proposal program riset desentralisasi dikti 2013

Transcription

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
5
INDIKATOR KEBERHASILAN (TARGET CAPAIAN).........................................................6
6
JADWAL PELAKSANAAN ..................................................................................................7
7
PETA JALAN (ROAD MAP) RISET ....................................................................................7
8
USULAN BIAYA RISET ......................................................................................................8
8.1
Belanja pegawai ......................................................................................................8
8.2
Belanja barang.........................................................................................................8
8.3
Belanja jasa..............................................................................................................8
9
CV TIM PENELITI...............................................................................................................9
10
LAMPIRAN BUKTI CAPAIAN OUTPUT TAHUN 2010-2012 ...........................................13
1
RINGKASAN PROPOSAL
Pencarian senyawa-senyawa antimikroba baru merupakan salah satu kegiatan riset yang penting,
karena dilatarbelakangi oleh adanya kenyataan obat-obat antibiotik yang tersedia tidak (cenderung
tidak) efisien lagi dalam pengobatan penyakit infeksi. Berbagai pendekatan dilakukan dalam mencari
sumber-sumber baru senyawa yang bersifat antimikroba, termasuk didalamnya adalah dari
tumbuhan obat tradisional. Tumbuhan dari kelompok Phylanthus (Euphorbiaceae) termasuk salah
satu yang menjadi target pencarian ini, dan beberapa kajian pendahuluan dalam tingkat ekstrak
menunjukkan adanya bukti-bukti kandungan komponen yang bersifat antimikroba. Berdasarkan
kajian fitokimia, kelompok tumbuhan ini merupakan penghasil beragam golongan senyawa alam,
diantaranya alkaloid, terpenoid, lignan, dan turunan asam galat. Namun demikian, komponen
spesifik yang bertanggung jawab terhadap sifat biologis tersebut belum mendapat perhatian para
peneliti. P. myrtifolius merupakan salah satu tumbuhan obat Indonesia dengan penyebaran yang
relatif luas, dan pada proposal ini diusulkan untuk dilakukan isolasi komponen kimia dari daun P.
myrtifolius, menentukan struktur molekul, dan menguji masing-masing komponen murni tersebut
sebagai antimikroba. Isolasi komponen kimia akan dilakukan melalui pendekatan fitokimia, yang
meliputi proses ekstraksi, fraksinasi cair-cair, fraksinasi dengan tenik kromatografi, dan pemurnian
fraksi dengan teknik kromatografi. Struktur molekul masing-masing senyawa murni tersebut akan
dilakukan dengan menggunakan data spektroskopi, yang meliputi spektrum UV, IR, NMR 1D dan 2D,
serta spektrum massa. Evaluasi sifat antimikroba akan dievaluasi dengan metoda disk diffusion
method terhadap mikroba-mikroba patogen Escherichia coli, Enterobacter aerogenes, Pseudomonas
aeruginosa, Salmonella thypii, Shigella dysentriae, Vibrio cholereae, Bacillus subtilis, Staphylococcus
aureus dan Streptococcus sp., serta terhadap beberapa jamur yaitu Aspergillus fumigates, Candida
albicans, Epidermophyton sp., Penicillium sp, dan Trichophyton rubrum. Hasil-hasil penelitian ini
diharapkan dapat menghasilkan kandidat senyawa antimikroba baru sebagai lead compound
senyawa antibiotik baru. Sesuai dengan roadmap KK Kimia Organik riset ini masuk ke dalam tahap 1
(Initial Stage) dalam rangka penggalian senyawa-senyawa berguna alami, dan pada gilirannya akan
menjadi masukan pada tahap 2 (Development Stage) untuk transformasi dan sintesa di laboratorium
dalam rangka optimasi sifat biologisnya.
2
PENDAHULUAN
2.1 Latar belakang masalah
Dalam bidang pengobatan, penemuan senyawa-senyawa yang bersifat antimikroba dari hasil
metabolisme merupakan salah satu terobosan penting dalam era pengembangan obat antibiotik.
Senyawa-senyawa tersebut meliputi kelompok β-laktam (penisilin), sefalosporin, dan karbapenem
(von Nussbaum, et al., 2006). Namun demikian, sejak kurun waktu akhir abad yang lalu,
kemampuan senyawa-senyawa antibiotik tersebut mulai berangsur-angsur menurun karena
mikroorganisme yang menjadi target ternyata mengembangkan kekebalan terhadap 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.
