PROPOSAL PROGRAM RISET DESENTRALISASI DIKTI...

PROPOSAL

PROGRAM RISET DESENTRALISASI DIKTI

2013

Ketua Tim Peneliti:

Prof. Dr. Yana Maolana Syah KK : Kimia Organik Fakultas : MIPA

INSTITUT TEKNOLOGI BANDUNG April, 2012

KAJIAN SIFAT ANTIMIKROBA DARI KOMPONEN KIMIA PHYLANTHUS MYRTIFOLIUS

DAFTAR ISI

Halaman IDENTITAS PROPOSAL ............................................................................................................1

1 RINGKASAN PROPOSAL..................................................................................................2

2 PENDAHULUAN.................................................................................................................2

2.1 Latar belakang masalah .........................................................................................2

2.2 Tujuan riset ..............................................................................................................3

3 METODOLOGI....................................................................................................................3

4 DAFTAR PUSTAKA............................................................................................................4

5 INDIKATOR KEBERHASILAN (TARGET CAPAIAN).........................................................6

6 JADWAL PELAKSANAAN..................................................................................................7

7 PETA JALAN (ROAD MAP) RISET ....................................................................................7

8 USULAN BIAYA RISET ......................................................................................................8

8.1 Belanja pegawai ......................................................................................................8

8.2 Belanja barang.........................................................................................................8

8.3 Belanja jasa..............................................................................................................8

9 CV TIM PENELITI...............................................................................................................9

10 LAMPIRAN BUKTI CAPAIAN OUTPUT TAHUN 2010-2012 ...........................................13

2

1 RINGKASAN PROPOSAL

Pencarian senyawa-senyawa antimikroba baru merupakan salah satu kegiatan riset yang penting,

karena dilatarbelakangi oleh adanya kenyataan obat-obat antibiotik yang tersedia tidak (cenderung tidak) efisien lagi dalam pengobatan penyakit infeksi. Berbagai pendekatan dilakukan dalam mencari

sumber-sumber baru senyawa yang bersifat antimikroba, termasuk didalamnya adalah dari tumbuhan obat tradisional. Tumbuhan dari kelompok Phylanthus (Euphorbiaceae) termasuk salah

satu yang menjadi target pencarian ini, dan beberapa kajian pendahuluan dalam tingkat ekstrak

menunjukkan adanya bukti-bukti kandungan komponen yang bersifat antimikroba. Berdasarkan kajian fitokimia, kelompok tumbuhan ini merupakan penghasil beragam golongan senyawa alam,

diantaranya alkaloid, terpenoid, lignan, dan turunan asam galat. Namun demikian, komponen spesifik yang bertanggung jawab terhadap sifat biologis tersebut belum mendapat perhatian para

peneliti. P. myrtifolius merupakan salah satu tumbuhan obat Indonesia dengan penyebaran yang

relatif luas, dan pada proposal ini diusulkan untuk dilakukan isolasi komponen kimia dari daun P. myrtifolius, menentukan struktur molekul, dan menguji masing-masing komponen murni tersebut

sebagai antimikroba. Isolasi komponen kimia akan dilakukan melalui pendekatan fitokimia, yang meliputi proses ekstraksi, fraksinasi cair-cair, fraksinasi dengan tenik kromatografi, dan pemurnian

fraksi dengan teknik kromatografi. Struktur molekul masing-masing senyawa murni tersebut akan dilakukan dengan menggunakan data spektroskopi, yang meliputi spektrum UV, IR, NMR 1D dan 2D,

serta spektrum massa. Evaluasi sifat antimikroba akan dievaluasi dengan metoda disk diffusion method terhadap mikroba-mikroba patogen Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Salmonella thypii, Shigella dysentriae, Vibrio cholereae, Bacillus subtilis, Staphylococcus aureus dan Streptococcus sp., serta terhadap beberapa jamur yaitu Aspergillus fumigates, Candida albicans, Epidermophyton sp., Penicillium sp, dan Trichophyton rubrum. Hasil-hasil penelitian ini diharapkan dapat menghasilkan kandidat senyawa antimikroba baru sebagai lead compound senyawa antibiotik baru. Sesuai dengan roadmap KK Kimia Organik riset ini masuk ke dalam tahap 1 (Initial Stage) dalam rangka penggalian senyawa-senyawa berguna alami, dan pada gilirannya akan

menjadi masukan pada tahap 2 (Development Stage) untuk transformasi dan sintesa di laboratorium dalam rangka optimasi sifat biologisnya.

2 PENDAHULUAN

2.1 Latar belakang masalah

Dalam bidang pengobatan, penemuan senyawa-senyawa yang bersifat antimikroba dari hasil metabolisme merupakan salah satu terobosan penting dalam era pengembangan obat antibiotik.

Senyawa-senyawa tersebut meliputi kelompok β-laktam (penisilin), sefalosporin, dan karbapenem

(von Nussbaum, et al., 2006). Namun demikian, sejak kurun waktu akhir abad yang lalu,

kemampuan senyawa-senyawa antibiotik tersebut mulai berangsur-angsur menurun karena mikroorganisme yang menjadi target ternyata mengembangkan kekebalan terhadap senyawa-

senyawa tersebut. Sebagai contoh, kloramfenikol (chloramphenicol), yang ditemukan di pertengahan abad-19 sebagai produk kimia dari organisme rendah Streptomyces venezuaelae, telah mampu

menurunkan tingkat kematian akibat penyakit tipes (thyphoid) (Van der Bergh, et al., 1999), tetapi sejak kurun waktu tahun 1970-an gejala kekebalan S. thypii terhadap obat ini mulai muncul (Lampe,

et al., 1974). Gejala yang sama juga terjadi pada obat-obat antibiotik lainnya, sehingga pencarian

senyawa-senyawa antimikroba baru sampai sekarang tetap menjadi pekerjaan para ilmuwan yang terkait.

Tumbuhan Phyllanthus (Euphorbiaceae) merupakan salah satu kelompok tumbuhan obat Indonesia.

Berdasarkan laporan yang tersedia, kelompok tumbuhan ini sebagai tumbuhan obat sudah dikenal

sejak 2000 tahun yang lalu, terutama terkenal dalam pengobatan penyakit kuning. Dalam laporan Heyne (1987), di Indonesia beberapa tumbuhan Phyllanthus, dari berbagai bagian tumbuhannya, telah digunakan untuk pengobatan sakit kepala, demam, mual, mulas (kolik), pencahar, diuretik, dan obat luka. Keterangan-keterangan tersebut dapat memberikan rasionalisasi keberadaan

senyawa-senyawa kimia dalam kelompok tumbuhan ini yang berkaitan dengan sifat antimikroba pada kelompok tumbuhan ini. Selain itu, beberapa kajian, seperti yang telah dilakukan oleh

Komuraiah dkk. (2009) dan Adegoke dkk. (2010), telah memperlihatkan kemampuan ekstrak dari

beberapa tumbuhan Phyllanthus sebagai antimikroba, termasuk mikroba yang resisten terhadap

3

obat yang ada. Dengan demikian, kajian pencarian komponen aktif dari tumbuhan Phyllanthus yang berfungsi sebagai antimikroba perlu dilakukan, sebagai kelanjutan dari penelitian-penelitian tersebut.

P. myrtifolius merupakan salah satu tumbuhan yang tersebar luas tumbuh di Indonesia. Kajian

komponen kimia menunjukkan tumbuhan ini merupakan penghasil senyawa-senyawa tanin turunan asam galat (Lin dkk., 1988), lignan (Lin dkk., 1995; Lee dkk., 1996), dan triterpen (Lee dkk., 2002). Kajian fungsi biologis dari senyawa-senyawa tersebut belum banyak dilakukan, kecuali dari turunan

lignan. Senyawa-senyawa dari turunan lignan tersebut ternyata mampu menghambat enzim HIV-1 reverse transcriptase cukup kuat (Chang dkk., 1995; Lee dkk., 1996). Berdasarkan pembahasan tersebut di atas, maka isolasi masing-masing komponen pada P. myrtifolius layak dilakukan, dalam rangka mencari kandidat baru (lead compounds) yang bersifat antibiotik.

2.2 Tujuan riset

Riset ini bertujuan mengisolasi dan menentukan struktur molekul komponen eksrrak metanol rimpang C. xanthorrhiza. Masing-masing komponen murni terpenoid tersebut kemudian dievaluasi

sifat antiimikrobanya terhadap Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Salmonella thypii, Shigella dysentriae, Vibrio cholereae, Bacillus subtilis, Staphylococcus aureus dan Streptococcus sp., serta terhadap beberapa jamur yaitu Aspergillus fumigates, Candida albicans, Epidermophyton sp., Penicillium sp, dan Trichophyton rubrum.

3 METODOLOGI

Sesuai dengan tujuan penelitian tersebut di atas, target akhir dari penelitian ini adalah memperoleh komponen kimia dari P. myrtifolius dan mengevaluasi masing-masing senyawa terpenoid tersebut

sebagai antimikroba. Oleh karena itu, penelitian ini akan menggunakan pendekatan fitokimia sehingga semua komponen yang terkandung pada P. myrtifolius dapat dilaksanakan Berdasarkan pendekatan ini, tahapan penelitiannya adalah sebagai berikut:

a. Pengumpulan bahan tumbuhan.

Bahan tumbuhan yang akan diteliti pada penelitian ini adalah daun P. myrtifolius.

b. Penyiapan serbuk kering bahan tumbuhan. Daun P. myrtifolius selanjutnya dikeringkan di bawah sinar matahari dan digiling halus.

c. Pembuatan ekstrak metanol. Ekstraksi akan dilakukan dengan metoda maserasi menggunakan metanol sebagai pelarut.

Lazimnya ekstraksi dilakukan tiga kali untuk masing-masing sampel tumbuhan untuk mencapai jumlah ekstrak yang maksimum. Gabungan ekstrak aseton kemudian dikeringkan dengan

penguapan pada tekanan rendah.

d. Analisis kromatografi lapis tipis terhadap ekstrak.

Analisis kromatografi lapis tipis (KLT) pada tahap ini dimaksudkan untuk mengetahui perkiraan jumlah komponen yang akan diisolasi, serta penentapan jenis-jenis eluen yang sesuai pada

tahapan fraksinasi.

e. Fraksinasi dan pemurnian komponen terpenoid.

Ekstrak aseton yang telah dikeringkan selanjutnya difraksinasi secara partisi kedalam faksi n-heksana, kloroform, dan etil asetat. Fraksi-fraksi tersebut akan difraksinasi lebih lanjut

menggunakan metoda kromatografi vakum cair (KVC). Eluen dipilih sedemikian rupa sehingga sesuai dengan pergerakan komponen di dalam kolom pada tekanan rendah tersebut. Hasil fraksinasi juga akan dimonitor oleh analisis KLT, dan selanjutnya dimurnikan dengan

menggunakan metoda kromatografi radial, sehingga diperoleh masing-masing komponen murni.

f. Verifikasi kemurnian hasil isolasi (isolat).

4

Kemurnian isolat akan ditetapkan berdasarkan hasil analisis KLT fasa diam silika gel. Isolat

dikatakan sudah (cukup) murni apabila pada tiga eluen yang berbeda tetap menunjukkan satu

noda.

g. Pembuatan spektrum UV, IR, NMR, dan spektrum massa. Analisis struktur terhadap isolat pada prinsipnya didasarkan atas hasil analisis spektrum NMR 1D: spektrum 1H, 13C, dan DEPT, dan NMR 2D: HMQC, HMBC dan NOESY. Pengukuran spektrum akan

dilakukan di ITB. Selain itu, data spektrum UV dan IR juga akan diukur untuk mendukung hasil analisis data NMR. Apabila dianggap perlu, spektrum massa untuk masing isolat juga akan

diukurkan.

h. Analisis data spektrum dan penentuan struktur.

Penentuan struktur terhadap isolat murni akan dilakukan berdasarkan metodologi yang sesuai untuk penentuan struktur senyawa alam. Data spektroskopi yang akan banyak dimanfatkan

adalah data NMR 1D (1H NMR dan 13C NMR), dan data NMR 2D (HMQC/HSQC, HMBC, dan NOESY). Metodologi interprestasi data NMR dan data spektrum lainnya telah dimiliki oleh tTim

Peneliti. Struktur molekul akan diusulkan sampai kepada aspek stereokimianya.

i. Penentuan sifat antimikroba.

Uji aktivitas antimikroba akan dilakukan secara in vitro dengan metode disk difusion method terhadap sejumlah mikroba patogen di antaranya adalah Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Salmonella thypii, Shigella dysentriae, Vibrio cholereae, Bacillus subtilis, Staphylococcus aureus dan Streptococcus sp., serta terhadap beberapa jamur

yaitu Aspergillus fumigates, Candida albicans, Epidermophyton sp., Penicillium sp, dan Trichophyton rubrum.

4 DAFTAR PUSTAKA

Adegoke, A.A., Iberi, P.A., Akinpelu, D.A., Aiyegoro, O.A., Mboto, C.I., (2010), Studies on

phytochemical screening and antimicrobial potentials of Phyllanthus amarus against multiple

antibiotic resistant bacteria, Int. J. Appl. Res. Nat. Prod., 3, 6-12. Chang, C.-W., Lin, M.-T., Lee, S.-S., Liu, K.C.S.C., Hsu, F.-L., Lin, J.-Y., Differential inhibition of

reverse transcriptase and cellular DNA polymerase-a activities by lignans isolated from Chinese herbs. Phyllanthus myrtifolius Moon. and tannins from Lonicera japonica Thumb. and

Castanopsis hystrix, Antiviral Res., 27, 367-374. Heyne, K., (1987), Tumbuhan Berguna Indonesia II, Badan Litbang Kehutanan, Jakarta. Komurairah, A., Bolla, K., Rao, K.N., Ragan, A., Raju, V.S., Charya, M.A.S., (2009), Antibacetrial

studies and phytochemical constituents of South Indian Phyllanthus species, Afr. J. Biotechnol., 8, 4991-4995.

Lee, S.-S., Lin, M.-T., Liu, C.-L., Lin, Y.-Y., Liu, K.C.S.C., (1996), Six lignans from Phyllanthus myrtifolius, J. Nat. Prod., 59, 1061-1065.

Lee, S.-S., Kishore, P.H., Chen, C.-H., (2002), Three novel triterpenoids from Phylanthus myrtifolius,

Helv. Chim. Acta, 85, 2403-2408. Lin, M.-T., Lee, S.-S., Liu, K.C.S.C., (1995), Phyllamyricins A-C, three novel lignans from Phylanthus

myrtifolius, J. Nat. Prod., 58, 244-249. Lin, M.-T., Lee, S.-S., Chen Liu, K.C.S.C., (1998), Polar constituents from Phyllanthus myrtifolius,

Chin. Pharm. J., 50, 327-336.

von Nussbaum, F., Brands, M., Hinzen, B., Weigand, S., Habich D., (2006), Antibacterial Natural Products in Medicinal Chemistry—Exodus or Revival?, Angew. Chem. Int. Ed., 45, 5072-5129.

5

Van den Bergh, E.T., Gasem, M.H., Keuter, M., Dolmans, M.V., (1999), Outcome in Three Groups of

Patients with Typhoid Fever in Indonesia between 1948 – 1990, Tropical Medicines and International Health, 4, 211-215.

6

5 INDIKATOR KEBERHASILAN (TARGET CAPAIAN)

No. Indikator Keberhasilan Deskripsi

1. Keluaran (output) Hasil Riset 2 (satu) publikasi internasional.

2. Dampak (outcome) Hasil Riset

Penguatan ITB sebagai pusat kajian senyawa alam yang unggul di Indonesia. Keterlibatan

mahasiswa S3 pada riset ini akan lebih menguatkan peran ITB dalam peningkatan

kapasitas sumber daya manusia untuk dapat

mengeksplorasi sumber daya alam Indonesia. Publikasi internasional yang

menjadi keluaran langsung pada riset ini juga menjadi bagian langsung dampak ke dalam

dari riset ini.

Riset ini membuka peluang ditemukannya

kandidat potensial senyawa bersifat

antimikroba. Senyawa potensial tersebut selanjutnya akan dijadikan objek sintesa organik untuk ditransformasi secara kimiawi sehingga memberikan keaktifan yang lebih

baik. Pada transformasi tersebut tidak

menutup kemungkinan juga memasukkan farmakofor tambahan. Proses ini merupakan

tahap lanjutan yang sesuai dengan roadmap KK Kimia Organik.

Riset ini juga dapat memberikan dampak yang penting bagi masyarakat, mengingat P. myrtifolius (Temulawak) merupakan

tumbuhan obat yang banyak dikonsumsi di Indonesia, dan sepengetahuan kami,

keterkaitan antara kandungan terpenoid dan sifat antibakteri secara lengkap belum pernah

dikaji sebelumnya.

3. Keterlibatan Mahasiswa S1, S2, S3 1 (satu) mahasiswa S3 dilibatkan dalam riset

ini, yaitu Neneng Handayani (NIM 30510002).

4. Pembinaan peer Pada pelaksanaannya, Keterlibatan dosen

muda merupakan bagian dari pembinaan staf di lingkungan KK Kimia Organik.

5.

Networking nasional dan internasional Pada proses pengujian antimikroba, riset ini akan bekerjasama dengan Departemen

Kesehatan (Akademi Analis Kesehatan, Cimahi)

7

6 JADWAL PELAKSANAAN

Jadual penelitian sebagaimana ditunjukkan pada tabel berikut

Bulan ke- Kegiatan

1 2 3 4 5 6 7 8 9 1

0

a. Pengumpulan bahan tumbuhan x

b. Penyiapan bahan x

c. Ekstraksi x

d. Analisis kromatografi x

e. Fraksinasi dan pemurnian x x x x x

f. Verifikasi kemurnian isolat x x x

g. Pembuatan spektrum x x x x

h. Analisis data spektrum x x x

i. Pengujian antimikroba x x x

j. Pembuatan publikasi internasional x x x

k. Pembuatan laporan x

7 PETA JALAN (ROAD MAP) RISET

Penelitian yang diusulkan merupakan bagian besar dari kegiatan yang bertujuan mendapatkan

molekul bioaktif baru berdasarkan model dari senyawa alam, yang merupakan bagian dari penelitian KK Kimia Organik. Kajian fitokimia dan pengujian sifat biologis dari senyawa-senyawa alam

merupakan TAHAP INISIASI dari target akhir mendapatkan obat baru yang potensial, yang

diwujudkan dalam bentuk paten.

Roadmap KK Kimia Organik sebagaimana tampak pada tabel berikut:

Short Term

(2011-2014)

Medium Term

(2015-2018)

Long Term

(2019-2020)

Final Stage Patent applications of unique and interesting

organic compound(s) that have proven to

have high biological

properties, as well as to have high corrosion

inhibitors properties or solar energy

conversion

Development Stage

Structure modification of potential organic

chemicals, tissue culture development, as well as

synthetic elaboration of

chemical analogs, to optimize their biological

or physical properties

Initial Stage Screening of organic compounds, mainly from

natural sources, but also includes the compounds

from synthetic origins, for their biological properties,

and for their physical

properties, such as corrosion inhibitors or

solar energy convertion

8

8 USULAN BIAYA RISET

8.1 Belanja pegawai

No. Pelaksana Kegiatan Jumlah Orang

Honor per Jam

Jumlah Jam/Bulan

Jumlah Bulan/Tahun

Jumlah Biaya (Rp)

1. Peneliti Utama 1 135.000 15 10 20.250.000

2. Anggota Peneliti 1 90.000 15 10 13.500.000

Jumlah total biaya honor (Rp) 33.250.000

8.2 Belanja barang

No. Peralatan/Bahan Volume Satuan Biaya Satuan

(Rp)

Jumlah Biaya

(Rp)

1. Aseton teknis 1 20 L 1.100.000 1.100.000

2. Kloroform p.a. 5 2,5 L 500.000 2.000.000

3. Diisopropil eter 2 2,5 L 2.500.000 5.000.000

4. Heksan teknis 1 20 L 700.000 700.000

5. Metanol teknis 1 20 L 450.000 450.000

6. Etil asetat teknis 1 20 L 700.000 700.000

7. Silika gel 1 1 kg 2.500.000 2.500.000

8. Pelat KLT silika gel 1 1 pak 2.500.000 2.500.000

8. Bahan tanaman 2 1 kg 50.000 100.000

Jumlah total biaya barang (Rp) 15.050.000

8.3 Belanja jasa

a. Honor pihak ketiga non PNS ITB dan ITB-BHMN atau asisten mahasiswa

No. Pelaksana Kegiatan Jumlah

Orang

Honor per

Jam

Jumlah

Jam/Bulan

Jumlah

Bulan/Tahun

Jumlah Biaya

(Rp)

2. Mahasiswa 1 20.000 60 10 12.000.000

Jumlah total biaya honor (Rp) 12.000.000

b. Perjalanan

No. Tujuan Volume Biaya Satuan (Rp) Jumlah Biaya

(Rp)

1. Tidak ada

Jumlah total biaya perjalanan (Rp)

c. Sewa Alat, Jasa Layanan dan Lain-lain

No. Nama Alat/Jasa Layanan Volume Biaya Satuan (Rp) Jumlah Biaya

(Rp)

1. Jasa analisis spektrum UV 6 100.000 600.000

2. Jasa analisis spektrum IR 6 150.000 900.000

3. Jasa analisis spektrum NMR 6 1.100.000 6.600.000

4. Jasa analisis spektrum MS 6 200.000 1.200.000

5. Jasa analisis evaluasi antimikroba 6 900.000 5.400.000

Jumlah total biaya sewa alat, jasa layanan, dll. (Rp) 14.700.000

9

9 CV TIM PENELITI

Ketua Peneliti:

(1) Nama : Dr. Yana Maolana Syah

(2) Tempat/tangal lahir : Karawang, 9-8-1962 (3) Program Studi/PT : Kimia/Institut Teknologi Bandung

(4) Alamat surat : Jalan Ganesha 10, Bandung 40132

~ Telpon/Faks : 022-2502103 pes. 2202/022-2504154 ~ E-mail : [email protected]

~ Telpon rumah : 022-91151768 (5) Satus akademik : Dosen Pembimbing

(6) Jabatan struktural : Ketua Program Studi Magister dan Doktor Kimia

(7) Pendidikan terakhir : Ph.D, 1998, Chemistry, University of Western Australia, Australia

(8) Pengalaman penelitian :

No. Judul Tahun, Sumber Dana

1 Sifat anti mikroba komponen terpenoid dari Curcuma xanthorrhiza

Program Riset Desentralisasi-ITB, 2012

2 Antimikroba dari komponen kimia Macaranga microcarpa

Program Riset dan Inovasi-

ITB, 2012 3 Kajian fitokimia, sifat sitotoksik, dan sifat antioksidan

senyawa-senyawa turunan fenol dari tumbuhan Macaranga indonesia

2009-2010, Diknas melalui

ITB.

4 Kajian Fitokimia dan Sifat Sitotoksik Senyawa Oligostilbenoid dari Tumbuhan Dipterocarpus Hasseltii

2006, Research Grant Fakultas MIPA, ITB

5 Cytotoxic Compounds from Lauraceous Plants 2004, Hibah B, Departe- men Kimia, FMIPA, ITB

6 Pemisahan Hopefenol sebagai Anti-HIV dan Senyawa-senyawa Sejenis dari Beberapa Tumbuhan

Meranti

2003-2004, Hibah Kompe-tisi XI, Dikti, Depdiknas

(9) Publikasi ilmiah : (dalam 5 tahun terakhir)

Internasional:

Syah, Y.M., Ghisalberti, E.L. (2012). “More Phenolic Derivatives with an Irregular Sesquiterpenyl Side Chain from Macaranga pruinosa”, Nat. Prod. J., (in print).

Agustina, W., Juliawaty, L.D., Hakim, E.H., Syah, Y.M. (2012). “Flavonoids from Macaranga lowii”, ITB J. Sci. (in print).

Tanjung, M., Mujahidin, D., Hakim, E.H., Darmawan, A., Syah, Y.M. (2010). “Geranylated

flavonols from Macaranga rhizinoides”, Nat. Prod. Commun., 5, 1209-1211. Syah, Y.M., Ghisalberti, E.L. (2010). “Phenolic Derivatives with an Irregular Sesquiterpenyl Side

Chain from Macaranga pruinosa”, Nat. Prod. Commun., 5, 219-222. Kurniadewi, F., Juliawaty, L.D., Syah, Y.M., Achmad, S.A., Hakim, E.H., Koyama, K., Kinoshita,

K., Takahashi, K. (2010). “Phenolic compounds from Cryptocarya konishii: their cytotoxic and tyrosine kinase inhibitory properties”, J. Natur. Med., 64, 121-125.

Mulyadi Tanjung, Euis H. Hakim, Didin Mujahidin, Muhammad Hanafi, Yana M. Syah. (2009).

“Macagigantin, a farnesylated flavonol from Macaranga gigantea”, J. Asian Nat. Prod. Res., 11, 929-932.

