PROPOSAL PROGRAM RISET DESENTRALISASI DIKTI 2013

PROPOSAL PROGRAM RISET DESENTRALISASI DIKTI 2013

KAJIAN SIFAT ANTIMIKROBA DARI KOMPONEN KIMIA PHYLANTHUS MYRTIFOLIUS

Ketua Tim Peneliti: Prof. Dr. Yana Maolana Syah

KK Fakultas

: Kimia Organik : MIPA

INSTITUT TEKNOLOGI BANDUNG April, 2012

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

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

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

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

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USULAN BIAYA RISET ......................................................................................................8 8.1

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

8.2

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

8.3

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

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

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

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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.

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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 senyawasenyawa 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 2

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.

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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 nheksana, 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). 3

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.

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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. 4

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.

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INDIKATOR KEBERHASILAN (TARGET CAPAIAN) No. 1.

Indikator Keberhasilan Keluaran (output) Hasil Riset

Deskripsi 2 (satu) publikasi internasional. 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.

2.

Dampak (outcome) Hasil Riset

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.

Keterlibatan Mahasiswa S1, S2, S3

1 (satu) mahasiswa S3 dilibatkan dalam riset ini, yaitu Neneng Handayani (NIM 30510002).

Pembinaan peer

Pada pelaksanaannya, Keterlibatan dosen muda merupakan bagian dari pembinaan staf di lingkungan KK Kimia Organik.

Networking nasional dan internasional

Pada proses pengujian antimikroba, riset ini akan bekerjasama dengan Departemen Kesehatan (Akademi Analis Kesehatan, Cimahi)

3.

4.

5.

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JADWAL PELAKSANAAN Jadual penelitian sebagaimana ditunjukkan pada tabel berikut Bulan ke-

Kegiatan 1 a. Pengumpulan bahan tumbuhan b. Penyiapan bahan c. Ekstraksi d. Analisis kromatografi e. Fraksinasi dan pemurnian f. Verifikasi kemurnian isolat g. Pembuatan spektrum h. Analisis data spektrum i. Pengujian antimikroba j. Pembuatan publikasi internasional k. Pembuatan laporan 7

2

3

4

5

6

x x x

x

x x

x x x

x x x x

7

8

9

x x x

x x x x

x x

1 0

x x

x x

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)

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

Initial Stage

Long Term (2019-2020)

Structure modification of potential organic chemicals, tissue culture development, as well as synthetic elaboration of chemical analogs, to optimize their biological or physical properties 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 7

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USULAN BIAYA RISET 8.1 Belanja pegawai No. 1. 2.

Pelaksana Kegiatan Peneliti Utama Anggota Peneliti

Jumlah Orang 1 1

Jumlah Honor per Jumlah Jam Jam/Bulan Bulan/Tahun 135.000 15 10 90.000 15 10 Jumlah total biaya honor (Rp)

Jumlah Biaya (Rp) 20.250.000 13.500.000 33.250.000

8.2 Belanja barang No. 1. 2. 3. 4. 5. 6. 7. 8. 8.

Peralatan/Bahan Aseton teknis Kloroform p.a. Diisopropil eter Heksan teknis Metanol teknis Etil asetat teknis Silika gel Pelat KLT silika gel Bahan tanaman

Biaya Satuan (Rp) 20 L 1.100.000 2,5 L 500.000 2,5 L 2.500.000 20 L 700.000 20 L 450.000 20 L 700.000 1 kg 2.500.000 1 pak 2.500.000 1 kg 50.000 Jumlah total biaya barang (Rp)

Volume 1 5 2 1 1 1 1 1 2

Satuan

Jumlah Biaya (Rp) 1.100.000 2.000.000 5.000.000 700.000 450.000 700.000 2.500.000 2.500.000 100.000 15.050.000

8.3 Belanja jasa a. Honor pihak ketiga non PNS ITB dan ITB-BHMN atau asisten mahasiswa Jumlah Honor per Jumlah Jumlah Jumlah Biaya No. Pelaksana Kegiatan Orang Jam Jam/Bulan Bulan/Tahun (Rp) 2. Mahasiswa 1 20.000 60 10 12.000.000 Jumlah total biaya honor (Rp) 12.000.000 b. Perjalanan No. 1.

Tujuan

Volume

Biaya Satuan (Rp)

Jumlah Biaya (Rp)

Tidak ada Jumlah total biaya perjalanan (Rp)

c. Sewa Alat, Jasa Layanan dan Lain-lain No. 1. 2. 3. 4. 5.

Nama Alat/Jasa Layanan Jasa Jasa Jasa Jasa Jasa

analisis analisis analisis analisis analisis

Volume

Biaya Satuan (Rp)

spektrum UV 6 100.000 spektrum IR 6 150.000 spektrum NMR 6 1.100.000 spektrum MS 6 200.000 evaluasi antimikroba 6 900.000 Jumlah total biaya sewa alat, jasa layanan, dll. (Rp)

Jumlah Biaya (Rp) 600.000 900.000 6.600.000 1.200.000 5.400.000 14.700.000

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CV TIM PENELITI Ketua Peneliti: (1) Nama (2) Tempat/tangal lahir (3) Program Studi/PT (4) Alamat surat ~ Telpon/Faks ~ E-mail ~ Telpon rumah (5) Satus akademik (6) Jabatan struktural (7) Pendidikan terakhir (8) Pengalaman penelitian No. 1

: : : : : : : : : :

Dr. Yana Maolana Syah Karawang, 9-8-1962 Kimia/Institut Teknologi Bandung Jalan Ganesha 10, Bandung 40132 022-2502103 pes. 2202/022-2504154 [email protected] 022-91151768 Dosen Pembimbing Ketua Program Studi Magister dan Doktor Kimia Ph.D, 1998, Chemistry, University of Western Australia, Australia

:

Judul

Tahun, Sumber Dana

Sifat anti mikroba komponen terpenoid dari Curcuma

Program Riset Desentralisasi-ITB, 2012 Program Riset dan InovasiITB, 2012 2009-2010, Diknas melalui ITB.

xanthorrhiza 2

Antimikroba dari komponen kimia Macaranga

microcarpa 3

4

Kajian fitokimia, sifat sitotoksik, dan sifat antioksidan senyawa-senyawa turunan fenol dari tumbuhan Macaranga indonesia Kajian Fitokimia dan Sifat Sitotoksik Senyawa Oligostilbenoid dari Tumbuhan Dipterocarpus

2006, Research Grant Fakultas MIPA, ITB

Hasseltii 5

Cytotoxic Compounds from Lauraceous Plants

6

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

(9) Publikasi ilmiah

2004, Hibah B, Departemen Kimia, FMIPA, ITB 2003-2004, Hibah Kompe-tisi XI, Dikti, Depdiknas

: (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.

9

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, 115. 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

10

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

1

Kajian Profil Kimiawi dan Hubungan Biogenesis Metabolit Sekunder Daun Sukun (Artocarpus communis) (Ketua Peneliti) Combinatorial Biosynthesis of Morus Diels-Alder Adduct (Kerjasama dengan The Tokyo University) (Ketua Peneliti) Evaluasi Senyawa Isoprenylflavonoid dari Tumbuhan Cempedak (Artocarpus champeden) untuk Obat Anti Malaria (Ketua Peneliti) Penyelidikan Intensif Metabolit Sekunder dariTumbuhan Murbei (Morus sp) sebagai Lead Compound Obat Anti Malaria (Anggota Peneliti) Chemical and Biological Evaluation of the Indigenous Artocarpus of Indonesia for Antimalarial (Anggota Peneliti)

2 3

4

5

6

Development for the Medicinal Chemistry Based on Biologically Active Natural Products in the Subtropical Zone (Anggota Peneliti)

Sumber dana Program Penguatan Riset Institusi, 2010 JSPS-DGHE Bilateral Joint Research, 2010-2013 Hibah Penelitian Strategis Nasional DIKTI, 2010 Hibah Publikasi International Batch III, DP2M, 2009 2007, TWAS (The Academy of Sciences for the Developing World) 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). 11

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) 12

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.

13

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

1

More Phenolic Derivatives with an Irregular Sesquiterpenyl Side Chain from Macaranga pruinosa Yana M. Syah1,* and Emilio L. Ghisalberti2 1

Natural 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.) Mll.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; 13 C, 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] 2210-3155/12 $58.00+.00

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 acetoneextract (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 F1F10. 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) © 2012 Bentham Science Publishers

2 The Natural Products Journal, 2012, Vol. 2, No. 1

8

HO

8a

Syah and Ghisalberti

4a

4

5 15"

OH

OH

3'

R1

1'

O 2

1"

4'

OH

OH HO

O

R1

O

OH OH

O

3"

1 R1 = H

3 R1 = 4"'

6"

10"

8" 1"

9" 11"

2 R1 =

1"'

7" 9"

10"

3"' 5"'

8"

3"

4"

4 R1 =

1"

3" 5"

13"

14"

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

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; 13C 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; 13C NMR (100 MHz) data, see Table 1; HREIMS m/z: [M]+ 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 (CHalkyl), 1643 (conj. C=O) cm-1; 1H NMR (400 MHz) data, see Table 1; 13C NMR (100 MHz) data, see Table 1; HREIMS m/z: [M]+ 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 13C 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

Phenolic Derivatives from Macaranga pruinosa

The Natural Products Journal, 2012, Vol. 2, No. 1

Table 1. 1H and 13C NMR Data of Compounds 1 – 3 in CDCl3 1 C. No.

2

3


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

3

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

Syah and Ghisalberti

Table 1. contd….

1 C. No.

2


H (mult., J in Hz)


C

3


H (mult., J in Hz)


C


H (mult., J in Hz)

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)

OH HO

OH

O

HO

O

OH OH


C

OH

O

OH

1

O

2

OH HO

O

OH OH

O

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

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].

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.

Received: September 12, 2011

REFERENCES [1]

[2] [3]

[4] [5]

[6]

[7] [8]

[9] [10]

ACKNOWLEDGEMENTS

5

[11]

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. Syah, Y.M.; Ghisalberti, E.L. Phenolic derivatives with an irregular sesquiterpenyl side chain from Macaranga pruinosa. Nat. Prod. Commun., 2010, 5, 219-222. Tanjung, M.; Mujahidin, D.; Hakim, E.H.; Darmawan, A.; Syah, Y.M. Geranylated flavonols from Macaranga rhizinoides. Nat. Prod. Commun., 2010, 5, 1209-1211. 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. 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. 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. Mabry, T.J.; Markham, K.R.; Thomas, M.B. The Systematic Identification of Flavonoids; Springer-Verlag, New York, 1970, pp. 41-164. Sutthivaiyakit, S.; Unganont, S.; Sutthivaiyakit, P.; Suksamrarn, A. Diterpenylated and prenylated flavonoids from Macaranga denticulata. Tetrahedron, 2002, 58, 3619-3622. Ravelonjato, B.; Kunesch, N.; Poisson, J.E. Neoflavonods from the stem bark of Calophyllum verticillatum. Phtochemistry, 1987, 26, 2973-2976. Cottiglia, F.; Dhanapal, B.; Sticher, O.; Heilmann, J. New chromanone acids with antibacterial activity from Calophyllum brasiliense. J. Nat. Prod., 2004, 67, 537-541. 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.

Revised: December 22, 2011

Accepted: January 10, 2012

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

13

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 1 E-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 P388 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’-Omethyl-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.

