktiviti-aktiviti penting (p ≤ 0.05) dalam DPPH dan TPC, bagaimanapun, tiada aktiviti penting yang diperhatikan dalam ujian TFC. Tambahan pula, ekstrak-ekstrak F. macrophylla menunjukkan daya pengurangan yang kuat. Keputusan ini mencadangkan bahawa F. macrophylla boleh bertindak sebagai agen chemopreventative, menyediakan sifat antioksidan dan menawarkan perlindungan yang berkesan dari radikal bebas. Selain itu, pelarut gliserol menunjukkan aktiviti antioksidan yang lebih baik dan ia cukup baik untuk menggantikan pelarut etanol, dengan itu menyokong penggunaan ekstrak pelarut yang lebih selamat. Sampel kering juga menunjukkan hasil pengekstrakan yang lebih baik terhadap sampel segar dalam ujian kuantifikasi. Sebagai kesimpulan, daun muda F. macrophylla adalah layak untuk terus menemui sebatian antioksidannya dan sebatian yang bersifat penyembuhan yang lain dengan menggunakan pelarut yang lebih selamat. Kata kunci: Flemingia Macrophylla (Willd.) Merrill (Leguminosae), aktiviti antioksidan, penangkapan radikal DPPH, pengekstrakan gliserol, pemeriksaan fitokimia. iii. FYP FIAT. Analisis Keupayaan dan Analisis Antioksidan Daun Muda Segar dan Kering Flemingia Macrophylla (Willd.) Merrill (Leguminosae).

(6) PAGE DECLARATION. ii. ACKNOWLEDGEMENT. iiii. ABSTRACT. iv. ABSTRAK. v. TABLE OF CONTENTS. vi. LIST OF TABLES. ix. LIST OF FIGURES. xi. LIST OF ABBREVIATION. xiii. LIST OF SYMBOLS. xv. CHAPTER 1 INTRODUCTION 1.1 Research Background 1.2 Problem Statement 1.3 Hypothesis 1.4 Objectives 1.5 Scope Of Study 1.6 Significance Of Study. 1 2 3 3 4 4. CHAPTER 2 LITERATURE REVIEW 2.1 Flemingia macrophylla (WILLD.) MERRILL 2.1.1 Taxonomy and Nomenclature 2.1.2 Plant Morphology 2.1.3 Ethnobotanical Uses 2.1.4 Phytochemical Study. 6 7 9 11. 2.2 ANTIOXIDANT 2.2.1 Free Radical Activity And Oxidative 2.2.2 Source Of Antioxidant 2.2.3 Type Of Antioxidant 2.2.4 Classification Of Antioxidant. Stress. 2.2.5 Effect On Application Antioxidant Therapy. iv. 12 13 14 15. FYP FIAT. TABLE OF CONTENTS.

(7) CHAPTER 3 MATERIALS AND METHODS 3.1 Chemical And Reagent 3.2 Instruments And Consumables 3.3 Plant Collection 3.4 Plant Preparation 3.5 Plant Extraction 3.6 Assessment of Phytochemical Screening 3.7 Assessment Of Antioxidant Activity 3.8 Estimation Of Total Phenolic Content 3.9 Estimation Of Total Flavonoid Content 3.10 Statistical Analysis. 16 17 18 19 20 21 21 22 22. 24 24 25 26 27 27 29 30 30 31. CHAPTER 4 RESULTS AND DISCUSSION 4.1 Extraction 4.2 Phytochemical Screening 4.3 Dpph Radical Scavenging Assay 4.4 Total Phenolic Content 4.5 Total Flavonoid Content. 33 35 39 44 47. CHAPTER 5 CONCLUSION 5.1 Conclusion 5.2 Recommendation. 51 52. REFERENCES. 53. APPENDIX A: CALCULATION. 57. APPENDIX B: SPSS OUTPUT. 64. APPENDIX C: PHOTOS OF EXPERIMENT. 68. v. FYP FIAT. 2.3 SOLVENT EXTRACTION 2.3.1 Raw Material Extraction Issue In Cosmetic Industry 2.3.2 Factors Considered When Selecting A Solvent 2.3.3 Organic Solvents 2.3.4 Green Solvents, The Agro- / Bio-Solvents 2.4 SAMPLE PREPARATION 2.4.1 Fresh Sample Condition 2.4.2 Dried Sample Condition 2.4.3 Non Thermal Drying Technique 2.4.4 Thermal Drying Technique 2.4.5 Effect Of Sample Preparation On Antioxidant Activity.

(8) NO.. PAGES. 4.1. Extract characteristic of different F. macrophylla sample condition. 33. 4.2. Phytochemical screening of F. macrophylla young leaves extract. 36. 4.3. Total Phenolic Content of F. macrophylla young leaves extract. 46. 4.4. Total Flavonoid Content of F. macrophylla young leaves extract. 49. A.2.1 IC50 value of ethanol extract of dried F. macrophylla young leaves. 58. (ED) A.2.2 IC50 value of ethanol extract of fresh F. macrophylla young leaves. 58. (EF) A.2.3 IC50 value of glycerol extract of dried F. macrophylla young leaves. 59. (GD) A.2.4 IC50 value of glycerol extract of fresh F. macrophylla young leaves. 59. (GF) A.2.5 IC50 value of standard Ascorbic Acid. 59. A.3.1 TPC of ethanol extract of dried F. macrophylla young leaves (ED). 60. A.3.2 TPC of ethanol extract of fresh F. macrophylla young leaves (EF). 60. A.3.3 TPC of glycerol extract of dried F. macrophylla young leaves (GD). 61. A.3.4 TPC of glycerol extract of freshF. macrophylla young leaves (GF). 61. A.4.1 TFC of ethanol extract of dried F. macrophylla young leaves (ED). 62. A.4.2 TFC of ethanol extract of fresh F. macrophylla young leaves (EF). 62. A.4.3 TFC of glycerol extract of dried F. macrophylla young leaves (GD). 63. A.4.4 TFC of glycerol extract of fresh F. macrophylla young leaves (GF). 63. B.1. Analysis of variance of Antioxidant Content and Antioxidant. 64. Activity B.2. Post hoc Test Total Phenolic Content. 64. B.3. Post hoc Test Total Flavonoid Content. 65. B.4. Post hoc Test DPPH Scavenging Assay. 65. B.5. Pearson Correlation between Antioxidant Content and Antioxidant. 66. Activity B.6. Test Normality of Assays. 66. vi. FYP FIAT. LIST OF TABLES.

(9) NO.. PAGE. 2.1. Flemingia macrophylla’s specimen. 5. 2.2. The structure of fleminigin compound. 10. 3.1. The young leaves of F. macrophylla plant grow parallel to gravity. 25. 3.2. a) Young and matured trifoliate leaves; b) Flower bract; c) Fruit pod. 25. 3.3. Flow chart of research activities. 32. 4.1. Calibration curve of Ascorbic Acid by DPPH assay. 40. 4.2. DPPH Radical Scavenging Activity of F. macrophylla young leaves. 41. extracts at different sample concentration. 4.3. IC50 value of F. macrophylla young leaves extract. 42. 4.4. Calibration curve of Gallic Acid by standard Folin-Ciocalteu method. 45. 4.5. Basic structure of flavonoid. 47. 4.6. Calibration curve of quercetin by Aluminium Chloride Colorimetric. 48. method C.1. Ground fresh and dried F. macrophylla leaves sample. 68. C.2. Extraction process in 90 % ethanol and 70 % glycerol solvent. 68. C.3. Filtering process. 68. C.4. Fresh leaves in glycerol extract (GF), dried leaves in glycerol extract. 69. (GD), dried leaves in ethanol extract (ED), fresh leaves in ethanol extract (EF) C.5. The appearance of bluish black colour in Ferric chloride test. 69. C.6. The formation of reddish-brown precipitate in Wagner test. 69. C.7. Intense yellow colour in solution before addition of hydrochloric acid. 70. in Alkaline Reagent test C.8. Disappearance of yellow colour in solution after addition of hydrochloric acid in Alkaline Reagent test vii. 70. FYP FIAT. LIST OF FIGURES.

