CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF THE FRUITS AND LEAVES OF Phaleria macrocarpa (SCHEFF.) BOERL. SITI NUR ATIQAH BINTI MD OTHMAN UNIVERSITI TEKNOLOGI MALAYSIA CHEMICAL CONSTITUENTS AND BIOACTIVITIES OF THE FRUITS AND LEAVES OF Phaleria macrocarpa (SCHEFF.) BOERL. SITI NUR ATIQAH BINTI MD OTHMAN A thesis submitted in fulfilment of the requirements for the award of the degree of Master of Science (Chemistry) Faculty of Science Universiti Teknologi Malaysia JULY 2014 iii Special dedication to My beloved bonda, Pn. Hajjah Siti Zarah Mahpok. My sisters and brothers, Saiful Nizam Fahrul Rozi Siti ‘Ainur Iza Hairani Shahrul Bazli Siti Nur ‘Aqila For all your love, prayers, support, and sacrifice. Thank you so much. iv ACKNOWLEDGEMENT First and foremost, I wish to express my sincere appreciation to my beloved supervisor, Dr Norazah Basar for her guidance, advices, patience, attention and continuous support on both academic and a personal level to accomplish throughout my project. My appreciation also goes to my co-supervisor, Dr Siti Pauliena Mohd Bohari for her guidance, and giving me opportunity to expand my research field onto tissue culture. Not forgotten to Assoc. Prof. Dr. Farediah binti Ahmad for her helpful suggestions and kindness, for which I am grateful. Completing this work would have been more difficult without the help, motivation and friendship provided by my lab members from both organic and biotechnology laboratories. I would like to express my gratitude to technical and laboratory staffs of Department of Chemistry, Faculty of Science, in particular Madam Zahratul „Ain Jalil, Mr Azmi Md Rais and Mr Rasyidi Abd Mubin for their assistance and guidance in the processing of NMR data and handling of certain instruments. I am indebted to them for their help. To my beloved mother Bonda Siti Zarah Mahpok, sisters and brothers, thank you so much for love, prayers, care, and endless support throughout my studies at Universiti Teknologi Malaysia. My research would not have been possible without their understanding and encouragement. Last but not least, I would like to acknowledge Ministry of Higher Education (MOHE) for financial support under Research Universiti Grant with vote number 05J90 and New Academic Staff Grant Scheme with vote number 4P036. v PREFACE This thesis is the results of my own work carried out at the Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia and Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia between July 2011 and January 2014 under the supervision of Dr. Norazah Basar and Dr. Siti Pauliena Mohd Bohari. Part of my work describe in this thesis has been reported in the following publication: 1. Othman, S.N.A.M and Basar, N. (2014). “Chemical Constituents and Antibacterial Activity of Phaleria macrocarpa (Scheff.) Boerl.”. International Journal of Pharmaceutical Science and Research. 5(8): 1000- 1005. E-ISSN 0975-8232 | P-ISSN 2320-5148. 2. Othman, S.N.A.M, Basar, N. and Bohari, S.P.M. (2013). “Cytotoxic Activity of Major Compounds from Phaleria macrocarpa (Scheff.) Boerl. Fruits”. Jurnal Teknologi. 62(2): 53-56. eISSN 2180-3722 | ISSN 0127-9696. 3. Othman, S.N.A.M and Basar, N. (2013). “Benzophenones from the Phaleria macrocarpa and Their Antimicrobial Properties”. In Ahmad, F., Assim, Z., Din, L. and Jantan, I. (Eds) Integrated Research in Natural Products Chemistry (pg 161-172). Kota Samarahan, Sarawak, Malaysia. Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (UNIMAS). 4. Othman, S.N.A.M, Basar, N. and Bohari, S.P.M. (2013). “Chemical Constituents and Cytotoxic Activity of Phaleria macrocarpa (Scheff.) Boerl. Fruits on MCF- 7 Cell Line”. Poster presented at International Conference on Natural Products 2013 (ICNP 2013), at Shah Alam Convention Centre, Selangor, Malaysia. 4 – 6 March 2013. vi 5. Othman, S.N.A.M and Basar, N. (2013). “Triterpenoids from Phaleria macrocarpa (Scheff.) Boerl.” Paper presented at the 3rd Academic Conference on Natural Science for Master and PhD Students from ASEAN Countries (CASEAN 2013), at Royal University of Phnom Penh, Cambodia.11 – 15 November 2013. vii ABSTRACT Phytochemicals and bioactivities of the fruits and the leaves of Phaleria macrocarpa have been studied. Successive extraction of the dried fruits using cold extraction followed by fractionation and purification afforded two benzophenones identified as 2,6,4'-trihydroxy-4-methoxybenzophenone and 6,4'-dihydroxy-4- methoxybenzophenone-2-O-β-D-glucopyranoside and two triterpenoids identified as 24-methylenecycloartan-3-one and 24-methyl-9,19-cyclolanost-25-en-3-ol. Meanwhile, cold extraction of the dried leaves followed by fractionation and purification yielded two sterols known as