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

14

2

W. Agustina, et al.

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 2,3dihydroflavonol 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

4"

15

5" 3"

OCH3

1"

OCH3

OCH3

4' 8

HO

8a

1'

O

HO

O

HO

O

2 4a

5

OH

4

OH

O

OH

1

O

OH

2

O

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

δH

2

5.09 (d, 11.5)

3 3-OH 4 4a 5 6 7 8 8a 1' 2'/6' 3'/5' 4' 1" 2" 3" 4" 5" 5-OH 4’-OCH3

4.61 (dd, 11.5, 4.0) 4.73 (d, 4.0) 6.06 (s) 7.52 (d, 9.0) 6.99 (d, 9.0) 3.19 (d, 7.5) 5.16 (tm, 7.5) 1.59 (s) 1.54 (s) 11.64 (s) 3.82 (s)

δC 84.0 73.2 198.4 101.5 162.6 96.6 165.4 108.6 160.9 131.3 130.0 114.4 160.8 22.0 123.3 130.5 25.8 17.8 55.5

HMBC ( 1H ⇔ 13C ) C-3, C-4, C-1', C-2'/C-6' C-2, C-4, C-1', C-4a, C-5, C-7,C-8 C-2, C-3'/5', C-4', C-6'/2' C-1', C-2'/6', C-4', C-5'/3' C-7, C-8, C-8a, C-2", C-3" C-1", C-4", C-5" C-2", C-3", C-5" C-2", C-3", C-4" C-4a, C-5, C-6 C-4'

16

W. Agustina, et al.

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; 13 C, 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]

[2] [3]

[4]

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. Airy Shaw, H.K., The Euphorbiaceae of Central Malesia (Celebes, Moluccas, Lesser Sunda Is.), Kew Bull., 37, 1-40, 1982. 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. 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.

18

[5]

W. Agustina, et al.

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, 867872, 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.

NPC

Natural Product Communications

Geranylated Flavonols from Macaranga rhizinoides

2010 Vol. 5 No. 8 1209 - 1211

Mulyadi Tanjunga,b, Didin Mujahidina, Euis H. Hakima, Ahmad Darmawanc and Yana M. Syaha * a

Natural Products Chemistry Research Group, Organic Chemistry Division, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia

b

Departement of Chemistry, Faculty of Science and Technology, Airlangga University, Surabaya 60115, Indonesia c

Research 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 1 H 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 13C 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

1210 Natural Product Communications Vol. 5 (8) 2010

Tanjung et al.

OCH3

OH

4' 8

HO

8a

O

1"

4a

5

OH

4

HO

O

1'

OCH3

OH

O

OH

3" 8"

O

4' 2

10"

OCH3

2'

1'

OH

1 7"

O

3

O

2

9"

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

δC

2 3 4 4a 5 6 7 8 8a 1’ 2’ 3’ 4’ 5’ 6’ 1” 2” 3” 4” 5” 6” 7” 8” 9” 10”

6.60 (s) 8.20 (d, 9.1) 7.10 (d, 9.1) 7.10 (d, 9.1) 8.20 (d, 9.1) 3.37 (br d, 6.7) 5.29 (tm, 6.7) 1.95 (br t, 7.0) 2.09 (m)* 5.06 (tm, 7.0) 1.59 (br s) 1.54 (br s) 1.79 (br s)

146.3 136.8 176.6 104.0 158.9 111.8 162.7 93.8 155.6 124.4 130.2 114.7 161.9 114.7 130.2 21.9 123.0 135.4 40.4 27.3 125.0 131.5 25.8 17.6 16.2

3-OH 5-OH 7-OH 3’-OH 4’-OCH3

8.08 (very br s) 12.11 (s) 9.69 (very br s)

-

3.88 (s)

55.7

2 HMBC ( H ⇔ C)

δH

C4a, C-6, C-7, C-8a C-2, C-4’, C-6’ C-1’, C-4’ C-1’, C-4’ C-2, C-2’, C-4’ C-5, C-6, C-7, C-2”, C-3” C-6, C-1”, C-4”, C-10” C-2”, C-3”, C-5”, C-10” C-4”, C-6”, C-7” C-8”, C-9” C-6”, C-7”, C-9” C-6”, C-7”, C-8” C-2”, C-3”, C-4”

6.26 (d, 1.8) 6.38 (d, 1.8) 6.96 (d, 8.5) 7.05 (d, 8.5) 3.47 (d, 6.7) 5.12 (tm, 6.7) 1.78 (br t, 7.0) 1.86 (br q, 7.0) 4.98 (tm, 7.0) 1.57 (br s) 1.49 (br s) 1.45 (br s)

150.2 137.7 177.0 104.7 162.5 99.1 164.8 94.4 158.3 124.4 128.5 145.2 149.4 109.3 122.5 26.4 123.5 135.3 40.3 27.3 125.0 131.6 25.7 17.6 16.2

C-4a, C-5, C-6 -

7.64 (very br s) 12.27 (s) 9.65 (very br s) 9.65 (very br s) 3.92 (s)

56.3

1

13

C-4’

δC

HMBC (1H ⇔ 13C) C-4a, C-5, C-7, C-8 C-4a, C-6, C-7 C-1’, C-3’ C-2’, C-4’ C-1’, C-2’, C-3’, C-2”, C-3” C-4”, C-10” C-5” C-4”, C-6” C-8”, C-9” C-6”, C-7”, C-9” C-6”, C-7”, C-8” C-2”, C-3”, C-4” C-4a, C-5, C-6 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 13C 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

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, precoated 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].

Natural Product Communications Vol. 5 (8) 2010 1211

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. 1 H NMR (500 MHz, acetone-d6): Table 1. 13 C 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. 1 H NMR (500 MHz, acetone-d6): Table 1. 13 C 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.