(10) The appearance of reddish-brown colour at the upper layer of. 70. Salkowski’s test C.10 Standard ascorbic acid for DPPH Antioxidant Activity Test. 71. C.11 Standard gallic acid for Total Phenolic Content (TPC) test. 71. C.12 Standard quercetin for Total Flavonoid Content (TFC) test. 71. viii. FYP FIAT. C.9.

(11) g min nm µg µl ml mM M rpm ft mg/ml ANOVA BHA BHT CABI DPPH EPPO GAE HAT IC50 MOPI QE R² ROS SET SD Sig SOP SPSS 10 TFC TPC UV-VIS VOC LC-MS. gram minute nanometre microgram microlitre millilitre millimolar molar revolutions per minute foot microgram per mililitre Analysis of variance Butylated hydroxy anisole Butylated hydroxy toulene Centre for Agriculture and Biosciences International 2,2-diphenyl-1-picryhydrazyl The European and Mediterranean Plant Protection Organisation Gallic acid equivalent Hydrogen-atom transfer Inhibitory concentration of sample to achieve 50% of DPPH inhibition Malaysian Organisation of Pharmaceutical Industries Quercetin equivalent Correlation coefficient Reactive oxygen species Single electron transfer Standard deviation Significant Standard operation procedure Statistical Package for the Social Science 10 Total flavonoid content Total phenolic content Ultraviolet visible Volatile organic compounds liquid chromatography-mass spectroscopy. ix. FYP FIAT. LIST OF ABBREVIATIONS.

(12) ºC % ≤ ± : µ. Degree Celcius Percent Less than or equal Plus-minus Ratio micro. x. FYP FIAT. LIST OF SYMBOLS.

(13) INTRODUCTION. 1.1 RESEARCH BACKGROUND. Flemingia macrophylla (Willd.) Merrill (Leguminosae) is one of the underutilised medicinal plant for various traditional treatment. F. macrophylla commonly known as serengan jantan among the Malaysian local people. It is belong to the Leguminosae family and widely found in tropical and subtropical area of Southeast Asian and African. F. macrophylla had been used as traditional treatment to treat smallpox spleen, cholera, dysentery, arthritis, rheumatism and influenza. The plant has been reported to possess estrogenic, anti-estrogenic, anti-inflammatory, cytotoxic, neuroprotective, analgesic, and anti-osteoporotic activity (Lai et al., 2013; Ghalot, Lal & Jha, 2012). The main constituents found in this plant by previous chemical studies are flemichin, flemiphilippinin, genistin, and a new compound, fleminigin was obtained from the roots part of F. macrophylla.. In Malaysia, the decoction of young leaves part of F. macrophylla had been used as smallpox treatment. Local people also claimed that the rubs of fresh young leaves to the face can give whitening effect. However, the potential of young leaves part of this plant as agent to be used in cosmetic composition has not yet being study. In present research, the antioxidant and partial chemical analysis will be conducted in order to determine the chemical constituent present in different state of dried and fresh F. macrophylla young leaves. Although the fresh leaves are frequently being use by local 1. FYP FIAT. CHAPTER 1.

(14) study will also compare the content and activity of chemical constituent extracted from two type of extraction solvent, glycerol and ethanol in order to determine the potential of glycerol as extracting solvent. As in cosmetic industry, glycerol base is widely being used in cosmetic composition due to its natural and safety properties.. 1.2 PROBLEM STATEMENT. Flemingia macrophylla (Willd.) Merrill (Leguminosae) or known as serengan jantan is one of the herbs that used by local people for smallpox treatment in Malaysia. Differ with traditional treatment at the other countries such as India that mostly used the root part of F. macrophylla, the local people in Malaysia used the leaves part of plant for treatment. However, there are still lack of scientific evidence on phytochemical and functional properties of the leaves part of F. macrophylla. Other than that, some local people in east coast of Malaysia also believe that the rubs of F. macrophylla leaves to the face can give whitening effect. Despite of all claimed, there are still lack of popularity about the potential of F. macrophylla plant as giving health potential in Malaysia.. Generally, previous studies on F. macrophylla were used alcohol as extraction solvent ( Orwa et al., 2009; Lai et al., 2013; Ghalot et al., 2012). However, TasioulaMargari & Tsabolatidou (2015) had reported that alcohol extraction resulted in incomplete recovery of some component. There is also an urge in searching for agro solvent such as glycerol which is safer to be used in extraction. Thus, this study will be using glycerol as extracting solvent to compare the effectiveness with alcohol solvent. 2. FYP FIAT. people, however dried leaves sample are more preferable for industrial application. This.

(15) H0 = There are no significant different of antioxidant activity, total phenolic and flavonoid content as well as phytochemical types of F. macrophylla extracted from dried and fresh young leaves as well as ethanol and glycerol as extracting solvent.. 1.4 OBJECTIVES. 1) To extract young leaves of F. macrophylla with glycerol and ethanol in fresh and dried sample condition. 2) To compare phytochemical types in fresh and dried sample condition of F. macrophylla extracts. 3) To determine antioxidant potential of F. macrophylla extracts using DPPH scavenging activity, total phenolic and total flavonoid assays in fresh and dried sample condition.. 1.5 SCOPE OF STUDY. This study was focus on the determination of antioxidant properties of F. macrophylla’s leaves by using glycerol and ethanol extraction. The sample part that was used in this study is the young leaves with different state; fresh and dried. The chemical profile of alkaloid, terpenoid, phenol and flavonoid in extract were identified by conducting phytochemical screening. Total phenolic and total flavonoid content in this. 3. FYP FIAT. 1.3 HYPOTHESIS.

(16) using DPPH radical scavenging assay.. 1.6 SIGNIFICANCE OF STUDY. This study was able to provide information on the antioxidant properties and preliminary phytochemical content of F. macrophylla’s leaves. The scientific information provided will encourage the society to fully utilize this indigenous plant as herbs treatment. It will also open the platform for the Malaysian Organisation of Pharmaceutical Industries (MOPI) to facilitate and encourage product developer to develop new product formulation based on the information of F. macrophylla’s antioxidant, phenolic and flavonoid content. As the rubs of F. macrophylla’s young leaves had been traditionally claimed as able to whiten the face, thus the cosmeceutical product such as soap, scrub and cream with the relaxing and whitening agent has potential to be commercialised. This study also able to give insight to future researcher in order to continuing the study on further detection chemical composition of F. macrophylla.. 4. FYP FIAT. plant also were analysed. In addition, antioxidant activity was also be determined by.

(17) LITERATURE REVIEW. 2.1 Flemingia macrophylla (WILLD.) MERRILL (LEGUMINOSAE). Figure 2.1: Flemingia macrophylla’s specimen Sources: Kews Herbarium (2017). Figure 2.1 shows the specimen of Flemingia macrophylla plant. F. macrophylla is a medicinal plant that has been use for traditional treatment especially for treatment of smalpox and dysentry. In Malaysia, F. macrophylla is popularly known as ‘pokok serengan jantan’ among the local people. F. macrophylla is considered as one of the underutilized plant species as their local and traditional uses are poorly documented and was only traditionally uses in localized areas. This genus consist over fourty species in the world and widely distributed into humid to subhumid tropics and subtropic region 5. FYP FIAT. CHAPTER 2.

(18) such as Malaysia, Taiwan, Brunei, Cambodia, China, India, Indonesia, Laos, Myanmar, Philippines, Sri Lanka, Province of China, Thailand and Vietnam. On the other hands, in several countries in Africa and south America, it was considered as exotic plant (Orwa et al., 2009). The habitat of F. macrophylla can be found mostly under trees along the watercourses and in grassland. Samkol and Ly (2001) had reported that F. macrophylla can tolerate acid and infertile soil that rich in soluble aluminium, thus it can easily be found on clay and lateritic soil area. The genus also was claimed that it can resist either long dry periods or slight flooding conditions.. 2.1.1 TAXONOMY AND NOMENCLATURE. The origin of word ‘macrophylla’ from the name Flemingia Macrophylla (Willd.) Merrill (Leguminosae) are from Greek which mean ‘large leaved’ (‘makros’ refer to ‘large’; ‘phyllon’ refer to ‘leaf’). According to Agroforestry Database version 4.0, the international common name or trade name for F. macrophylla is waras tree or warrus tree. Meanwhile, the local common name is serengan jantan in Malaysia, pokkepokan in Indonesia, khamin ling in Thailand, basa-salpan in India, barasalpan in Bangladesh, qianjinhong in China and cay dau ma in Vietnam (Orwa et al., 2009). The European and Mediterranean Plant Protection Organisation (EPPO) Global Database code for F. macrophylla is FLEMA (Centre for Agriculture and Biosciences International (CABI), 2017). According to Ghalot, Lal & Jha (2012), F. macrophylla is a venerable medicinal plant that belong to the family Fabaceae, which is also known as family Leguminosae. Its tribe are belong to Phaseoleae and subtribe are Cajaninae. Its subfamily is known as family Paplionaceae or Faboideae. This family consist of. 6. FYP FIAT. (Andersson et al., 2003). The species are consider as native in most Asian countries.

