PEMANFAATAN KULIT MANGGIS (GARCINIA MANGOSTANA)

SEBAGAI ANTIOKSIDAN DAN AGEN KEMOPREVENTIF PADA

PENYAKIT KANKER

MAKALAH
FARMAKOLOGI DALAM KEPERAWATAN

Oleh
Ayunda Puteri Rizanti (182310101160)
Nailatus Saadah (182310101201)

PROGRAM STUDI SARJANA KEPERAWATAN

FAKULTAS KEPERAWATAN
UNIVERSITAS JEMBER
2019
 PEMANFAATAN KULIT MANGGIS (GARCINIA MANGOSTANA)
SEBAGAI ANTIOKSIDAN DAN AGEN KEMOPREVENTIF PADA
PENYAKIT KANKER

MAKALAH
Disusun guna memenuhi tugas mata kuliah Farmakologi dalam Keperawatan
dengan Dosen Pembimbing :
Ns. Ana Nistiandani, M.Kep.

Oleh
Ayunda Puteri Rizanti (182310101160)
Nailatus Saadah (182310101201)

PROGRAM STUDI SARJANA KEPERAWATAN

FAKULTAS KEPERAWATAN
UNIVERSITAS JEMBER
2019

ii
 PRAKATA

Puji syukur kehadirat Allah SWT yang telah memberikan rahmat dan
karunia-Nya, sehingga penulis berhasil menyelesaikan makalah sederhana ini.
Sholawat dan salam semoga tetap tercurahkan kepada junjungan kita, Nabi
Muhammad SAW beserta keluarganya, sahabatnya dan pengikutnya hingga akhir
zaman.
Dalam penyusunan makalah ini, penulis banyak mendapat tantangan dan
hambatan akan tetapi dengan bantuan dari berbagai pihak tantangan itu bisa
teratasi. Olehnya itu, penulis mengucapkan terima kasih yang sebesar-besarnya
kepada semua pihak yang telah membantu dalam penyusunan makalah ini
1. Ns. Ana Nistiandani, M.Kep. selaku dosen pembimbing yang telah
membimbing kami sehingga makalah ini dapat terselesaikan dengan baik;
2. Ns.Wantiyah,M.. Kep selaku penanggung jawab mata kuliah farmakologi
dalam keperawatan; dan
3. Teman-teman mahasiswa Program Studi Sarjana Keperawatan Universitas
Jember kelas D yang telah membantu.
Makalah ini disusun untuk menjelaskan mengenai manfaat dari tanaman
obat atau hasil pertanian yang memiliki potensi besar sebagai obat obatan, serta
kandungan apa yang terdapat didalamnya. Penulis menyadari makalah ini masih
belum sempurna, oleh karena itu kritik dan juga saran dari berbagai pihak yang
bersifat membangun penulis harapkan demi kesempurnaan makalah ini dan kami
harapkan makalah ini bermanfaat dan menambah wawasan juga pengetahuan
pembaca.

Jember, 4 April 2019

Penulis

iii
 DAFTAR ISI

HALAMAN JUDUL ........................................................................................... ii

PRAKATA ........................................................................................................... iii
DAFTAR ISI ........................................................................................................ iv
BAB I. PENDAHULUAN ................................................................................... 1
1.1 Latar Belakang ................................................................................. 1
1.2 Tujuan .............................................................................................. 2
BAB II. KONSEP DASAR OBAT TRADISIONAL ........................................ 3
2.1 Definisi Obat Tradisional................................................................. 3
2.2 Tingkatan Obat Tradisional ............................................................. 4
2.3 Syarat-syarat Obat Tradisional ........................................................ 7
2.4 Peraturan Terkait Obat dan Pengobatan Tradisional ....................... 10
BAB III. ANALISA ARTIKEL ......................................................................... 12
3.1 Deskripsi Tanaman .......................................................................... 12
3.2 Kandungan Dalam Obat Tradisional ............................................... 13
3.3 Farmasetika dan Proses Pengolahan yang Dianjurkan .................... 15
3.4 Farmakokinetik ................................................................................ 16
3.5 Farmakodinamik .............................................................................. 17
3.6 Dosis ................................................................................................ 17
3.7 Indikasi dan Kontraindikasi ............................................................. 17
3.8 Efek Samping Obat .......................................................................... 18
3.9 Hal-hal yang Harus Diperhatikan .................................................... 19
3.10 Implikasi Keperawatan .................................................................... 19
BAB IV. PENUTUP ............................................................................................ 20
4.1 Kesimpulan ...................................................................................... 20
4.2 Saran ................................................................................................ 20
DAFTAR PUSTAKA .......................................................................................... 21

iv
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BAB I PENDAHULUAN

1.1 Latar Belakang

Obat tradisional di Indonesia memiliki peranan yang cukup besar dalam
pelayanan kesehatan masyarakat di Indonesia, sehingga obat tradisional
sangat berpotensi untuk dikembangkan di masa depan. Indonesia kaya akan
tanaman obat-obatan yang beraneka ragam, yang pemanfaatannya belum
dilakukan secara optimal untuk kesehatan. Indonesia diketahui memiliki
keragaman hayati terbesar kedua di dunia setelah Brasil . Obat tradisional
merupakan warisan budaya bangsa yang harus dilestarikan dan
dikembangkan untuk mendorong pembangunan dan peningkatan kualitas
kesehatan sekaligus untuk meningkatkan perekonomian rakyat. Obat
tradisional ini tentunya sudah diuji bertahun-tahun bahkan sejak dulu sesuai
dengan perkembangan kebudayaan bangsa Indonesia (Notoatmodjo, 2007).
Kanker menjadi salah satu penyebab kematian terbanyak setelah
kardiovaskular di dunia dan di Indonesia (Depkes RI, 2012). Hingga saat ini
pengobatan untuk kanker masih tergolong kurang memuaskan karena efek
samping yang ditimbulkan setelahnya. Sampai muncul salah satu tanaman
obat yang menjadi banyaknya objek penelitian dalam antikanker yaitu kulit
buah manggis (Ho, et al, 2002). Menurut beberapa penelitian yang
dilakukan kulit buah manggis mengandung senyawa alfa mangostin. alfa
mangostin memiliki perkembangan yang cukup pesat khususnya sebagai
antikanker. Mulai dari kanker hati, leukemia, kolon, pheocromocytoma,
hingga payudara. Potensi pada alfa mangostin mulai terlihat ketika
kandungannya memiliki efek menghambat poliferasi dan apoptosis hingga
melesat secara spesifik dapat memberikan efek antimetastasis dengan
berbagai macam pathway dan berbagai macam sel kanker, selain itu
kandungan xanton didalam kulit manggis adalah senyawa yang memiliki
antioksidan kuat yang amat dibutuhkan dalam penyeimbangan pro-oxidant
dalam tubuh dan lingkungan yang dikenal dengan sebutan radikal bebas.
Radikal bebas bermuatan listrik molekul, sehingga memiliki elektron yang
tidak berpasangan, yang mampu menangkap elektron dari zat lain untuk
 Page |2