NPC

Natural Product Communications

Phenolic Derivatives with an Irregular Sesquiterpenyl Side Chain from Macaranga pruinosa

2010 Vol. 5 No. 2 219 - 222

Yana M. Syaha * and Emilio L. Ghisalbertib a

Department of Chemistry, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia

b

Chemistry, 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)-3methylpent-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

220 Natural Product Communications Vol. 5 (2) 2010

Syah & Ghisalberti

OH HO

5'

4'

OH

4

β

1

1'

3

α

8

HO

OH

8a

1'

O

9"

10"

OH

2 1"

7"

R

R

3'

OH

5

OH

1

8" 7"

1"

12"

10"

11"

1

13

C no

δH

δC

HMBC ( H ⇔ C)

1 2 3 4 5 6

7.00 (d, 2.0) 6.76 (d, 8.1) 6.83 (dd, 8.1, 2.0) 6.81 (d, 16.5) 6.74 (d, 16.5) 6.54 (s) 3.35 (br d, 7.0) 5.30 (tm, 7.0) 1.94 (m) 1.83 (m) 1.59 (m) 1.43 (m) 1.57 (m) 2.50 (br t, 8.3)

130.8 113.6 146.1 145.8 116.2 119.7

C-4, C-6, C-α C-1, C-3, C6 C-4, C-α

128.3 126.9 137.1 105.7 156.9 115.2 23.0 123.9 134.8 38.7

C-1, C-2, C-6, C-β, C-1’ C-1, C-1’ C-4’, C-6’/2’, C-β C-3’/5’, C-4’, C-2”, C-3” C-1”, C-3”, C-4”, C-15” C-2”, C-3”, C-5”, C-6”, C-15”

2.05 (m) 1.50 (ddd, 11.3, 8.3, 3.9) 1.05 (s) 0.88 (s) 1.62 (br qi, 0.7) 4.76 (hept, 1.3) 4.58 (br s) 1.78 (br d, 1.1) 7.82 (br s) 7.96 (br s) 8.02 (s)

146.8 23.6 109.4

9” 10” 11” 12” 13” 14” 15” 3-OH 4-OH 3’/5’-OH

H H3C

CH3

C-3”, C-7”

41.7 40.4 48.7 25.6

C-9” C-6”, C-7”, C-9”, C-10”, C11”, C-12” C-5”, C-6”, C-8”, C-12”

25.2 24.6

C-6”, C-7”, C-8”, C-11” C-6”, C-7”, C-8”, C-10” C-8”, C-12”, C-14” C-8”, C-12”, C-13” C-2”, C-4” C-2, C-3, C-4 C-3, C-4, C-5 C-2’/6’, C-3’/5’, C-4’

CH3

OH

H3C H

H

30.2

16.3

H

H H

HO

O

11" 15"

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

6” 7” 8’

OH

8"

O

13"

15"

5”

OH

14"

6"

3"

α β 1’ 2’/6’ 3’/5’ 4’ 1” 2” 3’ 4”

4

3"

2 9"

R=

4a

H HO

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

OH OH

O

13" 14"

3

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. C no 2 3 4 4a 5 6 7 8 8a 1’ 2’ 3’ 4’ 5’ 6’ 1” 2” 3” 4” 5” 6” 7” 8” 9” 10” 11” 12” 13” 14” 15” 3-OH 5-OH 7-OH 3’-OH 4’-OH

2

3

HMBC (1H ⇔ 13C) C-4, C-2’/6’ C-4 C-4, C-4a, C-6, C-7, C-8a C-2, C-6’, C-4’ C-1’, C-4’, C-5’ C-1’, C-4’, C-3’ C-2, C-2’, C-4’ C-5, C-6, C-7, C-2”, C-3” C-6, C-1”, C-4”, C-15” C-2”, C-3”, C-5”, C-6”, C-15”

δH 6.38 (s) 6.92 (d, 8.3) 7.07 (d, 8.3) 3.36 (br d, 6.7) 5.34 (tm, 6.7) 2.06 (m)

29.3

C-6”, C-9”

2.09 (m)

40.9 39.9 48.0 25.0

C-7” C-7”, C-10”, C-11”, C-12”, C-14” C-7”, C-8”, C-5”, C-12”

5.02 (tm, 6.9) 1.68 (br s) 1.59 (br s)

24.9

C-6”, C-7”, C-8”

1.74 (br s)

C-6”, C-7”, C-8”’ C-8”, C-12”, C-14” C-8”, C-12”, C-13”

3.47 (br d, 7.0) 5.27 (tm, 7.0) 1.77 (br s)

C-2”, C-4” C-2, C-4 C-4a, C-5, C-6 C-6, C-7, C-8

1.84 (br s) 6.10 (br s) 12.11 (s) 6.30 (br s) 5.71 (br s)

17.9 -

-

5.84 (br s)

-

δH 4.98 (d, 11.9) 4.55 (d, 11.9) 5.98 (s) 7.38 (d, 8.6) 6.84 (d, 8.6) 6.84 (d, 8.6) 7.38 (d, 8.6) 3.37 (br d, 7.1) 5.24 (tm, 7.1) 2.01 (m) 1.91 (m) 1.64 (m) 1.44 (m) 1.56 (m) 2.50 (m) 2.06 (m) 1.50 (m) 1.05 (s)

δC 83.0 72.4 195.8 100.5 160.6 107.5 164.7 96.0 161.0 128.1 129.1 115.7 156.5 115.7 129.1 21.0 120.6 139.9 38.0

0.89 (s) 1.65 (br s) 4.81 (hept, 1.0) 4.61 (br s) 1.82 (br s) 3.55 (br s) 11.53 (s) 6.56 (br s)

24.2 146.3 23.4 108.9

5.61 (very br s)

-

16.4 -

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, precoated silica gel plates (Merck Kieselgel 60 GF254, 0.25 mm thickness) were used. Distilled technical grade solvents were used for extraction and preparative chromatography