(19) grow in a variety of climates and environment.. 2.1.2 PLANT MORPHOLOGY. F. macrophylla is a woody, deep-rooting erect shrub plant of Asiatic origin with range of 4-6 ft in height. The leaves are commonly 5-15 cm long, 2-8 cm wide in oblong shape and have prominent veins covered with silky hairs. The arrangement of leaves is trifoliate, which is having three leaflets in a group (Sa Ren & Gilbert, 2010; Orwa et al., 2009). The matured leaves are dark green in colour and it becomes papery when old. On the other hands, the young leaves are fluorescent and appeared to overlap one another. The flower bracts of F. macrophylla are racemes, which is having a flower cluster with the separate flower attached by short equal stalks along a central stem. It has 5 lanceolate lobes with 15-40 pea flowers and the flower at the apex are light purple in colour. F. macrophylla’s fruit pod are dark brown in colour which size about 11-15 mm. The pod structure is oblong, inflated and covered with fine glandular hairs and the fruit pods will explode when it’s ripe. There are also 2 tiny globular seeds in each fruit pod which mottled brown or shiny black in colour and the size are about 2-3 mm in diameter (Orwa et al., 2009).. 2.1.3 ETHNOBOTANICAL USAGE. Many literature reveals that F. macrophylla were widely used in traditional medication and lifestock production. In Asian countries, the roots, leaves, bracts, flowers and woods part were very useful in folk medicine for various health treatment. 7. FYP FIAT. perennial type of flowering plant in the legume category. Fabacea family are capable to.

(20) medication of smallpox spleen, cholera, dysentery and blindness. The most useful part of F. macrophylla for treatment is its root as the roots rich in secondary metabolite. The root part has been used in the treatment of fractures, trauma, arthritis, rheumatism and influenza. It also has been reported that some indigeneous groups in India had used the root part as an external application to ulcers and swellings, mainly in the neck area. Meanwhile in Malaysia and Indonesia, the leaves part is medicinally being used for treating smallpox treatment. The decoction or infusion of leaves part of this plant also have been indigenously used as remedy for sores and swellings in China. While in Taiwan, it is an antipyretic for treating postpartum fever and is used to treat paralysis and pain in the joints (Orwa et al., 2009; Lai et al., 2013). Despite of al the traditional medication, recent studies also had found out that F. macrophylla are effective in the treatment of osteoporosis and used as dietary supplements for postmenopausal women due to its estrogenic properties.. Other than medication, F. macrophylla also had been reported to widely being used as dyestuff material as the plant contain high proportion of tannins. As described by Orwa et al. (2009), in Middle East countries, one of the sources of the Arab dye called ‘waras’ or ‘warrus’ which is a coarse purple or orange-brown powder that consisting of the glandular hairs were derived from dried Flemingia’s fruit pods. The waras dye provide a brilliant orange dye for silk, however it is not capable to dying wool or cotton. Additionally, in African countries such as in Ghana, the leaves of F. macrophylla had been used as alternative for feeding livestock during the dry season because the plant was able to remain green throughout the year and retains most of its leaf during the dry season. However, the leaves are not preferred by livestock during the. 8. FYP FIAT. According to Ghalot, Lal & Jha (2012), various part of plant are reported to be used in.

(21) still considered as a poor forage as its not readily eaten by stock since it contains high fibre and condensed tannin concentrations (Kang, Wanapat, Phesatch, Norrapoke, Foiklang, Ampapon & Phesatcha, 2017).. 2.1.4 PHYTOCHEMICAL STUDY. Based on the potential economic value in health treatment of F. macrophylla, several studies has been conducted by researcher and it was reported that the crude extract of F. macrophylla exhibited estrogenic, anti-estrogenic, anti-inflammatory, cytotoxic, neuroprotective, analgesic, and anti-osteoporotic activity. Isoflavonoids compound that were isolated from the plant shows the act as both agonists and antagonists partial estrogen (Lai et al., 2013). All this activities were possessed by the various phytochemical constituent that presence in F. macrophylla.. Figure 2.2: The structure of fleminigin compound Source: Lai et al. (2013). 9. FYP FIAT. wet season as it reported to contain a high proportion of condensed tannins. The plant is.

(22) et al. (2012) showed that there are 23 main phytoconstituent that can be isolated from F. macrophylla are flemichin, flemiphilippinin, genistin, genistein, 2'-hydroxygenistein, cajanin,. 3'-isoprenylgenistein,. homoflemingin,. narigenin,. 7-(3,3-. dimethylallyl)genistein, prunetin, olmelin, erythrinin, 5,2',4'-trihydroxy-7-(3-methylbut2-enyloxy)isoflavone, neoraufurane, isoderrone, fleminone, an enantiomer of 5,2',4'trihydroxy-8,5'-di-(3-methylbut-2-enyl)-6,7-3,3-dimethylpyrano)flavanone, flemiflavanone, procyanidin, α- amyrin, flemingin, flemistricains, chalcone, lupeol, flemichin-D, 4'-O-methylgallocatechin, β-sitosterol, myricitrin robinin, flemiwallichin C, and stigmasterol. However, as shows in Figure 2.2, a new compound that posessed estrogenic activity was isolated from the roots methanolic extract of F. macrophylla were first time identified and reported by several researcher from Taiwan in 2013 (Lai et al., 2013). The compound was identified as 5,2',4'-trihydroxy-7,8-(1,2,2-trimethyldihydrofurano)isoflavone, and was named fleminigin. The reports on chemical composition in this plant are limited and further investigation on chemical composition need to be conducted to fully utilize this genus.. 2.2 ANTIOXIDANT. Antioxidant is a molecule or species that facilitate in inhibition of oxidation in other molecule. According to Antolovich, Prenzler, Patsalides, McDonald, & Robards (2001), antioxidant can be defined as any substance that can slow, delays or inhibits oxidative processes by interrupting the radical chain reaction of substrate even if it present at low concentration compared to the oxidizable substrate. The human body contain a variety of antioxidants for oxidative metabolism that serve to counterbalance. 10. FYP FIAT. Previous chemical studies from literature survey by Lai et al. (2013) and Ghalot.

(23) survival of human cells. In chemical reaction, antioxidant can be considered as reductant, which is acting as reducing agent that reduces the chemical compound by donating electrons to an oxidizing agent. Although antioxidants were considered as reductant, however, not all reductant must be an antioxidant (Craft, Kerrihard, Amarowicz, & Pegg, 2012). The electron-rich antioxidant molecule transfer or donate its electron to oxidizing agent, such as free radicals which undergoes a simultaneous reduction.. 2.2.1 FREE RADICAL ACTIVITY AND OXIDATIVE STRESS. A free radicals is an atom or molecules that can exist self-dependent with one or more unpaired electron in its outermost shell. Free radicals is an unstable compound and were highly reactive oxygen species that can cause oxidative changes in cells (Saha et al., 2008). High levels of free radicals cause by pollution, cigarette smoke, drugs, illnesses, and stress can disrupt homeostasis in human body. Cigarette smoke contains many oxidants, free radicals and organic compounds such as nitric oxide. Thus, inhalation of cigarette smoke into the lung can activate some endogenous mechanism and increase the oxidant injury in lung’s parenchymal cells. Biskup, Golonka, Gamian, & Sroka (2013) had reported that high level of free radicals also can overwhelm protective enzymes such as superoxide dismutase, catalase and peroxidase. It can cause enzymes destructive and lethal cellular effects by oxidizing DNA and enzymes, thus shutting down cellular respiration The consequent cellular damage can lead to the development of Alzheimer’s disease, Parkinson’s disease, dementia, atherosclerosis and asthma. Apart of human cellular damage, oxidation activity also can act as major cause. 11. FYP FIAT. the effect of oxidant. This metabolism is very essential to inactivate free radicals for the.