Juliawaty, L.D., Sahidin, Hakim, E.H., Achmad, S.A., Syah, Y.M., Latip, J., Said, I.M. (2009). “A

2-Arylbenzofuran Derivative from Hopea mengarawan”, Nat. Prod. Commun., 4, 947-950.

10

Musthapa, I., Latip, J., Takayama, H., Juliawaty, L.D., Hakim, E.H., Syah, Y.M. (2009).

“Prenylated flavones from Artocarpus lanceifolius and their cytotoxic properties”, Nat. Prod. Commun., 4, 927-300.

Musthapa, I., Juliawaty, L.D., Syah, Y.M., Hakim, E.H., Latip, L., Ghisalberti, E.L. (2009). “An

oxepinoflavone with cytotoxic activity against P-388 cells from Artocarpus elasticus”, Arch. Pharm. Res., 32, 191-194.

Syah, Y.M., Hakim, E.H., Ghisalberti, E.L., Jayuska, A., Mujahidin, D., and Achmad, S.A. (2009).

“A modified oligostilbenoid, diptoindonesin C, from Shorea pinanga”, Nat. Prod. Res., 23, 591-594.

Syah, Y.M., Hakim, E.H., Achmad, S.A., Hanafi, M., Ghisalberti, E.L. (2009). “Isoprenylated Flavanones and Dihydrochalcones from Macaranga trichocarpa”, Nat. Prod. Commun., 4, 63-67.

Wahyuningrum, D., Sadijah, A., Syah, Y.M., Buchari, Bundjali, B. (2008). “The correlation between structure and corrosion inhibition activity of 4,5-diphenyl-1-vinylimidazole

derivative compounds towards mild steel in 1% NaCl solution”, Inter. J. Electro. Sci., 3, 154-166.

Ferlinahayati, Hakim, E.H., Syah, Y.M., Juliawaty, L.D., Takayama, H., Said, I.M., Latip, L. (2008). “Phenolic constituents from the wood of Morus australis with cytotoxic activity”, Z. Naturforsch, 63c, 35-39.

Saroyobudiono, H., Juliawaty, L.D., Syah, Y.M., Achmad, S.A., Hakim, E.H. (2008). “Oligostilbenoids from Shorea gibbosa and their cytotoxic properties against P-388 cells”, J. Natur. Med., 62, 195-198.

Ahmat, N., Siad, I.M., Latip, J., Din, L.B., Syah, Y.M., Hakim, E.H. (2007). “New prenylated

dihydrostilbenes from Croton laevifolius”, Nat. Prod. Commun., 2, 1137-1140.

Nasional:

Tanjung, M., Hakim, E.H., Syah, Y.M. (2009). “Fitokimia dan sifat biologis senyawa-senyawa turunan fenol dari tumbuhan Macaranga”. Bull. Soc. Nat. Prod. Chem (Indonesian), 9, 1-15.

Siallagan, J., Hakim, E.H., Syah, Y.M., Juliawaty, L.D., Din, L.B., Latip, J. (2009). “Flavonoid dari tumbuhan Cryptocarya everettii Merr. (Lauraceae) serta sifat sitotoksiknya terhadap sel murine leukemia P388”. Bull. Soc. Nat. Prod. Chem (Indonesian), 9, 30-35.

Valentina, A.K., Murniati, A., Syah, Y.M., Sampana, A. (2006). “Kandungan Kimia Ekstrak Bangle (Zingiber purpureum Roxb.), Acta Pharm. Indonesia, 31, 127-130.

Sahidin, Hakim, E.H., Syah, Y.M., Juliawaty, L.D., Achmad, S.A., Latip, J. (2006). “Tiga oligomer resveratrol dari kulit batang Hopea gregaria (Dipterocarpaceae) dan sifat sitotoksiknya”, Majalah Farmasi Indonesia, 17, 109-115.

Syah, Y.M. (2006). “Fitokimia, biogenesis, dan sifat biologis senyawa-senyawa aromatik dari

tumbuhan Dendrobium, Bull. Soc. Nat. Prod. Chem (Indonesian), 6, 33-56. Syah, Y.M. and Ghisalberti, E.L. (2006). “Isolation of verbascoside and isoverbascoside from a

medicinal plant of Australia (Eremophila alternifolia)”, Bull. Soc. Nat. Prod. Chem (Indonesian), 6, 27-32.

Bandung, 22 September 2011

Dr. Yana Maolana Syah

11

Anggota Peneliti:

(1) Nama : Prof. Dr. Euis Holisotan Hakim, M.Si.

(2) Tempat & Tanggal Lahir : Garut, 10 Mei 1953 (3) Program Studi/PT : Kimia, FMIPA/ Institut Teknologi Bandung

(4) Alamat Surat : Jl. Ganesha no. 10 Bandung 40132

- Telpon/Faks : 022-2502103/022-2504154 - E-mail : [email protected]

(5) Status Akademik : Dosen

(6) Jabatan Struktural : -

(7) Pendidikan Terakhir : - S-3 (Cum Laude), 1994 Departemen, Institut Teknologi Bandung

(8) Riwayat pekerjaan : 1987 – sekarang , Staf Pengajar Kimia, FMIPA, ITB 2004, Professor di Kimia, FMIPA, ITB

(9) Keanggotaan Profesi :

1. Himpunan Kimia Indonesia (HKI)

2. Himpunan Kimia Bahan Alam Indonesia (HKBAI) 3. The American Society of Pharmacognosy (ASP)

(10) Pengalaman Penelitian (5 tahun terakhir) :

No

Judul

Sumber dana

1 Kajian Profil Kimiawi dan Hubungan Biogenesis Metabolit

Sekunder Daun Sukun (Artocarpus communis) (Ketua Peneliti)

Program Penguatan Riset

Institusi, 2010

2 Combinatorial Biosynthesis of Morus Diels-Alder Adduct

(Kerjasama dengan The Tokyo University) (Ketua Peneliti)

JSPS-DGHE Bilateral Joint

Research, 2010-2013

3 Evaluasi Senyawa Isoprenylflavonoid dari Tumbuhan Cempedak (Artocarpus champeden) untuk Obat Anti Malaria (Ketua Peneliti)

Hibah Penelitian Strategis Nasional DIKTI, 2010

4 Penyelidikan Intensif Metabolit Sekunder dariTumbuhan Murbei (Morus sp) sebagai Lead Compound Obat Anti

Malaria (Anggota Peneliti)

Hibah Publikasi International Batch III, DP2M, 2009

5 Chemical and Biological Evaluation of the Indigenous Artocarpus of Indonesia for Antimalarial (Anggota Peneliti)

2007, TWAS (The Academy of Sciences for the

Developing World)

6 Development for the Medicinal Chemistry Based on

Biologically Active Natural Products in the Subtropical Zone

(Anggota Peneliti)

2007, JSPS, Japan

(12) Buku

1. Sjamsul A. Achmad, Euis H. Hakim, Lukman Makmur, Yana M. Syah, Lia D. Juliawaty, Didin

Mujahidin, “Chemistry, Pharmacology and Uses: Indonesian Medicinal Plants”, Vol. 1, ITB Publisher, Indonesia (2008).

12

2. Sjamsul A. Achmad, Euis H. Hakim, Lukman Makmur, Yana M. Syah, Lia D. Juliawaty, Didin

Mujahidin,“Chemistry, Pharmacology and Uses: Indonesian Medicinal Plants”, Vol. 2, ITB Publisher,

Indonesia (2010).

(13) Publikasi (5 tahun terakhir)

Internasional 1. Ferlinahayati, Yana M. Syah, Lia D. Juliawaty, Sjamsul A. Achmad, Euis H. Hakim, Hiromitsu

Takayama, Ikram M. said, and Jalifah Latif, Phenolic Constituents from the Wood of Morus australis with Cytotoxic Activity, Z. Naturforsch, 63c. 35-39, 2008.

2. Haryoto Saroyobudiono, Lia D. Juliawaty, Yana M. Syah, Sjamsul A. Achmad, Euis H. Hakim,Jalifah Latip, Ikram M. Said, Oligostilbenoids from Shorea gibbosa and their cytotoxic propertiesagainst P-388 cell, J. Nat. Med, 62:195-198, 2008.

3. Iqbal Mustapha, Lia D. Juliawaty, Yana M. Syah, Euis H. Hakim, Jalifah Latif, and Emilio L.Ghisalberti,

An oxepinoflavone from Artocarpus elasticus with Cytotoxic Activity Against P-388 Cells,Arch. Pharm. Res. Vol. 32, No. 2, 191-194, 2009

4. Lia Dewi Juliawaty, Sahidin, Euis H. Hakim, Sjamsul A. Achmad, Yana M. Syah, Jalifah Latip, and

Ikram M. Said, "A 2-Arylbenzofuran Derivative from Hopea mengawaran", Natural Product Communications, Vol. 4, No. 7, 947-950, 2009

5. Iqbal Musthapa, Jalifah Latip, Hiromitsu Takayama, Lia Dewi Juliawaty, Euis Holisotan Hakim,and Yana M. Syah, "Prenylated Flavones from Artocarpus lanceifolius and their Cytotoxic Properties against P-388 cells", Natural Product Communications, Vol. 4, No. 7, 927-930, 2009

6. Iqbal Mustapha, Euis H. Hakim, Lia D. Juliawaty, Yana M. Syah, Sjamsul A. Achmad, PrenylatedFlavones from Some Indonesian Artocarpus and Their Antimalarial Properties, Medicinal

Plants, 2(2), 157-160, 2010 7. Fera Kurniadewi, Lia D. Juliawaty, Yana M. Syah, Euis H. Hakim, Kiyotaka Koyama, Kaoru Kinoshita,

Kunio Takahashi, Phenolic Compounds from Cryptocarya konishii : Their Cytotoxic and Tyrosine Kinase Inhibitroy Properties, J. Natur. Med., 64, 121-222, 2010

8. Hiroaki Sasaki, Kazuhiko Miki, Kaoru Kinoshita, Kiyotaka Koyama, Lia D. Juliawaty, Sjamsul A. Achmad, Euis H. Hakim, Miyuki Kaneda, Kunio Takahashi, β-Secretase (BACE-1) Inhibitory Effect of

Biflavonoids, Bioorganic and Medicinal Chemistry Letters, Vol. 20, 4558-4560 (2010)

Nasional 1. Hakim, E.H., Syah, Y.M., Juliawaty, L.D., dan Mujahidin, D., Aktivitas antioksidan dan inhibitor

tirosinase beberapa stilbenoid dari tumbuhan Moraceae dan Dipterocarpaceae yang potensial untuk

bahan kosmetik, Invited Review, JMS, 2008, vol. 13, No.2, 33-42 2. Sahidin, Hakim, E. H., Syah, Y.M., Juliawaty, L.D., Achmad, S.A., Din, L, dan Latif, J., Resveratrol

dimers from stembark of Hopea gregaria and their cytotoxic properties, Indonesian Journal of Chemistry, 2008, vol.8, no. 2

Semua data yang diisikan dan tercantum dalam curriculum vitae ini adalah benar. Demikian curriculum

vitae ini saya buat dengan sebenar-benarnya untuk memenuhi persyaratan pengajuan proposal Program Riset Desentralisasi DIKTI 2013

Bandung, 5 April 2012

(Prof. Dr. Euis Holisotan Hakim)

13

10 LAMPIRAN BUKTI CAPAIAN OUTPUT TAHUN 2010-2012

Publikasi internasional:

Syah, Y.M., Ghisalberti, E.L. (2012). “More Phenolic Derivatives with an Irregular Sesquiterpenyl Side

Chain from Macaranga pruinosa”, Nat. Prod. J., (in print). Agustina, W., Juliawaty, L.D., Hakim, E.H., Syah, Y.M. (2012). “Flavonoids from Macaranga lowii”, ITB J.

Sci. (in print). Tanjung, M., Mujahidin, D., Hakim, E.H., Darmawan, A., Syah, Y.M. (2010). “Geranylated flavonols from

Macaranga rhizinoides”, Nat. Prod. Commun., 5, 1209-1211. Syah, Y.M., Ghisalberti, E.L. (2010). “Phenolic Derivatives with an Irregular Sesquiterpenyl Side Chain

from Macaranga pruinosa”, Nat. Prod. Commun., 5, 219-222. Kurniadewi, F., Juliawaty, L.D., Syah, Y.M., Achmad, S.A., Hakim, E.H., Koyama, K., Kinoshita, K.,

Takahashi, K. (2010). “Phenolic compounds from Cryptocarya konishii: their cytotoxic and tyrosine kinase inhibitory properties”, J. Natur. Med., 64, 121-125.

Publikasi nasional:

Ferlinahayati, Juliawaty, L.D., Syah, Y.M., Hakim, E.H., Latip, J. (2011). Calkon dari kayu batang Morus nigra, Bull. Soc. Nat. Prod. Chem (Indonesian), 11, 12-16.

Syah, Y.M. (2010). Penentuan struktur senyawa aromatik. bagian 1: Papiriflavonol A dari Macaranga pruinosa, Bull. Soc. Nat. Prod. Chem (Indonesian), 10, 43-47.

Tanjung, M., Mujahidin, D., Juliawaty, L.D., Hakim, E.H., Achmad, S.A., Syah, Y.M. (2010). “Dua isomer flavonoid terprenilasi dari daun Macaranga rhizinoides”, Bull. Soc. Nat. Prod. Chem (Indonesian), 10, 9-13.

The Natural Products Journal, 2012, 2, 000-000 1

2210-3155/12 $58.00+.00 © 2012 Bentham Science Publishers

More Phenolic Derivatives with an Irregular Sesquiterpenyl Side Chain from Macaranga pruinosa

Yana M. Syah1,* and Emilio L. Ghisalberti

2

1Natural Products Chemistry Research Group, Organic Chemistry Division, Institut Teknologi Bandung, Jalan Ganesha

10, Bandung 40132, Indonesia; 2Chemistry, School of Biomedical, Biomolecular and Chemical Sciences, University of

Western Australia, Crawley WA 6009, Australia

Abstract: Three new flavonol derivatives, macapruinosins D-F (1-3), together with a known flavonoid glyasperin A, had

been isolated from the acetone extract of the leaves of Macaranga pruinosa. The structures of the new compounds were

identified based on their spectroscopic data, including UV, IR, 1D and 2D NMR, and HREIMS spectra. Compounds 1 – 2

were further examples of phenolic compounds having an irregular sesquiterpenyl side chain with a cyclobutane skeleton.

Keywords: Cyclobutane sesquiterpene, Euphorbiaceae, flavonoids, flavonols; macapruinosins D-F, Macaranga pruinosa, irregular sesquiterpene, structure elucidation.

INTRODUCTION

Macaranga is one of the large genera of the family Euphorbiaceae, with about 250 plant species, and is known to produce a variety of flavonoid and stilbene derivatives [1]. Recently, we have reported a stilbene and a dihydroflavonol derivatives containing an irregular sesquiterpenyl side chain with a cyclobutane skeleton from a polar fraction of the acetone extract of M. pruinosa (Miq.) M ll.Arg. [2]. In continuation of our phytochemical examination of the Macaranga plants growing in Indonesia [3-5], we now report the isolation and structure elucidation of three flavonol derivative, named macapruinosin D-F (1-3) (Fig. 1), from the less polar fraction of the extract of the title plant, along with a known isoprenylated flavonol derivative, glyasperin A (4) [6]. Compounds 1 and 2 are further examples of phenolic derivatives containing an irregular sesquiterpenyl side chain with a cyclobutane skeleton found in nature.

MATERIAL AND METHODS

General Experimental Procedures

UV and IR spectra were measured with a Varian 100 Conc and Perkin Elmer Spectrum One FTIR spectrometers, respectively.

1H and

13C NMR spectra were recorded in

CDCl3 with a Varian NMR System 400 MHz (1H, 400 MHz;

13C, 100 MHz). Mass spectra were measured with a VG

Autospec mass spectrometer (EI mode). VLC (vacuum liquid chromatography) and PCC (planar centrifugal chromatography) were carried out using Merck silica gel 60 GF254, respectively, and for TLC analysis, pre-coated silica gel plates (Merck Kieselgel 60 GF254, 0.25 mm thickness)

*Address correspondence to this author at the Natural Products Chemistry

Research Group, Organic Chemistry Division, Institut Teknologi Bandung,

Jalan Ganesha 10, Bandung 40132, Indonesia; Tel: +62-22-2502103;

Fax: +62-22-2504154; E-mail: [email protected]

were used. Solvents used for extraction and preparative chromatography were of technical grades that were distilled before use.

Plant Material

Samples of the leaves of M. pruinosa were collected from Kalimantan, Indonesia, in December 2007. The plant was identified by Mr. Ismail, Herbarium Bogoriense, Bogor, Indonesia, and the voucher specimen was deposited in the herbarium.

Extraction and Isolation

The dried and powdered leaves of M. pruinosa (1 kg) were macerated with acetone to give a dark green acetone-extract (40 g). A part of the extract (20 g) was fractionated by VLC on silica gel (150 g) eluted with petrol-EtOAc of increasing polarity (17:3, 7:3, 1:1) to give 10 fractions F1-F10. Purification of fraction F3 (0.4 g) by PCC (twice, eluent 1: petrol-diisopropyl ether = 1:3; eluent 2: petrol-EtOAc = 4:1) gave compound 2 (3 mg). Based on TLC analysis, fractions F4 and F5 were combined (F45, 1.14 g) and were subjected to fractionation by PCC (eluent: petrol-diisopropyl ether = 1:3, 160 mL; diisopropyl ether, 80 mL) to give a fraction (F45-13, 590 mg) containing phenolic compounds. Purification of this fraction by the same method (twice: eluent: petrol-EtOAc = 3:1) yielded compounds 1 (30 mg) and 3 (3 mg), and glyasperin A (4) (115 mg).

Macapruinosin D (1)

Yellow solid; [ ]D = -5.1 (c 1.7, CH3OH); UV (MeOH)

maks (log ): 203 (4.54), 229 (sh, 4.32), 254 (4.13), 271 (4.22), 294 (4.06), 333 (sh, 4.11), 367 (4.16) nm; (MeOH + NaOH): 203 (4.53), 222 (sh, 4.40), 273 (4.22), 299 (4.05), 337 (sh, 4.09), 379 (4.14) nm; (MeOH + AlCl3): 203 (4.54), 229 (4.31), 273 (4.30), 306 (sh, 3.89), 362 (3.91), 430 (4.30)

2 The Natural Products Journal, 2012, Vol. 2, No. 1 Syah and Ghisalberti

nm; IR (KBr) max: 3410, 3230 (OH), 3071 (=CH), 2921, 2854 (CH-alkyl), 1646 (conj. C=O) cm

-1;

1H NMR (400

MHz) data, see Table 1; 13

C NMR (100 MHz) data, see Table 1; HREIMS m/z: [M]

+ 490.2340 (calcd. for C30H34O6

490.2355).

Macapruinosin E (2)

Yellow solid. UV (MeOH) maks (log ): 203 (4.56), 230 (sh, 4.34), 272 (4.19), 301 (4.07), 367 (3.97) nm; (MeOH + NaOH): 212 (5.01), 282 (4.20), 328 (sh, 4.01), 415 (3.97) nm; (MeOH + AlCl3): 204 (4.56), 233 (sh, 4.30), 270 (4.23), 312 (3.97), 429 (3.96) nm; IR (KBr) max: 3412 (OH), 2921, 2851 (CH-alkyl), 1636 (conj. C=O) cm

-1;

1H NMR (400

MHz) data, see Table 1; 13


+ 558.2969 (calcd. for C35H42O6

558.2981).

Macapruinosin F (3)

Yellow solid. UV (MeOH) maks (log ): 203 (4.60), 227 (sh, 4.30), 271 (4.33), 368 (3.88) nm; (MeOH + NaOH): 211 (5.10), 275 (4.33), 337 (sh, 3.90), 413 (3.85) nm; (MeOH + AlCl3): 203 (4.59), 231 (4.26), 429 (3.89) nm; IR (KBr) max: 3413, 3236 (OH), 3076 (=CH), 2956, 2922, 2853 (CH-alkyl), 1643 (conj. C=O) cm

-1;

1H NMR (400 MHz) data, see

Table 1; 13


+ 490.2345 (calcd. for C30H34O6 490.2355).

RESULTS AND DISCUSSION

Macapruinosin D (1) was isolated as a yellow solid, and from its HREIMS spectrum a molecular formula C30H34O6 was deduced (found [M]

+ 490.2340, 1.5 mDa). This

compound exhibited UV absorptions typical of a flavonol structure [ max 203, 229 (sh), 254, 271, 294, 333 (sh), 367 nm] [7], and showed batochromic shifts on addition AlCl3 and NaOAc. The IR spectrum indicated the pesence of absorptions for hydroxyl (3410, 3230 cm

-1), aromatic (3071

cm-1

), and conjugated carbonyl (1646 cm-1

) groups. In the 13

C NMR and DEPT spectra (Table 1), 28 carbon signals representing for 30 carbon atoms were observed, including two signals at C 135.4 and 175.2 that are characteristics for C-3 and C-4 resonances of a flavonol structure [5, 8]. The aromatic region of the

1H NMR spectrum of 1 (Table 1)

showed a pair (2H) of doublets with an ortho-coupling ( H 8.10 and 6.96) and a singlet ( H 6.47, 1H), which together with five other oxyaryl carbon signals ( C 161.7, 157.6, 157.3, 154.9, and 145.5), suggesting that 1 is either an 8- or a 6-substituted kaempferol derivative with C15-side chain. The NMR parameters of the C15-side chain (Table 1) were very close to those the C15-side chain of macapruinosin A, namely an irregular sesquiterpenyl group containing a cyclobutane skeletone [2]. The identity of the sesquiterpenyl group was determined by extensive analysis of NMR spectra, particularly HSQC-DEPT and HMBC spectra. The characteristics proton signals at H 5.30 (1H, tm), 3.50 (2H, br d), and 2.52 (1H, br t) were due to H-2”, H-1”, and H-8”, respectively; the proton signals at H 4.82, 4.62 (each 1H, br s) and 1.68 (3H, br s) were allocated for the 2-propenyl group attached at C-8”; while the two singlets of methyl proton signals at H 1.09 and 0.92 were assigned for the geminal methyl groups at C-7”, and a broad methyl singlet at

H 1.88 was a methyl group at C-3”. Other proton signals that are part of the sesquiterpenenyl group were three methylene ( H 2.06 and 1.95, H-4”; 1.66 and 1.48, H-5”; 2.08 and 1.52, H-9”) and one methine ( H 1.60) signals. The attachment of the sesquiterpenyl group at C-6 was determined by the HMBC correlations from the methylene signal at H 3.50, which was correlated with a quarternary carbon signal at C 109.4 (C-6) and two oxyaryl carbon signals at C 161.7 (C-7) and 157.6 (C-5). The later carbon signal was correlated with a chelated –OH group at H 12.08. From these spectral analysis, therefore structure 1 was assigned as macapruinosin D. Other HMBC correlations supporting the structure 1 are shown in Fig. (2). The close agreement of NMR parameters and NOE correlations in the sesquiterpenyl unit between 1 and those macapruinosin A [2] allowed the relative stereochemistry at C-6” and C-8” to be determined as shown in structure 1.

Macapruinosin E (2), isolated as a yellow solid, showed UV and IR spectra similar to those 1, and based on its HREIMS measurement, this compound was found to have a molecular formula C35H42O6 (found [M]

+ 558.2969, 1.2

mDa). The 13

C NMR also disclosed carbon signals characteristics for a flavonol structure ( C 175.2 and 135.4). These spectral analysis suggested that 2 has a structure similar to those 1 with an additional C5-unit. The

1H NMR

O

O

HO

OH

OH

OH

2

45

88a

4a

1'

4'

R13'

1"

3"

15"

6"

8"

14"13"

10"

11"

9"

1 R1 = H

1"' 3"'

4"'

5"'

2 R1 =

O

O

HO

OH

OH

OHR1

3 R1 =1" 3" 7"

8"

9"10"

4 R1 =1" 3"

5"

4"

Fig (1). Structures of flavonoids isolated from M. pruinosa.

Phenolic Derivatives from Macaranga pruinosa The Natural Products Journal, 2012, Vol. 2, No. 1 3

Table 1. 1H and

13C NMR Data of Compounds 1 – 3 in CDCl3

1 2 3 C. No.