menetralisir sendiri. Untuk itu pada makalah ini dibahas mengenai manfaat
dan mekanisme kerja kandungan kulit manggis yang bermanfaat bagi obat-
obatan.
1.2 Tujuan
1.2.1 Tujuan Umum
Tujuan pembuatan makalah ini adalah sebagai sumber wawasan
mengenai pemanfaatan tanaman herbal dan bentuk sediaan obat yang
tersedia berdasarkan analisis yang dilakukan dari beberapa sumber
dan penelitian, sehingga mampu mengoptimalkan manfaat tanaman
yang memiliki potensi besar sebagai sumber obat kedepannya.
1.2.2 Tujuan Khusus
1.2.2.1 Mengetahu dan memahami definisi dan klasifikasi obat
tradisional
1.2.2.2 Mengetahui klasifikasi dan morfologi buah manggis dan
bentuk sediaan obat yang ada di masyarakat
1.2.2.3 Mengetahui beberapa penelitian terkait kulit manggis dan
pemanfaatannya
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BAB II KONSEP DASAR OBAT TRADISIONAL

2.1 Definisi Obat Tradisional

Obat tradisional adalah obat-obatan yang diolah dengan cara
tradisional, turun temurun, dan resepnya berasal dari nenek moyang, adat-
istiadat, kepercayaan, atau kebiasaan setempat, baik bersifat magic
maupun pengetahuan tradisional. Menurut beberapa penelitian, obat-
obatan tradisional memiliki banyak manfaat bagi kesehatan dan saat ini
penggunaannya cukup gencar dilakukan karena lebih mudah dijangkau
oleh masyarakat, baik dari sisi harga maupun ketersediaannya. Obat
tradisional pada saat ini banyak digunakan karena menurut beberapa
penelitian karena sedikit menyebabkan efek samping, karena masih bisa
dicerna oleh tubuh. Bagian dari obat tradisional yang sering digunakan
atau dimanfaatkan di masyarakat antara lain akar, rimpang, batang, buah,
daun dan bunga. adapun beberapa ciri-ciri obat herbal yaitu: tidak hanya
menyembuhkan satu gejala penyakit tapi juga menyembuhkan ke organ
tubuh lain dan sampai ke akarnya, diproduksi tanpa campuran bahan kimia
dan bebas toksin sehingga keasliannya terjamin, contohnya seperti
jamu,bersifat kuratif (benar-benar bersifat menyembuhkan),tidak
menimbulkan efek samping, asalkan diracik oleh herbalis yang ahli.selain
itu ciri utama obat tradisional yaitu terbuat dari rempah-rempah atau bahan
alami yang tentunya memiliki khasiat yang luar biasa, reaksinya juga
lambat tapi bersifat konstruktif dan biasanya hanya untuk mencegah,
pemulihan, dan mengobati penyakit yang memerlukan pengobatan yang
lama.
Menurut Peraturan Menteri Kesehatan Republik Indonesia Nomor
6 Tahun 2012 tentang Industri dan Usaha Obat Tradisional,, Obat
Tradisional adalah bahan baku atau ramuan bahan yang berupa bahan
tumbuhan, bahan hewan, bahan mineral, sediaan galenik atau campuran
dan bahan-bahan tersebut, yang secara traditional telah digunakan untuk
pengobatan berdasarkan pengalaman (Peraturan Menteri Kesehatan No.6,
2012). Menurut World Health Organization (WHO), pengobatan
 Page |4

tradisional adalah jumlah total pengetahuan, keterampilan, dan praktek-

praktek yang berdasarkan pada teori-teori, keyakinan, dan pengalaman
masyarakat yang mempunyai adat budaya yang berbeda, baik dijelaskan
atau tidak, digunakan dalam pemeliharaan kesehatan serta pencegahan,
diagnosa, perbaikan atau pengobatan penyakit secara fisik dan juga mental
(WHO, 2004).
2.2 Tingkatan Obat Tradisional
Obat bahan alami atau tradisional yang ada di Indonesia begitu
banyak dan hampir tak terhitung , namun obat tradisional dapat
dikategorikan menjadi 3 tingkatan golongan antara lain yaitu jamu, obat
herbal terstandar, dan fitofarmaka
2.2.1 Jamu (Empirical based herbal medicine)

gambar 2.1. logo jamu ,sumber : https://www.google.com.

Jamu merupakan obat tradisional yang disiapkan dan dibuat
secara tradisional. Berisi bahan Tanaman tertentu yang menjadi
penyusun jamu tersebut, higienis (bebas cemaran) dan biasanya
digunakan secara tradisional berdasarkan pengalaman. Jamu telah
digunakan secara turun-temurun selama berpuluh-puluh tahun
bahkan mungkin ratusan tahun yang lalu. Pada umumnya, jamu
dibuat dengan mengacu pada resep dari peninggalan leluhur atau
pengalaman leluhur. Sifat jamu pada umumnya belum terbukti
secara ilmiah (empirik) namun telah banyak dipakai oleh
masyarakat luas. Belum ada pembuktian ilmiah sampai dengan
klinis, tetapi digunakan dengan bukti empiris berdasarkan
pengalaman turun temurun. Perlu diperhatikan, jamu itu bisa
diartikan denga kata lain obat asli Indonesia. Jamu adalah obat-
obatan yang ramuannya masih khas dan sederhana, dan sering
dijumpai di masyarakat karena telah digunakan secara turun
 Page |5

temurun dan terbukti pada masyarakat bahwa jamu memiliki efek

yang baik.