(24) nutritional quality deterioration and safety of the foods (Biskup et al., 2013).. Oxidase stress is a term used to indicate the disturbance in the balance of the antioxidant status. It was arise from the imbalance between the reactive oxygen species (ROS) and defence and repair mechanism (Antolovich et al., 2001). ROS are produces. by normal cellular metabolism of living organism and environmental factor like air pollution and cigarette smoke. ROS can damage the structure and alter the function of important macromolecules cells such as carbohydrates, lipids, and proteins. Birben, Sahiner, Sackesen, Erzurum, & Kalayci (2012) has stated that the harmful effects of reactive oxygen species can effectively be block by enzymatic and non-enzymatic antioxidant.. 2.2.2 SOURCE OF ANTIOXIDANT. Various antioxidant can provide defence mechanism against the effect of excessive oxidation by interfering the production of free radicals and defusing them to limit the risk of oxidative stress. Antioxidant agents contained in topical creams and diet are helpful to eliminate the free radicals, thus provide protection against oxidative deterioration. Prakash, Upadhyay, & Pushpangadan (2011) has stated that regular consumption of fruits, vegetables and whole grains that contain antioxidant can reduces the risk of chronic diseases associated with oxidative damage. Phytochemicals constituent in food sources that shows capability toward antioxidant activity provide a very beneficial effects on human health. For example the main antioxidant compound in olive oil have ability to reduce the activity of xanthine oxidase enzyme that involved in 12. FYP FIAT. of chemical spoilage in food which effect in rancidity, colour, texture, flavour,.

(25) phase antioxidant capacity by scavenging oxygen free radicals (Birben et al., 2012). Medicinal plant such as green tea and turmeric also contain polyphenols and flavonoids which exhibit high antioxidant activity. Flavonoids and phenolic compounds are widely distributed in plants exert multiple biological effect, including antioxidant, free radical scavenging abilities, anti-inflammatory, and anti-carcinogenic (Procházková, Boušová, & Wilhelmová, 2011).. 2.2.3 TYPE OF ANTIOXIDANT. Antioxidant agent may occur naturally in plant and human body, or can be produced synthetically. Compounds such as carotenoid in carrots, tocopherol in walnuts, curcumin in turmeric and resveratrol in black grapes are examples of strong natural antioxidants (Prakash et al., 2011). The compounds are able to possess antioxidant activity and free radical scavenging activity similar to or even higher than synthetic antioxidants. Enzymatic antioxidant are natural antioxidant includes the superoxide dismutase, catalase and glutathione peroxidase, plus vitamin E, uric acid and serum albumins. Meanwhile non-enzymatic antioxidant can be the consumption of dietary antioxidants in daily diet such as anthocyanin in grapes and catechin in tea. The diet has a major role in contributing to both the source of free radicals and to fight the reactive nature of free radicals (Birben et al., 2012; Prakash et al., 2011).. Synthetic antioxidants like butylated hydroxy anisole (BHA), butylated hydroxy toulene (BHT) and the gallates also included in the human diet and were widely used in food and pharmaceutical industries (Amzad Hossain & Shah, 2015). Synthetic. 13. FYP FIAT. carcinogenesis. Ascorbic acid in water-soluble vitamin C provides cellular aqueous.

(26) to slow the rate of oxidation and extend the shelf life of food. For example, BHA and BHT commonly used in vegetable oils, dairy products and potato products. However, long term consumption of BHA can cause cancer in human as BHA able to interact with nitrites to form chemical that known to cause change in DNA of cells. It also can lead to behavioural problem as some people cannot metabolise it. Meanwhile, BHT are toxic to nervous system and liver. Synthetic antioxidant can suppress the immune system and disrupt the production of natural antioxidant in the body.. 2.2.4 CLASSIFICATION OF ANTIOXIDANT. Antioxidant can be divide into two classes which is primary antioxidant and secondary antioxidant. Primary antioxidant has been refer as chain breaking antioxidant as it active in inhibiting oxidation reaction. Primary antioxidant are able to retard the initiation step of free radical by reacting with a lipid radical even when it’s present in small trace amount. The chemical reaction of primary antioxidant mechanism can be classified into categories of hydrogen-atom transfer (HAT) and single electron transfer (SET). According to Craft et al. (2012), HAT mechanism occurs when an antioxidant compound such as phenolic eliminate free radical species by donating hydrogen atoms. Meanwhile, SET mechanism occurs when an antioxidant transfer a single electron to aid in the reduction of potential target compound.. Besides of primary antioxidant, secondary antioxidant which is the preventive antioxidant indirectly inhibit oxidation process by binding into free radical (Antolovich et al., 2001). For example, some of phenolic antioxidant were classified as secondary. 14. FYP FIAT. antioxidants usually derived from petroleum and were added to food products in order.

(27) Secondary antioxidant also can retard oxidation process by removal of substrate or single oxygen quenching. Secondary antioxidant often joint work together with primary antioxidant to yield stabilization effect.. 2.2.5 EFFECT ON APPLICATION ANTIOXIDANT THERAPY. The use of antioxidant orally or topical in short-term or long-term application also have different protection effect. Antolovich et al. (2001) has stated that the differentiation of a fast and slow acting group of antioxidants had occur based on the kinetic reaction and the rate of an antioxidant reacts with a specific radical versus the thermodynamics of the reaction. Khodr, Howard, Watson, & Khalil (2003) had conducting a study in order to identify the effect of short-term and long-term treatment of antioxidant therapy on an old rat that suffering hyperalgesia by being treated with 40 mg/kg vitamin E for 2 weeks and 10 g/kg vitamin E for 12 months respectively. It was resulted that both short and long term antioxidant therapy are equally effective in reducing oxidative stress markers in the plasma of old rats. However, the short-term antioxidant therapy treatment for 2 weeks was more effective in protecting against the development of hyperalgesia. This is because the imbalance between the oxidative damage and the local antioxidant defence system in old rats.. 15. FYP FIAT. antioxidant because of their capability to bind with potential metal ion free radical..

(28) Solvent is a substance that can dissolved a solute to form a solution. Solvent basically in liquid form, however it also can be in solid, gas and supercritical fluid form. In phytochemical extraction, usually the extracting solvent used are in liquid form to extract and dissolved the solid materials of constituent from the plant. The extracting solvent usually derived from petroleum and other synthetic sources (Rossol, 2010).. 2.3.1 RAW MATERIAL EXTRACTION ISSUE IN COSMETIC INDUSTRY. In cosmetic industry, extracting solvent are widely use during up stream processing in order to extract the desired constituent from plant materials. The most issue occurred are due to toxicity and incomplete recovery of extracting solvent. According to Rossol (2010), there are no safe solvent in upstream cosmetic industry either the solvent are natural or synthetic. The most widely used solvent are methanol which are highly toxic and hazardous when contact either direct contact with the solvent or inhalation of the vapour emitted. In perfume industry, the researcher and professional specialists found that at least 50% of the energy of the whole industrial process for the extraction of natural product can cause environmental impact. This is due to the high energy consumption and the large amount of solvent used during the extraction process, yet the yield are still very minimal. For example in extracting less than 1 gram of rose absolute, the extraction process required 1 kg of fresh roses as raw material, a large quantity of alcoholic solvent which basically methanol solvent, energy for extraction process and water as cooling agent during processing (Chemat et al., 2012). At the present time, all cosmetic, pharmaceutical, biofuel and food industry are using a variety 16. FYP FIAT. 2.3 SOLVENT EXTRACTION.