H (mult., J in Hz) C H (mult., J in Hz) C H (mult., J in Hz) C

2 - 145.5 - 145.6 - 145.6

3 - 135.4 - 135.4 - 135.4

4 - 175.2 - 175.2 - 175.2

4a - 103.5 - 103.5 - 103.5

5 - 157.6 - 157.7 - 157.6

6 - 109.4 - 109.2 - 109.2

7 - 161.7 - 161.6 - 161.8

8 6.47 (s) 94.3 6.48 (s) 94.3 6.49 (s) 94.4

8a - 154.9 - 155.0 - 155.0

1’ - 123.4 - 123.4 - 123.4

2’ 8.10 (d, 8.8) 129.6 7.98 (d, 2.3) 129.7 7.97 (d, 2.3) 129.7

3 6.96 (d, 8.8) 115.6 - 127.0 - 127.0

4’ - 157.3 - 156.3 - 156.3

5’ 6.96 (d, 8.8) 115.6 6.93 (d, 8.8) 116.0 6.93 (d, 8.5) 116.0

6’ 8.10 (d, 8.8) 129.6 7.99 (dd, 8.8, 2.3) 127.6 7.98 (dd, 8.5, 2.3) 127.6

1” 3.50 (br d, 7.1) 21.4 3.49 (br d, 7.1) 21.4 3.51 (d, 7.1) 21.4

2” 5.30 (tm, 7.1) 120.5 5.29 (tm, 7.1) 120.5 5.30 (tm, 7.1) 120.9

3” - 140.3 - 140.5 - 140.1

4” 2.06 (m); 1.95 (m) 38.0 2.05 (m); 1.93 (m) 38.0 2.12 (br t, 6.3) 39.7

5” 1.66 (m); 1.48 (m) 29.3 1.65 (m); 1.45 (m) 29.3 2.14 (br q, 6.3) 26.3

6” 1.60 (m) 40.9 1.58 (m) 40.9 5.07 (tm, 6.3) 123.6

7” - 39.9 - 39.9 - 132.2

8” 2.52 (br t, 8.3) 48.1 2.50 (m) 48.1 1.71 (br d, 1.0) 25.7

9” 2.08 (m); 1.52 (m) 25.0 2.08 (m); 1.50 (m) 25.0 1.63 (br d, 0.7) 17.7

10” 1.09 (s) 24.9 1.06 (s) 24.9 1.87 (br d, 1.2) 16.3

11” 0.92 (s) 24.2 0.90 (s) 24.2

12” - 146.3 - 146.3

13” 1.68 (br s) 23.4 1.65 (br s) 23.4

14” 4.82 (br s); 4.62 (br s) 108.9 4.81 (m); 4.61 (br s) 108.9

15” 1.88 (br s) 16.4 1.88 (br s) 16.5

1”’ 3.45 (br d, 7.1) 30.1 3.47 (d, 7.0) 30.1

2”’ 5.36 (tm, 7.1) 121.2 5.37 (tm, 7.0) 121.2

3”’ - 135.7 - 135.7

4”’ 1.80 (br s) 25.8 1.83 (br d, 1.2) 25.8

5”’ 1.82 (br s) 18.0 1.85 (br d, 0.9) 18.0

4 The Natural Products Journal, 2012, Vol. 2, No. 1 Syah and Ghisalberti

Table 1. contd….

1 2 3 C. No.

H (mult., J in Hz) C H (mult., J in Hz) C H (mult., J in Hz) C

3-OH 6.62 (s) 6.56 (br s) 6.56 (s)

5-OH 12.08 (s) 12.13 (br s) 12.11 (br s)

7-OH 6.34 (br s) 6.19 (br s) 6.21 (br s)

4’-OH 5.62 (br s) 5.53 (br s) 5.54 (br s)

O

O

OH

OH

HO

OH

1

O

O

OH

OH

HO

OH

2

O

O

HO

OH

OH

OH

3

Fig. (2). Selected HMBC correlations (1H

13C) in compounds 1-3.

spectrum of 2 (Table 1), together with 1H-

1H-COSY and

DEPT-HSQC spectra, also exhibited a high degree of

similarity with those 1, particularly for the presence of

signals belongs to the irregular sesquiterpenyl group, a singlet of an aromatic proton signal, and signals of four

phenolic –OH groups. It differed, however, from those 1 in

the presence of three aromatic proton signals of an ABX spin system ( H 7.99, 7.98, and 6.93), instead of a pair of an

ortho-coupled AA’XX’ spin system, and the proton signals

assignable to 3-methyl-2-butenyl group ( H 5.36, 3.45, 1.82, and 1.80). Therefore, the structure of macapruinosin

E (2) was determined to be 3’-(3”’-methyl-2”’-butenyl) macapruinosin D. Selected HMBC correlations supporting

the structure 2 are shown in Fig. (2). By comparison of the

NMR parameters between compounds 2 and 1, the relative stereochemistry at C-6” and C-8” of the sesquiterpenyl group

also follows to that of compound 1.

Macapruinosin F (3), also isolated as a yellow powder, gave absorptions properties of UV and IR light were very close to those compounds 1 and 2. The HREIMS measurement of this compounds showed [M]

+ ion at m/z

Phenolic Derivatives from Macaranga pruinosa The Natural Products Journal, 2012, Vol. 2, No. 1 5

490.2345, consistence to a molecular formula C30H34O6 ( 1.0 mDa), and thus it is an isomer of 1. The presence of six oxyaryl carbon signals ( C 175.2, 161.8, 157.6, 156.3, 145.6, and 135.4) (Table 1) also pointed to the presence of kaempferol structure in 3. In the

1H NMR spectrum (Table

1), four aromatic proton signals ( C 7.98, 7.97, 6.93, and 6.49) that were very close to those in 2 were observed, indicating that the C15-unit in 3 is in the form of a geranyl and an isoprenyl groups. This was corroborated by the presence of five methyl ( H 1.87, 1.85, 1.83, 1.71, and 1.63), four methylene ( H 3.51, 3.47, 2.14, and 2.12), and three vinyl methine ( H 5.37, 5.30, and 5.07) groups. By analysis of DEPT-HSQC and HMBC (Fig. 2), as well as by comparison of the NMR parameters with that of 2, the position of the geranyl and isoprenyl groups were deduced to be at C-6 and C-3’, respectively. Therefore, structure 3 was assigned to macapruinosin F.

Macapruinosins D (1) and E (2), along with macapruinosins A and B, are the first example of natural compounds having an irregular sesquiterpenyl side chain with a cyclobutane skeleton. The monoterpenyl and hemiterpenyl analogues have been reported to occur as the side chain of phenolic compounds isolated from Calophyllum verticillatum and C. brasiliense [9, 10], and as the metabolite of citrus mealybug, Planococcus citri [11].

ACKNOWLEDGEMENTS

Financial support from the office of the Ministry of National Education, Republic of Indonesia (Hibah Pasca Grant VII 2009) and from Endeavour Programme Australian Scholarships awarded to one of us (YMS) in 2008 (Award Contract No. 519-2008) are gratefully acknowledged.

REFERENCES

[1] Yoder, B.J.; Cao, S.; Norris, A.; Miller, J.S.; Ratovoson, F.;

Razafitsalama, J.; Andriantsiferana, R.; Rasamison, V.E.; Kingston, D.G.I. Antiproliferative prenylated stilbenes and flavonoids from

Macaranga alnifolia from the Madagascar Rainforest. J. Nat. Prod., 2007, 70, 342-346.

[2] Syah, Y.M.; Ghisalberti, E.L. Phenolic derivatives with an irregular sesquiterpenyl side chain from Macaranga pruinosa. Nat. Prod.

Commun., 2010, 5, 219-222. [3] Tanjung, M.; Mujahidin, D.; Hakim, E.H.; Darmawan, A.; Syah,

Y.M. Geranylated flavonols from Macaranga rhizinoides. Nat. Prod. Commun., 2010, 5, 1209-1211.

[4] Syah, Y.M.; Hakim, E.H.; Achmad, S.A.; Hanafi, M.; Ghisalberti, E.L. Isoprenylated flavanones and dihydrochalcones from

Macaranga trichocarpa. Nat. Prod. Commun., 2009, 4, 63-67. [5] Tanjung, M.; Hakim, E.H.; Mujahidin, D.; Hanafi, M.; Syah, Y.M.

Macagigantin, a farnesylated flavonol from Macaranga gigantea. J. Asian Nat. Prod. Res., 2009, 11, 929-932.

[6] Zeng, L.; Fukai, T.; Nomura, T.; Zhang, R.-Y.; Lou, Z.-C. Phenolic constituents of Glycyrrhiza species. 8. Four new prenylated

flavonoids, glyasperins A, B, C, and D from the roots of Glycyrrhiza aspera. Heterocycles, 1992, 34, 575-587.

[7] Mabry, T.J.; Markham, K.R.; Thomas, M.B. The Systematic Identification of Flavonoids; Springer-Verlag, New York, 1970,

pp. 41-164. [8] Sutthivaiyakit, S.; Unganont, S.; Sutthivaiyakit, P.; Suksamrarn, A.

Diterpenylated and prenylated flavonoids from Macaranga denticulata. Tetrahedron, 2002, 58, 3619-3622.

[9] Ravelonjato, B.; Kunesch, N.; Poisson, J.E. Neoflavonods from the stem bark of Calophyllum verticillatum. Phtochemistry, 1987, 26,

2973-2976. [10] Cottiglia, F.; Dhanapal, B.; Sticher, O.; Heilmann, J. New

chromanone acids with antibacterial activity from Calophyllum brasiliense. J. Nat. Prod., 2004, 67, 537-541.

[11] Bierl-Leonhardt, B.A.; Moreno, D.S.; Schwartz, M.; Fargerlund, J.; Plimmer, J.R. Isolation, identification and synthesis of the sex

pheromone of the citrus mealybug, Planococcus citri (Risso). Tetrahedron Lett., 1981, 22, 389-392.

Received: September 12, 2011 Revised: December 22, 2011 Accepted: January 10, 2012

ITB J. Sci., Vol. 44 A, No. 1, 2012, 13-18 13

Received May 26th, 2011, Revised July 28th, 2011, Accepted for publication July 29th, 2011.

Flavonoids from Macaranga lowii

Widiastuti Agustina, Lia D. Juliawaty, Euis H. Hakim & Yana M. Syah1

Organic Chemistry Division, Faculty of Mathematics and Natural Sciences, Institut

Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia 1E-mail: [email protected]

Abstract. A new isoprenylated dihydroflavonol derivative, macalowiinin (1),

together with two known flavonoids 4’-O-methyl-8-isoprenylnaringenin (2) and

4’-O-methyl-5,7,4’-trihydroxyflavone (3) (= acasetin), have been isolated from

the methanol extract of the leaves of Macaranga lowii. The structures of these

compounds were determined based on UV, NMR, and mass spectral data, and

optical rotation. Preliminary cytotoxic evaluation of compounds 1 – 3 against P-

388 cells showed that compound 3 was the most active with IC50 was 58.7 µM.

Keywords: acasetin; cytotoxicity; isoprenylated dihydroflavonol; Macaranga lowii;

macalowiniin; 4’-O-methyl-8-isoprenylnaringenin; P-388 cells.

1 Introduction

Macaranga is a large genus of Euphorbiaceae consisting of about 250 species and is distributed in the tropical region of the world, including Indonesia [1,2].

Phytochemical investigation has revealed that this genus is a rich source of

phenolic compounds, particularly the isoprenylated and geranylated flavonoids

and stilbenes [1,3]. In the course of our phytochemical study on Indonesian

Macaranga, recently we reported the isolation of isoprenylated flavanones and

dihydrochalcones from M. trichocarpa [4], isoprenylated, geranylated and

farnesylated flavonols from M. rhizinoides [5], M. pruinosa [6], and M.

gigantea [7], respectively, and a unique stilbene and dihydroflavonol

derivatives containing an irregular sesquiterpenyl side chain from M. pruinosa [6]. As part of this study, we have also examined a species collected from

Kalimantan island of Indonesia, M. lowii King ex. Hook.f., and successfully

isolated three flavonoids, including a new isoprenylated dihidroflavonol derivative, named macalowiinin (1), together with two known flavonoids 4’-O-

methyl-8-isoprenylnaringenin (2) [8] and 4’-O-methyl-5,7,4’-trihydroxyflavone

(3) (= acasetin) [9] (Figure 1), from the methanol extracts of the leaves of the plant. This paper reports the isolation and structure elucidation of the new

compound and cytoxic properties of compounds 1 - 3 against murine leukemia

P-388 cells.

14 W. Agustina, et al.

2 Results and Discussion

Macalowiinin (1) was isolated as an optically active pale yellow powder, and its

UV spectrum exhibited absorption maxima (296, 334 [sh] nm) typical for a

dihydroflavonol [6]. The UV absorption showed a bathochromic shift (37 nm) on addition NaOH solution, indicating that the compound contains one or more

free –OH phenolic groups. More spesifically, the presence of a free –OH

phenolic group at C-5 was also disclosed from the observation of a large

bathochromic shift (22 and 60 nm) on addition AlCl3 solution. However, on

addition of HCl, following AlCl3 addition, the UV spectrum was unchanged

indicating that the compound does not bear an 1,2-dihydroxyl group in the

aromatic rings. The HR-ESI-MS spectrum (negative mode) of 1 showed a quasimolecular [M-H]- ion (m/z 369.1340) consistent with a molecular formula

C21H22O6 (calculated [M-H]- 369.1338, ∆ 0.5 ppm), suggesting that 1 is a 2,3-

dihydroflavonol derivative containing an isoprenyl and a methoxyl groups. In

the 1H NMR spectrum (Tab. 1.) the presence of three proton signals at δH 5.09,

4.73, and 4.61, with multiplicities d (J = 11.5 Hz), d (J = 4.0 Hz), and dd (J =

11.5, 4.0 Hz), respectively, confirmed for the 2,3-dihydroflavonol skeleton in 1.

The 1H NMR spectrum of 1 also showed signals for an isoprenyl (δH 5.16, 1H;

3.19, 2H; 1.59 and 1.54, each 3H) and a methoxyl (δH 3.82, 3H) groups, and a

proton singlet signal at δH 11.64 that is consistent with an OH-phenolic at C-5. Further analysis of the 1H NMR spectrum in the aromatic region revealed the

presence of a pair of doublets of two-proton signals (δH 7.52 and 6.99) and a

singlet of one-proton signal (δH 6.06), suggesting that the isoprenyl group is either at C-6 or C-8. By analysis of HMQC and HMBC spectra of 1, the 5-OH

phenolic signal (δH 11.64) exhibited 1H-

13C long range correlations with the

signals of two aromatic quarternary (δC 162.6, C-5; 101.5, C-4a) and an

aromatic methine (δC 96.6, C-6) carbon atoms, and consequently these

correlations assign the isoprenyl group at C-8. Furthermore, the methoxyl

proton signal (δH 3.82) displayed a long range correlation with an oxyaryl

carbon signal (δC 160.8, C-4’) that does not have a correlation to the methylene

proton signal (δH 3.19) of an isoprenyl group, confirming that the methoxyl group is at C-4’. From these NMR data analysis, macalowiinin (1) was assigned

as 4’-O-methyl-5,7,4’-trihydroxy-8-isoprenyl-2,3-dihydroflavonol. Other

HMQC and HMBC correlations, as well as 13C NMR data assignment, that are

consistent with the structure 1 are shown in Tab. 1. The absolute

stereochemistry at C-2/C-3 was determined as shown in the structure 1, based

on the coupling constant (J = 11.5 Hz, trans) between H-2/H-3 and the optical rotation (+5.5o) [1].

Flavonoids from Macaranga lowii 15

1"

3"

O

O

HO

OH

OCH3

OH

2

45

8

4a

8a1'

4'

1

4" 5"

O

O

HO

OH

OCH3

2

O

O

HO

OH

OCH3

3

Figure 1 Structures of the flavonoids from M. lowii.

The occurrence of dihydroflavonol and flavone derivatives in the genus

Macaranga is very limited. To our knowledge the dihydroflavonol derivatives

have been isolated and identified only in three species, M. alnifolia [1], M. conifera [10], and M. pruinosa [6], while the presence of the flavone is the

second time after a similar compound has been isolated from M. gigantea [7].

Table 1 NMR (1H, 500 MHz; 13C 125 MHz) data of macalowiinin (1).

No C δδδδH δδδδC HMBC ( 1H ⇔⇔⇔⇔ 13C )

2 5.09 (d, 11.5) 84.0 C-3, C-4, C-1', C-2'/C-6'

3 4.61 (dd, 11.5, 4.0) 73.2 C-2, C-4, C-1',

3-OH 4.73 (d, 4.0) - -

4 - 198.4 -

4a - 101.5 -

5 - 162.6 -

6 6.06 (s) 96.6 C-4a, C-5, C-7,C-8

7 - 165.4 -

8 - 108.6 -

8a - 160.9 -

1' - 131.3 -

2'/6' 7.52 (d, 9.0) 130.0 C-2, C-3'/5', C-4', C-6'/2'

3'/5' 6.99 (d, 9.0) 114.4 C-1', C-2'/6', C-4', C-5'/3'

4' - 160.8 -

1" 3.19 (d, 7.5) 22.0 C-7, C-8, C-8a, C-2", C-3"

2" 5.16 (tm, 7.5) 123.3 C-1", C-4", C-5"

3" - 130.5 -

4" 1.59 (s) 25.8 C-2", C-3", C-5"

5" 1.54 (s) 17.8 C-2", C-3", C-4"

5-OH 11.64 (s) - C-4a, C-5, C-6

4’-OCH3 3.82 (s) 55.5 C-4'


Thus, the presence of these flavonoids could have a significant as a marker of a

certain group of Macaranga.

Compounds 1 – 3 were evaluated for their cytotoxicities against murine

leukemia P-388 cells, showing their IC50 were 119.3, 166.6, and 58.7 µM,

respectively.

3 Experimental

3.1 General

Optical rotation was measured with Polarimeter Perkin Elmer 341, while UV spectra were acquired with Varian 100 Conc spectrometer. 1H and 13C NMR

spectra were recorded with a Bruker Avance 500 spectrometer (1H, 500 MHz;

13C, 125 MHz), and mass spectra were measured with an ESI-TOF Water LCT

Premier XE (negative mode). VLC (vacuum liquid chromatography) and PCC

(planar centrifugal chromatography) were carried out using Merck silica gel 60

GF254, respectively, and for TLC analysis, pre-coated silica gel plates (Merck

Kieselgel 60 GF254, 0.25 mm thickness) were used. Solvents used for extraction

and preparative chromatography are technical grades that were distilled before

use.

3.2 Plant Materials

The leaves of M. lowii were collected from Kalimantan island, Indonesia, in

August 2008. The plant was identified by Mr. Ismail, Herbarium Bogoriense,

Bogor, Indonesia, and the voucher specimen was deposited in the herbarium.

3.3 Extraction and Isolation

The powdered and dried leaves of M. lowii (0.8 kg) were macerated in methanol

at room temperature (3x 5 L), and after evaporation of the solvent gave a

methanol extract as a semisolid residue (130 g). A portion of the extract (50 g)

was divided into acetone-soluble (22 g) and acetone-insoluble (28 g) fractions.

The acetone soluble fraction was fractionated through a VLC column, eluted

with n-hexane-EtOAc (17:3, 4:1, 7:3, and 1:1, each 450, 300, 300, and 600 mL,

respectively) to give ten fractions A-J. TLC analysis, monitored with UV lamp 254 nm, showed that the suspected flavonoid spots were contained in the

fraction D and H. Refractionation of the fraction D (1.08 g) by using the same

method (19:1, 9:1, 17:3, and 4:1, each 150, 150, 200, and 200 mL, respectively) afforded 14 fractions, and the fractions rich with flavonoids (175 mg) were

purified with sephadex LH-20 column eluted with MeOH to give a fraction

which on crystallization yielded compound 2 (50 mg) [8]. Fraction H (720 mg)

Flavonoids from Macaranga lowii 17

was also refractionated using PCC eluted with n-hexane-EtOAc (4:1 to 3:2) to

give two major fraction H1 and H2 containing flavonoids. Purification of

fraction H1 (140 mg) using the same method (n-hexane-EtOAc, 4:1) afforded compound 1 (25 mg). Fraction H2 (215 mg) was purified using PCC technique

(n-hexane-EtOAc, 9:1) and sephadex LH-20 (MeOH) to give compound 3 (5

mg) [9].

Macalowiinin (1)

Pale yellow powders; [α]D = + 5.5o (c 0.15, MeOH); UV (MeOH) λmax nm: 296,

334 (sh); UV (MeOH+NaOH) λmax nm: 333; UV (MeOH+AlCl3) λmax nm: 318,

394 (sh); UV (MeOH+AlCl3+HCl) λmax nm: 318, 394 (sh); 1H NMR (500 MHz,

acetone-d6) δ ppm: see Tab. 1.; 13C NMR (125 MHz, acetone-d6) δ ppm: see

Table 1.; HR-ESI-MS m/z: [M-H]- 369.1340 (calculated [M-H]- for C21H22O6

369.1338).

3.4 Cytotoxic Assay

The cytotoxic properties of compounds 1 – 3 were evaluated against murine leukemia P-388 cells, and were carried out by MTT assay according to the

method previously described [11].

Acknowledgement

The authors are grateful for the financial support from Hibah Pasca Grant VII 2009, Contract No. 0052f/K01.20/SPK-LPPM/I/2009. We also thank Prof. Peter

Proksch, the University of Dusseldorf, Germany, for NMR spectra

measurements.

References

[1] Yoder, B.J., Cao, S., Norris, A., Miller, J.S., Ratovoson, F.,

Razafitsalama, J., Andriantsiferana, R., Rasamison, V.E. & Kingston

D.G.I., Antiproliferative Prenylated Stilbenes and Flavonoids from

Macaranga Alnifolia from the Madagascar Rainforest, J. Nat. Prod., 25, 342-346, 2007.

[2] Airy Shaw, H.K., The Euphorbiaceae of Central Malesia (Celebes,

Moluccas, Lesser Sunda Is.), Kew Bull., 37, 1-40, 1982.

[3] Kawakami, S., Harinantenaina, L., Matsunami, K., Otsuka, H., Shinzato,

T. & Takeda Y., Macaflavanones A-G, Prenylated Flavanones from the

Leaves of Macaranga Tanarius, J. Nat.Prod., 71, 1872-1876, 2008.

[4] Syah, Y.M., Hakim, E.H., Achmad, S.A., Hanafi, M. & Ghisalberti, E.L.,

Isoprenylated Flavanones and Dihydrochalcones from Macaranga

Trichocarpa, Nat. Prod. Commun., 4, 63-67, 2009.


[5] Tanjung, M., Mujahidin, D., Hakim, E.H., Darmawan, A. & Syah, Y.M.,

Geranylated Flavonols from Macaranga Rhizinoides, Nat. Prod.

Commun., 5, 1209-1211, 2010. [6] Syah, Y.M. & Ghisalberti, E.L., Phenolic Derivatives with an Irregular

Sesquiterpenyl Side Chain from Macaranga Pruinosa, Nat. Prod.

Commun., 5, 219-222, 2010. [7] Tanjung, M., Hakim, E.H., Mujahidin, D., Hanafi, M., Syah, Y.M.,

Macagigantin, A Farnesylated Flavonol from Macaranga Gigantea, J.

Asian Nat. Prod. Res., 11, 929-932, 2009.

[8] Parson, I.C., Gray, A.I. & Waterman, P.G., New Triterpenes and

Flavonoids from The Leaves of Basistoa Brasii, J. Nat. Prod., 56, 46-53,

1993.

[9] Fujinori. H. & Neil, T.G.H., Flavones from Alnus rubra Bong. Coat Seed,

Bull. FFPRI., 2, 85-91, 2003.

[10] Jang, D.S., Cuendet, M., Hawthorne, M.E., Kardono, L.B.S., Kawanishi, K., Fong, H.H.S., Mehta, R.G., Pezzuto, J.M. & Kinghorn, A.D.,

Prenylated Flavonoids of The Leaves of Macaranga Conifera with

Inhibitory Activity Against Cyclooxygenase-2, Phytochemistry, 61, 867-

872, 2002.

[11] Sahidin, Hakim, E.H., Juliawaty, L.D., Syah, Y.M., Din, L.B.,

Ghisalberti, E.L., Latip, J., Said, I.M. & Achmad, S.A., Cytotoxic

Properties of Oligostilbenoids from The Tree Bark of Hopea Dryobalanoides, Z. Naturforsch. C., 60, 723-727, 2005.

Geranylated Flavonols from Macaranga rhizinoides

Mulyadi Tanjunga,b

, Didin Mujahidina, Euis H. Hakim

a, Ahmad Darmawan

c and Yana M. Syah

a *

aNatural Products Chemistry Research Group, Organic Chemistry Division, Institut Teknologi Bandung,

Jalan Ganesha 10, Bandung 40132, Indonesia

bDepartement of Chemistry, Faculty of Science and Technology, Airlangga University, Surabaya 60115,

Indonesia

cResearch Center for Chemistry, Indonesian Institute of Science, Serpong, 15310, Tangerang, Indonesia

[email protected]

Received: March 15th

, 2010; Accepted: July 15th

, 2010

Two geranylated and methylated flavonol derivatives, macarhizinoidins A (1) and B (2), along with a known phenolic

compound methyl 4-isoprenyloxycinnamate (3), have been isolated from the methanol extract of the leaves M. rhizinoides. The

structures of these compounds were identified based on their spectroscopic data. On cytotoxic evaluation against murine

leukemia P-388 cells, compounds 1-2 showed IC50 values of 11.4 and 13.9 μM, respectively, while compound 3 was inactive.

Keywords: Macarhizinoidins A and B, flavonol, geranyl group, Macaranga rhizinoides, Euphorbiaceae.