3
METODOLOGI
Sesuai dengan tujuan penelitian tersebut di atas, target akhir dari penelitian ini adalah memperoleh
komponen kimia dari P. myrtifolius dan mengevaluasi masing-masing senyawa terpenoid tersebut
sebagai antimikroba. Oleh karena itu, penelitian ini akan menggunakan pendekatan fitokimia
sehingga semua komponen yang terkandung pada P. myrtifolius dapat dilaksanakan Berdasarkan
pendekatan ini, tahapan penelitiannya adalah sebagai berikut:
a. Pengumpulan bahan tumbuhan.
Bahan tumbuhan yang akan diteliti pada penelitian ini adalah daun P. myrtifolius.
b. Penyiapan serbuk kering bahan tumbuhan.
Daun P. myrtifolius selanjutnya dikeringkan di bawah sinar matahari dan digiling halus.
c. Pembuatan ekstrak metanol.
Ekstraksi akan dilakukan dengan metoda maserasi menggunakan metanol sebagai pelarut.
Lazimnya ekstraksi dilakukan tiga kali untuk masing-masing sampel tumbuhan untuk mencapai
jumlah ekstrak yang maksimum. Gabungan ekstrak aseton kemudian dikeringkan dengan
penguapan pada tekanan rendah.
d. Analisis kromatografi lapis tipis terhadap ekstrak.
Analisis kromatografi lapis tipis (KLT) pada tahap ini dimaksudkan untuk mengetahui perkiraan
jumlah komponen yang akan diisolasi, serta penentapan jenis-jenis eluen yang sesuai pada
tahapan fraksinasi.
e. Fraksinasi dan pemurnian komponen terpenoid.
Ekstrak aseton yang telah dikeringkan selanjutnya difraksinasi secara partisi kedalam faksi 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.
4
DAFTAR PUSTAKA
Adegoke, A.A., Iberi, P.A., Akinpelu, D.A., Aiyegoro, O.A., Mboto, C.I., (2010), Studies on
phytochemical screening and antimicrobial potentials of Phyllanthus amarus against multiple
antibiotic resistant bacteria, Int. J. Appl. Res. Nat. Prod., 3, 6-12.
Chang, C.-W., Lin, M.-T., Lee, S.-S., Liu, K.C.S.C., Hsu, F.-L., Lin, J.-Y., Differential inhibition of
reverse transcriptase and cellular DNA polymerase-a activities by lignans isolated from Chinese
herbs. Phyllanthus myrtifolius Moon. and tannins from Lonicera japonica Thumb. and
Castanopsis hystrix, Antiviral Res., 27, 367-374.
Heyne, K., (1987), Tumbuhan Berguna Indonesia II, Badan Litbang Kehutanan, Jakarta.
Komurairah, A., Bolla, K., Rao, K.N., Ragan, A., Raju, V.S., Charya, M.A.S., (2009), Antibacetrial
studies and phytochemical constituents of South Indian Phyllanthus species, Afr. J. Biotechnol.,
8, 4991-4995.
Lee, S.-S., Lin, M.-T., Liu, C.-L., Lin, Y.-Y., Liu, K.C.S.C., (1996), Six lignans from Phyllanthus
myrtifolius, J. Nat. Prod., 59, 1061-1065.
Lee, S.-S., Kishore, P.H., Chen, C.-H., (2002), Three novel triterpenoids from Phylanthus myrtifolius,
Helv. Chim. Acta, 85, 2403-2408.
Lin, M.-T., Lee, S.-S., Liu, K.C.S.C., (1995), Phyllamyricins A-C, three novel lignans from Phylanthus
myrtifolius, J. Nat. Prod., 58, 244-249.
Lin, M.-T., Lee, S.-S., Chen Liu, K.C.S.C., (1998), Polar constituents from Phyllanthus myrtifolius,
Chin. Pharm. J., 50, 327-336.
von Nussbaum, F., Brands, M., Hinzen, B., Weigand, S., Habich D., (2006), Antibacterial Natural
Products in Medicinal Chemistry—Exodus or Revival?, Angew. Chem. Int. Ed., 45, 5072-5129.
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.
5
5
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.
6
6
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
8
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
8
9
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].
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)
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
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
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
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
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.
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.
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
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Macaranga trichocarpa. Natural Product Communications, 4, 63-67.