(29) current extraction, percolation, decoction, infusion and Soxhlet extraction (Handa, Khanuja, Longo, & Rakesh, 2008). However, recent trends in every industry is to develop an extraction technique that are able to minimize the use of solvent and minimise the environmental impact by practising a greener extraction concept.. 2.3.2 FACTORS CONSIDERED WHEN SELECTING A SOLVENT. Every solvent has different characteristic and group of polarity from polar to non-polar. In selecting an extracting solvent, the physico-chemical property and polarity of desired extracting compound in plant also need to be considered. Jones & Flemming (2001) had stated that the extraction that use polar solvent can effectively dissolved polar compound, meanwhile extraction using non-polar solvent can effectively dissolved non-polar compound from plant sample. Example of polar solvent are methanol, ethanol, glycerol and water; mid polar solvent are ethyl acetate, chloroform and acetone; and non-polar solvent are petroleum ether, hexane and toluene (Sadek, 2002). Other than polarity, the factors to be considered when selecting a solvent also must depends on its solvent selectivity, boiling temperature, reactivity, viscosity, safety, cost, vapour pressure and recovery. Compound with high selectivity or power are very important in order to extract only the active desired constituents from the plant material. The boiling point of the solvent must be as low as possible in order to facilitate removal of the solvent from the extraction product (Smallwood, 1996). The extracting solvent also must have low reactivity, which is must not easily react chemically with the extract or altering the constituent’s characteristic. For viscosity factor, solvent with low 17. FYP FIAT. of extraction process such as maceration, steam distillation, hydro distillation, counter-.

(30) eliminating the solvent from extract mixture. The ideal vapour pressure must be low at operating temperature in order to prevent loss of solvent by evaporation during conducting experiment. The solvent also should be readily available at low cost and the solvent must easily separate from the extract for complete recovery of the extract. Most importantly, the solvent must be safe for the user, non-flammable and non-corrosive, and are not toxic which the trace will not cause environmental pollution (Mourtzinos et al., 2016; Rossol, 2010).. 2.3.3 ORGANIC SOLVENTS. Current regulation are strictly diminishing the usage of petrochemical organic solvent and volatile organic compounds (VOCs) in industry in order to minimize the environmental pollution and the greenhouse effect. According to Chemat et al. (2012), manufacturer that need to use organic solvent in large scale must show that there are no risk occur during extraction and must ensure the safety of ingredients as solvent traces. Example of organic solvent that widely used in industry are ethyl acetate, dimethyl ether, methanol and ethylene glycol. The characteristic of organic solvent are mostly flammable, volatile and toxic. In phytochemical extraction, methanol solvent are widely used due to its polarity. It is very ideal for extracting certain phytochemicals of both hydrophilic and lipophilic molecules from plant parts. Due to its highly volatile characteristic, methanol can be remove at low temperature by distillation after extraction. However, Tasioula-Margari & Tsabolatidou (2015) had mention that methanol extraction resulted in incomplete recovery of some component.. 18. FYP FIAT. viscosity can leads to low pressure drop and good heat and mass transfer during.

(31) or personnel are the adverse effects of chemicals that may cause toxicity (Smallwood, 1996). Highly toxic solvent such as methanol may cause toxicity effect when come into contact with body surface such as skin, eyes or respiratory tract. When having direct contact to the skin, the solvent can cause effect such as severe burn, irritation, drying and chapping on the skin. Some other solvent may cause no visible symptom upon exposure. However it may penetrate to the skin barrier, entering the bloodstream, and damaging the other organs in the body. Other than that, the inhalation exposure of solvent vapour can primarily irritate and damage the sensitive membrane around the nose and throat area, and also may cause chronic lung disease after a long term exposure (Rossol, 2010). Typically, the workers or users are unaware about the sub chronic toxicity symptom, however they can take a precaution step by practising the laboratory standard operation procedure (SOP) by using mask and glove during handling the solvent.. 2.3.4 GREEN SOLVENTS, THE AGRO- / BIO-SOLVENTS. The agro- and bio-based solvent had been introduced in order to protect both the environment and consumer. Example of typical and widely used bio-based solvent used in industry are glycerol, ethanol acetone, lactic acid, acetic acid, propylene glycol and limonene. The most common bio-based solvent are ethanol. It can be obtained by fermentation of sugar-rich materials such as sugar beet (Płotka-Wasylka, Rutkowska, Owczarek, Tobiszewski, & Namieśnik, 2017). Ethanol is widely used on a large scale in industry because of its availability in high purity, has a low price and the trace is completely biodegradable which cam minimise the environment impact. However, 19. FYP FIAT. The major risk of extraction process using organic solvent towards the workers.

(32) high selectivity in dissolving phytoconstituent such as alkaloid and phenolic compound (Chemat et al., 2012). On the other hand, the most common solvent that used on a large scale for maceration of herbs and spice in cosmetic industry are glycerol solvent. It can be derived from the by-product of vegetable oils trans-esterification (Płotka-Wasylka et al., 2017). On the other hands, glycerol has high chemical and thermal stability, nonflammable and non-volatile organic compound (non-VOC). Glycerol also has a very high polarity in extracting polar compound from plant parts. However, due to its low selectivity power, some of compound cannot fully dissolved and extracted with glycerol. Thus, there is a challenge for the industries to develop processing method using green solvent which able to give similar or better extraction effect as organic solvent.. 2.4 SAMPLE PREPARATION. The quality of herbal based product is associated with the quality of raw materials used. There are many factor during herbal sample preparation that contributed to the quality of herbal bioactive metabolites compositions. According to Dulski (2016), sample preparation can be describe as a processes in which a representative piece of compound is extracted from a larger amount of sources and properly prepared for further analysis. The most common factor during sample preparation that affect the quality of herbs product are the condition of sample either fresh or dried and also the drying method used.. 20. FYP FIAT. ethanol are quite flammable and potentially explosive. It also has a high polarity and.

(33) In phytochemical studies, both fresh and dried plant sample are being used for undergo extraction process. However, in most cases, dried plant sample are more preferable to be used by considering the time of experimental design. This is because most leaves takes at most 3 hours period between harvest and experimental work to maintain its freshness. Fresh sample also are fragile and tend to deteriorate faster than dried sample (Azwanida, 2015). This is because after plucking, the leaves will having biochemical change by increasing the enzymatic activity and soluble amino acids, which lead to faster deterioration of leaves.. 2.4.2 DRIED SAMPLE CONDITION. Drying of herbal raw plant can be done using two drying method, either natural or artificial drying. Most of the Malaysian herbal producer are using natural drying as standard practice in industrial application. However, according to Abdullah, Shaari, & Azimi (2012), artificial method are more applicable than natural method because it provides more hygienic drying condition due to operational activity in closed chamber and higher drying rate by using heat. Generally, the ideal temperature for drying that most herbs can tolerate for various post-harvest drying techniques are around 32-37 °C. Reduces drying time and the use of lower temperatures can prevent the degradation of chemical constituents during the drying process as well as can preserving the colour, aroma, oil content and medicinal properties in plant. In phytochemical extraction process, the most common drying technique used by researcher are non-thermal and thermal drying such as air-drying, microwave-drying and oven-drying.. 21. FYP FIAT. 2.4.1 FRESH SAMPLE CONDITION.

(34) Air-drying is a common non-thermal drying technique that used in plant material sample preparation. Plant sample will be exposed to the air at ambient temperature in controlled environment like laboratory for naturally dried. The process usually takes time from 3-7 days to months depends on the types of plant part to be dried either leaves, roots, bark or seed. This method can preserved the heat- labile compound contained in the plant sample as the sample are not force dried by using high temperature. However, air-drying method is very time consuming compared to the other drying method. Plant sample also may be subjected to contamination and mould growth if stored or exposed at unstable temperature condition (Azwanida, 2015).. 2.4.4 THERMAL DRYING TECHNIQUE. The thermal drying technique that usually used in plant sample preparation are oven-drying. Oven-drying is considered as the easiest and rapid thermal processing as it only takes 3 hours to days for drying the sample depends on the amount and plant part of leaves, roots, bark or seed (Azwanida, 2015). This method are widely used due to its high accuracy to dry a large sample volume at the same time. This technique can preserving the phytochemical constituent in the plant from degrade even it use thermal energy to remove moisture from plant sample. On the other hand, oven-drying cannot preserve the other liquid components such as alcohol, flavour and acetic acid from evaporate during the drying process.. 22. FYP FIAT. 2.4.3 NON THERMAL DRYING TECHNIQUE.