The genus Macaranga (Euphorbiaceae) contains about

250 species which are distributed from Africa and

Madagascar in the West to tropical Asia, north

Australia, and the Pacific islands in the East [1]. This

genus has been shown to produce a number of

phenolic compounds, particularly flavonoids and

stilbenoids [2,3]. Recently, we reported the isolation of

isoprenylated flavanones and dihydrochalcones from

M. trichocarpa [4], a farnesylated and a geranylated

flavonol from M. gigantea [5] and M. pruinosa [6],

respectively, and a stilbene and a dihydroflavonol

derivative containing an irregular sesquiterpenyl side

chain from M. pruinosa [6]. In continuation of our work

on the Indonesian Macaranga, the present paper report

the isolation of two geranylated flavonols, trivially

named macarhizinoidins A (1) and B (2), along with the

known compound methyl 4-isoprenyloxycinnamate (3)

[7], from the methanol extract of the leaves of

M. rhizinoides (Blume) Muell Arg. Cytotoxic properties

of compounds 1-3 against murine leukemia P-388 cells

are also briefly described.

Macarhizinoidin A (1) was isolated as a yellow solid

and the molecular formula C26H28O6 was deduced

by combined analysis of HR-EIMS ([M]+ peak at

m/z 436.1879, Δ 2.1 ppm) and NMR data (Table 1). The

UV spectrum showed absorption maxima (λmax 203,

217 sh, 271, 297, 335 sh, 368 nm) typical of a flavonol

chromophore, while the IR spectrum disclosed the

presence of a conjugated carbonyl group (1622 cm-1

).

Signals for a methoxyl group were clearly seen in the 1H and

13C NMR data (δH 3.88, δC 55.7) of 1, and

together with proton signals characteristic of a geranyl

group (δH 5.29, 5.06, 3.37, 2.09, 1.95, 1.79, 1.59 and

1.54) suggested that 1 is a geranyl derivative of a

methylated kaempferol. The presence of a pair of

doublets at δH 8.20 and 7.10 (each 2H, J = 9.1 Hz) and a

singlet at δH 6.60 in the aromatic region of the 1H NMR

spectrum pinpointed the geranyl group to the A ring of a

kaempferol structure. The 13

C NMR spectrum of 1

showed 24 carbon signals representing 26 carbon atoms

and their assignment were made from HMQC and

HMBC spectra. The long range 1H-

13C correlations in

the HMBC spectrum between a chelated –OH signal (δH

12.11) and three quaternary carbon signals (δC 104.0,

111.8, 158.9) established that the geranyl group is at

C-6. Furthermore, the presence of 1H-

13C long range

correlations between the signals of an aromatic doublet

(δH 8.20) and the methoxyl signal (δH 3.88) with the

same oxyaryl carbon signal (δC 161.9) secured the

position of the methoxyl group at C-4’. Macarhizinoidin

A (1), therefore, was assigned as 6-geranyl-4’-O-methyl

kaempferol. Complete HMBC correlations in support of

structure 1 are shown in Table 1.

Macarhizinoidin B (2), isolated also as a yellow solid,

showed UV (λmax 205, 256, 296, 349 nm) and IR (1637

cm-1

) absorptions very similar to those of 1, suggesting

NPC Natural Product Communications 2010

Vol. 5

No. 8

1209 - 1211

1210 Natural Product Communications Vol. 5 (8) 2010 Tanjung et al.

O

O

HO

OH

OCH3

OH

OH

2

1"

3"

7"

8"

9"

10"

O

O

HO

OH

OCH3

OH

2

45

8

4a

8a 1'4'

1

2'

1'4'

OCH3

O

O3

Table 1: NMR spectroscopic data of macarhizinoidins A (1) and B (2) in acetone-d6.

1 2

δH δC HMBC (1H ⇔

13C) δH δC HMBC (

1H ⇔

13C)

2 - 146.3 - - 150.2 -

3 - 136.8 - - 137.7 -

4 - 176.6 - - 177.0 -

4a - 104.0 - - 104.7 -

5 - 158.9 - - 162.5 -

6 - 111.8 - 6.26 (d, 1.8) 99.1 C-4a, C-5, C-7, C-8

7 - 162.7 - - 164.8 -

8 6.60 (s) 93.8 C4a, C-6, C-7, C-8a 6.38 (d, 1.8) 94.4 C-4a, C-6, C-7

8a - 155.6 - - 158.3 -

1’ - 124.4 - - 124.4 -

2’ 8.20 (d, 9.1) 130.2 C-2, C-4’, C-6’ - 128.5 -

3’ 7.10 (d, 9.1) 114.7 C-1’, C-4’ - 145.2 -

4’ - 161.9 - - 149.4 -

5’ 7.10 (d, 9.1) 114.7 C-1’, C-4’ 6.96 (d, 8.5) 109.3 C-1’, C-3’

6’ 8.20 (d, 9.1) 130.2 C-2, C-2’, C-4’ 7.05 (d, 8.5) 122.5 C-2’, C-4’

1” 3.37 (br d, 6.7) 21.9 C-5, C-6, C-7, C-2”, C-3” 3.47 (d, 6.7) 26.4 C-1’, C-2’, C-3’, C-2”, C-3”

2” 5.29 (tm, 6.7) 123.0 C-6, C-1”, C-4”, C-10” 5.12 (tm, 6.7) 123.5 C-4”, C-10”

3” - 135.4 - - 135.3 -

4” 1.95 (br t, 7.0) 40.4 C-2”, C-3”, C-5”, C-10” 1.78 (br t, 7.0) 40.3 C-5”

5” 2.09 (m)* 27.3 C-4”, C-6”, C-7” 1.86 (br q, 7.0) 27.3 C-4”, C-6”

6” 5.06 (tm, 7.0) 125.0 C-8”, C-9” 4.98 (tm, 7.0) 125.0 C-8”, C-9”

7” - 131.5 - - 131.6 -

8” 1.59 (br s) 25.8 C-6”, C-7”, C-9” 1.57 (br s) 25.7 C-6”, C-7”, C-9”

9” 1.54 (br s) 17.6 C-6”, C-7”, C-8” 1.49 (br s) 17.6 C-6”, C-7”, C-8”

10” 1.79 (br s) 16.2 C-2”, C-3”, C-4” 1.45 (br s) 16.2 C-2”, C-3”, C-4”

3-OH 8.08 (very br s) - - 7.64 (very br s) - -

5-OH 12.11 (s) - C-4a, C-5, C-6 12.27 (s) - C-4a, C-5, C-6

7-OH 9.69 (very br s) - - 9.65 (very br s) - -

3’-OH 9.65 (very br s) - -

4’-OCH3 3.88 (s) 55.7 C-4’ 3.92 (s) 56.3 C-4’

*overlapping with residual solvent peaks.

that it is also a flavonol derivative. The HR-EIMS of 2

gave a [M]+ peak at m/z 452.1839 that, together with

NMR data (Table 1), corresponds to the molecular

formula C26H28O7 (Δ 0.9 ppm). The 13

C NMR spectrum

of 2 showed 26 carbon signals and their assignments

were determined by HMQC and HMBC spectra. From

NMR analysis, compound 2 also contained a methoxyl

(δH 3.92, δC 56.3) and a geranyl (δH 5.12, 4.98, 3.47,

1.86, 1.78, 1.57, 1.49 and 1.45) group. These spectral

data suggested that 2 is a geranyl derivative of a

methylated quercetin. The location of the geranyl group

was deduced to be at C-2’ by the observation in the 1H

NMR spectrum of a pair of meta-coupled (J = 1.8 Hz)

doublets (δH 6.38 and 6.26) and a pair of ortho-coupled

(J = 8.5 Hz) doublets (δH 7.05 and 6.96). Analysis of

HMBC correlations originating from the signals of H-5’

(δH 6.26) and H-6’ (δH 6.38) allowed identification of

carbon signals C-1’, C-2’, C-3’ and C-4’. These carbon

signals were used to confirm the placement of the

geranyl and the methoxyl groups at C-2’ and C-4’,

respectively, from the 1H-

13C long range correlations

observed from the methylene of the geranyl group

(δH 3.47) and methoxyl (δH 3.92) signals, as shown in

Table 1. Macarhizinoidin B (2), therefore, was

determined as 2’-geranyl-4’-O-methylquercetin.

Preliminary cytotoxic evaluation of compounds 1-3 was

carried out against murine leukemia P-388 cells

according to the MTT assay, as previously described

[8]. Compounds 1-2 showed moderate cytotoxicity

with IC50 values of 11.4 ± 1.5 and 13.9 ± 0.5 μM,

respectively, while compound 3 was inactive.

Experimental

General: UV and IR spectra were measured with

Varian 100 Conc and Perkin Elmer Spectrum One FTIR

Geranylated flavonols from Macaranga rhizinoides Natural Product Communications Vol. 5 (8) 2010 1211

spectrometers, respectively. 1H and

13C NMR spectra

were recorded with a JEOL ECA 500 spectrometer

(1H, 500 MHz;

13C, 125 MHz). MS were measured with

a Finnigan MAT 95 spectrometer (EI mode). VLC

(vacuum liquid chromatography) and PCC (planar

centrifugal chromatography) were carried out using

Merck silica gel 60 GF254, and for TLC analysis, pre-

coated silica gel plates (Merck Kieselgel 60 GF254, 0.25

mm thickness) were used. Solvents utilized for

extraction and preparative chromatography were

technical grades that were distilled before use.

Plant materials: The leaves of M. rhizinoides were

collected from Salak Mt., Bogor, Indonesia, in June

2008. The plant was identified by Mr Ismail, Herbarium

Bogoriense, Bogor, Indonesia, and the voucher

specimen was deposited in the herbarium.

Extraction and isolation: The powdered and dried

leaves of M. rhizinoides (0.8 kg) were macerated in

methanol at room temperature (3x), and, after

evaporation of the methanol extract, gave a dark

residue (100 g). The methanol extract was partitioned

into n-hexane and EtOAc fractions. The EtOAc fraction

(18 g) was further fractionated by VLC on silica gel

(150 g) eluted with n-hexane-EtOAc of increasing

polarity (9:1, 4:1; 7:3, 1:1, and 1:4) to give 5 major

fractions A-D. On TLC analysis, the phenolic

constituents were observed only in fractions B and C.

Fraction B (380 mg) was purified by PCC eluted

with n-hexane-CHCl3 (4:1 to 1:1) to give compound 1

(8 mg). Using the same method [PCC, eluted with

n-hexane-CHCl3 (4:1) and CHCl3], purification of

fraction C (480 mg) afforded compounds 2 (10 mg) and

3 (15 mg) [7].

Macarhizinoidin A (1)

Yellow solid.

IR (KBr): νmax = 3300, 2922, 2850, 1622, 804 cm-1

.

UV/Vis (MeOH): λmax (log ε) = 203 (4.57), 217 (sh,

4.50), 271 (4.30), 297 (4.11), 335 (sh, 4.18), 368 (4.21)

nm; (MeOH+NaOAc) 203 (4.60), 272 (4.25), 358

(4.09), 425 (3.87) nm; (MeOH+AlCl3) 204 (4.58), 228

(sh, 4.38), 273 (4.39), 306 (sh, 3.93), 359 (4.01), 428

(4.37) nm. 1H NMR (500 MHz, acetone-d6): Table 1.

13C NMR (125 MHz, acetone-d6): Table 1.

HRMS-EI: m/z [M]+ calcd. for C26H28O6: 436.1886;

found: 436.1879.

Macarhizinoidin B (2)

Yellow solid.

IR (KBr): νmax = 3409, 2922, 2850, 1637, 802 cm-1

.

UV/Vis (MeOH): λmax (log ε) = 205 (4.54), 256 (4.07),

296 (3.95), 349 (3.82) nm; (MeOH+NaOAc) 204 (4.62),

261 (3.98), 300 (3.83), 336 (3.76), 418 (3.38) nm;

(MeOH+AlCl3) 205 (4.55), 226 (sh, 4.29), 266 (4.14),

312 (3.89), 413 (3.85) nm. 1H NMR (500 MHz, acetone-d6): Table 1.

13C NMR (125 MHz, acetone-d6): Table 1.

HRMS-EI: m/z [M]+ calcd. for C26H28O7: 452.1835;

found: 452.1839.

Acknowledgments - The authors are grateful for the

financial support from Hibah Pasca Grant VII 2009,

Contract No. 0052f/K01.20/SPK-LPPM/I/2009. We

also thank Prof. Sven Doye, the University of

Oldenburg, Germany, for mass spectra measurements.

References

[1] Blattner FR, Weising K, Banfer G, Maschwitz U, Fiala B. (2001) Molecular analysis of phylogenetic relationships among

Myrmecophytic Macaranga species (Euphorbiaceae). Molecular Phylogenetics and Evolution, 19, 331-334.

[2] Yoder BJ, Cao S, Norris A, Miller JS, Ratovoson F, Razafitsalama J, Andriantsiferana R, Rasamison VE, Kingston DGI. (2007) Antiproliferative prenylated stilbenes and flavonoids from Macaranga alnifolia from the Madagascar rainforest. Journal of Natural

Products, 70, 342-346.

[3] Kawakami S, Harinantenaina L, Matsunami K, Otsuka H, Shinzato T, Takeda Y. (2008) Macaflavanones A-G, prenylated

flavanones from the leaves of Macaranga tanarius. Journal of Natural Products, 71, 1872-1876.

[4] Syah YM, Hakim EH, Achmad SA, Hanafi M, Ghisalberti EL. (2009) Isoprenylated flavanones and dihydrochalcones from

Macaranga trichocarpa. Natural Product Communications, 4, 63-67.

[5] Tanjung M, Hakim EH, Mujahidin D, Hanafi M, Syah YM. (2009) Macagigantin, a farnesylated flavonol from Macaranga

gigantea. Journal of Asian Natural Products Research, 11, 929-932.

[6] Syah YM, Ghisalberti EL. (2010) Phenolic derivatives with an irregular sesquiterpenyl side chain from Macaranga pruinosa.

Natural Product Communications, 5, 219-222.

[7] Delle Monache F, Delle Monache G, De Moraes e Souza MA, Cavalcanti MS, Chiappeta A. (1989) Isopentenylindole derivatives

and other components of Esenbeckia leiocarpa. Gazzetta Chimica Italiana, 119, 435-439.

[8] Sahidin, Hakim EH, Juliawaty LD, Syah YM, Din LB, Ghisalberti EL, Latip J, Said IM, Achmad SA. (2005) Cytotoxic properties

of oligostilbenoids from the tree bark of Hopea dryobalanoides. Zeitschrift für Naturforschung, 60C, 723-727.

Phenolic Derivatives with an Irregular Sesquiterpenyl Side Chain from Macaranga pruinosa Yana M. Syaha * and Emilio L. Ghisalbertib aDepartment of Chemistry, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia bChemistry, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, Crawley WA 6009, Australia [email protected] Received: August 18th, 2009; Accepted: October 12th, 2009

A stilbene and two flavonoid derivatives, macapruinosins A-C (1-3), together with two known flavonoids, papyriflavonol A and nymphaeol C, have been isolated from the acetone extract of the leaves of Macaranga pruinosa. The structures of these compounds were identified based on spectral data analysis. Compounds 1 and 2 are the first examples of natural compounds containing an irregular sesquiterpenyl side chain with a cyclobutane skeleton. Keywords: Macapruinosins A-C, Stilbene, Flavonol, Dihydroflavonol, Irregular sesquiterpenyl group, Macaranga pruinosa, Euphorbiaceae. In the Euphorbiaceae, Macaranga is one of the large genera with about 250 species, and is known to produce a variety of terpenoids and isoprenylated flavonoids and stilbenes [1]. Recently, we have reported four isoprenylated flavonoids from M. trichocarpa [2]. In continuation of a phytochemical examination of Macaranga plants growing in Indonesia, we now report the isolation and structure elucidation of a stilbene and flavonoid derivatives (1-3) from the acetone extract of the leaves of M. pruinosa (Miq.) Műll.Arg. Compounds 1 and 2 are the first example of natural compounds containing an irregular sesquiterpenyl side chain with a cyclobutane skeleton. The HR-EIMS of macapruinosin A (1) gave a [M]+ peak at m/z 448.2611, which, together with NMR data, corresponds to the molecular formula C29H36O4. The UV spectrum of 1 showed absorptions (λmax 203, 224 sh, 299 sh, and 330 nm) typical for a stilbene chromophore. Proton signals in the 1H NMR spectrum corresponding to a trans-vinyl group (δH 6.81 and 6.74, J = 16.5 Hz) supported the presence of a trans-stilbene structure in 1. Further analysis of the 1H NMR spectrum (Table 1) revealed that the compound has the structure of a C-4’ substituted piceatannol [3]. The substituent must have the formula C15H25, and by the observation of alkenyl proton signals at δH 5.30 (=CH), 4.76, and 4.58 (=CH2), it is a monocyclic C15-unit. From extensive analysis of NMR spectral data, including 13C NMR,

COSY, DEPT-HSQC and HMBC spectra, the substituent was identified as an irregular sesquiterpenyl, E-5-(2,2-dimethyl-3-(prop-1-en-2-yl) cyclobutyl)-3-methylpent-2-en-1-yl group. Salient 1H-1H COSY cross peaks were observed for vicinal couplings between H2-1”/H-2”, H2-4”/H2-5”, H-6”/H2-9”, and H-8”/H2-9”, as well as long range couplings between H2-1”/H3-15”, H2-1”/H2-4”, and H-8”/H3-13”. These COSY data, together with HMBC correlations, in particular between H2-4”/ C-15” and H2-5”/C-3”, ruled out a cyclopentane or cyclohexane skeleton in the sesquiterpenyl group. Complete HMBC correlations in support of structure 1 are shown in Table 1. The relative stereochemistry at C-6” and C-8” was determined from the NOESY spectrum. Important NOE correlations, as shown in Figure 1, established a trans relationship at these chiral carbon atoms. Structure 1, therefore, was assigned to macapruinosin A. By comparison of the NMR data (1H and 13C NMR, COSY, DEPT-HSQC, HMBC and NOESY spectra) (Table 2), macapruinosins B (2) also contained the same C15-side chain as that of compound 1. The presence of a dihydroflavonol skeleton in 2 was suggested from its UV (λmax 205, 291 and 352 nm) and IR absorptions (νmax 1639 cm-1), as well as from the presence of a pair of oxygenated methines (δH 4.98 and 4.55, each d) in the 1H NMR spectrum. The presence of proton signals of an aromatic singlet at δH 5.98, a pair of aromatic

NPC Natural Product Communications 2010 Vol. 5 No. 2

219 - 222

220 Natural Product Communications Vol. 5 (2) 2010 Syah & Ghisalberti

OH

OHHO

OH

R

1

3

4

α

β

1'

3'

5' O

O

HO

OH

OH

OH

2

45

88a

4a

1'

4'

R

O

O

HO

OH

OH

OH

OH

1"

3"

6"7"

8"

9"

12"

10" 11"

13"

14"

15"

1 2

3

R =

1"

3"7"

8"

9" 10"

11"

13"

14"

15"

Table 1: NMR spectroscopic data of macapruinosin A (1) in acetone-d6.

C no δH δC HMBC (1H ⇔ 13C)1 - 130.8 - 2 7.00 (d, 2.0) 113.6 C-4, C-6, C-α 3 - 146.1 - 4 - 145.8 - 5 6.76 (d, 8.1) 116.2 C-1, C-3, C6 6 6.83 (dd, 8.1,

2.0) 119.7 C-4, C-α

α 6.81 (d, 16.5) 128.3 C-1, C-2, C-6, C-β, C-1’ β 6.74 (d, 16.5) 126.9 C-1, C-1’ 1’ - 137.1 - 2’/6’ 6.54 (s) 105.7 C-4’, C-6’/2’, C-β 3’/5’ - 156.9 - 4’ - 115.2 - 1” 3.35 (br d, 7.0) 23.0 C-3’/5’, C-4’, C-2”, C-3” 2” 5.30 (tm, 7.0) 123.9 C-1”, C-3”, C-4”, C-15” 3’ - 134.8 - 4” 1.94 (m)

1.83 (m) 38.7 C-2”, C-3”, C-5”, C-6”, C-15”

5” 1.59 (m) 1.43 (m)

30.2 C-3”, C-7”

6” 1.57 (m) 41.7 C-9” 7” - 40.4 - 8’ 2.50 (br t, 8.3) 48.7 C-6”, C-7”, C-9”, C-10”, C-

11”, C-12” 9” 2.05 (m)

1.50 (ddd, 11.3, 8.3, 3.9)

25.6 C-5”, C-6”, C-8”, C-12”

10” 1.05 (s) 25.2 C-6”, C-7”, C-8”, C-11” 11” 0.88 (s) 24.6 C-6”, C-7”, C-8”, C-10” 12” - 146.8 - 13” 1.62 (br qi, 0.7) 23.6 C-8”, C-12”, C-14” 14” 4.76 (hept, 1.3)

4.58 (br s) 109.4 C-8”, C-12”, C-13”

15” 1.78 (br d, 1.1) 16.3 C-2”, C-4” 3-OH 7.82 (br s) C-2, C-3, C-4 4-OH 7.96 (br s) C-3, C-4, C-5 3’/5’-OH 8.02 (s) C-2’/6’, C-3’/5’, C-4’

HO

OHCH3

H3C

CH3

H

H

H

H

H3CH

HH

Figure 1: Important NOE correlations in compound 1.

doublets at δH 7.38 and 6.84 (each 2H), and four –OH groups (δH 11.53, 6.56, 5.61 and 3.55) pinpointed that the dihydroflavonol part of 2 has the same structure as that of bonanniol A [4]. The HMBC spectrum of 2 (Table 2) revealed 1H-13C correlations between

H2-1”/C-5, C-6 and C-7, confirming the attachment of the C15-side chain at C-6, while the coupling constant (11.9 Hz) of H-2 and H-3 secured the trans relationship between these hydrogens. From these spectroscopic data analysis, structure 2 was assigned to macapruinosin B. Compound 3 showed UV (λmax 207, 232 sh, 258, 274, 295, 374 nm) and IR absorptions (νmax 1636 cm-1) typical of a flavonol derivative. The HR-EIMS [M]+ peak at m/z 506.2305 showed that this compound has the molecular formula C30H34O7. The 1H NMR spectrum of 3, together with COSY and NOESY spectra, showed signals for isoprenyl (δH 5.27, 3.47, 1.84 and 1.77) and geranyl (δH 5.34, 5.02, 3.36, 2.09, 2.06, 1.74, 1.68 and 1.59) groups. Further analysis of the 1H NMR spectrum in the aromatic region revealed the presence of a singlet at δH 6.38 and a pair of ortho coupled signals (J = 8.3 Hz) at δH 7.07 and 6.92, suggesting that compound 3 has the structure of quercetin substituted at either C-6/C-2’ or C-8/C-2’ by the isoprenyl and geranyl groups. The 13C NMR of 3 showed 30 carbon signals and their multiplicities were determined from a DEPT-HSQC spectrum. The HMBC correlations observed between a chelated –OH group (δH 12.11) with carbon signals of an oxyaryl (δC 157.9) and two quarternary C-sp2 (δC 109.6 and 104.0) carbon atoms established that C-8 is unsubstituted. Further analysis of the HMBC spectrum allowed identification of the signal of the methylene protons attached to C-6 as a doublet at δH 3.47 (H2-11”). In the COSY spectrum, long range couplings between this methylene and two methyl signals (H3-14” and H3-15”) were observed, while the second methylene doublet (δH 3.36, H2-1”) showed a long range correlation with only one methyl signal (H3-10”). These correlations secured the attachment of the isoprenyl and geranyl groups at C-6 and C-2’, respectively, which were corroborated with the DEPT-HSQC and HMBC correlations, as shown in Table 2. Structure 3, therefore, is assigned to macapruinosin C. Compounds 1 and 2 are the first examples of natural products with an irregular s esquiterpenyl side chain

Phenolic derivatives from Macaranga trichocarpa Natural Product Communications Vol. 5 (2) 2010 221

Table 2: NMR spectroscopic data of macapruinosins B (2) and C (3) in CDCl3.