a b c
gambar 2.2. contoh jamu, a.jamu beras kencur, b.jamu asam
jawa, c.sediaan jamu dalam kemasan, sumber:
https://www.google.com.
gambar diatas merupakan beberapa contoh dari jamu seperti
jamu asam jawa yang dipercayai memiliki khasiat untuk
menghilangkan nyeri pada saat haid sampai pada jamu yang telah
dikemas dalam bentuk kemasan dan dipasarkan ke berbagai apotek
atau toko toko di Indonesia. Dalam menkonsumsi jamu dalam
kemasan tersebut sebaiknya juga memperhatikan keperluan dan
fungsi dari jamu itu sendiri agar dengan mengkonsumsinya bisa
menghasilkan sesuatu yang diharapakan.
2.2.2 Obat Herbal Terstandar (Scientific based herbal medicine)

gambar 2.3. Logo Obat Herbal terstandar (OHT), sumber :

https://www.google.com.
Obat Herbal Terstandar (OHT) adalah obat tradisional yang
diproduksi dari ekstrak atau penyarian bahan alam (dapat berupa
tanaman obat, binatang, maupun mineral). Untuk melakukan proses
ini membutuhkan peralatan yang lebih rumit dan berharga mahal,
ditambah dengan tenaga kerja yang mumpuni dan ahli dalam bidang
pengetahuan maupun ketrampilan pembuatan ekstrak. Selain
diproses dengan teknologi maju, jenis ini telah ditunjang dengan
 Page |6

adanya pembuktian ilmiah berupa penelitian-penelitian pre-klinik

(uji pada hewan) dengan mengikuti standar kandungan dari bahan-
bahan berkhasiat, standar proses pembuatan ekstrak tanaman obat,
standar pembuatan obat tradisional yang higienis, dan telah
dilakukan uji toksisitas akut maupun kronis, yang mana tentunya
OHT sudah terstandardisasi komposisinya, dan sudah diujikan dan
terbukti berkhasiat lewat penelitian pada bahan bahannya baik
hewan maupun tanaman.
2.2.3 Fitofarmaka (Clinical based herbal medicine)

Gambar 2.4. logo farmakosetika, sumber :

https://www.google.com.

Fitofarmaka merupakan obat tradisional dari bahan alam yang

dapat disetarakan dengan obat modern karena :
a) Proses pembuatannya yang telah terstandar,
b) Ditunjang bukti ilmiah s/d uji klinik pada manusia dengan
criteria- memenuhi syarat ilmiah,
c) Protokol uji yang telah disetujui,
d) Dilakukan oleh pelaksana yang kompeten,
e) Memenuhi prinsip etika,
f) Tempat pelaksanaan uji memenuhi syarat.
Dengan dilakukan uji klinik, maka akan lebih meyakinkan
para praktisi medis ilmiah untuk menggunakan obat herbal sebagai
salah satu sarana pelayanan kesehatan. Masyarakat juga bisa
menggunakan obat herbal karena manfaatnya jelas dengan
pembuktian telah dilakukan secara ilmiah. Pada intinya, fitofarmaka
itu obat dari bahan alam yang secara penelitian dan khasiat sudah
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bisa disetarakan dengan obat-obatan sintesis/modern. Penelitiannya

sudah dilakukan melalui uji klinis (pada manusia).
2.3 Syarat-syarat Obat Tradisional
Pengobatan dengan senyawa tunggal (single entity) atau senyawa
isolate murni maupun sintesis belum memberikan kesembuhan optimal dan
paripurna. Maka masyarakat berupaya untuk mencari obat alternatif,
misalnya obat tradisional atau herbal. Obat tradisional memiliki peran
penting dalam bidang kesehatan bahkan bisa menjadi produk andalan
Indonesia maka perlu dilakukan upaya penetapan standar mutu dan
keamanan ekstrak tanaman obat.Pada prinsipnya standardisasi suatu bahan
obat/sediaan obat dilakukan mulai dari bahan baku sampai dengan sediaan
jadi. Berdasarkan hal inilah standarisasi obat tradisional dikelompokkan
menjadi 3 kelompok yaitu :
1. Standarisasi bahan sediaan (simplisia atau ekstrak terstandar/bahan aktif
diketahui kadarnya)
2. Standarisasi produk dengan kandungan bahan aktif stabil atau tetap
3. Standarisasi proses dari metode, proses dan peralatan dalam pembuatan
sesuai dengan pedoman Cara Pembuatan Obat Tradisional (CPOBT)
Berdasarkan Keputusan Menteri Kesehatan Republik Indonesia
Nomor : 661/Menkes/Sk/Vii/ 1994 Tentang Persyaratan Obat Tradisional
ada beberapa syarat obat tradisional yang disesuaikan dengan bentuk dari
obat tradisional itu sendiri.
1. Rajangan
Rajangan adalah sediaan obat tradisional berupa potongan
simplisia, campuran simplisia, atau campuran simplisia dengan sediaan
galenik, yang penggunaannya dilakukan dengan pendidihan atau
penyeduhan dengan air panas. Kadar air tidak lebih dari 10 %,
Aflatoksin.tidak lebih dari 30 bagian per juta (bpj ) Wadah dan
penyimpanan, dalam wadah tertutup baik disimpan pada suhu kamar,
ditempat kering dan terlindung dari sinar matahari.
 Page |8

2. Serbuk
Serbuk adalah sediaan obat tradisional berupa butiran homogen
dengan derajat halus yang cocok ; bahan bakunya berupa simplisia
sediaan galenik, atau campurannya.Kadar air, tidak lebih dari 10 %,
Pengawet Serbuk dengan bahan baku simplisia dilarang ditambahkan
bahan pengawet Serbuk dengan bahan baku sediaan galenik dengan
penyari air atau campuran etanol air bila diperlukan dapat
ditambahkan bahan pengawet. Jenis dan kadar pengawet harus
memenuhi persyaratan.
3. Pil
Pil adalah sediaan pada obat tradisional berupa massa bulat, bahan
bakunya berupa serbuk simplisia, sediaan galenik, atau campurannya.
Waktu hancur tidak lebih dari 60 menit, Aflatoksin.tidak lebih dari 30
bpj.
4. Dodol Atau Jenang
Dodol atau jenang adalah sediaan padat obat tradisional bahan
bakunya berupa serbuk simplisia, sediaan galenik atau
campurannya.Mikroba patogen, negatif. Penetapan dilakukan menurut
cara yang tertera pada Metode Analisis Direktorat Jenderal Pengawasan
Obat dan Makanan Departemen Kesehatan Republik lndonesia.
Aflatoksin.Tidak lebih dari 30 bpj.
5. Pastiles
Pastiles adalah sediaan padat obat tradisional berupa lempengan
pipih umumnya berbentuk segi empat; bahan bakunya berupa campuran
serbuk simplisia, sediaan galenika, atau campuran keduanya. Kadar air
tidak lebih dari 10 %.
6. Kapsul
Kapsul adalah sediaan obat tradisional yang terbungkus cangkang
keras atau lunak, bahan bakunya terbuat dari sediaan galenik dengan
atau tanpa bahan tambahan.Waktu hancur tidak lebih dari 15 menit.
Penetapan dilakukan menurut cara yang tertera pada Farmakope
lndonesia. Kadar air isi kapsul, tidak lebih dari 10 %, Aflatokin, tidak
 Page |9