δC 147.7 136.3 175.3 104.0 157.9 109.6 161.6 94.3 155.6 121.9 127.2 142.6 146.7 113.1 123.1 27.9 121.4 139.9 39.6 26.2 123.5 136.0 25.7 17.7 16.1 21.4 120.9 136.0 25.8

HMBC (1H ⇔ 13C) C-4a, C-6, C-7, C-8a C-1’, C-3’ C-2, C-2’, C-4’ C-1’, C-2’, C-3’, C-2”, C-3” C-2’, C-1”, C-4”, C-10” C-2”, C-3”, C-6” C-6”, C-7” C-5”, C-9” C-6”, C-7”, C-9” C-6”, C-7”, C-8” C-2”, C-3” C-5, C-6, C-7, C-12”, C-13” C-11”, C-14”, C-15” C-12”, C-13”, C-15” C-12”, C-13”, C-14” C-2, C-3, C-4 C-4a, C-5, C-6 C-6, C-7, C-8 C-2’, C-3’, C-4’ C-3’, C-4’, C-5’

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

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. 1 H NMR (600 MHz, acetone-d6): Table 1. 13 C 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).

Syah & Ghisalberti 1

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

13

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. 1 H NMR (400 MHz, CDCl3): Table 2. 13 C 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.

J Nat Med (2010) 64:121–125 DOI 10.1007/s11418-009-0368-y

ORIGINAL PAPER

Phenolic compounds from Cryptocarya konishii: their cytotoxic and 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 Ó 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), cryptocaryone (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 cytotoxic and tyrosine kinase inhibitory activities of compounds 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

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.

Introduction The genus Cryptocarya (Lauraceae) contains at least 200 species distributed mainly in the tropical region of the

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

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), conjugated 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

123

122

J Nat Med (2010) 64:121–125

Fig. 1 Structures of compounds isolated from C. konishii

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 -hydroxychalcone. 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 connectivity) 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 absorptions for hydroxyl (3341 cm-1), alkyl C–H (2924 cm-1),

123

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 30

– 6.99 (dd, 8.0, 1.8)

164.4 118.9

– C-10 , C-50

40

7.56 (td, 8.0, 1.3)

137.4

C-20 , C-60

0

7.00 (td, 8.0, 1.8)

119.8

C-10 , C-30

0

6

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

0

12.88 (s)

5

2 -OH

C-10 , C-20 , C-30

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 infectocaryone (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

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 support 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 consistent 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

1

dC

HMBC ( H– C)

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 5

– 3.56 (ddd, 8.7, 5.0, 4.3)

172.6 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) 2.64 (dd, 15.9, 4.3)

C-5, C-6, C-10, C-12

12

–

177.5

–

10 20 /60

– 6.75 (m)

135.3 128.0

– C-2, C-60 /C-20 , C-40

30 /50

7.36 (m)

128.9

C-10 , C-50 /C-30

4

7.36 (m)

129.2

C-20 /C-60

4-OH

16.14 (s)

0

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 centrifugal 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.

13

dH (multiplicity, J in Hz)

39.7

Experimental

Extraction and isolation

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

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.

C-10, C-4, C-3

The dried and powdered wood of C. konishii (5.1 kg) was macerated with MeOH (39, each 15 L) at room temperature. 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 infectocaryone (3) (6 mg) [10], cryptocaryone (4) (390 mg) [14] and pinocembrin (6) (33 mg) [15]. After a series of separation and purification procedures using centrifugal planar chromatography (eluents CHCl3), fraction E (17.8 g) afforded desmethylinfectocaryone (2) (23 mg) and kurzichalcolactone A (5) (45 mg) [16]. Using a similar procedure, trans-N-feruloyltyramine (7) (25 mg) [17] was obtained from fraction F (8.6 g).

123

124 Table 3 Cytotoxic and tyrosine kinase inhibitory properties of compounds 1–7

J Nat Med (2010) 64:121–125

Compounds 20 -Hydroxychalcone (1)

P-388 (IC50, lM)b 64.26 ± 8.10

Inactive

2.17 ± 0.20

Inactive

0.8 ± 0.03

Inactive

Desmethylinfectocaryone (2) Infectocaryone (3) Cryptocaryone (4) Kurzichalcolactone A (5)

a b c

Positive controls Measured in triplicate Single measurement

0.04 ± 0.01

47.4

12.73 ± 0.57

Inactive

Pinocembrine (6)

231.64 ± 9.75

Inactive

trans-N-Feruloyltyramine (7)

119.50 ± 5.23

Inactive

Artonin Ea HV-1

a

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]20 D = ? 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-acetone): 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 bromide) 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

123

Tyrosine kinasec (% inhibition at 100 ppm)

1.1 ± 0.03 –

– 50.4

(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 Discovery 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 supplying 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 crassinervia. 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 oubatchensis. Org Lett 8:3825–3828 4. Lin F-W, Wang J-J, Wu T-S (2002) New pavine N-oxide alkaloids 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 Cryptocarya 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) Cryptofolione derivatives from Cryptocarya alba fruits. J Pharm Pharmacol 53:563–567 8. Juliawaty LD, Kitajima M, Takayama H, Achmad SA, Aimi N (2000) A 6-substituted-5,6-dihydro-2-pyrone from Cryptocarya strictifolia. Phytochemistry 54:989–993

J Nat Med (2010) 64:121–125 9. Usman H, Hakim EH, Harlim T, Jalaluddin MN, Syah YM, Achmad SA, Takayama H (2006) Cytotoxic chalcones and flavanones 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 acuminatissimus. 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

125 14. Govindachari TR, Parthasarathy PC, Desai HK, Shanbhag MN (1973) Structure of cryptocaryone. Constituent of Cryptocarya bourdilloni. Tetrahedron 29:3091–3094 15. Wagner H, Chari VM, Sonnenbichler J (1976) 13C-NMR-spektren 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 (Capsicum annuum 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