2 3 C no δH δC HMBC (1H ⇔ 13C) δH δC HMBC (1H ⇔ 13C)

2 4.98 (d, 11.9) 83.0 C-4, C-2’/6’ - 147.7 - 3 4.55 (d, 11.9) 72.4 C-4 - 136.3 - 4 - 195.8 - - 175.3 - 4a - 100.5 - - 104.0 - 5 - 160.6 - - 157.9 - 6 - 107.5 - - 109.6 - 7 - 164.7 - - 161.6 - 8 5.98 (s) 96.0 C-4, C-4a, C-6, C-7, C-8a 6.38 (s) 94.3 C-4a, C-6, C-7, C-8a 8a - 161.0 - - 155.6 - 1’ - 128.1 - - 121.9 - 2’ 7.38 (d, 8.6) 129.1 C-2, C-6’, C-4’ - 127.2 - 3’ 6.84 (d, 8.6) 115.7 C-1’, C-4’, C-5’ - 142.6 - 4’ - 156.5 - - 146.7 - 5’ 6.84 (d, 8.6) 115.7 C-1’, C-4’, C-3’ 6.92 (d, 8.3) 113.1 C-1’, C-3’ 6’ 7.38 (d, 8.6) 129.1 C-2, C-2’, C-4’ 7.07 (d, 8.3) 123.1 C-2, C-2’, C-4’ 1” 3.37 (br d, 7.1) 21.0 C-5, C-6, C-7, C-2”, C-3” 3.36 (br d, 6.7) 27.9 C-1’, C-2’, C-3’, C-2”, C-3” 2” 5.24 (tm, 7.1) 120.6 C-6, C-1”, C-4”, C-15” 5.34 (tm, 6.7) 121.4 C-2’, C-1”, C-4”, C-10” 3” - 139.9 - - 139.9 - 4” 2.01 (m)

1.91 (m) 38.0 C-2”, C-3”, C-5”, C-6”, C-15” 2.06 (m) 39.6 C-2”, C-3”, C-6”

5” 1.64 (m) 1.44 (m)

29.3 C-6”, C-9” 2.09 (m) 26.2 C-6”, C-7”

6” 1.56 (m) 40.9 C-7” 5.02 (tm, 6.9) 123.5 C-5”, C-9” 7” - 39.9 - - 136.0 - 8” 2.50 (m) 48.0 C-7”, C-10”, C-11”, C-12”, C-14” 1.68 (br s) 25.7 C-6”, C-7”, C-9” 9” 2.06 (m)

1.50 (m) 25.0 C-7”, C-8”, C-5”, C-12” 1.59 (br s) 17.7 C-6”, C-7”, C-8”

10” 1.05 (s) 24.9 C-6”, C-7”, C-8” 1.74 (br s) 16.1 C-2”, C-3” 11” 0.89 (s) 24.2 C-6”, C-7”, C-8”’ 3.47 (br d, 7.0) 21.4 C-5, C-6, C-7, C-12”, C-13” 12” - 146.3 - 5.27 (tm, 7.0) 120.9 C-11”, C-14”, C-15” 13” 1.65 (br s) 23.4 C-8”, C-12”, C-14” - 136.0 - 14” 4.81 (hept, 1.0)

4.61 (br s) 108.9 C-8”, C-12”, C-13” 1.77 (br s) 25.8 C-12”, C-13”, C-15”

15” 1.82 (br s) 16.4 C-2”, C-4” 1.84 (br s) 17.9 C-12”, C-13”, C-14” 3-OH 3.55 (br s) - C-2, C-4 6.10 (br s) - C-2, C-3, C-4 5-OH 11.53 (s) - C-4a, C-5, C-6 12.11 (s) - C-4a, C-5, C-6 7-OH 6.56 (br s) - C-6, C-7, C-8 6.30 (br s) - C-6, C-7, C-8 3’-OH - 5.71 (br s) - C-2’, C-3’, C-4’ 4’-OH 5.61 (very br s) - - 5.84 (br s) - C-3’, C-4’, C-5’

containing a cyclobutane skeleton. The monoterpenyl and hemiterpenyl analogues have been reported to occur in the metabolites of Calophyllum verticillatum and C. brasiliense [5,6], and in the citrus mealybug, Planococcus citri [7], respectively. Experimental

General: UV and IR spectra were measured with a Varian 100 Conc and Perkin Elmer Spectrum One FTIR spectrometers, respectively. 1H and 13C NMR spectra were recorded with either a Varian NMR System 400 MHz (1H, 400 MHz; 13C, 100 MHz) or a Bruker Avance 600 MHz (1H, 600 MHz). Mass spectra were measured with a VG Autospec mass spectrometer (EI mode). VLC (vacuum liquid chromatography) and PCC (planar centrifugal chromatography) were carried out using Merck silica gel 60 GF254, and for TLC analysis, pre-coated silica gel plates (Merck Kieselgel 60 GF254, 0.25 mm thickness) were used. Distilled technical grade solvents were used for extraction and preparative chromatography

Plant materials: Samples of the leaves of M. pruinosa were collected from Kalimantan, Indonesia, in December 2007. The plant was identified by Mr Ismail, Herbarium Bogoriense, Bogor, Indonesia. Extraction and isolation: The dried and powdered leaves of M. pruinosa (1 kg) were macerated with acetone to give a dark green extract (40 g). Part of this (20 g) was fractionated by VLC on silica gel (150 g) eluted with light petrol-EtOAc of increasing polarity (17:3, 7:3, 1:1) to give 10 fractions. From TLC analysis, the major fraction was contained in fraction-7 (F7, 1.1 g). This fraction was refractionated into two fractions, F7-23 (360 mg) and F7-46 (450 mg) by PCC eluting with light petrol-diisopropyl ether (1:3). Purification of fraction F7-46 by the same method (PCC, eluents CHCl3-acetone 37:3, 9:1, and 17:3) afforded macapruinosin A (1) (100 mg) and a fraction, which on further purification (PCC, CHCl3-acetone 37:3) gave macapruinosin B (2) (6 mg). Purification of F7-23 (PCC twice, light petrol-EtOAc 4:1 to 13:7; CHCl3-acetone 9:1 to 17:3) yielded macapruinosin C (3) (4 mg). Using

222 Natural Product Communications Vol. 5 (2) 2010 Syah & Ghisalberti

the same methodology, fraction 6 (F6, 600 mg) afforded papyriflavonol A (4) [8] (20 mg) and nymphaeol C (5) [9] (5 mg). Macapruinosin A (1)

Brownish-yellow solid. [α]D: -2.0 (c 0.4, MeOH). IR (KBr) νmax: 3400, 3078, 2923, 2854, 1616, 1516, 1442, 1280, 1191, 1158, 1033, 958, 823, 809 cm-1. UV/Vis (MeOH) λmax (log ε): 203 (4.51), 224 (sh, 4.35), 299 (sh, 4.15), 330 (4.24) nm; (MeOH+NaOH) 203 (4.63), 224 (sh, 4.35), 338 (4.21) nm. 1H NMR (600 MHz, acetone-d6): Table 1. 13C NMR (100 MHz acetone-d6): Table 1. HRMS-EI: m/z [M+] calcd. for C29H36O4: 448.2614; found: 448.2611. Macapruinosin B (2)

Pale yellow solid. [α]D: +1.8 (c 0.6, MeOH). IR (KBr) νmax: 3414, 3076, 2956, 2926, 2857, 1639, 1615, 1497, 1453, 1274, 1159, 1113, 1087, 828 cm-1. UV/Vis (MeOH): λmax (log ε): 205 (4.49), 291 (4.10), 352 (sh, 3.65) nm; (MeOH+NaOH) 209 (4.68), 243 (4.23), 325 (4.14), 408 (3.57).

1H NMR (400 MHz, CDCl3): Table 2. 13C NMR (100 MHz CDCl3): Table 2. HRMS-EI: m/z [M+] calcd. for C30H36O6: 492.2512; found: 492.2533. Macapruinosin C (3)

Greenish-yellow solid. IR (KBr) νmax: 3412, 3082, 2961, 2923, 2854, 1636, 1617, 1599, 1482, 1449, 1367, 1314, 1290, 1314, 1290, 1188, 1156, 1086, 809 cm-1. UV/Vis (MeOH) λmax (log ε): 207 (4.57), 232 (sh, 4.26), 258 (4.12), 274 (4.02), 295 (3.95), 374 (4.08) nm; (MeOH+NaOH) 206 (4.54), 258 (4.08), 275 (4.03), 328 (4.05), 388 (4.02) nm. 1H NMR (400 MHz, CDCl3): Table 2. 13C NMR (100 MHz CDCl3): Table 2. HRMS-EI: m/z [M+] calcd. for C30H34O7: 506.2304; found: 506.2305. Acknowledgments - Financial support from Endeavour Programme Australian Scholarships awarded to one of us (YMS) in 2008 is gratefully acknowledged (Award Contract No. 519-2008).

References

[1] Yoder BJ, Cao S, Norris A, Miller JS, Ratovoson F, Razafitsalama J, Andriantsiferana R, Rasamison VE, Kingston DGI. (2007) Antiproliferative prenylated stilbenes and flavonoids from Macaranga alnifolia from the Madagascar rainforest. Journal of Natural Products, 70, 342-346.

[2] Syah YM, Hakim EH, Achmad SA, Hanafi M, Ghisalberti EL. (2009) Isoprenylated flavanones and dihydrochalcones from Macaranga trichocarpa. Natural Product Communications, 4, 63-67.

[3] Belofsky G, French AN, Wallace DR, Dodson SL. (2004) New geranyl stilbenes from Dalea purpurea with in vitro opioid receptor affinity. Journal of Natural Products, 67, 26-30.

[4] Bruno M, Savona G, Lamartina L, Lentini F. (1985) New flavonoids from Bonannia graeca (L.) Halacsy. Heterocycles, 23, 1147-1153.

[5] Ravelonjato B, Kunesch N, Poisson JE. (1987) Neoflavonoids from the stem bark of Calophyllum verticillatum.Phytochemistry, 26, 2973-2976.

[6] Cottiglia F, Dhanapal B, Sticher O, Heilmann J. (2004) New chromanone acids with antibacterial activity from Calophyllum brasiliense. Journal of Natural Products, 67, 537-541.

[7] Bierl-Leonhardt BA, Moreno DS, Schwartz M, Fargerlund J, Plimmer JR. (1981) Isolation, identification and synthesis of the sex pheromone of the citrus melybug, Planococus citri (Risso). Tetrahedron Letters, 22, 389-392.

[8] Son KH, Keon SJ, Chang HW, Kim HP, Kang SS. (2001) Papyriflavonol A, a new prenylated flavonol from Broussonetia papyrifera. Fitoterapia, 72, 456-458.

[9] Yakushijin K, Shibayama K, Murata H, Furukawa H. (1980) New prenylflavanones from Hernandia nymphaefolia (Presl) Kubitzki. Heterocycles, 14, 397-402.

ORIGINAL PAPER

Phenolic compounds from Cryptocarya konishii: their cytotoxicand tyrosine kinase inhibitory properties

Fera Kurniadewi • Lia D. Juliawaty • Yana M. Syah •

Sjamsul A. Achmad • Euis H. Hakim • Kiyotaka Koyama •

Kaoru Kinoshita • Kunio Takahashi

Received: 1 June 2009 / Accepted: 24 September 2009 / Published online: 11 December 2009

� The Japanese Society of Pharmacognosy and Springer 2009

Abstract Two chalcone derivatives, 20-hydroxychalcone

(1) and desmethylinfectocaryone (2), together with five

known phenolic compounds infectocaryone (3), crypto-

caryone (4), kurzichalcolactone A (5), pinocembrin (6) and

trans-N-feruloyltyramine (7), were isolated from the

methanol extract of the wood of Cryptocarya konishii. The

structures of the new compounds were determined based on

the analysis of spectroscopic data, including UV, IR, 1D

and 2D NMR, and mass spectra. Evaluation of the cyto-

toxic and tyrosine kinase inhibitory activities of com-

pounds 1–7 showed that compounds 2–4 strongly inhibited

the growth of murine leukemia P-388 cells, whereas

compound 4 significantly inhibited the enzyme.

Keywords 20-Hydroxychalcone �Desmethylinfectocaryone � Chalcone � Cryptocarya

konishii � Lauraceae � Cytotoxicity � Tyrosine kinase �P-388 cells

Introduction

The genus Cryptocarya (Lauraceae) contains at least 200

species distributed mainly in the tropical region of the

world [1]. Phytochemical studies have revealed that this

genus produces alkaloids, 2-pyrones and flavonoids as the

main secondary metabolite constituents, e.g. see [2–10].

Recently, we reported chalcone and flavanone derivatives

from C. costata that exhibited cytotoxic activity against

murine leukemia P-388 cells [9]. In continuation of our

work on phytochemistry and biological evaluation of the

metabolites from lauraceous plants, we examined the

activity of the MeOH extract of the tree bark of C. konishii

Hayata grown in Indonesia against P-388 cells and tyrosine

kinase, showing it significantly inhibited both the cells

(IC50 6.5 lg/mL) and the enzyme (% inhibition of 48.9 at

100 lg/mL). This plant has been shown to contain a

number of alkaloid derivatives [11–13]. In this paper,

we report the isolation of two new chalcone derivatives,

20-hydroxychalcone (1) and desmethylinfectocaryone (2),

along with five known phenolic derivatives 3–7 (Fig. 1),

from the wood of the title plant, as well as their cytotoxic

and inhibitory properties against P-388 cells and tyrosine

kinase.

Results and discussion

Compound 1, isolated as a yellowish powder, exhibited a

molecular ion at m/z 224.0840 in the high resolution (HR)

electron ionization mass spectrum (EIMS), corresponding

to a molecular formula C15H12O2 (calcd. 224.0837). The

UV spectrum of 1 showed maxima (kmax 203 and 315 nm)

that were comparable with a chalcone chromophore, and

the IR spectrum exhibited absorptions for hydroxyl

(3429 cm-1), aromatic or alkenyl C–H (3063 cm-1), con-

jugated carbonyl (1639 cm-1) and aromatic (1574 cm-1)

groups. In the 13C NMR spectrum (APT, attached proton

test) (Table 1), 1 showed 13 carbon signals representing 15

F. Kurniadewi � L. D. Juliawaty � Y. M. Syah �S. A. Achmad � E. H. Hakim (&)

Natural Products Research Group, Department of Chemistry,

Bandung Institute of Technology, Jalan Ganeca 10,

Bandung 40132, Indonesia

e-mail: [email protected]

K. Koyama � K. Kinoshita � K. Takahashi

Department of Pharmacognosy and Phytochemistry,

Meiji Pharmaceutical University, 2-522-1 Noshio,

Kiyose, Tokyo 204-8588, Japan

123

J Nat Med (2010) 64:121–125

DOI 10.1007/s11418-009-0368-y

carbon atoms, all of them having chemical shifts of sp2

carbon, in which two of the signals were assignable to a

conjugated carbonyl (dC 194.1) and an oxyaryl (dC 163.6)

carbon atom. These spectroscopic data suggested that 1 is a

simple monohydroxylated chalcone derivative. The 1H

NMR spectrum of 1 (Table 1) showed a characteristic

signal of a chalcone structure by the presence of a pair of

doublets at dH 7.94 and 8.07 with a trans coupling constant

(J = 15.3 Hz). The phenolic –OH group was determined to

be at C-20 by the observation in the 1H NMR spectrum of a

chelated –OH signal at dH 12.88 and four aromatic signals

(dH 6.99, 7.00, 7.56, 8.29) with multiplicities typical for a

1,2-disubstituted benzene. Consequently, the ring B in 1

was an unsubstituted phenyl group (dH 7.47, 3H and 7.90,

2H). Compound 1, therefore, was assigned as 20-hydroxy-

chalcone. Further support for the structure 1 was obtained

from the one- and two/three-bond 1H–13C correlations

found in the heteronuclear multiple quantum coherence

(HMQC) and HMBC (heteronuclear multiple bond con-

nectivity) spectra of 1 as shown in Table 1. A literature

search disclosed that this compound has been synthesized

by Guidugli et al. in order to study its mass spectroscopic

behaviour [14], but this paper is the first report of its

occurrence from natural sources.

Compound 2, isolated as a brownish solid, had a

molecular formula C17H16O4 based on its high resolution

EIMS which showed a molecular ion at m/z 284.1045

(calcd. 284.1049). The IR spectrum of 2 exhibited absorp-

tions for hydroxyl (3341 cm-1), alkyl C–H (2924 cm-1),

carboxylic and conjugated carbonyl (1709 and 1632 cm-1),

and aromatic (1562 cm-1) groups. The UV spectrum (kmax

203, 282, 348 and 383 nm) was very close to those of in-

fectocaryone (3) and cryptocaryone (4), which were also

isolated from the title plant. Comparison of the 1H and 13C

NMR spectra of the compounds 2 and 3 also revealed a

structural similarity of these two compounds. The most

significant differences observed are the appearance of a

proton signal of a methoxyl ester group in 3, which is absent

Fig. 1 Structures of compounds isolated from C. konishii

Table 1 NMR data (d6-acetone) of compound 1

Position dH (multiplicity,

J in Hz)

dC HMBC (1H–13C)

a 8.07 (d, 15.3) 121.4 C-b, C-1, C=O

b 7.94 (d, 15.3) 146.2 C-a, C-2/C-6, C=O

C=O – 194.9 –

10 – 120.4 –

20 – 164.4 –

30 6.99 (dd, 8.0, 1.8) 118.9 C-10, C-50

40 7.56 (td, 8.0, 1.3) 137.4 C-20, C-60

50 7.00 (td, 8.0, 1.8) 119.8 C-10, C-30

60 8.29 (dd, 8.0, 1.3) 131.4 C-20; C-40, C=O

1 – 135.7 –

2/6 7.90 (m) 129.8 C-4

3/5 7.47 (m) 129.9 C-1, C-5/C-3

4 7.47 (m) 131.8 C-2/C-6

20-OH 12.88 (s) C-10, C-20, C-30

122 J Nat Med (2010) 64:121–125

123

in compound 2, as well as the presence of the carbon signal

characteristic for a carboxylic group at dC 177.5 in 2,

instead of a signal of dC 172.6 for the ester carbon of 3 [10].

In addition, the low resolution (LR) EIMS of compound 2

gave the same base peak at m/z 131 and a strong peak at m/z

225. The latter peak is very indicative for the presence of

carboxylic group in 2, which can be rationalized as a loss of

a –CH2COOH radical (mass of 59) from the molecular ion.

Moreover, the presence of a peak at m/z 266, due to a loss of

water molecule from the molecular ion, gave further sup-

port for the presence of this group in 2. Thus, structure 2

was assigned as desmethylinfectocaryone. The one- and

two/three-bond 1H–13C correlations observed in the HMQC

and HMBC spectra of compound 2 (Table 2) were consis-

tent with the structure of desmethylinfectocaryone. By

comparison of the optical rotation values, compound 2 was

assumed to have the same stereochemistry as those of

compound 3. Compounds 2–4 represent the members of

naturally occurring chalcone containing a reduced A ring at

C-5 and C-6. Compound 3 had previously been isolated

from C. infectoria [10], whereas compound 4 was initially

isolated in 1973 from C. bourdilloni [14].

The isolated compounds 1–7 were evaluated for their

cytotoxicities against P-388 cells and their inhibitory

properties against tyrosine kinase (Table 3). From the

bioactivity data shown in the table, compounds 2–4 showed

strong cytotoxic properties, whereas compound 4 was the

only compound with significant inhibitory effect against

tyrosine kinase. Thus, compounds 2–4 could be promising

lead compounds for cancer treatment.

Experimental

General

UV and IR spectra were measured with Varian 100 Conc

and FTIR Spectrum One Perkin-Elmer instruments,

respectively. 1H and 13C NMR spectra were recorded with

a JEOL ECA 500 spectrometer operating at 500 (1H) and

125 (13C) MHz, using residual and deuterated solvent

peaks as reference standards. MS spectra were obtained

with a JEOL JMS-700 mass spectrometer (EI mode).

Vacuum column liquid chromatography (VLC) and cen-

trifugal planar chromatography (ChromatotronTM, Harrison

Research, USA) were carried out using Si gel 60 G and Si

gel GF254, respectively, and, for TLC analysis, precoated Si

gel plates (Merck Kieselgel 60 GF254, 0.25 mm) were used.

Plant material

Samples of the wood of C. konishii were collected in 2007

from Cibodas Botanical Garden, West Java, Indonesia.

Extraction and isolation

The dried and powdered wood of C. konishii (5.1 kg) was

macerated with MeOH (39, each 15 L) at room tempera-

ture. After evaporation under reduced pressure, the dried

MeOH extract (110 g) was redissolved in MeOH/H2O,

partitioned using hexane/EtOAC (3:7) (39) to give a

hexane/EtOAc extract (40 g). The hexane/EtOAc extract

was fractionated using VLC [Si gel, n-hexane, n-hexane/

EtOAc (9:1 ? 3:7), EtOAc, EtOAc/MeOH (9:1)] into six

major fractions A–F. Fraction B (220 mg) was purified

using centrifugal planar chromatography (hexane/CHCl3,

9:1) to provide 20-hydroxychalcone (1) (7 mg). Fraction C

(3.8 g) was refractionated by VLC using a step gradient of

hexane/CHCl3 (1:1 ? 1:9), CHCl3, CHCl3/MeOH (99:1 ?98:2) and repeated purification by centrifugal planar

chromatography (hexane/CHCl3, 9:1) to provide infecto-

caryone (3) (6 mg) [10], cryptocaryone (4) (390 mg) [14]

and pinocembrin (6) (33 mg) [15]. After a series of sepa-

ration and purification procedures using centrifugal planar

chromatography (eluents CHCl3), fraction E (17.8 g)

afforded desmethylinfectocaryone (2) (23 mg) and kurz-

ichalcolactone A (5) (45 mg) [16]. Using a similar proce-

dure, trans-N-feruloyltyramine (7) (25 mg) [17] was

obtained from fraction F (8.6 g).

Table 2 NMR data (d6-acetone) of compound 2

Position dH (multiplicity,

J in Hz)

dC HMBC (1H–13C)

2 7.68 (d, 15.3) 140.5 C-3, C-4, C-10,C-20/C-60

3 7.01 (d, 15.3) 117.7 C-2, C-4, C-10

4 – 172.6 –

5 3.56 (ddd, 8.7, 5.0, 4.3) 29.4 C-6, C-7, C-9,

C-10, C-11

6 2.46 (dd, 17.5, 6.5) 29.3 C-5, C-7, C-10

2.64 (dd, 17.5, 8.7)

7 6.69 (dd, 9.8, 6.5) 143.9 C-6, C-9

8 6.19 (d, 9.8) 129.9 C-6, C-10

9 – 188.2 –

10 – 108.6 –

11 2.41 (dd, 15.9, 5.0) 39.7 C-5, C-6, C-10,

C-122.64 (dd, 15.9, 4.3)

12 – 177.5 –

10 – 135.3 –

20/60 6.75 (m) 128.0 C-2, C-60/C-20,C-40

30/50 7.36 (m) 128.9 C-10, C-50/C-30

40 7.36 (m) 129.2 C-20/C-60

4-OH 16.14 (s) C-10, C-4, C-3

J Nat Med (2010) 64:121–125 123

123

20-Hydroxychalcone (1)

Yellowish solid. UV (MeOH) kmax nm (log e): 203 (3.67),

315 (3.64); IR (KBr) mmax cm-1: 3429, 3063, 1639,

1574, 1485, 1439, 1339, 1203, 1153, 1026, 976; 1H

NMR (d6-acetone): see Table 1; 13C NMR (d6-acetone):

see Table 1; HREIMS m/z: [M]? 224.0840 (calcd. for

C15H12O2: 225.0837).

Desmethylinfectocaryone (2)

Brownish yellow solid. [a]D20 = ? 51 (c 0.02, MeOH); UV

(MeOH) kmax nm (log e): 203 (4.11), 282 (3.98), 348

(3.89), 383 (4.05); IR (KBr) mmax cm-1: 3341, 2924, 1709,

1632, 1562, 1416, 1281, 1157, 1030; 1H NMR (d6-ace-

tone): see Table 2; 13C NMR (d6-acetone); see Table 2;

LREIMS m/z (% rel. int.): [M]? 284 (30), 266 (5), 225

(68), 131 (100), 121 (30), 103 (27); HREIMS m/z: [M]?

284.1045 (calcd. for C17H16O4: 284.1049).

Cytotoxic evaluation

Cytotoxic properties of the isolated compounds 1–7 against

murine leukemia P-388 cells was evaluated using the MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide) assay as previously described [18].

Tyrosine kinase inhibitor assay

The assay was carried out according to the supplied manual

of the Universal Tyrosine Kinase Assay Kit, purchased

from Takara Bio, Japan. Briefly, the kit contains the

96-well plate coated with a solid tyrosine peptide

(PTK substrate immobilized microplate). A suspension of

HUVECs lysate and the samples dissolved in DMSO are

diluted with kinase reacting solution and added with

40 mM of ATP into each well. Then, the plate is incubated

in a humidified atmosphere at 37�C. After 2 h, each well is

washed with 0.05% of Tween-PBS and incubated with the

blocking solution, after which the anti-phosphotyrosine

(PY20) HRP is probed. After 30 min, the immunoreactive

tyrosine is detected by addition of HRP substrate solution

(TMBZ) and 1 N H2SO4 as a stop solution. The absorbance

of the solution is measured at 450 nm. The inhibition ratio

was obtained by the following equation: inhibition

(%) = (1 - sample OD/DMSO OD) 9 100%, where OD

is the optical density.

Acknowledgments This study was supported by a JSPS (The Japan

Society for the Promotion of Science) grant to one of us (FK) through

Meiji Pharmaceutical University Asia/Africa Centre for Drug Dis-

covery Program, Japan. Financial assistance from a Doctoral

Research Grant, Higher Education Bureau, National Education

Department of Republic Indonesia, is also gratefully acknowledged.