lebih dai 30 bpj, Bahan tambahan, jenis dan kadar pengawet yang
diperbolehkan sesuai dengan persyaratan pengawet yang tertera pada
persyaratan Wadah dan penyimpanan
7. Tablet
Tablet adalah sediaan obat tradisional padat kompak, dibuat secara
kempa cetak, dalam bentuk tabung pipih, silindris, atau bentuk lain,
kedua permukaannya rata atau cembung, terbuat dari sediaan galenik
dengan atau tanpa bahan tambahan.Waktu hancur, tidak lebih dari 20
menit untuk tablet tidak bersalut dan tidak lebih dari 60 menit untuk
tablet bersalut. Penetapan dilakukan menurut cara yang tertera pada
Farmakope Indonesia. Kadar air, tidak lebih dari 10 % . Allatoksin
tidak lebih dari 30 bpj.
8. Sari Jamu
Sari jamu adalah cairan obat dalam dengan tujuan tertentu
diperbolehkan mengandung etanol .
9. Parem, Pilis Dan Tapel
Parem, pilis dan tapel adalah sediaan padat obat tradisional bahan
bakunya berupa serbuk simplisia, sediaan galenik, atau campurannya
dan digunakan sebagai obat luar. Kadar air tidak lebih dari 10 %.
Penetapan dilakukan menurut cara yang tertera pada Farmakope
lndonesia atau Materia Medika lndonesia.Mikroba pathogen, negatif.
10. Koyok
Koyok adalah sediaan obat tradisional berupa pita kain yang cocok
dan tahan air yang dilapisi dengan serbuk simplisia dan atau sediaan
galenik, digunakan sebagai obat luar dan pemakainya ditempelkan pada
kulit.Mikroba pathogen, negatif.
11. Cairan Obat Luar
Cairan obat luar adalah sediaan obat tradisional berupa larutan
suspensi atau emulsi, bahan bakunya berupa simplisia, sediaan galenik
dan digunakan sebagai obat luar.Mikroba pathogen, negative.Penetapan
dilakukan menurut cara yang tertera pada Metode Analisis Direktorat
 P a g e | 10

Jenderal Pengawasan Obat dan Makanan Departemen Kesehatan

Republik lndonesia
Parameter terkait ketentuan mutu dan keamanan obat tradisional
dibuat dalam dokumen resmi pemerintah seperti Materia Medika Indonesia,
Monografi Ekstrak, Farmakope, Farmakope Herbal yang merupakan standar
resmi pemerintah. Adapun parameter- parameter tersebut dikelompokkan
menjadi dua yaitu :
1) Aspek parameter spesefik : yakni berfokus pada senyawa atau golongan
senyawa yang bertanggung jawab terhadap aktivitas farmakologis.
Analisis kimia yang dilibatkan ditujukan untuk analisa kualitatif dan
kuantitatif terhadap senyawa aktif.
2) Aspek parameter non spesifik : yakni berfokus pada aspek kimia,
mikrobiologi, dan fisis yang akan mempengaruhi keamanan konsumen
dan stabilitas, meliputi : kadar air, cemaran logam berat, aflatoksin, dan
lain-lain.
2.4 Peraturan Terkait Obat dan Pengobatan Tradisional
Di Indonesia terdapat beberapa peraturan yang dibuat pemerintah
melalui Menteri kesehatan dan beberapa instansi guna mengawasi
pengembangan obat tradisional mulai dari bahan baku hingga pemasaran
yang diatur oleh pemerintah baik berupa Undang-Undang, Peraturan
Pemerintah, dan lain sebagainya. Adapun beberapa aturan tersebut
diantaranya :
1. RENSTRA Kementrian Kesehatan RI dengan PP 17/1986 tentang
Kewenangan Pengaturan Obat Tradisional di Indonesia
2. Peraturan Menteri Kesehatan RI Nomor : 246/Menkes/Per/V/1990,
Izin Usaha Industri Obat Tradisional dan Pendaftaran Obat
Tradisional
3. Undang Undang No. 23 Tahun 1992 tentang Kesehatan
4. Peraturan Menteri Kesehatan RI No. 760/MENKES/PER/IX/1992
tentang Fitofarmaka
5. Peraturan Menteri Kesehatan RI No. 761/MENKES/PER/IX/1992
tentang Pedoman Fitofarmaka
 P a g e | 11

6. GBHN 1993 tentang Pemeliharaan & Pengembangan Pengobatan

tradisional sebagai warisan budaya bangsa (ETNOMEDISINE).
7. Keputusan Menteri Kesehatan RI No. 661/Menkes/SK/VII/1994
tentang Persyaratan Obat Tradisional
8. Keputusan Menteri Kesehatan RI No. 56/Menkes/SK/I/2000 tentang
Pedoman Pelaksanaaan Uji Klinik Obat Tradisional
9. Peraturan Menteri Kesehatan RI No. 949/MENKES/PER/VI/2000
tentang Pengertian Obat Tradisional

\
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BAB III ANALISA ARTIKEL

3.1 Deskripsi Tanaman

Nama Ilmiah : Garcinia Mangostana

Gambar 3.1.buah manggis

Klasifikasi tanaman Manggis:

 Kingdom : Plantae
 Divisi : Spermatophyte
 Sub-divisi : Angiospermae
 Kelas : Dicotyledoneae
 Ordo : Guttiferanales
 Family : Guttiferae
 Genus : Garcinia
 Spesies : Garcinia mangostana L
Manggis merupakan tanaman buah berupa pohon yang berasal dari
hutan tropis yang teduh di kawasan Asia tenggara, yaitu hutan belantara
Kalimantan Timur di Indonesia atau semenanjung Malaya. Tanaman ini
tumbuh subur pada daerah yang mendapat banyak sinar matahari,
kelembaban tinggi, serta musim kering yang pendek (untuk menstimulasi
perbungaan). Pada kondisi kering, diperlukan irigasi untuk menjaga
kelembapan tanah. Tanaman ini dapat ditanam hingga ketinggian 1000 m di
atas permukaan laut (20-40 ) di daerah tropis, namun biasanya pertumbuhan
maksimal berlangsung di daerah dataran rendah. Ciri-ciri buah manggis
yang sudah matang adalah kulit buahnya berwarna ungu kemerahan,
bentuknya bulat agak pipih, tangkainya bertekstur lunak, dan diameter
buahnya sekitar 4-7 cm. Tingkat kematangan buah sangat berpengaruh
 P a g e | 13

terhadap mutu dan daya simpan buah. Semakin matang maka semakin
singkat daya simpannya.
Adapun ciri ciri tumbuhannya yaitu batang tegak, kulit batang coklat
memiliki getah kuning, daun tunggal, posisi daun berhadapan atau bersilang
berhadapan. helai daun mengkilat di permukaan, permukaan atas hijau gelap
dengan permukaan bawah hijau terang, berbentuk elips memanjang, ukuran
12-23 cm 4,5-10 cm, tangkai 1,5-2 cm. c) Bunga Bunga betina 1-3 di ujung
batang, susunan menggarpu, garis tengah 5-6 cm. Mempunyai 4 daun
kelopak, dua daun kelopak yang t erluar hijau kuning, dua yang terdalam
lebih kecil bertepi merah, melengkung kuat, tumpul. d) Buah Bentuk bola
tertekan, garis tengah 3,5-7 cm, ungu tua dengan kepala putik duduk (tetap),
kelopak tetap, dinding buah tebal, berdaging, ungu, dengan getah kuning. e)
Biji Memiliki biji 1-3 butir, diselimuti oleh selaput biji yang tebal berair,
putih, dapat dimakan (termasuk biji yang gagal tumbuh sempurna)
(Nugroho, 2009).
Bentuk sediaan obat dari buah manggis yang banyak dijual di
masyarakat adalah berupa kapsul dan obat tersebut termasuk dalam obat
tradisional terstandar contohnya seperti mastin

Gambar 3.2. obat tradisional terstandar dari kulit manggis

3.2 Kandungan Dalam Obat Tradisional
Kandungan Nutrisi Buah Manggis per 100 gam yaitu, Jumlah Kalori
63,00 Kkal, Karbohidrat 15,60 g , Lemak 0,60 g, Protein 0,60 g, Kalsium
8,00 mg, Vitamin C1 2,00 mg, Vitamin B1 0,03 mg, Fosfor 12,00 mg, Zat
Besi 0,80 mg Bagian yang dapat dimakan 29,00% (Hasyim dan Iswari,
2012). Adapun kandungan kimia yang ada di kulit buah manggis adalah
xanthon, mangostin, garsion, flavonoid, dan tannin, dan senyawa lainnya.
Metabolit sekunder utama dari kulit buah manggis adalah inti xanton.
Xanton merupakan derivate dari campuran polifenol yang mempunyai
 P a g e | 14

aktivitas biologis yang sangat signifikan dalam sistem in vitro (Linuma et al.
1996). Sebagian besar kandungan xanton ditemukan dalam tumbuhan yang
tinggi yang dapat diisolasi dari empat suku, yaitu Guttiferae, Moraceae,
Polygalaceae, dan Gentianaceae (Sluis, 1985). Senyawa utama dari xanthon
adalah α-mangostin dan γ-mangostin (Jung et al., 2006). Pada kulit buah
manggis juga mengandung senyawa lain diantaranya mangostenol,
mangostinon, trapezifolixanton, caloxanton dan lain sebagainya. Kemudian
terdapat beberapa fungsi yang ditemukan dalam kandungan ekstrak kulit
manggis antara lain :
a. Antikanker, Banyak penelitian yang telah dilakukan untuk memecahkan
masalah kanker salah satunya adalah tanaman yang menjadi objek
penelitian yaitu kulit manggis. hal ini dibuktikan oleh beberapa penelitian
salah satunya oleh tim dari Tumor Pathology University di Okinawa,
Jepang. Dalam penelitian tersebut dilakukan percobaan terhadap mencit
untuk melihat kemampuan alpha mangostin dalam menghambat
pertumbuhan selsel kanker kolon selama lima minggu perlakuan, ternyata
hasilnya menunjukanbahwa senyawa dari bagian senyawa xanthone
tersebut sangat berpotensi untuk digunakan sebagai penghambat
pertumbuhan kanker. (Setiawan, 2011), dan menurut artikel terkait
Singkatnya, 14 senyawa diisolasi dan diidentifikasi dari G. mangostana.
Senyawa baru diuji dengan garis sel tujuh kanker dan pertumbuhan
penduduk sisi CNE-2, yang menunjukkan bahwa senyawa tersebut
memiliki sifat anti-kanker yang potensial. Perlu dicatat bahwa konstituen
bioaktif ini juga dapat dideteksi dalam manggis. Ada beberapa
kemungkinan bahwa makan manggis berguna untukpencegahan dan
pengobatan kanker (Renyue Yang,2017)
b. Antioksidan, Xanton sendiri adalah senyawa yang memiliki antioksidan
kuat yang amat dibutuhkan dalam penyeimbangan pro-oxidant dalam
tubuh dan lingkungan yang dikenal dengan sebutan radikal bebas.
Radikal bebas bermuatan listrik molekul, sehingga memiliki elektron
yang tidak berpasangan, yang mampu menangkap elektron dari zat lain
untuk menetralisir sendiri. Hal ini karena saat metabolisme disamping
 P a g e | 15