(35) A study by Vongsak et al. (2013) had determined that the comparison between fresh and dried sample extract of Moringa oliefera leaves showed that dried sample has higher flavonoid content than fresh sample and no significant in total phenolic content of both fresh and dried sample. Other study conducted by Abdullah et al. (2012) on Orthosiphon stamineus leaves using different types of drying also showed a significant different on the types of compound obtained. An active antioxidant compound in O. stamineus leaves, sinensetin was only detected al a very low concentration from the sample that undergo oven drying, meanwhile for sample dried in open sunlight or shade, sinensetin cannot be detected. The situation showed that the compound is sensitive to the high temperature and direct sunlight during drying process. The total phenolic content of O. stamineus leaves sample in oven drying also showed a significantly low content as compared to the amount of total phenolic that obtained from the sample that used shade drying (Abdullah et al., 2012).. 23. FYP FIAT. 2.4.5 EFFECT OF SAMPLE PREPARATION ON ANTIOXIDANT ACTIVITY.

(36) MATERIALS AND METHODS. 3.1 CHEMICAL AND REAGENT. The chemicals and reagents that were used in this study are 70 % glycerol, 95 % ethanol, concentrated sulphuric acid, distilled water, 5 % ferric chloride, 1 M sodium hydroxide, hydrochloric acid, chloroform, quercetin, iodine pearl, potassium iodide, ascorbic acid, Folin-Ciocalteu phenol reagent, 7.5 % sodium carbonate, gallic acid monohydrate, iron (III) chloride, 1 mM 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) solution, 0.3 M aluminium chloride, and 0.5 M sodium nitrite solution. All the chemical are provided by the FIAT’s laboratory and it were obtained from Sigma Aldrich, United State. All the chemical used were analytical grade.. 3.2 EQUIPMENTS AND CONSUMABLES. The equipment and consumables that were used in this research are tray, polyethylene zipper bags, scissors, chopping board, airtight glass bottle, magnetic stirrer, 250 ml screw-cap bottle, parafilm, convection oven, 100 ml media bottle, glassstoppered container, 0.125 µm filter paper, fume hood, glove, 250 ml beaker, 50 ml beaker, knife, vacuum pump, buchner funnel, aluminium foil, tissue paper, 10 ml test tube, test tube rack, dropper, 250 ml conical flask, filter funnel, glass rod, spatula, 25 ml measuring cylinder, 10 ml measuring cylinder, micro pipette, pipette tips, spectrophotometer cuvettes, analytical balance (Sartorius, Germany), electrical grinder 24. FYP FIAT. CHAPTER 3.

(37) United Kingdom), and UV-VIS spectrophotometer (Merck & Co, Germany). All the equipment were provided by the FIAT’s laboratory.. 3.3 PLANT COLLECTION AND IDENTIFICATION. Figure 3.1: The red arrow shows that the young leaves of F. macrophylla plant grow parallel to gravity. Figure 3.2: a) Young and matured trifoliate leaves; b) Flower bract; c) Fruit pod 25. FYP FIAT. (Panasonic, Japan), vortex mixer (Smith & Nephew, United State), hot plate (Stuart,.

(38) morning at two local village area (Coordinates: N 5º57’09.4”, E 102º15’21.6”, N 5º57’19.4”, E 102º15’11.8”) in Ketereh, Kelantan on July 2018. Figure 3.1 shows that the young leaves of F. macrophylla grow parallel to gravity and the plant also located at the bushy area near to the lake. As shown in Figure 3.2, the leaves are identified as trifoliate with three leaflets in each groups, having large vein and grow parallel to gravity. The flower bract has purplish pink sepals with yellow filament and the young leaves were identicated as soft and flourescent in colour. The sample were packed instantly in polyethylene bags after harvest to avoid decomposition of some bioactive compound. The sample were authenticated by Dr Shamsul Muhammad and the villagers.. 3.4 PLANT PREPARATION. Pre-extraction of F. macrophylla were conducted according to the method done by Madan, Singh, Kumar, & Kohli (2010) with modification. The plant sample were divided into two groups, fresh and dried. Each leaves sample were washed thoroughly under the running tap water to remove impurities. The leaves sample were blot dried and cut into smaller pieces. Then, the dried sample group were placed on a tray and undergo oven-drying process for 24 hours at 45 ºC temperature. The dryness was determined by constant weight after 2 times measuring at 2 hours interval. Both of the fresh and dried leaves sample were ground until turn into powder form. The samples were placed in zipper bag and stored at 4 ºC temperature for further use. The percentage yield of dried sample were calculated as follow. % Yield =. Dried Fresh. 26. x 100. FYP FIAT. The fresh young leaves of the plant F. macrophylla were collected in the.

(39) F. macrophylla extraction method were conducted according to Apostolakis, Grigorakis, & Makris, (2014); Shehata, Grigorakis, Loupassaki, & Makris (2015) with modification. Amount of 5 g of grounded fresh and dried leaves sample were mixed with 125 ml of 70 % glycerol with ratio 1:25 in different beaker. The extraction process undergoe stirring with a teflon-coated magnetic stirrer at 400 rpm for 120 min at 23 ºC temperature. The extracts were filtered through 0.125 µm filter paper and placed in a screw-cap bottle with parafilm sealed. The extraction method for fresh and dried leaves sample were repeated using 95 % ethanol absolute as extracting solvent. The concentration of glycerol and ethanol solvent were chosed based on the experimental design of previous study by Shehata et al (2015). All extracts were stored at -20 °C until further analysis. Concentrated of the filtrates considered as 40 mg/ml concentration.. 3.6 ASSESSMENT OF PHYTOCHEMICAL SCREENING. Several test were conducted for determining the presence of phenolic, flavonoid, terpenoid and alkaloid compound in the samples extracted with glycerol and ethanol. The method followed as described by Khanam, Wen, & Bhat (2015).. i. Ferric chloride test. Amount of 5 ml extract were dissolved in 0.5 ml distilled water and 0.05 µl of 5% iron (III) chloride were added. The presence of phenolic compounds in sample were indicated by the appearance of bluish black colour. 27. FYP FIAT. 3.5 PLANT EXTRACTION.

(40) Amount of 0.2 ml sodium hydroxide were added into 5 ml extracts to give intense yellow colour to the solution. The presence of flavonoid were showed by disappearance of yellow colour after addition of 0.2 ml hydrochloric acid.. iii. Salkowski’s test. Amount of 1 ml chloroform were added into 2 ml of extract followed by the addition of 0.5 ml concentrated sulphuric acid to form a layer. The presence of terpenoids were identified by the appearance of reddish brown colour at the upper layer.. iv. Wagner’s test. Amount of 5 ml extracts were stirred with 0.25 ml of dilute hydrochloric acid and filtered. Then, 0.10 ml of Wagner’s reagent were added at the side of the test tube. The presence of alkaloids were showed by the formation of reddish-brown precipitate.. 28. FYP FIAT. ii. Alkaline reagent test.

(41) Antioxidant activity of sample extracted with ethanol and glycerol were assessed by using DPPH free radical scavenging assay as describe by Kumar & Veere Gowda (2011); Madan et al. (2010) with modification. Sample solution were diluted by serial dilution to final concentrations of 200, 100, 50, 25, 12.5 and 6.25 µg/mL in ethanol. One millilitre of a 1mM DPPH ethanol solution were added to 2.5 mL sample solutions of different concentrations. The mixture were shaken vigorously and allowed to react in dark place for 30 min. The absorbance value were measured at a 517 nm using UV-VIS spectrophotometer against corresponding test blanks. Meanwhile, 1.0 mL of 1 mM DPPH solution were used as a negative control. One millilitre of different concentration of ascorbic acid (AA) served as positive control. The percentage antioxidant activity (AA %) was calculated using the following equation:. AA % =. 1 –. Abs sample Abs control. x 100%. The EC50 values of DPPH scavenging activity was calculated by graph of scavenging activity at 50% scavenging effect.. 29. FYP FIAT. 3.7 ASSESSMENT OF ANTIOXIDANT ACTIVITY.