We thank Cibodas Botanical Garden, West Java, Indonesia, for sup-

plying and identifying the sample.

References

1. Cronquist A (1981) An integrated system of classification

of flowering plants. Columbia University Press, New York,

pp 74–78

2. Awang K, Hadi AHA, Saidi N, Mukhtar MR, Morita H, Litaudon

M (2008) New phenantrene alkaloids from Cryptocarya crass-inervia. Fitoterapia 79:308–310

3. Toribio A, Bonfils A, Delannay E, Prost E, Harakat D, Henon E,

Richard B, Litaudon M, Nuzillard J-M, Renault J-H (2006) Novel

seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchen-sis. Org Lett 8:3825–3828

4. Lin F-W, Wang J-J, Wu T-S (2002) New pavine N-oxide alka-

loids from the stem bark of Cryptocarya chinensis Hemsl. Chem

Pharm Bull 50:157–159

5. Dumontet V, Van Hung N, Adeline M-T, Riche C, Chiaroni A,

Sevenet T, Gueritte F (2004) Cytotoxic flavonoids and a-pyrones

from Cryptocarya obovata. J Nat Prod 67:858–862

6. Chan Y-Y, Wu C-H, Wu S-J, Wu T-S (2002) The constituents

and synthesis of cryptamygin-A from the stem bark of Crypto-carya amygdalina. J Chin Chem Soc 49:263–268

7. Schmeda-Hirschmann G, Astudillo L, Bastida J, Codina C, De

Arias AR, Ferreira ME, Inchaustti A, Yaluff G (2001) Crypto-

folione derivatives from Cryptocarya alba fruits. J Pharm Phar-

macol 53:563–567

8. Juliawaty LD, Kitajima M, Takayama H, Achmad SA, Aimi N

(2000) A 6-substituted-5,6-dihydro-2-pyrone from Cryptocaryastrictifolia. Phytochemistry 54:989–993

Table 3 Cytotoxic and tyrosine

kinase inhibitory properties of

compounds 1–7

a Positive controlsb Measured in triplicatec Single measurement

Compounds P-388 (IC50, lM)b Tyrosine kinasec

(% inhibition at 100 ppm)

20-Hydroxychalcone (1) 64.26 ± 8.10 Inactive

Desmethylinfectocaryone (2) 2.17 ± 0.20 Inactive

Infectocaryone (3) 0.8 ± 0.03 Inactive

Cryptocaryone (4) 0.04 ± 0.01 47.4

Kurzichalcolactone A (5) 12.73 ± 0.57 Inactive

Pinocembrine (6) 231.64 ± 9.75 Inactive

trans-N-Feruloyltyramine (7) 119.50 ± 5.23 Inactive

Artonin Ea 1.1 ± 0.03 –

HV-1a – 50.4

124 J Nat Med (2010) 64:121–125

123

9. Usman H, Hakim EH, Harlim T, Jalaluddin MN, Syah YM,

Achmad SA, Takayama H (2006) Cytotoxic chalcones and flav-

anones from the tree bark of Cryptocarya costata. Z Naturforsch

61c:184–188

10. Dumontet V, Gaspard C, Van Hung N, Fahy J, Tchertanov L,

Sevenet T, Gueritte F (2001) New cytotoxic flavonoids from

Cryptocarya infectoria. Tetrahedron 57:6189–6196

11. Lu S-T (1966) Alkaloids of Formosan lauraceous plants. IX.

Alkaloids of Cryptocarya chinensis and C. konishii. Yakugaku

Zasshi 86:296–299

12. Lu S-T (1967) Alkaloids of Formosan lauraceous plants. XII.

Alkaloids of Cryptocarya konishii and Machilus acuminatissi-mus. Yakugaku Zasshi 87:1278–1281

13. Lee SS, Lin YJ, Chen CK, Liu KCS, Chen CH (1993) Quaternary

alkaloids from Litsea cubeba and Cryptocarya konishii. J Nat

Prod 56:1971–1976

14. Govindachari TR, Parthasarathy PC, Desai HK, Shanbhag MN

(1973) Structure of cryptocaryone. Constituent of Cryptocaryabourdilloni. Tetrahedron 29:3091–3094

15. Wagner H, Chari VM, Sonnenbichler J (1976) 13C-NMR-spek-

tren naturlich vorkommender flavonoide. Tetrahedron Lett 17:

1799–1802

16. Fu X, Sevenet T, Hamid A, Hadi A, Remy F, Pais M (1993)

Kurzilactone from Cryptocarya kurzii. Phytochemistry 33:1272–

1274

17. Yoshihara T, Yamaguchi K, Takamatsu S, Sakamura S (1981) A

new lignan amide, grossamide, from bell pepper (Capsicumannuum var. grossum). Agric Biol Chem 45:2593–2598

18. Sahidin HEH, Juliawaty LD, Syah YM, Din LB, Ghisalberti EL,

Latip J, Said IM, Achmad SA (2005) Cytotoxic properties of

oligostilbenoids from the tree bark of Hopea dryobalanoides. Z

Naturforsch 60c:723–727

J Nat Med (2010) 64:121–125 125

123

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4558–4560

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

b-Secretase (BACE-1) inhibitory effect of biflavonoids

Hiroaki Sasaki a, Kazuhiko Miki b, Kaoru Kinoshita b, Kiyotaka Koyama b, Lia D. Juliawaty c,Sjamsul A. Achmad c, Euis H. Hakim c, Miyuki Kaneda a, Kunio Takahashi b,*

a School of Pharmacy, Shujitsu University, Nishigawara 1-6-1, Naka-ku, Okayama 703-8516, Japanb Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japanc Natural Products Research Group, Department of Chemistry, Bandung Institute of Technology, Jalan Ganeca 10, Bandung 40132, Indonesia

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 March 2010Revised 27 May 2010Accepted 3 June 2010Available online 8 June 2010

Keywords:b-SecretaseBACE-1AlzheimerAmentoflavoneBiflavonoid2,3-Dihydroamentoflavone2,3-Dihydro-6-methylginkgetin

0960-894X/$ - see front matter � 2010 Elsevier Ltd.doi:10.1016/j.bmcl.2010.06.021

* Corresponding author. Tel./fax: +81 424 95 8912.E-mail address: [email protected] (K. Tak

Here, we describe amentoflavone-type biflavonoids, which were isolated from natural sources and werefound to inhibit b-secretase (BACE-1). The structure–activity relationship was studied, and compounds1–8, 10, 17, and 18 showed BACE-1 inhibitory activity. Among these compounds, 2,3-dihydroamentoflav-one 17 and 2,3-dihydro-6-methylginkgetin 18 exhibited potent inhibitory effects with IC50 values of 0.75and 0.35 lM, respectively.

� 2010 Elsevier Ltd. All rights reserved.

O

OO

HO

OCH3

HOOHOCH3

H3C

O

OO

HO

OH

HOOHOH

2S

3

6

2S

3

1817

The most common form of dementia is Alzheimer’s disease (AD),which now affects over 30 million people worldwide.1 AD is a neu-rodegenerative disorder characterized by accumulation and deposi-tion of amyloid b (Ab) peptides, which are generated from thecleavage of the b-amyloid precursor protein (APP) by consecutiveaction of b-secretase (BACE-1: b-site APP cleaving enzyme-1) andc-secretase.2–4 c-Secretase affects the Notch cleavage, while b-secretase demonstrates no compensatory mechanism for APPcleavage.5 The young BACE knockout mice were found to be healthyand fertile.5 Hence, the discovery of a BACE-1 inhibitor could be aneffective and safe therapeutic strategy for AD.

Biflavonoids are well known as constituents of gymnospermousplants and are flavonoid dimers connected by C–C or C–O–C bonds.Recently, these plants were found to exhibit anti-influenza,6,7 anti-inflammatory,8 and anti-malarial9 activities.

In this Letter, we report the isolation of biflavonoids from a vari-ety of plants and study their BACE-1 inhibitory activities and struc-ture–activity relationships.

Acetone or CHCl3 extracts of a variety of plants were subjectedto silica gel column chromatography, Sephadex LH-20 columnchromatography, and HPLC to afford compounds 1–21. All isolatedbiflavonoids were identified on the basis of their spectroscopicdata as well as by comparison with published data. Compounds

All rights reserved.

ahashi).

1–18 were amentoflavone-type biflavonoids with the flavonoidmoieties connected by a C30–C800 bond. Among them, 17 and 18were 2,3-dihydro structures (Fig. 1). Amentoflavone 1 andsequoiaflavone 2 were isolated from Cunninghamia lanceolata,10,11

bilobetin 3, ginkgetin 6, 7,700,40-tri-O-methylamentoflavone 12, sci-adopitysin 13, amentoflavone-7,700,40,40 0 0-tetramethyl ether 16 and2,3-dihydro-6-methylginkgetin 18 from Cephalotaxus harringtoniavar. fastigiata,12–14 amentoflavone-7,700-dimethyl ether 7 fromCephalotaxus harringtonia var. harringtonia,15 sotetsuflavone 4,40,700-di-O-methylamentoflavone 9 and kayaflavone 15 from Tor-reya nucifera,16–18 podocarpusflavone A 5, podocarpusflavone B 8and isoginkgetin 10 from Podocarpus macrophyllus var. macrophyl-

OH OOH O

Figure 1. Structures of 2,3-dihydroamentoflavone 17 and 2,3-dihydro-6-meth-ylginkgetin 18.

http://dx.doi.org/10.1016/j.bmcl.2010.06.021

mailto:[email protected]

http://dx.doi.org/10.1016/j.bmcl.2010.06.021

http://www.sciencedirect.com/science/journal/0960894X

http://www.elsevier.com/locate/bmcl

OO

OHO

OH

HO

OH O

OH

OH

OHO

OH O

OH

OHO

OH O

OH

OHO

OH O

OH

OO

OOH

HO

3'

6''

8

8''

4'

6''

19

20

21

Figure 2. Structures of robustaflavone 19, cupressuflavone 20 and hinokiflavone21.

Table 2BACE-1 inhibitory assay results for compounds 1–21

Compound BACE-1 inhibition IC50 (lM)

1 1.542 1.403 2.024 1.585 0.996 4.187 6.258 4.219 >10.0

10 3.0111 >10.012 >10.013 >10.014 >10.015 >10.016 >10.017 0.7518 0.3519 >10.0

H. Sasaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4558–4560 4559

lus,19–21 700,40 0 0-dimethylamentoflavone 11 and heveaflavone 14from Hevea brasiliensis,15,22 and 2,3-dihydroamentoflavone 17 fromCycas revoluta10 (Table 1). Robustaflavone 19 with the flavonoidmoieties connected by a C30–C600 bond was isolated from Selaginellamoellendorffii,19 cupressuflavone 20, with the flavonoid moietiesconnected by a C8–C800 bond was isolated from Cupressus macro-carpa ‘Goldcrest’,23 and hinokiflavone 21 with the flavonoid moie-ties connected by a C40–O–C600 bond was isolated fromMetasequoia glyptostoboides20,24 (Fig. 2).

Compounds 1–21 were all tested using the BACE-1 FRET assaykit.25 Several amentoflavone-type biflavonoids showed inhibitoryactivity, whereas robustaflavone 19, cupressuflavone 20, and hin-okiflavone 21 did not. Amentoflavone 1 and its monomethoxy ana-logues 2–5 showed strong inhibitory activity with IC50 values of1.54, 1.40, 2.02, 1.58, and 0.99 lM, respectively. Compounds 6–8and 10 showed lower activities than 1–5 with IC50 values of 4.18,6.25, 4.21, and 3.01 lM, respectively. The dimethoxy compounds9 and 11, trimethoxy compounds 12–15, and tetramethoxy com-pound 16 exhibited no inhibitory activity. Compound 17, a 2,3-dihydro analogue of 1, showed an increase in inhibitory activity,while compound 18 showed the strongest inhibitory activity ofBACE-1 among amentoflavone-type biflavonoids (Table 2).

These results indicate that the amentoflavone-type biflavonoidsconsisting of two apigenin molecules linked at the C30–C800 positionare important for BACE-1 inhibitory activity. The data also suggestthat more than two hydroxyl groups at the R1–R4 position areneeded for inhibitory activity. The results with compounds 17and 18 show that the presence of a flavanone moiety in the amen-toflavone biflavonoid is advantageous for inhibitory activity. More-over, the presence of a methyl at the C6 position increases theinhibitory effect.

Some amentoflavone-type biflavonoids exhibited neuroprotec-tive effects on oxidative stress-induced and amyloid b peptide-in-duced cell death in neuronal cells.26 In addition, we found thatamentoflavone-type biflavonoids have significant BACE-1 inhibi-tory activity. These results suggest that amentoflavone-type bifl-

Table 1Structures of amentoflavone-type biflavonoids 1–16

O

O

O

HO

OR1

R3O

OH O

OR4OR23'

8''

Compounds 1–16 R1 R2 R3 R4

1 H H H H2 CH3 H H H3 H CH3 H H4 H H CH3 H5 H H H CH3

6 CH3 CH3 H H7 CH3 H CH3 H8 CH3 H H CH3

9 H CH3 CH3 H10 H CH3 H CH3

11 H H CH3 CH3

12 CH3 CH3 CH3 H13 CH3 CH3 H CH3

14 CH3 H CH3 CH3

15 H CH3 CH3 CH3

16 CH3 CH3 CH3 CH3

20 >10.021 >10.0b-Secretase inhibitor 0.07

avonoids could be multiple targets for the development of noveltherapeutic strategies for Alzheimer’s disease.

Acknowledgments

This research was partially supported by the Japan Society forthe Promotion of Science (JSPS) AA Scientific Platform Programand a Grant from the High-Tech Research Center Project, Ministryof Education, Culture, Sports, Science and Technology (MEXT), Ja-pan (S0801043).

References and notes

1. Selkoe, D. J. Ann. Intern. Med. 2004, 140, 627.2. Crouch, P. J.; Harding, S.-M. E.; White, A. R.; Camakaris, J.; Bush, A. I.; Masters, C.

L. Int. J. Biochem. Cell Biol. 2008, 40, 181.3. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.4. Tanzi, R. E.; Bertram, L. Cell 2005, 120, 545.5. Citron, M. Trends Pharmacol. Sci. 2004, 25, 92.6. Miki, K.; Nagai, T.; Suzuki, K.; Tsujimura, R.; Koyama, K.; Kinoshita, K.;

Furuhata, K.; Yamada, H.; Takahashi, K. Bioorg. Med. Chem. Lett. 2007, 17, 772.7. Miki, K.; Nagai, T.; Nakamura, T.; Tuji, M.; Koyama, K.; Kinoshita, K.; Furuhata,

K.; Yamada, H.; Takahashi, K. Heterocycles 2008, 75, 879.8. Kwak, W.-J.; Han, C. K.; Son, K. H.; Chang, H. W.; Kang, S. S.; Park, B. K.; Kim, H.

P. Planta Med. 2002, 68, 316.9. Ichino, C.; Kiyohara, H.; Soonthornchareonnon, N.; Chuakul, W.; Ishiyama, A.;

Sekiguchi, H.; Namatame, M.; Otoguro, K.; Omura, S.; Yamada, H. Planta Med.2006, 72, 611.

4560 H. Sasaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4558–4560

10. Anhut, S.; Seeger, T.; Zinsmeister, H. D.; Geiger, H. Z. Naturforsch. 1989, 44c,189.

11. Krauze-Baranowska, M.; Mardarowicz, M.; Wiwart, M. Z. Naturforsch. 2002,57c, 998.

12. Yook, C.-S.; Jung, J.-H.; Jeong, J.-H.; Nohara, T.; Chang, S.-Y. Nat. Prod. Sci. 2000,6, 1.

13. Mai, V. T.; Phan, T. S.; Duong, A. T.; Duong, N. T. Tap Chi Hoa Hoc 2002, 40, 24.14. Sasaki, H.; Miki, K.; Koyama, K.; Kinoshita, K.; Takahashi, K. Heterocycles 2008,

75, 939.15. Gu, Y.; Xu, Y.; Fang, S.; He, Q. Zhiwu Xuebao 1990, 32, 631.16. Lopez-Saez, J. A.; Perez-Alonso, M. J.; Velasco, N. A. Z. Naturforsch. 1994, 49, 267.17. Khan, N. U.; Ansari, W. H.; Rahman, W.; Okigawa, M.; Kawano, N. Chem. Pharm.

Bull. 1971, 19, 1500.18. Sun, C.-M.; Syu, W.-J.; Huang, Y.-T.; Chen, C.-C.; Ou, J.-C. J. Nat. Prod. 1997, 60,

382.19. Xu, L.; Chen, Z.; Sun, N. Zhiwu Xuebao 1993, 35, 138.20. Markham, K. R.; Sheppard, C.; Geiger, H. Phytochemistry 1987, 26, 3335.21. Pan, J.-X.; Zhang, H.-Y.; Yang, X.-B. J. Plant Res. Environ. 1995, 4, 17.22. Zhang, Y.; Tan, N.; Huang, H.; Jia, R.; Zeng, G.; Ji, C. Yunnan Zhiwu Yanjiu 2005,

27, 107.23. Maatooq, G. T.; El-Sharkawy, S. H.; Afifi, M. S.; Rosazza, J. P. N. Nat. Prod. Sci.

1998, 4, 9.24. Geiger, H.; Markham, K. R. Z. Naturforsch. 1996, 51c, 757.25. BACE-1 assays were performed on 384-well black plates using a BACE-1 FRET

assay kit (Invitrogen Co., USA). The assay was carried out according to thesupplied manual with modifications. Samples were dissolved in the assay

buffer (50 mM sodium acetate, pH 4.5) with DMSO (final concentrations were10%). 10 lL of test samples, 10 lL of BACE-1 substrate (750 nM Rh-EVNLDAEFK-Quencher, in 50 mM ammonium bicarbonate), and tenmicrolitre of BACE-1 enzyme (1.0 U/mL) were mixed in the wells, andincubated 60 min in the dark at 25 �C. The fluorescence intensities of themixtures were measured by fluoroskan ascent (Thermo Scientific) forexcitation at 544 nm and emission at 590 nm. The inhibition ratio wascalculated by the following equation: inhibition (%) = [1 � {(S � S0) � (B � B0)/(C � C0) � (B � B0)}] � 100, where C was the fluorescence of a control [enzyme,substrate, and assay buffer concentration with DMSO (final concentrationswere 10%)] after 60 min of incubation, C0 was the initial fluorescence of acontrol [enzyme, substrate, and assay buffer concentration with DMSO (finalconcentrations were 10%)], B was the fluorescence of a control [substrate andassay buffer concentration with DMSO (final concentrations were 10%)] after60 min of incubation, B0 was the initial fluorescence of a control [substrate andassay buffer concentration with DMSO (final concentrations were 10%)], S wasthe fluorescence of the tested samples (enzyme, sample solution, andsubstrate) after 60 min of incubation, S0 was the initial fluorescence of thetested samples (enzyme, sample solution, and substrate). To check thequenching effect of the tested samples, the sample solution was added toreaction mixture C, and any reduction in fluorescence by the sample wasinvestigated. b-Secretase inhibitor (Wako, Japan) was used as a positivecontrol.

26. Kang, S. S.; Lee, J. Y.; Choi, Y. K.; Song, S. S.; Kim, J. S.; Jeon, S. J.; Han, Y. N.; Son,K. H.; Han, B. H. Bioorg. Med. Chem. Lett. 2005, 15, 3588.

.

12 Bull. Soc. Nat. Prod. Chem. (Indonesia), 2011, 11,12-16

ARTIKEL PENELITIAN

CALKON DARI KAYU BATANG MORUS NIGRA

Ferlinahayati†‡, Lia D. Juliawaty

†, Yana M. Syah, Euis H. Hakim

†∗, Jalifah Latip

† Kelompok Penelitian Kimia Organik Bahan Alam, Kelompok Keahlian Kimia Organik, Institut Teknologi Bandung,

Jalan Ganesha 10, Bandung, 40132, Indonesia

‡ Jurusan Kimia Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Sriwjaya, Jalan Raya Palembang

Prabumulih Km 32, Ogan Ilir, Sumatera Selatan, Indonesia

§ Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia

Abstrak

Dua senyawa calkon, isobavacalkon (1) dan moracalkon A (2), telah diisolasi untuk pertamakalinya

dari ekstrak metanol kayu batang Morus nigra. Struktur kedua senyawa tersebut telah ditetapkan

berdasarkan data-data spektroskopi yang meliputi spektrum UV, IR dan NMR. Sitotoksisitas kedua

senyawa tersebut terhadap sel murine leukemia P-388 memperlihatkan nilai IC50 berturut-turut 8,8

dan 6,1 µg/mL.

Kata kunci: Calkon, isobavacalkon, moracalkon A, Morus nigra, sitotoksisitas, sel P-388.

Abstract

Chalcones from the heartwood of Morus nigra

Two chalcone derivatives, isobavachalcone (1) and morachalcone A (2), had been isolated for the

first time from the methanol extract of the heartwood of Morus nigra. The structures of these

compounds were determined based on spectral evidence, including UV, IR and NMR. The

cytotoxicity of these compounds was evaluated against murine leukemia P-388 cells showing their

IC50 were 8.8 dan 6.1 µg/mL respectively.

Keywords: chalcone, cytotoxicity, isobavachalcone, moracalcone A, Morus nigra, P-388 cells.

∗ Alamat untuk korespondensi. E-mail: [email protected].

PENDAHULUAN

Morus, atau lebih dikenal dengan nama

“murbei”, merupakan salah satu genus penting

disamping Artocarpus dan Ficus dari famili

Moraceae. Genus ini tumbuh di daerah

beriklim sedang dan subtropis di Asia, Eropa,

Afrika, Amerika Utara dan Selatan, dan

ditanam di Asia Timur, Tengah dan Selatan

sebagai makanan ulat sutra.1,2 Selain itu, buah

Morus dapat dimakan dan kayunya digunakan

sebagai bahan bangunan.1 Beberapa spesies

Artikel Penelitian

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2011, 11, 12-16 13

Calkon dari Morus nigra

Morus, seperti M. alba, M. bombycis, M. lhou

dan M. multicaulis, telah lama digunakan di

sejumlah negara sebagai bahan obat tradisional

untuk menyembuhkan berbagai penyakit,

seperti batuk, asma, hipertensi, arteriosklerosis,

influenza, rematik, artritis, hepatitis dan

anemia.3 Di Indonesia, walaupun sebelumnya

hanya terdapat dua spesies Morus, yaitu M.

alba dan M. macroura,4, tetapi dewasa ini

beberapa spesies lainnya, seperti M. australis,

M. nigra, M. cathayana dan M. multicaulis

telah ditanam di beberapa daerah di Indonesia

untuk keperluan sebagai pakan ulat sutra.

Berdasarkan studi literatur, Morus

dilaporkan menghasilkan senyawa turunan

fenol dari kelompok stilben, 2-arilbenzofuran,

flavonoid, dan berbagai turunannya sebagai

hasil penggabungan Diels Alder. Umumnya

senyawa yang dilaporkan tersebut berasal dari

bagian kulit batang dan kulit akar tumbuhan

genus ini. Sebagai contoh, mulberosida A

(kelompok stilben) dari M. lhou,5 macrourin A

(kelompok 2-arilbenzofuran) dari M.

macroura,6 kuwanon A dan B (kelompok

flavonoid) dari M. alba,7 serta sanggenon C

(kelompok pengabungan Diels Alder) dari M.

cathayana8. Struktur senyawa turunan fenol

yang terdapat pada genus Morus, lazimnya

mempunyai gugus-gugus hidroksil yang

berposisi meta satu dengan lainnya dan dapat

tersubstitusi oleh gugus isoprenil atau geranil.

Senyawa turunan fenol dari genus Morus

mempunyai beragam bioaktivitas diantaranya

adalah sebagai antinematodal, antiviral,

antiplatelet, antiinflammasi, sitotoksik dan anti

HIV.9-13 Sebelumnya, kami telah melaporkan

kajian fitokimia dari M. australis14 dan telah

berhasil mengisolasi senyawa turunan fenol

dari kelompok stilben, 2-arilbenzofuran,

flavonoid dan dimer stilben. Pada kesempatan

ini akan dilaporkan penemuan dua senyawa

calkon, yaitu isobavacalkon (1) dan

moracalkon A (2), dari ekstrak metanol kayu

batang tumbuhan M. nigra. Selain itu juga

akan dilaporkan sitotoksisitas kedua senyawa

tersebut terhadap sel murine leukemia P-388.

OH

OH

HO

O

1

4

1 '

2'

4'

7'

9'

10 '

11'

1 R = H

2 R = OH

B

A

R

PERCOBAAN

Umum. Spektrum UV diukur dengan

spektrometer Varian Conc sedangkan spektrum

IR diukur dengan spektrometer Perkin Elmer

FTIR Spectrum One menggunakan pelet KBr.