menghasilkan energi juga akan dihasilkan radikal bebas, sedangkan

faktor eksternal antara lain dari asap rokok dan pabrik, emisi kendaraan
bermotor maupun mengkonsumsi alkohol (pervical,1998). Aktivitas
antioksidan sendiri dapat menstabilkan radikal bebas dengan cara
melengkapi kekurangan elektron bebas dan menghambat terjadinya
reaksi berantai dari pembentukan radikal bebas yang akan menimbulkan
stres oksidatif (Maria,2014).
c. Antiinflamasi, Ada bukti tentang antialergi dan anti-inflamasi dari
manggis dalam model in vitro yang berbeda, seperti sel RBL-2H3, dan
sel glioma tikus C6. penelitian menunjukkan bahwa mangostin diisolasi
dari manggis menghambat anafilaksis sistemik, imunositoadheren pada
marmot dan tikus, dan penghambatan primer dan sekunder dari tikus
yang diinduksi arthritis.
d. Antibakteri, Bakteri adalah mahluk hidup paling kecil bersel tunggal
yang dapat berkembang biak sangat cepat, penelitian menunjukan bahwa
kandungan xanthone dalam kulit manggis mampu menghambat
reproduksi beberapa jenis bakteri. Salah satu bakteri yang dihancurkan
adalah salmonella,bakteri penyebab keracunan makanan, semakin
bertambah usia seeorang secara alamiah semakin berkurangnya zat asam
didalam perut.
3.3 Farmasetika dan Proses Pengolahan yang Dianjurkan
Banyak bentuk obat herbal yang dihasilkan dari ekstrak kulit
manggis,melihat khasiatnya yang begitu banyak kulit manggis terus diteliti
agar mampu terus dikembangkan pemanfaatannya ekstrak kulit manggis
yang sering kita jumpai biasanya berupa suplemen, sirup dan ada beberapa
bentuk gel yang dibuat dari kulit manggis pula. Warna ungu pada kulit
manggis mengandung senyawa xantone adalah komponen antioksidan
paling penting pada manggis, untuk antikanker, antibakteri, antiinflamasi,
memperkuat sistem imun, keseimbangan mikroba meningkatkan kelenturan
sendi dan lain-lain. Banyak cara yang bisa dilakukan untuk pengolahan obat
herbal ini yaitu membuatjus kulit manggis, teh kulit manggis, tepung kulit
manggis dan lainnya. Ada beberapa industri pangan yang memang membuat
 P a g e | 16

minuman jenis jus berbahan dasar kulit buah manggis serta teh kulit buah
manggis.
Banyaknya kekeliruan dalam pengolahan kulit manggis karena salah
caranya yang malah menyebabkan diare dan merasa tidak ada ada efek
positifnya terhadap kesehatan. Hal tersebut disebabkan cara pengolahan
yang keliru, yaitu dengan merebus kulit manggis dan yang di minum
langsung adalah air rebusannya padahal air rebusan justru banyak
mengandung getah, tanin dan saponin yang dapat beresiko diare dan
menutup pori usus. Selain dengan merebus banyak yang memprosesnya
dengan cara di jemur di terik matahari secara berulang-ulang karena tidak
bisa kering dalam satu kali penjemuran padahal itu sangat rentan terhadap
tumbuhnya jamur disamping itu juga terpapar oleh banyaknya bakteri
karena tidak seteril. Cara pengolahan yang keliru bisa jadi menjadi masalah
dan menimbulkan dampak negatif bagi kesehatan kita, adapun anjuran
dalam pengolahan secara alami kulit manggis sebagai berikut:
1. Ambil kulit manggis dari masa petik selama 3-7 hari (masa petik
mempengaruhi kandungan xanthon dan getah)
2. Rebus kedalam air yang mendidih selama 10 menit kemudian buang
airnya.
3. Masukkan kulit manggis dalam es batu selama 10 menit ( untuk
mengurangi Tanin dan Saponin yang berlebih)
4. Ambil kulit manggisnya kemudian di blender tanpa di beri campuran
apapun
5. Saring/peras ambil airnya. (1 kg Manggis menghasilkan 100 ml )
3.4 Farmakokinetik
Proses farmakokinetik pada kulit manggis tergantung pada obat apa ia
dicampurkan pada kelas polifenol yang terdapat pada xanthone memiliki
kemampuan memberi atom hidrogen dengan mekanisme memutus rantai
pembentuk radikal dan mengikat ion logam transisi sehingga menghambat
pembentukan radikalbebas. Senyawa xanthone, mangostin, garsinon,
flavonoid dan tanin yang terkandung dalam kulit buah manggis merupakan
senyawa-senyawa bioaktif fenolik. Senyawa-senyawa ini diduga berperan
 P a g e | 17

dalam menentukan aktivitas antioksidan pada kulit buah manggis. Kulit

buah manggis yang mengandung senyawa xanthone memiliki fungsi
antioksidan tinggi yang dapat dimanfaatkan untuk melindungi dan
mengurangi kerusakan sel terutama yang diakibatkan oleh radikal bebas
(Soedibyo, 2008)
3.5 Farmakodinamik
Kandungan xantone yang terdapat dalam kulit manggis mengikat
oksigen bebas atau yang biasa kita sebut sebagai radikal bebas,inilah yang
mengakibatkan kerusakan jaringan sel sehingga dengan adanya xantone
mampu menghambat proses degenerasi (kerusakan sel) xantone juga
merangsang perbaikan jaringan sel atau regenerasi sel -sel yang rusak.
Selain itu xantone juga efektif mengatasi sel kanker dengan sistem apoptosis
yaitu dengan memaksa sel unruk membunuh dirinya dengan memuntahkan
cairan yang ada didalam mitokondria sehingga sel tersebut mati dan xantone
juga berperan dalam mengaktifkan sistem kekebalan tubuh dengan
merangsang aktifnya sel pembunuh alami (natural killer cell atau NK Cell),
Nk Cell itulah yang nantinya membunuh sel kanker dan virus yang masuk
kedalam tubuh. Adapun efek samping dari ektrak kulit manggis ini tidak ada
apabila konsumen tidak mengalami gejala merugikan seperti mual, muntah,
diare, tremor atau kejang bahkan pingsan ketika mengonsumsi ekstrak kulit
manggis tersebut dalam dosis normal, maka dapat dikatakan ekstrak tersebut
aman untuk dikonsumsi,dan jika hal diatas terjadi mungkin ada reaksi alergi
dari dalam tubuh, Namun walau demikian kulit manggis tidak disarankan
untuk dikonsumsi secara berlebihan agar tetap aman dikosumsi.
3.6 Dosis
Dosis dari Mastin sendiri ialah dapat dikonsumsi secara teratur 2 X 2
kapsul per hari. Dosis juga dapat ditingkatkan menjadi 4 X 2 kapsul per hari
sesuai kebutuhan.
3.7 Indikasi dan Kontraindikasi
3.7.1 Indikasi
Kapsul herbal dengan ekstrak kulit manggis yang berkhasiat sangat
tinggi bagi kesehatan tubuh. Dalam kapsul mastin mengandung 100%
 P a g e | 18