(42) Phenolic quantification assay was determined based on standard Folin-Ciocalteu method. The method was conducted according to. Madan et al. (2010) with. modification. An amount of 100 μl of extract solution was mixed with 2 ml of prepared Folin-Ciocalteu reagent and 1.6 ml of 7.5 % sodium carbonate solution in a test tube. The mixture were then shaken vigorously and incubate for 2 hours at room temperature to complete the reaction. Then, the absorbance of standards, blanks and samples were measured at 700 nm using spectrophotometer (Wollgast & Anklam, 2007). Standard curve was prepared by using various concentration of gallic acid monohydrate as the reference standard phenol. The total phenolic content was determined from the standard curve and the result was expressed as concentration of µg gallic acid equivalent (GAE) / mg fresh weight of sample.. 3.9 ESTIMATION OF TOTAL FLAVONOID CONTENT. The total flavonoid content of F. macrophylla extract were determined by using aluminium chloride colorimetric method as described by Arulmozhi & Wilson (2015) with modification. An amount of 0.3 ml of diluted extracts were mixed with 150 µl of 0.5 M sodium nitrite solution and 150 µl of a freshly prepared 0.3 M aluminium chloride solution. After rest at room temperature for 5 min, 1 ml of 1M sodium hydroxide were added before the absorbance of the reaction mixture were measured at 506 nm using spectrophotometer against blank. The total flavonoid content was determined from the standard curve and the result was expressed as mg quercetin equivalents (QE) / mg fresh weight of sample. 30. FYP FIAT. 3.8 ESTIMATION OF TOTAL PHENOLIC CONTENT.

(43) All tests were done in triplicate and mean values was taken for calculation. The experimental results were expressed using statistical analysis as mean ± standard deviation (SD) of three parallel measurements by using Statistical Package for the Social Science 10 (SPSS 10) (Amzad Hossain & Shah, 2015). Student’s t-test was used for comparison between two means and a one-way analysis of variance (ANOVA) was used for comparison of more than two means. A difference is considered statistically significant when p ≤ 0.05.. 31. FYP FIAT. 3.10 STATISTICAL ANALYSIS.

(44) Flemingia macrophylla's young leaves sample were obtained from local area in Kelantan. Young leaves sample were separated into two group of fresh and dried condition. Dried group sample were undergo oven drying at 45°C for 24 hours.. Dried and fresh sample of young leaves were extracted using 70% glycerol solvent and 95% ethanol solvent. Chemical analysis. Ferric chloride test. Determination of antioxidant activity. Determination of antioxidant content. DPPH radical scavenging assay. Alkaline reagent test. Total Phenolic Content. Total Flavonoid Content. Salkowski’s test. Wagner’s test. Figure 3.3: Flow chart of research activities. 32. FYP FIAT. FLOWCHART OF RESEARCH ACTIVITIES.

(45) RESULTS AND DISCUSSION. 4.1 EXTRACTION. F. macrophylla leaves of fresh and dried sample conditions were extracted by using two different solvents, which are 70 % glycerol and 90 % ethanol. The extract characteristic of each fresh and dried leaves extract is showed in Table 4.1.. Table 4.1 Extract characteristic of different F. macrophylla sample condition Sample condition. Solvent. Fresh. 90 % Ethanol. Light green, less concentrate. 70 % Glycerol. Light brown, concentrate. 90 % Ethanol. Dark green, less concentrate. 70 % Glycerol. Dark brown, highly concentrate. Dried. Extract characteristic. Preliminary extraction showed significant different in extract yield of F.macrophylla that extracted from different state of dried and fresh young leaves by using ethanol and glycerol as extracting solvent as shown by different filtrate colour. As can be observed in Table 4.1, in terms of extract colour, the fresh young leaves samples of F. macrophylla produced lighter colour than dried leaves samples. The ethanol extract of fresh leaves (EF) showed light green colour, the glycerol extract of fresh leaves (GF) showed light brown colour, the ethanol extract of dried leaves (ED) showed dark green, and the glycerol extract of dried leaves (GD) showed dark brown colour. 33. FYP FIAT. CHAPTER 4.

(46) extract colour is due to the presence of chlorophyll pigment in the leaves extract. In general, chlorophyll consist of several pigment and the major pigment are chlorophyll a and b. A writing by Waghulde (2008) had mentioned that chlorophyll a has a methyl group (Y=CH3) in a position where chlorophyll b has an aldehyde (Y=CHO). Thus, the different structure makes chlorophyll b is slightly more polar than chlorophyll a. Futhermore, the visual green colour in chlorophyll were possesed by the less polar chloropyll a. The study were correspond with the extraction result that the less polar solvent, which is ethanol solvent produced green extract rather than glycerol, a more polar solvent. Lan et al. (2010) also had stated that alcoholic solvent such as ethanol has a great efficiency to extract chlorophyll pigment that give the green colour to the extract. This is because less polar compound are best dissolved in less polar solvent.. Other than polarity aspect, another factor that affect the colour is the degradable of some pigment or compound in plant sample after undergoe 45 °C drying process. This is because chlorophyll especially chlorophyll b is a heat sensitive compound that will degrade under strong thermal treatment (Erge, Karaden, Koca & Soyer, 2008). On the other hands, in terms of extract concentration, the leaves sample that extracted with ethanol has less concentration and the sample that extracted with glycerol has much thicker extract concentration. This is because the nature of glycerol that has high viscosity due to having many hydrogen bonding between molecules (Płotka-Wasylka et al., 2017).. 34. FYP FIAT. The filtrates colour were shown in Appendix C; Figure C.4. The different in.

(47) due to the different concentration of solvent. 70% glycerol solvent has thick concentration than 90% ethanol make it required more time to get the extract. For 150 ml extracting solvent, both of fresh and dried sample that extract using ethanol solvent only take utmost 2 hours to complete the filtration. On the other hands, same amount of the fresh leaves sample extracted with 70% glycerol solvent take 2 days to get all the filtrate and the dried leaves sample of 70% glycerol solvent has the longest filtration time which took 3 days to complete the process. The alternative filtration method had been done by using vacuum pump to filter glycerol, however it was failed due to high consistency of solvent. Hence, glycerol solvent required more extraction time compared to ethanol solvent in extracting plant material thus it will be considered as less practicality and less suitable for industrial application due to time consuming.. 4.2 PARTIAL CHEMICAL ANALYSIS. The leaves of F. macrophylla are traditionally able to cure several diseases such as smallpox. The curative properties of the leaves extract are perhaps due to the presence of various phytochemical such as alkaloids, flavonoids, glycoside, phenols, saponins, steroids and tannins. The phytochemical analysis of the ethanol and glycerol extract from fresh and dried young leaves of F. macrophylla is shown in Table 4.2 respectively.. 35. FYP FIAT. The time taken to complete the extraction process are differ between samples.

(48) Test. Phytochemical. 90 % Ethanol. 70 % Glycerol. Fresh. Dried. Fresh. Dried. Ferric chloride. Phenolic. +. +. +. +. Alkaline reagent. Flavonoid. +. +. +. +. Salkowski. Terpenoid. -. -. +. +. Wagner. Alkaloid. +. +. +. +. *Note: + = present; - = absent. Table 4.2 shows the preliminary phytochemical screening, where the present of phenolic, flavonoid and alkaloid compounds were detected in all sample extract which are fresh leaves in ethanol extract, dried leaves in ethanol extract, fresh leaves in glycerol extract and dried leaves in glycerol extract. On the other hand, terpenoids compound are present in both fresh and dried leaves sample that extracted with 70% glycerol but absent in ethanol extracts.. Relatively, all the four preliminery test were found positive with aqueous solvent of glycerol and this indicates that the solvent are more effective than ethanol solvent to isolate active compounds due to its higher in polarity. In fact, glycerol have relative polarity of 0.812 which is higher than relative polarity of ethanol, 0.654 (Murov, 2010). According to Jones & Flemming (2001), polar solvent can effectively dissolved polar substances and non-polar solvent can effectively dissolved non-polar substances.. 36. FYP FIAT. Table 4.2 Phytochemical screening of F. macrophylla young leaves extract.