stigmasterol and β-sitosterol. Fractionation and purification process were done using vacuum liquid chromatography and column chromatography, respectively. The structures of isolated compounds were elucidated on the basis of their spectral data including 1D and 2D Nuclear Magnetic Resonance (1H, 13C, DEPT, COSY, HMQC, HMBC), Infrared, Ultraviolet spectroscopies and mass spectrometry. The antibacterial activities of all crude extracts and isolated compounds were performed using disc diffusion method followed by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against two Gram-positive bacterial strains, Bacillus subtilis and Staphylococcus aureus together with two Gram-negative bacterial strains including Escherichia coli and Pseudomonas putida. The results have demonstrated that all extracts and isolated compounds exhibited weak activity against all tested bacteria with the MBC values exceeded 1000 μg/mL. Evaluation of antioxidant activity using 2,2-diphenyl-1- picrylhydydrazyl (DPPH) assay has revealed strong free radical scavenging properties of ethyl acetate and ethanol extracts from the fruits with SC50 of 14.00 and 19.97 μg/mL, respectively. The chloroform extract from the fruits and all extracts from the leaves exhibited moderate antioxidant activities with SC50 ranging between 58.07 – 94.10 μg/mL. Among the isolated compounds, 2,6,4'-trihydroxy-4-methoxy- benzophenone exhibited high free radical scavenging activity with SC50 of 29.73 μg/mL whereas the other isolated compounds were inactive against DPPH. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out to evaluate the cytotoxic activity of crude extracts and isolated compounds. MTT assay of CHO, MCF-7, MDA-MB-231 and MDA-MB-468 cells were treated with all extracts and isolated compounds whereas the cell viability of 3T3, HeLa and HT-29 were treated by extracts of the fruits and two isolated benzophenones. The result has demonstrated good activity of dichloromethane extract from the leaves against MDA-MB-231 cell line (IC50 70 μg/mL) and moderate cytotoxic effect of ethanol extract from the fruits towards MDA-MB-468 and HT-29 cell lines with the same IC50 of 100 μg/mL. Chloroform extract from the fruits was found to have moderate cytotoxic properties against HT-29 cell line (IC50 100 μg/mL). All isolated compounds were found to have weak cytotoxic activity towards all tested cell lines. viii ABSTRAK Fitokimia dan bioaktiviti terhadap buah dan daun Phaleria macrocarpa telah dikaji. Pengekstrakan ke atas buah kering menggunakan pengekstrakan sejuk diikuti dengan fraksinasi dan penulenan telah berjaya menghasilkan dua benzofenon yang dikenal pasti sebagai 2,6,4'-trihidroksi-4-metoksibenzofenon dan 6,4'-dihidroksi- 4-metoksibenzofenon-2-O-β-D-glukopiranosida dan dua triterpenoid yang dikenal pasti sebagai 24-metilensikloartanon-3-on dan 24-metil-9,19-siklolanost-25-en-3-ol. Manakala, pengekstrakan sejuk ke atas sampel daun kering diikuti dengan fraksinasi dan penulenan telah menghasilkan dua sebatian sterol iaitu stigmasterol dan β-sitosterol. Proses fraksinasi dan penulenan telah dilakukan menggunakan kromatografi cecair vakum dan kromatografi turus. Struktur bagi semua sebatian tulen telah dikenal pasti berdasarkan data spektoskopi termasuk 1D dan 2D Resonan Magnetik Nuklear (1H, 13C, DEPT, COSY, HMQC, HMBC), Inframerah, Ultralembayung dan spektrometri jisim. Aktiviti antibakterial semua ekstrak mentah dan sebatian tulen telah dijalankan menggunakan kaedah pembauran cakera diikuti dengan penentuan kepekatan perencatan minimum (MIC) dan kepekatan bakterisida minimum (MBC) terhadap dua bakteria Gram-positif, Bacillus subtilis and Staphylococcus aureus bersama-sama dengan dua bakteria Gram-negatif termasuk Escherichia coli and Pseudomonas putida. Keputusan menunjukkan bahawa semua ekstrak mentah dan sebatian tulen menunjukkan aktiviti lemah terhadap semua bakteria yang diuji dengan nilai MBC yang melebihi 1000 μg/mL. Penilaian aktiviti antioksidan menggunakan asai 2,2-difenil-1-pikrilhidrazil (DPPH) telah mendedahkan sifat penangkapan radikal bebas yang kuat oleh ekstrak etil asetat dan etanol dari buah dengan SC50 masing-masing ialah 14.00 dan 19.97 μg/mL. Ekstrak kloroform daripada buah dan semua ekstrak dari daun menunjukkan aktiviti antioksidan yang sederhana dengan SC50 antara 58.07–94.10 μg/mL. Antara semua sebatian tulen, 2,6,4'-trihidroksi-4-metoksibenzofenon menunjukkan aktiviti penangkapan radikal bebas yang tinggi dengan SC50 29.73 μg/mL manakala sebatian tulen yang lain tidak aktif terhadap DPPH. Asai MTT (3-(4,5-dimetiltiazol-2-il)-2,5- difeniltetrazolium bromida) telah dijalankan untuk menilai aktiviti sitotoksik ekstrak mentah dan sebatian tulen. Asai MTT