kulit manggis dengan kualitas yang tinggi. Berdasarkan hasil

penelitian yang ada menunjukkan bahwa kulit manggis kaya akan
antioksidan yang baik bagi kesehatan tubuh terutama antosianin,
tannin, xanthone maupun asam fenolat. Mastin berkhasiat sebagai anti
kanker dan tumor, memelihara kesehatan tubuh, anti bakteri,
mengobati sariawan, disentri, hipertensi, memelihara kesehatan kulit,
anti obesitas, antiobik, anti jamur, dan mengobati TBC.
3.7.2 Kontraindikasi
Manggis merupakan antioksidan yang mampu melindungi sel
tubuh dari kerusakan akibat oksidasi. Efek pelindung dari ekstrak kulit
manggis ini ternyata dapat mengganggu kinerja obat-obatan
kemoterapi dan terapi radiasi, sehingga beberapapengobatan
kankersecaramedis justru jalan di tempat atau malah memperburuk
kondisi pasien.
3.8 Efek Samping Obat
3.8.1 Endapan yang mengganggu ginjal
Meski kulit manggis berguna untuk menangkal radikal bebas anti
diabetes, anti inflamasi, mampu mengusir bakteri, hingga mencegah
perkembangan sel kanker, namun pada kenyataannya nilai plus di atas
juga dibarengi dengan dampak yang cukup serius. Endapan yang bisa
dikatakan sebagai efek samping mastin ini dapat membahayakan
organ ginjal dan usus lantaran sulit untuk diserap. Apabila senyawa
tidak bisa dicerna oleh ginjal dengan sempurna, maka kinerja ginjal
akan terhambat. Jika meminum air seduhan kulit manggis, maka harus
mengimbanginya dengan memperbanyak minum air putih, juga bisa
mengatasinya dengan jintan atau daun sembung yang bersifat diuretik.
3.8.2 Kembung
Efek samping mastin yang kerap dirasakan oleh customer ialah
perut kembung. Kondisi ini timbul karena kecenderungan bahwa
pasien memiliki jenis penyakit lain yang muncul seiring dengan
penyakit utama yang mereka miliki.Sebagai contohnya, Anda
menderita maag dan merasa perutnya kembung sehabis menelan
 P a g e | 19

suplemen mastin ini. Tidak menutup kemungkinan jika Anda juga

memiliki gangguan pencernaan lain yang riskan terhadap sifat asam.
Solusinya,bisa meracik ramuan menggunakan kunyit atau temulawak.
3.9 Hal-hal yang Harus Diperhatikan
3.9.1 Tidak disarankan bagi ibu hamil dan menyusui. Keamanan ekstrak
kulit mangis belum dievaluasi secara menyeluruh bagi para wanita
hamil. Oleh karena itu, baik wanita hamil maupun menyusui
sebaiknya menghindari konsumsi suplemen dengan kandungan
ekstrak kulit manggis.
3.9.2 Beberapa praktisi kesehatan di the Memorial Sloan-Kettering Cancer
Center, or MSKCC, menemukan terjadinya kasus lactic acidosis
dimana pasien telah mengonsumsi jus manggis setiap hari selama satu
tahun terakhir.Lactic Acidosis sendiri merupakan kondisi fisiologis
yang ditandai dengan pH rendah pada jaringan tubuh dan darah serta
disertai dengan terjadinya penumpukan laktat. Tanda-tanda lactic
acidosis adalah sesak nafas, muntah, dan sakit pada bagian perut. Jika
tidak ditanggulangi segera, tumpukan asam dalam tubuh dapat
membahayakan jiwa. Hal ini tidak menutup kemungkinan juga bisa
terjadi jika terlalu banyak mengonsumsi ekstrak kulit manggis.
3.10 Implikasi Keperawatan
Implikasi keperawatan yang dapat diambil dari hasil analisis ini terhadap
profesi keperawatan diantaranya dapat digunakan oleh penderita kanker
untuk lebih banyak mendapatkan informasi tentang manfaat kulit manggis
sebagai pengobatan alternatif secara tradisional. Hasil analisis ini dapat
diterapkan pada pelayanan kesehatan sebagai proses informasi pendidikan
kesehatan bagi penderita kanker yang menggunakan obat tradisional.
 P a g e | 20

BAB IV PENUTUP

4.1 Kesimpulan
Obat tradisional adalah obat-obatan yang diolah dengan cara
tradisional, turun temurun, dan resepnya berasal dari nenek moyang, adat-
istiadat, kepercayaan, atau kebiasaan setempat, baik bersifat magic maupun
pengetahuan tradisional. Menurut beberapa penelitian, obat-obatan
tradisional memiliki banyak manfaat bagi kesehatan dan saat ini
penggunaannya cukup gencar dilakukan karena lebih mudah dijangkau oleh
masyarakat salah satunya adalah Manggis yang merupakan tanaman buah
berupa pohon yang berasal dari hutan tropis yang teduh di kawasan Asia
tenggara, yaitu hutan belantara Kalimantan Timur di Indonesia atau
semenanjung Malaya. Adapun kandungan kimia yang ada di kulit buah
manggis adalah xanthon, mangostin, garsion, flavonoid, dan tannin, dan
senyawa lainnya. Metabolit sekunder utama dari kulit buah manggis adalah
inti xantonmanfaat manggis begitu banyak diantaranya yaitu anti oksidan,
anti kanker, anti inflamasi dan masih banyak lagi. Beberapa bentuk sediaan
obat yang dibuat dari kulit manggis antara lain yaitu mastin, sirup, gel dll.
4.2 Saran
Perkembangan zaman sudah modern dan teknologi semakin canggih.
Hal tersebut juga membawa warna baru bagi dunia kesehatan. Salah satu
contohnya ialah obat. Berbagai macam obat sudah semakin banyak dijual
dipasaran. Jenis obat dibagi menjadi obat tradisional/herbal dan obat
kimia/sintesis. Tentunya kedua jenis tersebut memiliki manfaat untuk
mengobati suatu penyakit. Kita sebagi konsumen pastinya jangan sampai
tergiur dengan masalah harga obat yang penting demi kesembuhan diri
sendiri, jangan meminum obat berlebihan. Minumlah obat sesuai dosis yang
telah dianjurkan dan jangan menyepelekan sebuah gejala penyakit. Karena
akan lebih baik mencegah sebuah penyakit daripada mengobati.
 P a g e | 21