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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, Japan Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japan c Natural Products Research Group, Department of Chemistry, Bandung Institute of Technology, Jalan Ganeca 10, Bandung 40132, Indonesia b

a r t i c l e

i n f o

Article history: Received 12 March 2010 Revised 27 May 2010 Accepted 3 June 2010 Available online 8 June 2010 Keywords: b-Secretase BACE-1 Alzheimer Amentoflavone Biflavonoid 2,3-Dihydroamentoflavone 2,3-Dihydro-6-methylginkgetin

a b s t r a c t Here, we describe amentoflavone-type biflavonoids, which were isolated from natural sources and were found to inhibit b-secretase (BACE-1). The structure–activity relationship was studied, and compounds 1–8, 10, 17, and 18 showed BACE-1 inhibitory activity. Among these compounds, 2,3-dihydroamentoflavone 17 and 2,3-dihydro-6-methylginkgetin 18 exhibited potent inhibitory effects with IC50 values of 0.75 and 0.35 lM, respectively. Ó 2010 Elsevier Ltd. All rights reserved.

The most common form of dementia is Alzheimer’s disease (AD), which now affects over 30 million people worldwide.1 AD is a neurodegenerative disorder characterized by accumulation and deposition of amyloid b (Ab) peptides, which are generated from the cleavage of the b-amyloid precursor protein (APP) by consecutive action of b-secretase (BACE-1: b-site APP cleaving enzyme-1) and c-secretase.2–4 c-Secretase affects the Notch cleavage, while bsecretase demonstrates no compensatory mechanism for APP cleavage.5 The young BACE knockout mice were found to be healthy and fertile.5 Hence, the discovery of a BACE-1 inhibitor could be an effective and safe therapeutic strategy for AD. Biflavonoids are well known as constituents of gymnospermous plants and are flavonoid dimers connected by C–C or C–O–C bonds. Recently, these plants were found to exhibit anti-influenza,6,7 antiinflammatory,8 and anti-malarial9 activities. In this Letter, we report the isolation of biflavonoids from a variety of plants and study their BACE-1 inhibitory activities and structure–activity relationships. Acetone or CHCl3 extracts of a variety of plants were subjected to silica gel column chromatography, Sephadex LH-20 column chromatography, and HPLC to afford compounds 1–21. All isolated biflavonoids were identified on the basis of their spectroscopic data as well as by comparison with published data. Compounds

1–18 were amentoflavone-type biflavonoids with the flavonoid moieties connected by a C30 –C800 bond. Among them, 17 and 18 were 2,3-dihydro structures (Fig. 1). Amentoflavone 1 and sequoiaflavone 2 were isolated from Cunninghamia lanceolata,10,11 bilobetin 3, ginkgetin 6, 7,700 ,40 -tri-O-methylamentoflavone 12, sciadopitysin 13, amentoflavone-7,700 ,40 ,4000 -tetramethyl ether 16 and 2,3-dihydro-6-methylginkgetin 18 from Cephalotaxus harringtonia var. fastigiata,12–14 amentoflavone-7,700 -dimethyl ether 7 from Cephalotaxus harringtonia var. harringtonia,15 sotetsuflavone 4, 40 ,700 -di-O-methylamentoflavone 9 and kayaflavone 15 from Torreya nucifera,16–18 podocarpusflavone A 5, podocarpusflavone B 8 and isoginkgetin 10 from Podocarpus macrophyllus var. macrophyl-

OH HO

H 3C O

O

0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2010.06.021

O O

OH

2S

3

OCH 3

OH

O OH O

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

OCH 3

HO

2S

3

HO

6

HO 17

OH

O OH O

18

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

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

lus,19–21 700 ,4000 -dimethylamentoflavone 11 and heveaflavone 14 from Hevea brasiliensis,15,22 and 2,3-dihydroamentoflavone 17 from Cycas revoluta10 (Table 1). Robustaflavone 19 with the flavonoid moieties connected by a C30 –C600 bond was isolated from Selaginella moellendorffii,19 cupressuflavone 20, with the flavonoid moieties connected by a C8–C800 bond was isolated from Cupressus macrocarpa ‘Goldcrest’,23 and hinokiflavone 21 with the flavonoid moieties connected by a C40 –O–C600 bond was isolated from Metasequoia glyptostoboides20,24 (Fig. 2). Compounds 1–21 were all tested using the BACE-1 FRET assay kit.25 Several amentoflavone-type biflavonoids showed inhibitory activity, whereas robustaflavone 19, cupressuflavone 20, and hinokiflavone 21 did not. Amentoflavone 1 and its monomethoxy analogues 2–5 showed strong inhibitory activity with IC50 values of 1.54, 1.40, 2.02, 1.58, and 0.99 lM, respectively. Compounds 6–8 and 10 showed lower activities than 1–5 with IC50 values of 4.18, 6.25, 4.21, and 3.01 lM, respectively. The dimethoxy compounds 9 and 11, trimethoxy compounds 12–15, and tetramethoxy compound 16 exhibited no inhibitory activity. Compound 17, a 2,3dihydro analogue of 1, showed an increase in inhibitory activity, while compound 18 showed the strongest inhibitory activity of BACE-1 among amentoflavone-type biflavonoids (Table 2). These results indicate that the amentoflavone-type biflavonoids consisting of two apigenin molecules linked at the C30 –C800 position are important for BACE-1 inhibitory activity. The data also suggest that more than two hydroxyl groups at the R1–R4 position are needed for inhibitory activity. The results with compounds 17 and 18 show that the presence of a flavanone moiety in the amentoflavone biflavonoid is advantageous for inhibitory activity. Moreover, the presence of a methyl at the C6 position increases the inhibitory effect. Some amentoflavone-type biflavonoids exhibited neuroprotective effects on oxidative stress-induced and amyloid b peptide-induced cell death in neuronal cells.26 In addition, we found that amentoflavone-type biflavonoids have significant BACE-1 inhibitory activity. These results suggest that amentoflavone-type bifl-