Spektrum 1H and

13C NMR diukur

menggunakan JEOL ECP400 yang bekerja

pada 400 (1H) and 100 (

13C) MHz dengan

menggunakan sinyal residu pelarut (1H) dan

sinyal pelarut terdeuterasi (13C) sebagai standar

nilai geseran kimia. Kromatografi Cair Vakum

(KCV) dan kromatografi radial dilakukan

masing-masing menggunakan silika gel Merck

60 GF254 (230 – 400 mesh) dan silika gel

Merck PF254, kolom sephadex menggunakan

sephadex LH-20, sedangkan analilis

kromatografi lapis tipis (KLT) pada pelat

alumunium berlapis Si gel Merck Kieselgel 60

GF254 0,25 mm. Pelarut yang digunakan

semuanya berkualitas teknis yang didestilasi.

Bahan tanaman. Bahan tumbuhan berupa

kayu batang M. nigra dikumpulkan dari Desa

Cibeureum, Kecamatan Cisurupan, Kabupaten

Garut, Jawa Barat pada bulan Juli 2005.

Identitas tumbuhan ditetapkan oleh Herbarium

Bogoriensis, Lembaga Ilmu Pengetahuan

Indonesia (LIPI), Cibinong, Indonesia dan

spesimen tumbuhan disimpan di herbarium

tersebut.

Ekstraksi dan isolasi. Serbuk kayu batang

M. nigra yang telah kering (4,1 kg) diekstraksi

dengan cara maserasi (3x 24 jam) dengan

pelarut metanol dan menghasilkan ekstrak

metanol sebanyak 153 g. Sebagian (5 x 20 g)

ekstrak metanol tersebut difraksinasi dengan

KCV (eluen n-heksana:EtOAc = 7:3 sampai

EtOAc dan EtOAc:MeOH = 9:1)

Artikel Penelitian .


Ferlinahayati et.al

menghasilkan enam fraksi utama A-F (1,2; 2,1;

17,2; 7,2; 20,0; dan 7,7 g). Selanjutnya, fraksi

C (17,2 g) difraksinasi lebih lanjut dengan

metoda yang sama (eluen n-heksana:EtOAc =

7:3 sampai 4:6, EtOAc dan EtOAc:MeOH =

9:1) menghasilkan enam fraksi C1-C6. Fraksi

C2 (1,8 g) dipisahkan dengan kromatografi

radial (eluen n–heksana:EtOAc = 7:3, 1:1 dan

3:7) menghasilkan delapan fraksi C2.1-C2.8.

Pemisahan terhadap gabungan fraksi C2.5 dan

C2.6 (230 mg) dengan kromatografi radial

(eluen CHCl3:MeOH = 98:2) yang dilanjutkan

dengan cara yang sama (eluen n-

heksana:EtOAc = 7:3 sampai 1:1), diperoleh

senyawa 2 (9 mg). Selanjutnya pemisahan

terhadap fraksi C1 (1,2 g) dengan kromatografi

radial (eluen n-heksana:EtOAc = 9:1 sampai

6:4) dan kolom sephadex (eluen MeOH),

diperoleh senyawa 1 (12 mg).

Isobavacalkon (1), diperoleh berupa

padatan kuning. UV (MeOH) λmax nm (log ε):

203 (4,34), 227 (bahu, 4,11) dan 368 (4,20),

UV (MeOH+NaOH) λmax nm (log ε): 203

(4,66), 238 (bahu, 4,03) dan 432 (4,32),

penambahan pereaksi geser AlCl3 ataupun

NaOAc tidak mengakibatkan terjadinya

pergeseran; IR (KBr) νmaks cm-1: 3380 (OH),

2956 dan 2920 (C-H alifatik), 1620 (C=O

terkonyugasi), 1605, 1551, 1513 dan 1445

(C=C aromatik). Spektrum 1H NMR (aseton-

d6, 400 MHz): lihat tabel 1. Spektrum 13C

NMR (aseton- d6, 100 MHz): lihat Tabel 1.

Moracalkon A (2), diperoleh berupa

padatan jingga, t.l. 122-125 oC. UV (MeOH)

λmax nm (log ε): 203 (4,20), 318 (3,74) dan 386

(3,87); UV (MeOH+NaOH) λmax nm (log ε):

203 (4,40), 333 (3,71) dan 440 (3,94); UV

(MeOH+AlCl3) λmaks nm (log ε): 203 (4,38),

317 (3,80) dan 388 (3,87); UV

(MeOH+NaOAc) λmax nm (log ε): 205 (4,74),

318 (3,76) dan 388 (3,85); IR (KBr) νmaks cm-1:

3403 (OH), 2920 dan 2855 (C-H alifatik),

1607, 1544, 1512, 1486 dan 1453 (C=C

aromatik). Spektrum 1H NMR (aseton-d6, 400

MHz) : lihat Tabel 1.

Penentuan sifat sitotoksik. Sifat sitotoksik

kedua senyawa hasil isolasi diuji terhadap sel

murine leukemia P-388 mengikuti metode

MTT [3-(4,5-dimetiltiazo-2-il)2,5-difeniltetra-

zolium bromida] sebagaimana telah

dikemukakan pada laporan terdahulu.15

PEMBAHASAN

Senyawa 1 berhasil dimurnikan berupa

padatan berwarna kuning. Spektrum UV dalam

metanol memperlihatkan serapan maksimum

pada λmax 203, 227 (bahu) dan 368 nm yang

khas untuk senyawa turunan calkon,

penambahan pereaksi geser NaOH

menyebabkan terjadinya pergeseran

batokromik, yang menunjukkan adanya fenol

bebas pada senyawa ini. Spektrum IR senyawa

ini konsisten dengan senyawa calkon yang

tersubsitusi oleh gugus hidroksi, dengan

adanya serapan pada νmaks 3380 cm-1 untuk

gugus hidroksi, 1620 cm-1 untuk gugus

karbonil terkonyugasi, dan 1605-1445 cm-1

untuk C=C aromatik. Selain itu, pada spektrum

IR terdapat pula serapan pada 2956-2920 cm-1

untuk C-H alifatik yang lazimnya berasal dari

gugus isoprenil. Spektrum 13C NMR senyawa

1 memperlihatkan adanya 18 sinyal yang

mewakili 20 karbon, yang dapat ditetapkan

sebagai berasal dari satu karbon C=O tak jenuh

(δC 192,7 ppm), tiga karbon =C-O-, empat C-

kuarterner, tujuh sinyal untuk sembilan =CH-,

satu karbon –CH2-, dan dua karbon –CH3.

Sinyal-sinyal tersebut menunjukkan bahwa

pada senyawa turunan calkon tersebut terdapat

subsituen berupa isoprenil. Spektrum 1H NMR

senyawa 1 memperlihatkan adanya satu sinyal

singlet yang sesuai untuk gugus hidroksil

terkelasi (δΗ 14,01 ppm), dua sinyal dari trans-

1,2-disubsitusi etena (δΗ 7,83 dan 7,75 ppm, J

= 15,4 Hz), dua sinyal proton doblet yang khas

untuk gugus p-hidroksifenil (δΗ 7,73 dan 6,92

ppm, J = 8,4 Hz), dan dua sinyal proton

aromatik doblet lainnya untuk unit 1,2,3,4-

tetrasubsitusifenil (δΗ 7,98 dan 6,52 ppm, J =

8,8 Hz). Spektrum 1H NMR juga menunjukkan

adanya sinyal-sinyal yang khas untuk gugus

isoprenil, yaitu dua sinyal metil vinilik berupa

singlet (δΗ 1,76 dan 1,63 ppm), sinyal doblet

dari gugus metilen (δΗ 3,36 ppm, J = 7,0 Hz)

Artikel Penelitian


Calkon dari Morus nigra

Tabel 1. Data NMR senyawa 1 dan 2 dalam aseton-d6

δH (multiplisitas, J dalam Hz) δC No. C 1 2 1

1 - - 127,5

2 7,73 (d, 8,4) - 131,7

3 6,92 (d, 8,4) 6,52 (d, 2,6) 116,7

4 - - 160,9

5 6,92 (d, 8,4) 6,45 (dd, 2,6 & 8,8) 116,7

6 7,73 (d, 8,4) 7,69 (d, 8,8) 131,7

α 7,75 (d, 15,4) 7,80 (d, 15,4) 118,3

β 7,83 (d, 15,4) 8,22 (d, 15,4) 144,9

C=O - - 192,7

1’ - - 114,3

2’ - - 162,7

3’ - - 116,1

4’ - - 165,1

5’ 6,52 (d, 8,8) 6,51 (d, 8,8) 108,0

6’ 7,98 (d, 8,8) 7,89 (d, 8,8) 130,2

7’ 3,36 (d, 7,0) 3,36 (d, 7,3) 22,5

8’ 5,27 (t, 7,0) 5,27 (t, 7,3) 123,2

9’ - - 131,4

10’ 1,63 (s) 1,64 (s) 17,9

11’ 1,76 (s) 1,77 (s) 26,0

2’-OH 14,01 (s) 14,16 (s) -

dan sinyal triplet dari olefin (δΗ 5,27 ppm, J =

7,0 Hz). Sinyal-sinyal tersebut sesuai untuk

senyawa turunan calkon yang teroksigenasi

pada C-4, C-2’ dan C-4’ serta tersubsitusi oleh

gugus isoprenil pada posisi C-3’. Berdasarkan

data tersebut di atas dan data NMR

pembanding16 maka disimpulkan bahwa

senyawa 1 merupakan senyawa 3’-isoprenil-

4,2’4’-trihidroksicalkon yang dikenal dengan

nama trivial isobavacalkon (1).

Senyawa 2 diisolasi berupa padatan

berwarna jingga, yang memiliki pola spektrum

UV dan spektrum IR sangat mirip dengan

senyawa 1. Perbedaan yang muncul terletak

pada spektrum 1H NMR, dimana spektrum

senyawa 2 tersebut memperlihatkan adanya

sinyal-sinyal untuk unit 1,2,4-trisubsitusifenil

yang muncul sebagai sistem ABX pada δΗ 6,45

(dd, J = 2,6 & 8,8 Hz), 6,52 (d, J = 2,6 Hz) dan

7,69 ppm (d, J = 8,8 Hz), menggantikan unit

p-hidroksifenil pada senyawa 1. Berdasarkan

ciri-ciri struktur tersebut, dapat disimpulkan

bahwa struktur senyawa 2 adalah moracalkon

A. Perbandingan data NMR senyawa 2 dengan

data yang sama dari moracalkon A17

menunjukkan kesesuaian yang tinggi.

Sitotoksisitas senyawa 1 dan 2 terhadap sel

murine leukemia P-388 memperlihatkan nilai

IC50 masing-masing 8,8 dan 6,1 µg/mL.

Berdasarkan data tersebut tampak bahwa

adanya gugus hidroksi dengan orientasi meta

pada cincin A senyawa calkon dapat

meningkatkan sitotoksisitas senyawa tersebut.

UCAPAN TERIMA KASIH

Terima kasih disampaikan kepada staf

Herbarium Bogoriense, PP-Biologi LIPI

Cibinong, yang telah mengindentifikasi

spesimen tumbuhan.



Ferlinahayati et.al

Daftar Pustaka

1. Venkataraman, K.. “Wood phenolics in the

chemotaxonomy of the Moraceae”, Phytochemistry,

1972, 11, 1571-1586.

2. Weiguo, Z.; Yile, P.; Shihai, Z.Z.J.; Xuexia, M.;

Yongping, H. “Phylogeny of the Genus Morus

(Urticales: Moraceae) inferred from ITS and trnL-F

sequences”, African J. Biotechnol., 2005, 4, 563-56.

3. Kimura, T. ”International Collation of Traditional

and Folk Medicine” Part I: Northeast Asia, World

Scientific, Singapore, 1996, hal. 12 – 13.

4. Heyne, K. “Tumbuhan Berguna Indonesia II”, Badan

Litbang Kehutanan, Jakarta, 1987, 659-660.

5. Hirakura, K.; Fujimoto, Y.; Fukai, T.; Nomura, T.

“Constituents of the cultivated Mulberry tree. 30.

Two phenolic glycosides from the root bark of the

cultivated Mulberry tree (Morus lhou)”, J. Nat.

Prod., 1986, 49, 218-224.

6. Sun, S.G.; Chen, R.Y.; Yu, D.Q. “Structures of two

new benzofuran derivatives from the bark of

Mulberry tree (Morus macroura Miq.)”, J. Asian

Nat. Prod. Res, 2001, 3, 253-259.

7. Nomura, T.; Fukai, T.; Katayanagi, M. “Studies on

constituen of cultivated Mulberry tree III, Isolation

of four new flavones kuwanon A, B, C and

oxydihydromorusin from the root bark of Morus

alba L”, Chem. Pharm. Bull, 1978, 26, 1453-1458.

8. Shen, R.; Lin, M. “Diels -Alder type adduct from

Morus cathayana”, Phytochemistry, 2001, 57, 1231-

1235.

9. Syah, Y.M.; Achmad, S.A.; Ghisalberti, E.L.;

Hakim, E.H.; Iman, M.Z.N.; Makmur, L.;

Mujahiddin D. “Andalasin A, a new stilbene dimer

from Morus macroura”, Fitoterapia, 2000, 71, 630-

635.

10. Oh, H.; Ko, E.K.; Jun, J.Y.; Oh, X.H.; Park, A.U.;

Kang, K.H.; Lee, H.S.; Kim, Y.C. “Hepatoprotective

and free radical scavenging activities of

prenylflavonoids, coumarins and stilbene from

Morus alba”, Planta Med., 2002, 68, 932-934.

11. Du, J.; He, Z.D.; Jiang R.W.; Ye, W.C.; Xu, H.X;

But, P.P.H. “Antiviral flavonoids from the root bark

of Morus alba L.”, Phytochemistry, 2003, 62(8),

1235-1238.

12. Ko, H.Y.; Yu, S.M.; Ko, F.N.; Teng, C.M.; Lin, C.N.

“Bioactive constituents of Morus australis and

Broussonetia papyfera”, J. Nat. Prod, 1997, 60,

1008-1011.

13. Ko, H.Y.; Wang, J.J.; Lin, H.C.; Wang, J.P.; Lin,

C.N. “Chemistry and biological activities of

constituents from Morus australis”, Biochem.

Biophysic. Acta, 1999, 1428, 293-299.

14. Ferlinahayati; Syah, Y.M.; Juliawaty, L.D.; Achmad,

S.A.; Hakim, E.H.; Takayama, H.; Said, I.M.; Latip,

J., “Phenolic constituents from the wood of Morus

australis with cytotoxic activity”, Z. Naturforsch.,

2008, 63c, 35-39.

15. Saroyobudiono, H.; Hakim, E.H.; Juliawaty, L.D.;

Latip, J. “Trimerstilbenoid dari kulit batang Shorea

rugosa” Bull. Soc. Nat. Prod. Chem (Indonesia),

2006, 6, 13-18.

.


ARTIKEL PENELITIAN

PENENTUAN STRUKTUR SENYAWA AROMATIK. BAGIAN 1:

PAPIRIFLAVONOL A DARI MACARANGA PRUINOSA

Yana M. Syah∗

Kelompok Penelitian Kimia Organik Bahan Alam, Kelompok Keahlian Kimia Organik, Institut Teknologi Bandung,


Abstrak

Satu turunan kuersetin terdisioprenilasi, yaitu papiriflavonol A (1), telah berhasil diisolasi dari

ekstrak aseton daun Macaranga pruinosa. Penetapan struktur molekul senyawa tersebut dilakukan

berdasarkan hasil analisis lengkap data spektroskopi yang meliputi spektrum UV, IR, NMR 1D,

NMR 2D, spektrum massa ESI-TOF dan ESI-IT. Makalah ini menyajikan metodologi penentuan

truktur senyawa turunan flavonol tersebut.

Kata kunci: Elusidasi struktur, ESI-TOF, ESI-IT, Flavonol terprenilasi, Macaranga pruinosa,

NMR 1D dan 2D, Papiriflavonol A.

Abstract

Strucure elucidation of aromatic compounds. Part 1: Papyriflavonol A from Macaranga

pruinosa

A diisoprenylaed quercetin derivative, namely papyriflavonol A (1), has been isolated from the

acetone extract of the leaves of Macaranga pruinosa. The structure of the compound was

determined by extensive analysis of spectroscopic data, including UV, IR, NMR 1D and 2D, ESI-

TOF, and ESI-IT spectra. This paper discussed the methodology of structure elucidation of the

compound.

Keywords: ESI-TOF, ESI-IT, Macaranga aleuritoides, NMR 1D and 2D, Papyriflavonol A, Prenylated

flavonol, Structure elucidation.



44 Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 43-47

Y.M. Syah

PENDAHULUAN

Macaranga merupakan salah satu genus

terbesar dari famili Euphorbiaceae, terdiri dari

300 spesies, dengan penyebarannya meliputi

wilayah Afrika, Madagaskar, Asia, pantai

timur Australia, dan kepulauan Pasifik.1 Di

Indonesia kelompok tumbuhan ini dikenal

dengan nama lokal “mahang”,2 dan merupakan

tumbuhan endemik, sehingga dapat dijumpai di

seluruh kawasan negeri ini. Secara fitokimia,

Macaranga merupakan penghasil senyawa-

senyawa fenol golongan flavonoid dan stilben.

Karakteristik dan keunikan senyawa-senyawa

flavonoid dan stilbenoid adanya substituen dari

berbagai jenis terpenoid yang meliputi turunan

prenil (C5), geranil (C10) dan geranil-geranil

(C20). Baru-baru ini kami telah melaporkan

kajian fitokimia dari M. aleuritoides,3 M.

gigantea,4 M. pruinosa,

5 M. rhizinoides,

6 dan

M. trichocarpa,7 dan telah berhasil mengisolasi

berbagai turunan dihidrocalkon, flavanon,

flavonol, 2,3-dihidroflavonol, dan stilben yang

terisoprenilasi, tergeranilasi, dan terfarnesilasi,

termasuk juga yang mengandung gugus

samping seskuiterpen yang tidak lazim. Pada

kajian fitokimia terhadap M. pruinosa, telah

pula diisolasi turunan diisopenilflavonol, yaitu

papiriflavonol A (1), yang pertamakali

ditemukan pada tumbuhan Macaranga.

Papiriflavonol A (1) pertamakali ditemukan

oleh dua kelompok peneliti, yaitu Zhang dkk.

dari tumbuhan Broussonetia kazinoki8 dan Son

dkk. dari B. Papyrifera.9 Penemuan kedua

kelompok ini dilaporkan pada tahun yang

sama. Nama papiriflavonol A (=

papyriflavonol A) diberikan oleh Son dkk.,

sedangkan kelompok Zhang memberi nama

trivial untuk senyawa ini broussonol E. Pada

kesempatan ini akan dilaporkan penentuan

struktur senyawa ini. Pembahasan akan

difokuskan pada metodologi penentuan

struktur senyawa flavonoid jenis flavonol.

Selain itu, sifat-sifat biologis dari senyawa ini

juga akan dibahas.

PERCOBAAN

Umum. Spektrum UV dan IR ditetapkan

dengan spektrometer Cary Varian 100 Conc.

dan Perkin Elmer FT-IR Spectrum One.

Spektrum 1H dan

13C NMR ditentukan dengan

spectrometer Varian NMR System 400 MHz

(1H, 400 MHz;

13C, 100 MHz) menggunakan

residu pelarut aseton-d6 (δH 2,04 ppm) dan

pelarut aseton-d6 terdeuterasi (δC 29,8 ppm)

sebagai referensi. Spektrum massa diukur

dengan spektrometer ESI-TOF Waters LCT

Premier XE dan ESI-IT Bruker HCT.

Kromatografi vakum cair (KVC)

menggunakan Si-gel 60 GF254 (Merck),

kromatografi radial menggunakan Si-gel 60

PF254 (Merck Art. 7749), dan analisa

kromatografi lapis tipis (KLT) menggunakan

plat Kieselgel 60 F254 0,25 mm (Merck).

Pelarut yang digunakan semuanya berkualitas

teknis yang didestilasi.

Bahan tanaman. Bahan tumbuhan berupa

daun M. pruinosa dikumpulkan dari

Kalimantan. Spesimen tumbuhan diidentifikasi

di Herbarium Bogoriense, Lembaga Ilmu

Pengetahuan Indonesia, Cibinong.

Ekstraksi dan isolasi. Serbuk daun M.

pruinosa (1 kg) dimaserasi dengan aseton

sebanyak dua kali. Ekstrak aseton yang

diperoleh dipekatkan dengan alat penguap

bertekanan rendah sehingga diperoleh ekstrak

berupa semi padat (40 g). Sebagian dari

ekstrak tersebut (20 g) selanjutnya difraksinasi

dengan metoda KVC yang dielusi dengan

campuran petrol-EtOAc (17:3, 7:3, 1:1)

menghasilkan 10 fraksi (F1-F10). Komposisi

fraksi F6 (800 mg) disederhanakan dengan

kromatografi radial (eluen petrol-diisopropil

eter = 1:3) menghasilkan satu fraksi yang

relatif bersih. Pemurnian fraksi ini dengan

metoda yang sama (eluen CHCl3-EtOAc = 9:1)

menghasilan papiriflavonol A (1) (20 mg).

Senyawa 1, padatan berwarna kuning; UV

(MeOH) λmaks (log ε): 207 (4,57), 232 (bh,

4,26), 258 (4,12), 274 (4,02), 295 (3,95), 374

(4,08) nm; (MeOH+NaOH) 206 (4,54), 258

(4,08), 275 (4,03), 328 (4,05), 388 (4,02) nm;

(MeOH + AlCl3) 207 (4,53), 271 (4,18), 310

(bh, 3,84), 444 (4,16) nm; IR (KBr) υmax: 3390,

Artikel Penelitian


Penentuan struktur senyawa aromatik: Papiriflavonol A

3076, 2961, 2923, 2857, 1646, 1626, 1562,

1482, 1443, 1372, 1316, 1264, 1196, 1153,

1087, 1048, 807 cm-1

; 1H NMR (400 MHz,

aseton-d6): lihat Tabel 1; 13

C NMR (100 MHz,

aseton-d6): lihat Tabel 1; HRESIMS m/z:

[M+H]+ 439.1761 (pehitungan [M+H]

+ utuk

C25H26O7 439.1757); LRESIMS/MSn m/z:

439,2 [M+H]+, 461,1 [M+Na]

+, 477,1 [M+K]

+,

383,1 [M+H-56]+ (MS

2), 327,1 [M+H-56-56]

+

(MS3), 299,1 [M+H-56-56-28]

+ (MS

4), 271,1

[M+H-56-56-28]+ (MS

4) (mode positif).

PEMBAHASAN

Senyawa 1 diperoleh sebagai padatan

berwarna kekuningan. Spektrum massa ESIMS

memberikan ion kuasimolekul [M+H]+ resolusi

rendah pada m/z 439,2; 461,1; dan 477,1;

berturut-turut sesuai untuk [M+H]+, [M+Na]

+,

dan [M+K]+, sehingga senyawa ini dapat

dipastikan memiliki massa molekul 438. Pada

pengukuran spektrum massa ESIMS resolusi

tinggi menghasilkan ion [M+H]+ pada m/z

439,1761 yang sesuai dengan rumus molekul

C25H26O7 (∆ 0,4 Da, 0,9 ppm). Senyawa ini

menyerap sinar UV dengan puncak-puncak

maksimum pada 207, 258, 274, 295, dan 374

nm. Karakteristik serapan tersebut sesuai

dengan kromofor flavonoid dari jenis

flavonol.4,8,9

Adanya gugus –OH fenol bebas,

termasuk gugus –OH di C-5, dapat disarankan

dari pergeseran puncak serapan UV akibat

penambahan pereaksi geser larutan NaOH dan

AlCl3. Pada spektrum 13

C NMR (Gambar 1),

tampak kemunculan 25 sinyal karbon,

termasuk dua sinyal karbon pada δC 136,5 dan

176,3 ppm, yang karakteristik untuk C-3 dan

C-4 pada struktur flavonol.4,8,9

Sinyal-sinyal

lainnya adalah 17 sinyal karbon-sp2, termasuk

enam sinyal oksiaril (δC 162,5; 158,8; 155,4;

146,7; 146,2; 144,9 ppm), dan enam sinyal

karbon alifatik, yang meliputi dua sinyal

karbon metilena (δC 29,0 dan 21,9 ppm) dan

empat sinyal karbon metil (δC 25,8; 25,8; 17,9;

17,8 ppm). Berdasarkan data spektroskopi

tersebut, maka dapat disarankan bahwa

senyawa 1 merupakan turunan flavonol

kuersetin yang mengikat dua gugus isoprenil

(C5). Dukungan terhadap adanya dua gugus

isopreni juga diperoleh dari spektrum 1H NMR

dengan kemunculan empat sinyal metil singlet

(δH 1,76; 1,74; 1,73; 1,63 ppm), dua sinyal

metilena doblet (δH 3,39, J = 7,3 Hz; dan 3,34,

J = 7,2 Hz), dan dua sinyal vinil berupa tripel

multiplet (δH 5,35, J = 7,3 Hz; dan 5,25, J =

7,2 Hz).

