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UNIVERSITI PUTRA MALAYSIA PHYTOCHEMICAL STUDIES AND BIOACTIVITY TESTS OF MURRAYA PANICULATA JACK, AEGLE MARMELOS CORREA AND ZINGIBER AMARICANS BLUME SUGENG RIYANTO FSAS 2003 3 PHYTOCHEMICAL STUDIES AND BIOACTIVITY TESTS OF MURRYA PANICULATA JACK AEGLE MARMELOS CORREA AND ZIGIBER AMARICANS BLUME By SUGENG RIY ANTO Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulflment of the Requirements for the Degree of Doctor of Philosophy January 2003 DEDICATION %i tfesi i tccatea to: my son, Iffsan)c :urofo ( I)) ana my aaugfter )sefn :ur{ Ifftiarni (}) ana my 6ewvea wife NanifSufarani 11 Abstract of the thesis presented to the Senate ofUniversiti Puta Malaysia in flflment of the requirement for the degee of Doctor of Philosophy PHYTOCHEMICAL STUDIES BIOACTIVITY TESTS OF MURAYA PANICULATA JACKAEGLE MARMELOS CORREA ZINGIBER AMARICANS BLUME By SUGENG RIYANTO January 2003 Chairman: Associate Professor Mohd. Aspollah Hj. Sukari, Ph.D. Faculty: Science and Environmental Studies South East Asian countries are very rich in biodiversity wit thousands of the plats species of which only a small proportion of the species have been investigated in detail. This research was cared out to investigate the chemical constituents ad bioactivities of three well kown medicinal plants Muraya paniculata Jack, Aegle marmelos Corea (Rutaceae) and Zingiber amaricans Blume (Zingiberaceae). Leaves ad stem bak of paniculata; leaves, bak ad root of A. marmelos and rhizomes of Z. amaricans were sepaately extracted wit vaous solvents. The extracts and isolated pure compounds were exained for their bioactivity against microbes ad cacer cell line. Cytotoxic assay was perfoned III against CEM-SS (huma T-Iymphoblastic leukaemia) cell line wit MIT reaget, while atimicrobial tests were conducted by diffsion method against a list of the patogenic bacteria and fngi. The structures of the isolated compounds were elucidated by using spectroscopic methods. Eight compounds were isolated fom M. paniculata ad identifed as auraptene, stigasterol, -sitosterol, sucrose ad methoxylated favones including 3, 3' ,4' ,5,5' ,6,7 ,8-octamethoxyfavone, 3,3',4' ,5,5' ,6,7 -heptaetoxyfavone, 3,3' , 4',5,5',7 -hexaethoxyavone and 3' ,4',5,5',7, -pentaetoxyfavone. The study on A. marmelos afforded eleven compounds; -sitosterol, stigasterol ad hopae triterenes including zeorin ad dustanin; alkaloids aegeline and skimmiain; epilupeol, lupenone ad couman derivates; auraptene, eoxyauraptene ad mamin. By employing te sae procedures, the extracts of Z amaricans aforded zerumbone as the major constituent, togeter with cholesterol, capesterol, stigasterol ad -sitosterol. The results of atimicrobial ad cytotoxic tests revealed that some of the extracts showed strong activities. Nevertheless, most of te isolated compounds showed only weak activity. L the other hand, the cytotoxic test results on dustain ad epilupeol showed moderate activity with ICso values of 1 . 19 1 0-2 Imol/ml and 1 .43 1 0-2 Ilmol/ml, respectively. The results obtained for other compounds revealed weak cytotoxic activity as indicated by their ICso values; zermbone (6.88 1 0-2 Jol/ml), zeorin (3.76 1 0-2 Imol/ml), lupenone (4.08 1 0-2 I mol/ml), main (6.66 1 0-2 Imol/ml) ad aegeline (7. 58 1 0-2 Jollml). IV Abstrak tesis yang dikemukakan kepada Senat Universiti Puta Malaysia bagi memenuhi keperlua ijaah Doktor Falsafa KAJIAN FITOKIMIA DAN UJIAN KAKIFAN BIOLOGI DARIPADA MURRAYA PANICULATA JACKAEGLE MARMELOS CORA AND ZINGIBER AMARICANS BLUME Oleh SUGENGRANTO Januari 2003 Pengerusi: Profesor Madya Mohd. Aspollah Hj. Sukari, Ph.D. Fakulti: Sains dan Pengajian Alam Sekitar Negara-negaa di Asia tenggara mempunyai kepelbagaia biologi yang kaya, ribuan spesies pokok tumbuh-tumbuha wujud di daera ini, tetapi hanya sebaagian kecil spesies dikaji secara mendala. Kajia ini tela dijalankan untuk menyelidiki kadungan kimia da keaktifa biolog daipada tiga taaan ubata yang terkenal iaitu : Murraya paniculata Jack, Aegle marelos Corea dan Zingiber amaricans Blume. Daun dan kulit batang daipada paniculata; daun, kulit batag da aka dapada A. marmelos dan rizom daripada Z amaricans diekstak secara berasinga dengan berbagai pelat. Ekstak da sebatia yag beraya dipencilkan diperiksa keatifan biologinya terhadap sel kaser dan mikob. Ujian sitotoksik dilakukan ke atas sel leukaemia T -lymfoblast manusia (CEM-SS) v denga menggunaa reagen MT, sedaga untk mengesa keatifan antimikob dilauka denga kaeda peresapa caera ke atas senarai bateria patogen dan fngi. St molekul daripada sebatia yag telah dipencilkan dikenal pasti denga kaedah spekoskopi. Lapan sebatian