OH HO

OH HO

O O

6'' 3'

OH

O

4'

O

19

OH O

HO

8''

O OH OH

OH

HO HO

OH O

O

O 6''

HO

OH O

8

O

OH O

20

21 OH O Figure 2. Structures of robustaflavone 19, cupressuflavone 20 and hinokiflavone 21.

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

BACE-1 inhibition IC50 (lM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 b-Secretase inhibitor

1.54 1.40 2.02 1.58 0.99 4.18 6.25 4.21 >10.0 3.01 >10.0 >10.0 >10.0 >10.0 >10.0 >10.0 0.75 0.35 >10.0 >10.0 >10.0 0.07

Table 1 Structures of amentoflavone-type biflavonoids 1–16

OR1 HO

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

O

O 3'

R 3O

8''

OR 2

Acknowledgments OR

4

O

OH O Compounds 1–16

R1

R2

R3

R4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

H CH3 H H H CH3 CH3 CH3 H H H CH3 CH3 CH3 H CH3

H H CH3 H H CH3 H H CH3 CH3 H CH3 CH3 H CH3 CH3

H H H CH3 H H CH3 H CH3 H CH3 CH3 H CH3 CH3 CH3

H H H H CH3 H H CH3 H CH3 CH3 H CH3 CH3 CH3 CH3

This research was partially supported by the Japan Society for the Promotion of Science (JSPS) AA Scientific Platform Program and a Grant from the High-Tech Research Center Project, Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (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.

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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 the supplied manual with modifications. Samples were dissolved in the assay

buffer (50 mM sodium acetate, pH 4.5) with DMSO (final concentrations were 10%). 10 lL of test samples, 10 lL of BACE-1 substrate (750 nM RhEVNLDAEFK-Quencher, in 50 mM ammonium bicarbonate), and ten microlitre of BACE-1 enzyme (1.0 U/mL) were mixed in the wells, and incubated 60 min in the dark at 25 °C. The fluorescence intensities of the mixtures were measured by fluoroskan ascent (Thermo Scientific) for excitation at 544 nm and emission at 590 nm. The inhibition ratio was calculated 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 concentrations were 10%)] after 60 min of incubation, C0 was the initial fluorescence of a control [enzyme, substrate, and assay buffer concentration with DMSO (final concentrations were 10%)], B was the fluorescence of a control [substrate and assay buffer concentration with DMSO (final concentrations were 10%)] after 60 min of incubation, B0 was the initial fluorescence of a control [substrate and assay buffer concentration with DMSO (final concentrations were 10%)], S was the fluorescence of the tested samples (enzyme, sample solution, and substrate) after 60 min of incubation, S0 was the initial fluorescence of the tested samples (enzyme, sample solution, and substrate). To check the quenching effect of the tested samples, the sample solution was added to reaction mixture C, and any reduction in fluorescence by the sample was investigated. b-Secretase inhibitor (Wako, Japan) was used as a positive control. 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.

.

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.

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

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

12

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

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. 6 macroura, 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.

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

Artikel Penelitian R

OH 4

B HO

4'

1

A

1'

7' 2' 10 '

OH

O

9' 11'

1 R=H 2 R = OH

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) 13

Ferlinahayati et.al .

Artikel Penelitian 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 nheksana: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 (asetond6, 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 14

MTT [3-(4,5-dimetiltiazo-2-il)2,5-difeniltetrazolium 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 Ckuarterner, 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 trans1,2-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 1,2,3,4tetrasubsitusifenil (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) Bull. Soc. Nat. Prod. Chem. (Indonesia), 2011, 11,12-16

Calkon dari Morus nigra

Artikel Penelitian

Tabel 1. Data NMR senyawa 1 dan 2 dalam aseton-d6 No. C 1 2 3 4 5 6 α β C=O 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’ 11’ 2’-OH

δH (multiplisitas, J dalam Hz) 1 7,73 (d, 8,4) 6,92 (d, 8,4) 6,92 (d, 8,4) 7,73 (d, 8,4) 7,75 (d, 15,4) 7,83 (d, 15,4) 6,52 (d, 8,8) 7,98 (d, 8,8) 3,36 (d, 7,0) 5,27 (t, 7,0) 1,63 (s) 1,76 (s) 14,01 (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’-isoprenil4,2’4’-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 Bull. Soc. Nat. Prod. Chem. (Indonesia), 2011, 11, 12-16

2 6,52 (d, 2,6) 6,45 (dd, 2,6 & 8,8) 7,69 (d, 8,8) 7,80 (d, 15,4) 8,22 (d, 15,4) 6,51 (d, 8,8) 7,89 (d, 8,8) 3,36 (d, 7,3) 5,27 (t, 7,3) 1,64 (s) 1,77 (s) 14,16 (s)

δC 1 127,5 131,7 116,7 160,9 116,7 131,7 118,3 144,9 192,7 114,3 162,7 116,1 165,1 108,0 130,2 22,5 123,2 131,4 17,9 26,0 -

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.

15

Artikel Penelitian 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, 12311235.

Ferlinahayati et.al .

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, 630635. 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.

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

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, Jalan Ganesha 10, Bandung, 40132, Indonesia

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, ESITOF, 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.

∗

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

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 43-47

43

Y.M. Syah .

Artikel Penelitian PENDAHULUAN

PERCOBAAN

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 senyawasenyawa 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.

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,

44

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 43-47

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; 13C 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]+ (MS2), 327,1 [M+H-56-56]+ (MS3), 299,1 [M+H-56-56-28]+ (MS4), 271,1 [M+H-56-56-28]+ (MS4) (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 13C 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

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 43-47

Artikel Penelitian 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 1 H-1H COSY sangat diperlukan. Dengan memperhatikan hubungan kopling dari masing45

Y.M. Syah .