Dengan memperhatikan jumlah atom

oksigen pada rumus molekul dan tambahan

enam atom karbon oksiaril, juga dapat

disarankan senyawa ini merupakan turunan

diisoprenil dari kuersetin (5,7,3’,4’-

tetrahidroksi. Pada spektrum 1H NMR di

daerah aromatik yang lebih deshielding

teramati adanya sepasang sinyal doblet

kopling-meta (J = 2,2 Hz) pada δH 7,67 dan

7,57 ppm, dan satu sinyal singlet yang lebih

shielding pada δH 6,51 ppm. Kemunculan

sinyal-sinyal tersebut menunjukkan bahwa

salah satu gugus isoprenil haruslah terletak di

C-5’, sementara satu gugus isoprenil lainnya

dapat berada di C-6 atau di C-8. Untuk

menentukan posisi gugus isoprenil yang kedua

tersebut hanya dapat dilakukan dengan

memanfaatkan korelasi 1H-

13C jarak jauh.

Salah satu sinyal proton metilena (δH 3,34

ppm) memberikan korelasi dengan dua sinyal

karbon oksiaril pada δC 162,5 dan 158,8 ppm,

sementara sinyal karbon δC 158,8 ppm

berkorelasi dengan sinyal proton –OH terkelasi

pada δH 12,40 ppm. Dengan cara yang sama,

maka dapa dibuktikan juga posisi gugus

isoprenil di C-5’ (Gambar 3). Korelasi 1H-

13C

jarak jauh yang lain, yang mendukung kepada

struktur 5’,6-diisoprenilkuersetin, dapat dilihat

pada Gambar 3. Dengan korelasi HMBC

tersebut, maka semua sinyal proton dan karbon

pada struktur kuersetin dapat dialokasikan

sesuai dengan posisinya pada struktur 1.

Pada kesempatan ini, akan dikemukakan

juga metodologi penetapan masing-masing

sinyal proton atau karbon pada kedua gugus

isoprenil. Untuk dapat melakukan ini dengan

baik, maka bantuan dari spektrum NMR 2D 1H-

1H COSY sangat diperlukan. Dengan

memperhatikan hubungan kopling dari masing-



Y.M. Syah

O

O

HO

OH

OH

OH

OH

6.519.56

3.34

5.251.76

1.63

3.397.57

5.351.74

1.73

7.67

7.83

7.76

8.70

12.40

O

O

HO

OH

OH

OH

OH

93.7

111.6 103.9

162.5 155.4

158.8

123.1

21.9

131.5

17.8

17.9

25.8

25.8

123.3

132.8

29.0

146.2

129.0

121.7

113.2

136.5

146.7

144.9

122.8

176.3

Gambar 1. Data 1H dan

13C NMR senyawa 1.

Gambar 2. Spektrum

1H-

1H COSY senyawa 1 di daerah sinyal alifatik. Tampak hubungan

kopling jarak jauh antara sinyal pada δH 5,25 dan 3,25 ppm dengan dua sinyal metil

pada δH 1,63 dan 1,76 ppm, dan antara sinyal pada δH 5,35 dan 3,39 ppm dengan

dua sinyal metil pada δH 1,73 dan 1,74 ppm.

3.23.33.43.53.63.73.83.94.04.14.24.34.44.54.64.74.84.95.05.15.25.35.4f2 (ppm)

1.50

1.55

1.60

1.65

1.70

1.75

1.80

1.85

f1 (ppm)

5,35

1,73 1,74

1,76

5,25

3,39 3,34

1,63

Artikel Penelitian


Penentuan struktur senyawa aromatik: Papiriflavonol A

O

O

HO

OH

OH

OH

OH

Gambar 3. Korelasi 1H-

13C jarak jauh pada senyawa 1.

masing sinyal vinil, yaitu pada δH 5,35 dan

5,25 ppm (Gambar 2), maka dapat

diidentifikasi sinyal-sinyal proton dari salah

satu gugus isoprenil adalah sebagai berikut: δH

5,25; 3,34; 1,76, dan 1,63 ppm, dan untuk

gugus isoprenil lainnya: δH 5,35; 3,39; 1,74;

dan 1,73 ppm.

UCAPAN TERIMA KASIH

Ucapan terima kasih disampaikan kepada

Endeavour Sholarship Awards tahun 2011 atas

beasiswa yang telah diberikan kepada penulis

untuk melakukan penelitian di University of

Western Australia. Ucapan terima kasih juga

disampaikan kepada Prof. Emiio L. Ghisalberti

yang telah memberikan kesempatan kepada

penulis melakukan penelitian tersebut di atas

pada Maret-Agustus 2008.

Daftar Pustaka

1. Blattner, F.R.; Weising, K.; Banfer, G.; Maschwitz,

U.; Fiala, B. “Molecular analysis of phylogenetic

relationships among myrmecophytic Macaranga

species (Euphorbiaceae)”, Mol. Phylogen. Evol,.

2001, 19, 331-334.

2. Heyne, K. Tumbuhan Berguna Indonesia, Jilid I,

1987, Yayasan Sarana Wanajaya, Jakarta.

3. Tanjung, M.; Mujahidin, D.; Juliawaty, L.D.; Hakim,

E.H.; Achmad, S.A.; Syah. Y.M. “Dua isomer

flavonoid terprenilasi dari daun Macaranga

aleuritoides”, Bull. Soc. Nat. Prod. Chem

(Indonesia), 2010, 10, 9-13.

4. Tanjung, M.; Hakim, E.H.; Mujahidin, D.; Hanafi,

M.; Syah YM. “Macagigantin, a farnesylated

flavonol from Macaranga gigantea”, J. Asian Nat.

Prod. Res., 2009, 11, 929-932.

5. Syah, Y.M.; Ghisalberti, E.L. “Phenolic derivatives

with an irregular sesquiterpenyl side chain from

Macaranga pruinosa”, Nat. Prod. Commun., 2010,

5, 219-222.

6. Tanjung, M.; Mujahidin, D.; Hakim, E.H.;

Darmawan, A.; Syah, Y.M. “Geranylated flavonols

from Macaranga rhizinoides”, Nat. Prod. Commun.,

2010, 5, 1209-1211.

7. Syah, Y.M.; Hakim, E.H.; Achmad, S.A.; Hanafi,

M.; Ghisalberti EL. “Isoprenylated flavanones and

dihydrochalcones from Macaranga trichocarpa”,

Nat. Prod. Commun., 2009, 4, 63-67.

8. Zg, P-C.; Wang, S.; Wu, Y.; Chen, R-Y.; Yu, D-Q.

“Five new diprenylated flavonols from the leaves

Broussonetia kazinoki. J. Nat. Prod., 2001, 64, 1206-

1209.

9. Son, K.H.; Kwon, S.J.; Chang, H.W.; Kim, H.P.;

Kang, S.S. “Papyriflavonol A, a new prenylated

flavonol from Broussonetia papyrifera”, Fitoterapia,

2001, 71, 456-458.


ARTIKEL PENELITIAN

DUA ISOMER FLAVONOL TERPRENILASI DARI DAUN MACARANGA

ALEURITOIDES (EUPHORBIACEAE)

Mulyadi Tanjung,†‡ Didin Mujahidin,† Lia D. Juliawaty,† Euis H. Hakim,† Sjamsul A. Achmad,† dan Yana M. Syah†∗ † Kelompok Penelitian Kimia Organik Bahan Alam, Kelompok Keahlian Kimia Organik, Institut Teknologi Bandung,


‡ Jurusan Kimia, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Airlangga, Surabaya 60115, ,

Indonesia

Abstrak

Dua isomer flavonol terprenelasi, gliasperin A (1) dan broussoflavonol F (2), telah diisolasi untuk pertamakalinya dari ekstrak metanol daun Macaranga aleuritoides. Struktur kedua senyawa tersebut ditetapkan berdasarkan data spektroskopi, yang meliputi data spektrum UV, IR, 1D dan 2D NMR. Sifat sitotoksik kedua senyawa tersebut terhadap sel murine leukemia P-388 memperlihatkan nilai IC50 berturut-turut 6,0 and 5,1 μg/ml.

Kata kunci: Flavonol terprenilasi, Macaranga aleuritoides, Sitotoksisitas, Sel P-388.

Abstract

Two isomeric prenylated flavonols from the leaves of Macaranga aleuritoides (Euphorbiaceae)

Two isomeric prenylated flavonols, glyasperin A (1) and broussoflavonol F (2), had been isolated for the first time from the methanol extract of the leaves of Macaranga aleuritoides. Structures of both compounds were determined based on spectroscopic data, inlcuding UV, IR, 1D and 2D NMR, and mass spectra. Compounds 1 and 2 were evaluated for their cytotoxicities against murine leukemia P-388 cells showing their IC50 were 6.0 and 5.1 μg/ml, respectively.

Keywords: Cytotoxicity, Macaranga aleuritoides, P-388 cells, prenylated flavonol.


PENDAHULUAN

Macaranga merupakan salah satu genus terbesar dari famili Euphorbiaceae, terdiri dari 300 spesies dengan nama lokal “mahang”. Tumbuhan ini merupakan salah satu tumbuhan

endemik Indonesia dan dijumpai di seluruh kawasan negeri ini. Tumbuhan Macaranga penyebarannya relatif luas, selain di Indonesia, dijumpai di wilayah Afrika, Madagaskar, Asia, pantai timur Australia, dan kepulauan Pasifik.1 Umumnya tumbuhan Macaranga berupa



M. Tanjung et al

O

OOH

OH

HO

OH

R1

R2

1 R1 = . R2 = H

2 R1 = H, R2 = .

2

3

44a

8a7

1'3'

4'

1"

3"

4"

5"

6" 8"

9"

10"

semak atau pohon, dan tumbuh pada tempat yang banyak mendapat sinar matahari di hutan sekunder atau hutan yang sudah rusak. Kelompok tumbuhan ini memiliki fungsi ekologi yang unik, salah satunya sebagai tumbuhan pelopor, yang dapat membuka hutan yang sudah rusak dapat tertanami secara alamiah. Selain tumbuhan pelopor, Macaranga bersimbiosis dengan sekelompok semut tertentu sehingga tumbuhan ini sering disebut Macaranga-semut.1 Tumbuhan ini banyak dimanfaatkan masyarakat untuk keperluan bahan bangunan, seperti tiang, dan atap rumah, bahan pewarna, dan pengobatan tradisional. Penggunaan obat tradisional dari tumbuhan ini, antara lain digunakan sebagai obat diare, luka, dan batuk.2 Secara fitokimia, Macaranga

merupakan penghasil senyawa-senyawa fenol golongan flavonoid dan stilben. Karakteristik dan keunikan senyawa-senyawa flavonoid dan stilbenoid adanya substituen dari berbagai jenis terpenoid yang meliputi turunan prenil (C5), geranil (C10) dan geranil-geranil (C20). Senyawa-senyawa flavonoid dan sttilbenoid dari tumbuhan Macaranga memperlihatkan berbagai bioaktivitas seperti antitumor, antikanker, antivirus, antimikroba, dan antioksidan.3 Baru-baru ini kami telah melaporkan kajian fitokimia dari M.

trichocarpa,4 M. gigantea,5 dan M. pruinosa6

dan telah berhasil mengisolasi jenis-jenis dihidrocalkon dan flavanon terprenilasi, flavonol tergeranilasi dan terfarnesilasi, serta stilben dan dihidroflavonol yang mengandung gugus samping seskuiterpen yang tidak lazim.

Pada kesempatan ini akan dilaporkan penemuan dua isomer flavonol terprenilasi, yaitu gliasperin A (1) dan broussoflavonol F (2), dari ekstrak metanol daun M. aleuritoides. Sifat sitotoksik kedua senyawa tersebut terhadap sel murin leukemia P-388 juga akan disinggung pada makalah ini.

PERCOBAAN

Umum. Spektrum UV dan IR ditetapkan dengan spektrometer Cary Varian 100 Conc. dan Perkin Elmer FT-IR Spectrum One. Spektrum 1H dan 13C NMR ditentukan dengan spectrometer Varian NMR System 400 MHz (1H, 400 MHz; 13C, 100 MHz) menggunakan residu pelarut aseton-d6 (δH 2,04 ppm) dan pelarut aseton-d6 terdeuterasi (δC 29,8 ppm) sebagai referensi. Spektrum massa diukur dengan spektrometer ESI-TOF Waters LCT Premier XE. Kromatografi vakum cair (KVC) menggunakan Si-gel 60 GF254 (Merck), kromatografi radial menggunakan Si-gel 60 PF254 (Merck Art. 7749), dan analisa kromatografi lapis tipis (KLT) menggunakan plat Kieselgel 60 F254 0,25 mm (Merck). Pelarut yang digunakan semuanya berkualitas teknis yang didestilasi.

Bahan tanaman. Bahan tumbuhan berupa daun M. aleuritoides dikumpulkan dari kawasan konservasi hutan Sorong, Papua. Spesimen tumbuhan diidentifikasi di Herbarium Bogoriense, Lembaga Ilmu Pengetahuan Indonesia, Cibinong.

Ekstraksi dan isolasi. Serbuk daun M. aleuritoides (1,8 kg) dimaserasi dengan MeOH sebanyak dua kali. Ekstrak MeOH yang diperoleh dipekatkan dengan alat penguap bertekanan rendah sehingga diperoleh ekstrak kental (200g), yang selanjutnya dipartisi dengan n-heksan dan EtOAc. Ektrak EtOAc (20 g) selanjutnya difraksinasi dengan metoda KVC yang dielusi dengan campuran n-heksan-EtOAc yang ditingkatkan kepolarannya sehingga menghasilkan empat fraksi utama A-D. Fraksi C dimurnikan dengan kromatografi radial dan dielusi dengan eluen campuran n-

Artikel Penelitian


Dua flavonol terprenilasi dari Macaranga aleuritoides

heksan-CHCl3 (4:1, 7:3, dan 3:2) menghasil-kan senyawa 1 dan 2.

Penentuan sifat sitotoksik. Sifat sitotoksik dari ketiga senyawa hasil isolasi diuji terhadap sel murine leukemia P388 mengikuti metode MTT [3-(4,5-dimetiltiazo-2-il)2,5-difeniltetra-zolium bromida] assay sebagaimana telah dikemukan pada laporan terdahulu.7

Senyawa 1, padatan berwarna kuning; UV (MeOH) λmaks (log ε): 205 (4,34), 232 (bh, 4,12), 253 (4,08), 270 (4,07), 336 (bh, 3,98), 369 (4,02) nm; (MeOH+NaOH) 204 (4,81), 235 (bh, 4,14), 277 (4,06), 323 (3,91), 412 (4,07) nm; (MeOH + AlCl3) 205 (4,30), 232 (bh, 4,05), 266 (4,10), 307 (bh, 3,67), 359 (3,72), 434 (4,12) nm; (AlCl3 + HCl): tidak berubah dari spektrum dengan +AlCl3; (NaOAc): 205 (4,89), 267 (4,08), 307 (bh, 3,72), 352 (3,72), 435 (4,09) nm; IR (KBr) υmax: 3321, 2964, 2912, 1645, 1606-1448 cm-1; 1H NMR (400 MHz, aseton-d6): lihat Tabel 1; 13C NMR (100 MHz, aseton-d6): lihat Tabel 1.

Senyawa 2, padatan berwarna kuning; Spektrum UV dan IR memperlihatkan pola serapan yang hampir sama dengan senyawa 1; 1H NMR (400 MHz, aseton-d6): lihat Tabel 2; 13C NMR (100 MHz, aseton-d6): lihat Tabel 2.

PEMBAHASAN

Senyawa 1 diperoleh sebagai padatan berwarna kuning. Spektrum UV dalam metanol memperlihatkan serapan-serapan pada λmaks 205, 232, 253, 270, 336, dan 369 nm, yang merupakan ciri khas turunan flavonol,5 dan memberikan efek batokromik pada penambahan AlCl3, NaOH, dan NaOAc. Spektrum IR menunjukkan pita serapan untuk gugus –OH (3321 cm-1), C-H alkil (2964, 2912, dan 2854 cm-1), C=O terkonyugasi (1645 cm-1), dan aromatik (1568-1448 cm-1). Pada spektrum 13C NMR (Tabel 1) tampak adanya 25 sinyal karbon, yang disertai dengan kemunculan sinyal-sinyal yang khas untuk suatu turunan flavonol, yaitu δC 136,5 dan 176,4 ppm yang sesuai untuk sinyal-sinyal C-3 dan C-4 flavonol. Adanya dua gugus samping prenil terlihat dengan jelas pada sinyal-sinyal

1H NMR (Tabel 1) pada δH 5,37 (1H), 5,27 (1H), 3,35 (2H), 3,37 (2H), 1,77 (3H), 1,74 (6H), 1,64 (3H). Berdasarkan data spektroskopi tersebut, maka dapat disarankan bahwa senyawa 1 merupakan turunan flavonol terdiprenilasi. Pada spektrum 13C NMR, selain adanya sinyal oksiaril C-3, juga tampak muncul lima sinyal oksiaril lainnya (δC 146,9; 155,5; 157,9; 158,9; dan 162,6 ppm), yang berarti senyawa ini merupakan turunan flavonol dengan cincin B termonohidroksilasi di C-4’. Selaras dengan ciri struktur ini adalah kemunculan empat sinyal –OH fenol pada δH 7,85; 8,89; 9,59; dan 12,40 ppm. Selanjutnya, adanya tiga sinyal di daerah aromatik berupa sistem spin ABX pada δH 6.98; 7,95; dan 8,03 ppm menyarankan salah satu gugus prenil berada di C-3’, sementara kemunculan satu singlet sinyal aromatik pada δH 6.57 ppm memberi kemungkinan gugus prenil lainnya di C-6 atau C-8. Kepastian posisi gugus prenil yang kedua tersebut ditetapkan berdasarkan hasil analisis spektrum NMR 2D HMBC dan HMQC. Hasil analisis spektrum HMBC menunjukkan bahwa sinyal –OH fenol terkelasi (5-OH) memberikan korelasi jarak jauh dengan tiga sinyal karbon kuarterner aromatik (δC 104,0; 111,6; dan 158,9 ppm), yang berarti gugus prenil yang kedua berada di C-6. Berdasarkan hasil analisis NMR tersebut, maka senyawa 1 disarankan memiliki struktur sebagai 3,5,7,4’-tetrahidroksi-3’,6-diprenilfla-von atau gliasperin A. Korelasi HMBC lain yang penting dalam mendukung struktur gliasperin ditunjukkan pada Gambar 1. Bukti lebih lanjut terhadap struktur 1 diperoleh dengan perbandingan data NMR senyawa ini dengan data pustaka untuk gliasperin A.8

Senyawa 2 juga diisolasi sebagai padatan berwarna kuning. Spektrum UV dan IR senyawa ini sangat mirip dengan data yang sama dari senyawa 1. Spektrum NMR senyawa 2 (Tabel 1) juga memperlihatkan kemiripan yang tinggi dengan spektrum yang sama dari senyawa 1, terutama yang berhubungan dengan nilai geseran kimia proton dan karbon pada unit-unit cincin B dan C, serta dua gugus prenil. Perbedaan parameter NMR yang berar-



M. Tanjung et al

Tabel 1. Data 1H dan 13C NMR senyawa 1 dan 2 dalam aseton-d6.

δH (multiplisitas, J dalam Hz) δC C

1 2 1 2

2 - - 146,9 147,1 3 - - 136,5 136,4 4 - - 176,4 176,7 4a - - 104,0 104,1 5 - - 158,9 159,8 6 - 6,34 (s) 111,6 98,9 7 - - 162,6 161,9 8 6,57 (s) - 93,8 107,1 8a - - 155,5 154,9 1’ - - 123,4 123,7 2’ 8,03 (d, 2,0) 8,04 (d, 2,4) 130,1 129,8 3’ - - 128,9 129,1 4’ - - 157,9 157,7 5’ 6,98 (d, 8,4) 7,01 (d, 7,2) 115,8 115,7 6’ 7,95 (dd, 8,4; 2,0) 8,05 (dd, 7,2; 2,4) 127,9 128,2 1” 3,37 (d, 7,2) 3,40 (d, 7,6) 29,0 29,0 2” 5,37 (tm, 7,2) 5,31 (tm, 7,6) 123,1 123,0 3” - - 133,1 132.0 4” 1,74 (s) 1,74 (s) 25,7 25,9 5” 1,74 (s) 1,74 (s) 17,9 17,8 6” 3,35 (d, 7,2) 3,55 (d, 7,4) 21,9 22,2 7” 5,27(tm, 7,2) 5,39 (d, 1,6) 123,2 123,3 8” - - 131,6 133,3 9” 1,64 (s) 1,65 (s) 25,7 25,9

10” 1,77 (s) 1,80 (s) 17,8 18,1 3-OH 7,85 (br,s) 7,89 (br,s) 5-OH 12,40 (br, s) 12,09 (br, s) 7-OH 9,59 (br, s) 6,58 (br, s) 4’OH 8,89 (br, s) 8,96 (br, s)

ti tampak pada sinyal-sinyal proton dan karbon untuk cincin A, sehingga menyarankan bahwa senyawa ini memiliki struktur sebagai 3,5,7,4’-tetrahidroksi-3’,8-diprenilflavon. Bukti bahwa posisi salah satu gugus prenil di C-8 selanjutnya diperoleh dari korelasi 1H-13C jarak jauh sebagaimana dinyatakan pada Gambar 2. Dengan demikian senyawa 2 ditetapkan sebagai 3,5,7,4’-tetrahidroksi-3’,8-diprenilflavon atau broussoflavonol F. Perbandingan data NMR senyawa ini dengan data yang sama yang telah dipublikasikan

memperlihatkan kesesuaian yang tinggi pada parameter-parameter NMRnya.8

Hasil uji sitotoksik senyawa 1 dan 2 terhadap sel murine P-388 memperlihatkan nilai IC50 masing-masing 6,0 and 5,1 μg/ml, yang tergolong berkeaktifan sedang. Tampak bahwa adanya penempatan gugus prenil di cincin A sedikit berpengaruh pada keaktifan sitotoksik.

Artikel Penelitian


Dua flavonol terprenilasi dari Macaranga aleuritoides

UCAPAN TERIMA KASIH

Terima kasih disampaikan kepada Direktorat Jenderal Pendidikan Tinggi, Departemen Pendidikan Nasional Republik Indonesia yang telah memberikan beasiswa BPPs kepada salah satu dari kami (MT). Sebagian dari penelitian ini juga terlaksana berkat bantuan biaya penelitian Hibah Pasca VII 2009 (No. Kontrak 0052f/K01.20/SPK-LPPM/I/2009). Ucapan terima kasih juga disampaikan kepada Prof. Emilio L. Ghisalberti, University of Western Australia, Australia, atas fasilitas yang diberikan pada pengukuran spektrum NMR.

Daftar Pustaka

1. Blattner, F.R.; Weising, K.; Banfer, G.; Maschwitz, U.; Fiala, B. “Molecular analysis of phylogenetic relationships among myrmecophytic Macaranga

species (Euphorbiaceae)”, Mol. Phylogen. Evol,. 2001, 19, 331-334.

2. Heyne, K. Tumbuhan Berguna Indonesia, Jilid I, 1987, Yayasan Sarana Wanajaya, Jakarta.

3. Tanjung, M.; Hakim, E.H.; Syah, Y.M. “Fitokimia dan sifat biologis senyawa-senyawa turunan fenol dari tumbuhan Macaranga”, Bull. Soc. Nat. Prod.

Chem. (Indonesia), 2009, 9, 1-15. 4. Syah, Y.M.; Hakim, E.H.; Achmad, S.A.; Hanafi,

M.; Ghisalberti EL. “Isoprenylated flavanones and dihydrochalcones from Macaranga trichocarpa”, Nat. Prod. Commun., 2009, 4, 63-67.

5. Tanjung, M.; Hakim, E.H.; Mujahidin, D.; Hanafi, M.; Syah YM. “Macagigantin, a farnesylated flavonol from Macaranga gigantea”, J. Asian Nat.

Prod. Res., 2009, 11, 929-932. 6. Syah, Y.M.; Ghisalberti, E.L. “Phenolic derivatives

with an irregular sesquiterpenyl side chain from Macaranga pruinosa”, Nat. Prod. Commun., 2010, 5, 219-222.

7. Saroyobudiono, H; Hakim, E.H.; Juliawaty, L.D.; Latip, J. “Trimerstilbenoid dari kulit batang Shorea

rugosa”, Bull. Soc. Nat. Prod. Chem. (Indonesia), 2006, 6, 13-18.

8. Fang, S-C.; Shieh, B-C.; Wu, R-R.; Lin, C-N. “Isoprenylated flavonols of Formosan Broussonetia

papyripera”, Phytochemistry, 1995, 38, 535-537.

PROPOSAL PROGRAM RISET DESENTRALISASI DIKTI...

Documents

Transcript of PROPOSAL PROGRAM RISET DESENTRALISASI DIKTI...