yag telah beraya dipencilkan da paniculata dikenal pasti sebagai: auraptene, stigasterol, -sitosterol, sukos ad avon tenetoksi iaitu 3,3' ,4' ,5,5' ,6,7 ,8-oktaetoksiavon, 3,3' ,4' ,5,5' ,6,7 -hepta-metoksiavon, 3, 3' ,4' ,5,-5' ,7-heksaetoksiavon ad 3' ,4' ,5,5' , 7 -pentaetoksi-avon. Penyelidikan ke atas A. marmelos memperolehi sebelas sebatian, yaki: sitosterol, stigasterol dan triterena hop aa iaitu zeorin da dustanin; alkaloid, aegeline and skimmiain; epilupeol, lupenon dan terbitan koumain; auraptene, epoxyauraptene ad main. Z amaricans diselidiki menggnaa kaedah yag saa menghasilkan zerumbone sebagai kadunga utaa, bersaa denga cholesterol, capesterol, stigasterol and -sitosterol. Keutusa ujian antimikob dan sitotoksik menunjukka bahawa beberapa ekstra sagat berkesa. Imm begitu, kebayaka sebatia yag tela dipencilkan didapati mempunyai keatifa lemah. Maaala keputsa ujia sitotoksik mendapati baawa dustain da epilupeol meunjukka kesa sederhaa dengan nila ICso masing-masing 1 . 1 9 1 0-2 JmoVml dan 1 .43 1 0-2 J moVml,. Keutusa yang diperolehi daipada sebatian lain menujuka kesan sitotoksik lemah dengan nilai ICsQ, yaitu: zermbon (6.88 1 0-2 JmoVml), zeorin V (3. 76 10-2 /lmolml), lupenon (4.08 lO-2 /lmolml), marin (6.66 110-2 /molml) da aegelin (7. 58 lO-2/lmol/ml). Vll ACKOWEDGEMENTS This work was car ied out with a hope to contbute towads te expansion of our cur ently limited kowledge on compounds isolated fom medicinal plants ad its bioactivities. The completion of tis thesis would have been impossible without the assistance ad direct involvement of so may kindheaed individual. Thus, I a very much indebted to my previous mentors ad I have no way of repaying such a debt except to express my sincerest gatitude. First and foremost, I a very gatefl to my adviser Associate Professor Mohd. Aspolla HJ.Sukai for his stong support, guidace, patience ad for the very enriching ad thought provoking discussions ad lectures which helped to shape the thesis. He was always there to provide everying I needed in the laboratory. I would also like to thank him for prviding fnacial support during the period of study trough the IP A research fnd. I a also gatefl to Professor Mawadi Raa and Professor Abdul Manaf Ali in their capacities as mebers of the Superisory Committee. Tha you for the comments ad suggestion which contbuted a lot towads the improvement of te fnal mauscript. I a also indebted to the staf of the Deament of Chemistry Universiti Puta Malaysia for their help ad cooperation. My thas are extended to Professor Norio Am ad Associate Professor Moriko Kitajima of Chiba University Japa ad Professor Watera fom Australia for teir collaboration in spectoscopic aalysis. Vlll Acknowledgent is also made for the thesis gat gven by te SEAMEO SEARCA of Los Baos, Philippines which has helped in certain ways to accomplish my thesis. In addition, I would like to express my appreciation to the Dea of Faculty of Pharmacy ad Rector of Gadjah Mada University for study leave which enable me to pusue Ph.D. degee ad gave me aid when I faced fnacial problem in Malaysia. My thaks also go to my colleagues in the Depament of Phaaceutical Chemistry for taing over my job since I started this study. I wish to express thak to Prof. Dr. Mulyadi, Director of " Pasca Saaa" UGM ad Drs. Edy Praono, President Director ofPT Indofaa for the fnacial support. IX cf t a Ei on L00me o ZMJaua Z W cduc te f eton of Sugg Rnto o m M of Plosphy teis O"Pytoeic Stdie ad Bioacvit 1m of Mur piclata Ja Aegle marelos Cre ad Zing/her amarca Blue" acrdac wt Unvesit Pe Maasia (Hge Dee ) Ac 1980 ad Univet Pea Maaia Hge Dee) Relaon 1981. Te omrmes t t cddae b ae te reet dege. Mebe of te EiComa a fllows: MM..Ph.D. As ae Pfesr Faclt of Scec ad Environetl Stdie Univeit 0Malaysia (C) MOI. APOLLA M.SU Ph.D. Asae Pofesr Faclt of Scec ad Evionet Stdie Unive P Malasia (Mebe) R .PofsFaclt of Scec and Envinetl Stdie Univeit 0Masia (ebe) ADUL MAA AI Ph.D. Pfsor Fa o Fo Scec ad Bioolog Univei P Msa (Mebe) Wm&,PhD. Pfes Faclt of Scece ad Tenolog Unveit KeagsaNWW(dedet Eae) , MNMRI Ph.D. PofsDt Scol of G Stie Univet P Masia L.a 7 JAN 2003 x This thesis submitted to te Senate ofUniversiti Putra Malaysia has been acceted a flflment of the requirements to the degee of Doctor of Philosophy. The members of te Superisory Committee ae a follows: MORD. ASPOLLAH RJ. SUKI, Ph.D. Associate Professor Faculty of Science ad Environmental Studies Universiti Putra Malaysia. (Chaira) ARDI RAHMANI, Ph.D. Professor Faculty of Science ad Environmental Studies Universiti Putra Malaysia (Member) ABDUL MANAF ALI, Ph.D. Professor Faculty of Food Science and Biotechnology Univer