(49) the experiment are polar substances. Thus, antioxidant compounds such as flavonoid, terpenoid, alkaloid and phenolic are best extracted with more polar solvents which is glycerol for more extraction yield. Other than that, based on study conducted by Apostolakis, Grigorakis, Makris (2017), there are significantly different selectivity between ethanol and glycerol solvent based on comparison of polyphenolic profile of same sample by using liquid chromatography-mass spectroscopy (LC-MS) analysis.. For Ferric Chloride test, the appearance of bluish black colour in extract indicate the presence of phenolic compound. All of the young leaves’ extracts were resulted in positive for phenolic compounds. The phenolic compounds are secondary metabolites that able to promote several health effects such as reduced risk of heart and cardiovascular diseases. Phenolic compounds also are known to have activity against diseases and therefore may suggest the leaves to possess antioxidant, antiviral, anticancer and anti-inflammatory activities (Khanam et al., 2015). The result are accordance with the claimed by native that the plant can treat inflamatory disease such as dysentry.. Similarly, alkaline reagent test for flavonoid compound detection also showed a positive result in all of the F. macrophylla sample extract. According to Bargah (2015), the presence of flavonoids indicates the natural occurring phenolic compound, with beneficial effects in the human diet as antioxidants and neutralizing free radicals. Generally, the flavonoids can be divided into six major subtypes, which include chalcones, flavones, isoflavonoids, flavanones, anthoxanthins and anthocyanins. Example of flavonoid compound that present in F. macrophylla is an antibiotic named flemiflavanone from flavanones group (Lai et al., 2013). 37. FYP FIAT. The physico-chemical property of the antioxidant compounds to be extracted in.

(50) all F. macrophylla’s glycerol and ethanol extract. Khanam et al. (2015) have been reported that alkaloid possess antispasmodic, bactericidal, antimalarial and analgesic properties. In fact, the extract of plant that contain alkaloid have been used throughout human history as remedies, poisons and psychoactive drugs. Alkaloid are widely used in medicines for reducing headache and fever, thus compatible with the traditional usage of F. macrophylla decoction by the native as postpartum fever treatment. (Orwa et al., 2009; Lai et al., 2013).. As can be observed in Table 4.2, terpenoid compounds in Salkowski test were detected in both dried and fresh young leaves sample that extracted with glycerol. However, there are no sign of terpenoid compound presence in F. macrophylla sample that extracted with 90% ethanol solvent. The absence of alkaloid compounds in fresh leaves and dried leaves sample extracted with ethanol are quite expected as Malik, Ahmad & Khan (2017) had mention that the extraction of plant using ethanolic solvent may lead to highly oxygenated mostly polar bioactive compounds like triterpenes. The variation in yield extract also may due to the solubility rates and reactivity of terpenoid compound in ethanol at room temperature. Example of bioactive compounds in terpenoid classes are triterpenes, sesquiterpenes and diterpenes. Terpenoid compound have been referred to as antibiotics, insecticidal, anthelmintic antimicrobial, antioxidant, anticancer, neuro-protective and chemo-protective properties (Rimjhim, Kumari & Jainendra (2014); Malik et al. (2017)). Hence, the high percentage of total terpenoid contents in the plant extract endorse their use as a therapeutic medicine against deadly diseases like cancer and Alzheimer.. 38. FYP FIAT. Alkaloid compounds detection with Wagner test also shows a positive result for.

(51) The antioxidant activity of the young leaves of F. macrophylla samples were determined by using quantitative analysis of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Generally, the mechanism of DPPH assay was performed based on the ability of DPPH free radical to decolorize in the presence of antioxidants. DPPH is an unstable radical containing an odd electron that responsible for a visible deep purple colour. When DPPH solution were added into the extract solution that contain antioxidant, the antioxidant molecule will neutralize the free radical by donating an electron into DPPH free radical molecule to make it a stable molecule, thus resulting in decolorization of DPPH purple colour (Saha et al., 2008). Therefore, the decolorization activity of DPPH can be quantitatively measured from the changes under UV absorbance.. Standard calibration curve to determine the free radical scavenging activity of F. macrophylla young leaves samples were prepared by using ascorbic acid as reference compound. The choice of reference compounds is considered as a critical control point in antioxidant activity assessment. In fact, ascorbic acid is an oxidative compound that has a strong antioxidant activity. According to Nenadis, Lazaridou & Tsimidou (2007), ascorbic acid are quite less unstable due to fast degradation kinetics. However, ascorbic acid is widely used as standard in DPPH assay as it is convenience, cheaper and easy to get. For this assay, different concentration of ascorbic acid that range from 10, 5, 2.5, 1.25, 0.625 and 0.313 µg/ml had been used as standard antioxidant compound. Figure 4.1 shows the calibration curve of ascorbic acid with correlation coefficient (R²) value obtained are 0.9904 and the equation of the graph of scavenging activity at 50%. 39. FYP FIAT. 4.3 DPPH RADICAL SCAVENGING ASSAY.

(52) detection, the IC50 value that had achieved by ascorbic acid is 4.693927109 g/ ml.. % Scavenging at 517 nm. 120 100. 80 60 40. 20 0 0. 2. 4. 6. 8. 10. 12. Concentration of Ascorbic Acid (µg/ml). Figure 4.1: Calibration curve of Ascorbic Acid by DPPH assay. According to Antolovich et al. (2001) the absorbance of DPPH solution at 517 nm must be in range of 0.7 to 0.9. Lower absorbance value of sample solution against the reference compound (range 0.1 - 0.7) of DPPH solution indicate higher radical scavenging potential in plant sample to scavenge the DPPH free radicals. Apparently, all the F. macrophylla extracts has shown higher radical scavenging potential with absorbance below 0.4. in this experiment, each extract samples of F. macrophylla were diluted into several concentration that range from 200, 100, 50, 25, 12.5 and 6.25 μg/ml in order to perform the DPPH inhibitory activity. The antioxidant capacity of ethanolic and glycerol extracts of all F. macrophylla young leaves sample was concentration dependent which displaying a maximal antioxidant capacity at 200 μg/ml. As can be. 40. FYP FIAT. scavenging effect are y = 9.8421x + 3.8019. In this case, at 517 nm spectrophotometer.

(53) concentration had achieved the highest scavenging capacity (94.57547 %), followed by dried leaves in ethanol extract (ED) (91.98113 %), and fresh leaves in glycerol extract (GF) (88.4434 %). Meanwhile, the lowest value of antioxidant activity of extract was fall on the fresh leaves sample in ethanol extract (EF) (62.9717 %). All antioxidant scavenging activity values were statistically significant at (p ≤ 0.05).. Free radical scavenging activity (%). 100 90 80. Dried leaves in ethanol extract (ED). 70 60. Fresh leaves in ethanol extract (EF). 50 Dried leaves in glycerol extract (GD). 40 30. Fresh leaves in glycerol extract (GF). 20 10 0. 0. 50. 100 150 Concentration (µg/ml). 200. 250. Figure 4.2: DPPH Radical Scavenging Activity of F. macrophylla young leaves extracts at different sample concentration.. 41. FYP FIAT. observed in Figure 4.2, the dried leaves sample in glycerol extract (GD) at 200 µg/ml.

(54) a. 180 160 140. b. IC50. 120 100. c d. 80 60. 40 20 0 Ethanol-Dried. Ethanol-Fresh. Glycerol-Dried. Glycerol-Fresh. Sample Figure 4.3. : IC50 value (µg / ml) of F. macrophylla young leaves extract. *Note. : Spectrophotometric detection was at 517 nm. The standard error bar shows on graph were representing the standard deviation. Different superscript letters represent significant different (p ≤ 0.05) among sample by Duncan test.. From the present study, the IC50 value of each sample tested were obtained based on the percentage free radical scavenging of sample. The IC50 is a quantitative measure and the value was defined as the concentration of compound that gives halfmaximal response, where percent inhibition is equal to 50 and was the mean from at least two independent experiments (Fassy et al., 2016). The IC50 determinations were performed in the presence of serial dilutions of samples extract. Apparently, all of the sample of F. macrophylla young leaves extracts had achieved 50 % DPPH inhibition (IC50). The ethanolic extract of fresh F. macrophylla young leaves (EF) had showed the highest IC50 value (164.3995 μg/ml) followed by the glycerol extract of fresh leaves (GF) and ethanolic extract of dried young leaves (ED) with the value of 96.96836 μg/ml. 42. FYP FIAT. 200.