Artikel Penelitian

OH

OH 8.70

9.56

6.51

HO

144.9

OH

7.67

113.2

7.76

HO

O

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3.34

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1.76

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122.8 146.7 136.5

1.73

17.9

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158.8 176.3 OH 121.7 O 123.1 OH

17.8

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93.7

162.5 111.6 21.9

OH

146.2 129.0 29.0

132.8 25.8

131.5 25.8

1.63

Gambar 1. Data 1H dan 13C NMR senyawa 1.

3,39

3,34

5,35 5,25

1.50

1.55

1.60

1.65

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1,63

1.70

1,73 1,74 1,76

1.75

1.80

1.85

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5.3

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4.8

4.7

4.6

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4.1

4.0

3.9

3.8

3.7

3.6

3.5

3.4

3.3

3.2

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.

46

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 43-47

Penentuan struktur senyawa aromatik: Papiriflavonol A

Artikel Penelitian OH OH

HO

O

OH OH

O

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.

2. 3.

4.

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.

5.

6.

7.

8.

Daftar Pustaka 1. Blattner, F.R.; Weising, K.; Banfer, G.; Maschwitz, U.; Fiala, B. “Molecular analysis of phylogenetic relationships among myrmecophytic Macaranga

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 43-47

9.

species (Euphorbiaceae)”, Mol. Phylogen. Evol,. 2001, 19, 331-334. Heyne, K. Tumbuhan Berguna Indonesia, Jilid I, 1987, Yayasan Sarana Wanajaya, Jakarta. 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. 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. Syah, Y.M.; Ghisalberti, E.L. “Phenolic derivatives with an irregular sesquiterpenyl side chain from Macaranga pruinosa”, Nat. Prod. Commun., 2010, 5, 219-222. Tanjung, M.; Mujahidin, D.; Hakim, E.H.; Darmawan, A.; Syah, Y.M. “Geranylated flavonols from Macaranga rhizinoides”, Nat. Prod. Commun., 2010, 5, 1209-1211. 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. 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, 12061209. 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.

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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, Jalan Ganesha 10, Bandung, 40132, Indonesia

‡

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

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

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 9-13

9

Artikel Penelitian

M. Tanjung et al . 4'

R2 HO

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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. 10

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-heksanEtOAc yang ditingkatkan kepolarannya sehingga menghasilkan empat fraksi utama AD. Fraksi C dimurnikan dengan kromatografi radial dan dielusi dengan eluen campuran n-

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 9-13

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Dua flavonol terprenilasi dari Macaranga aleuritoides

heksan-CHCl3 (4:1, 7:3, dan 3:2) menghasilkan 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-difeniltetrazolium 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; 1 H NMR (400 MHz, aseton-d6): lihat Tabel 1; 13 C 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; 1 H NMR (400 MHz, aseton-d6): lihat Tabel 2; 13 C 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 Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 9-13

1

H 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-diprenilflavon 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 berar11

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M. Tanjung et al .

Tabel 1. Data 1H dan 13C NMR senyawa 1 dan 2 dalam aseton-d6. C

1 2 3 4 4a 5 6 7 8 8a 1’ 2’ 3’ 4’ 5’ 6’ 1” 2” 3” 4” 5” 6” 7” 8” 9” 10” 3-OH 5-OH 7-OH 4’OH

6,57 (s) 8,03 (d, 2,0) 6,98 (d, 8,4) 7,95 (dd, 8,4; 2,0) 3,37 (d, 7,2) 5,37 (tm, 7,2) 1,74 (s) 1,74 (s) 3,35 (d, 7,2) 5,27(tm, 7,2) 1,64 (s) 1,77 (s) 7,85 (br,s) 12,40 (br, s) 9,59 (br, s) 8,89 (br, s)

2 6,34 (s) 8,04 (d, 2,4) 7,01 (d, 7,2) 8,05 (dd, 7,2; 2,4) 3,40 (d, 7,6) 5,31 (tm, 7,6) 1,74 (s) 1,74 (s) 3,55 (d, 7,4) 5,39 (d, 1,6) 1,65 (s) 1,80 (s) 7,89 (br,s) 12,09 (br, s) 6,58 (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’,8diprenilflavon atau broussoflavonol F. Perbandingan data NMR senyawa ini dengan data yang sama yang telah dipublikasikan 12

δC

δH (multiplisitas, J dalam Hz) 1

2

146,9 136,5 176,4 104,0 158,9 111,6 162,6 93,8 155,5 123,4 130,1 128,9 157,9 115,8 127,9 29,0 123,1 133,1 25,7 17,9 21,9 123,2 131,6 25,7 17,8

147,1 136,4 176,7 104,1 159,8 98,9 161,9 107,1 154,9 123,7 129,8 129,1 157,7 115,7 128,2 29,0 123,0 132.0 25,9 17,8 22,2 123,3 133,3 25,9 18,1

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.

Bull. Soc. Nat. Prod. Chem. (Indonesia), 2010, 10, 9-13

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/SPKLPPM/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

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species (Euphorbiaceae)”, Mol. Phylogen. Evol,. 2001, 19, 331-334. Heyne, K. Tumbuhan Berguna Indonesia, Jilid I, 1987, Yayasan Sarana Wanajaya, Jakarta. 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. 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. 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. Syah, Y.M.; Ghisalberti, E.L. “Phenolic derivatives with an irregular sesquiterpenyl side chain from Macaranga pruinosa”, Nat. Prod. Commun., 2010, 5, 219-222. 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. Fang, S-C.; Shieh, B-C.; Wu, R-R.; Lin, C-N. “Isoprenylated flavonols of Formosan Broussonetia papyripera”, Phytochemistry, 1995, 38, 535-537.

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