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[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]
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[8]
Son KH, Keon SJ, Chang HW, Kim HP, Kang SS. (2001) Papyriflavonol A, a new prenylated flavonol from Broussonetia
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[9]
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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
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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
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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
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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
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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
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Naturforsch 60c:723–727
123
Bioorganic & Medicinal Chemistry Letters 20 (2010) 4558–4560
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
b-Secretase (BACE-1) inhibitory effect of biflavonoids
Hiroaki Sasaki a, Kazuhiko Miki b, Kaoru Kinoshita b, Kiyotaka Koyama b, Lia D. Juliawaty c,
Sjamsul A. Achmad c, Euis H. Hakim c, Miyuki Kaneda a, Kunio Takahashi b,*
a
School of Pharmacy, Shujitsu University, Nishigawara 1-6-1, Naka-ku, Okayama 703-8516, 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.
4559
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.
4560
H. Sasaki et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4558–4560
10. Anhut, S.; Seeger, T.; Zinsmeister, H. D.; Geiger, H. Z. Naturforsch. 1989, 44c,
189.
11. Krauze-Baranowska, M.; Mardarowicz, M.; Wiwart, M. Z. Naturforsch. 2002,
57c, 998.
12. Yook, C.-S.; Jung, J.-H.; Jeong, J.-H.; Nohara, T.; Chang, S.-Y. Nat. Prod. Sci. 2000,
6, 1.
13. Mai, V. T.; Phan, T. S.; Duong, A. T.; Duong, N. T. Tap Chi Hoa Hoc 2002, 40, 24.
14. Sasaki, H.; Miki, K.; Koyama, K.; Kinoshita, K.; Takahashi, K. Heterocycles 2008,
75, 939.
15. Gu, Y.; Xu, Y.; Fang, S.; He, Q. Zhiwu Xuebao 1990, 32, 631.
16. Lopez-Saez, J. A.; Perez-Alonso, M. J.; Velasco, N. A. Z. Naturforsch. 1994, 49, 267.
17. Khan, N. U.; Ansari, W. H.; Rahman, W.; Okigawa, M.; Kawano, N. Chem. Pharm.
Bull. 1971, 19, 1500.
18. Sun, C.-M.; Syu, W.-J.; Huang, Y.-T.; Chen, C.-C.; Ou, J.-C. J. Nat. Prod. 1997, 60,
382.
19. Xu, L.; Chen, Z.; Sun, N. Zhiwu Xuebao 1993, 35, 138.
20. Markham, K. R.; Sheppard, C.; Geiger, H. Phytochemistry 1987, 26, 3335.
21. Pan, J.-X.; Zhang, H.-Y.; Yang, X.-B. J. Plant Res. Environ. 1995, 4, 17.
22. Zhang, Y.; Tan, N.; Huang, H.; Jia, R.; Zeng, G.; Ji, C. Yunnan Zhiwu Yanjiu 2005,
27, 107.
23. Maatooq, G. T.; El-Sharkawy, S. H.; Afifi, M. S.; Rosazza, J. P. N. Nat. Prod. Sci.
1998, 4, 9.
24. Geiger, H.; Markham, K. R. Z. Naturforsch. 1996, 51c, 757.
25. BACE-1 assays were performed on 384-well black plates using a BACE-1 FRET
assay kit (Invitrogen Co., USA). The assay was carried out according to 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)
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
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.
.
16
ARTIKEL PENELITIAN
PENENTUAN STRUKTUR SENYAWA AROMATIK. BAGIAN 1:
PAPIRIFLAVONOL A DARI MACARANGA PRUINOSA
Yana M. Syah∗
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.
∗
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.
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
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
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
3.39
7.57
1.74
5.35
3.34
OH
7.83
1.76
5.25 OH
12.40
O
155.4
103.9
122.8
146.7
136.5
1.73
17.9
123.3
158.8
176.3 OH
121.7
O
123.1 OH
17.8
O
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
f1 (ppm)
1,63
1.70
1,73
1,74
1,76
1.75
1.80
1.85
5.4
5.3
5.2
5.1
5.0
4.9
4.8
4.7
4.6
4.5
4.4 4.3 4.2
f2 (ppm)
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
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
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.
47
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†∗
†
‡
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
9
Artikel Penelitian
M. Tanjung et al
.
4'
R2
HO
8a
O
OH
3'
2
1"
1'
7
5"
3
R1
4a
OH
4
OH
3"
O
4"
10"
1 R1 =
.
6"
2 R1 = H, R2 =
8"
R2 = H
9"
.
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.
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-
Artikel Penelitian
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
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
Artikel Penelitian
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.
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
2.
3.
4.
5.
6.
7.
8.
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.
13

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