DAFTAR PUSTAKA

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dan J. Yan. 2017. Xanthones from the pericarp of garcinia mangostana.
Molecules. 22(683): 1-10.
Zhang, C., G. Yu, dan Y. Shen. 2018. The naturally occurring xanthone α-
mangostin induces ROS-mediated cytotoxicity in non-small scale lung
cancer cells. Saudi Journal of Biological Sciences. 25: 1090-1095.
Laksmiani, N. P. L. 2019. Ethanolic extract of mangosteen (garcinia mangostana)
pericarp as sensitivity enhancer of doxorubicin on MCF-7 cells by
inhibiting P-glycoprotein. Nusantara Bioscience. 11(1): 49-55.
Kritsanawong, S., S. Innajak, M. Imoto, dan R. Watanapokasin. 2016.
Antiproliferative and apoptosis induction of α-mangostin in T47D breast
cancer cells. International Journal of Oncology. 48: 2155-2165.
Saifudin, A., V. Rahayu, dan H. Y. Teruna. 2011. Standarisasi Bahan Obat Alam.
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Gunawan, D. dan S. Mulyani. 2010. Ilmu Obat Alam (Farmakognosi) Jilid I.
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Lokal Indonesia. Jakarta: PT Gramedia Pustaka Utama.
Raffi, P. 2010. Dahsyatnya Manggis Untuk Menumpas Penyakit. Jakarta Selatan:
PT Agro Media Pustaka.
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Hasyim, A., dan K. Iswari. 2012. Manggis Kaya Antioksidan. http://hortikultura.
Litbang.deptan.go.id/IPTEK/Hasyim_manggis.pdf. [Diakses pada 1 april
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https://www.kompasiana.com/ambarsari/54f92293a33311ed068b47c1/cara-
mengolah-kulit-manggis-yang-keliru-beresiko-bagi-kesehatan
 molecules
Article
Xanthones from the Pericarp of Garcinia mangostana
Renyue Yang 1,† , Ping Li 1,† , Nana Li 1 , Qian Zhang 1 , Xue Bai 1 , Lishuo Wang 1 , Yiying Xiao 1 ,
Lirong Sun 2, *, Quan Yang 3, * and Jian Yan 1, *
1 Key Laboratory of Tropical Agro Environment, Ministry of Agriculture and Guangdong Engineering
Research Centre for Modern Eco-Agriculture, South China Agricultural University,
Guangzhou 510642, China; renyueyang123@126.com (R.Y.); liping2016@scau.edu.cn (P.L.);
lnnaxtj@163.com (N.L.); chermmon@163.com (Q.Z.); baixue0816@gmail.com (X.B.);
wulidamonpalm@yahoo.com (L.W.); xyy15966@126.com (Y.X.)
2 Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University,
Guangzhou 510515, China
3 Laboratory of State Administration of Traditional Chinese Medicine for Production and Development of
Cantonese Medicinal Materials, School of Chinese Materia Medica, Guangdong Pharmaceutical University,
Guangzhou 510006, China
* Correspondence: slr0807@fimmu.com (L.S.); yangquan7208@vip.163.com (Q.Y.);
yanjian78@scau.edu.cn (J.Y.); Tel.: +86-20-6278-9020 (L.S.); +86-20-3935-2353 (Q.Y.); +86-20-3834-8099 (J.Y.)
† These authors contributed equally to this work.

Academic Editor: Derek J. McPhee

Received: 20 March 2017; Accepted: 18 April 2017; Published: 25 April 2017

Abstract: Mangosteen (Garcinia mangostana L.) is one of the most popular tropical fruits (called
the “Queen of Fruits”), and is a rich source of oxygenated and prenylated xanthone derivatives.
In the present work, phytochemical investigation has resulted in one new prenylated xanthone and
13 known xanthones isolated from the pericarp of G. mangostana. Their structures were established by
spectroscopic data analysis, including X-ray diffraction. The new one was further tested for cytotoxic
activity against seven cancer cell lines (CNE-1, CNE-2, A549, H490, PC-3, SGC-7901, U87), displaying
the half maximal inhibitory concentration (IC50 ) values 3.35, 4.01, 4.84, 7.84, 6.21, 8.09, and 6.39 µM,
respectively. It is noteworthy that the new compound can promote CNE-2 cells apoptosis in late
stage, having a remarkable inhibition effect on the side population growth of CNE-2 at 1.26 µM.
The bioactive compound was also detected in extract from fresh mangosteen flesh, which indicated
that the popular fruit could have potential cytotoxic activity for cancer cell lines.

Keywords: Garcinia mangostana; xanthone; cell lines; cytotoxic

1. Introduction
The genus Garcinia belongs to the Clusiaceae family, and a total of 21 species of the genus
have been recorded to date in China [1]. Garcinia mangostana (mangosteen) is a tropical fruit native
to the Malay Archipelago, the Sunda Islands, and the Moluccas. In Southeast Asia, mangosteen
fruit shell is a traditional folk medicine used for the treatment of diarrhea, sprains, typhoid, ulcers,
skin infections, and is used as an anti-inflammatory and for sterilization. In the previous extensive
investigations on the phytochemical constituents of G. mangostana [2], G. hanburyi [3], G. bracteata [4],
G. cowa [5], etc., Garcinia species are known to be rich in a variety of benzophenones and xanthones,
some of which showed a wide range of biological and pharmacological activities including cytotoxic,
anti-inflammatory, antimicrobial, and antifungal activity, as well as immune regulation and the
amelioration of experimental autoimmune encephalomyelitis [6–13]. A dietary α-mangostin isolated
from genus Garcinia had anticancer and antiproliferative properties in leukemia as well as prostate,
breast, colorectal, and brain cancers [14], and another caged polyprenylated xanthone—gambogic

Molecules 2017, 22, 683; doi:10.3390/molecules22050683 www.mdpi.com/journal/molecules

Molecules 2017, 22, 683 2 of 10

acid—was exhibited to have anti-proliferative and pro-apoptotic effects on hepatocarcinoma, gastric

carcinoma, lung carcinoma, breast cancer, and glioma in vivo and in vitro [15].
In China, G. mangostana—also named as Shan Zhu, mountain bamboo imported from Thailand,
Malaysia and other Southeast Asian countries—was one of the most popular fruits, called the “Queen
of Fruits” for its sweetness and juiciness as well as its importance in enhancing a person’s health [16].
In recent years, increasing evidence has supported that diet plays an important role in preventing the
development of cancer [8,17]. Fruits are one of the main dietary components for daily consumption.
It is popularly believed that increasing fruit consumption will contribute to reduced risk of cancers
of oral cavity, pharynx, larynx, esophagus, stomach and lung [18] due to the intake of some specific
active substances as we consume fruit every day.
In the course of our scanning for bioactive constituents from mangosteen fruits, one new
prenylated xanthone named as 7-O-demethyl mangostanin (1) and 13 known xanthones (2–14) were
isolated from the pericarp, and their structures were established using nuclear magnetic resonance
(NMR), high resolution electrospray ionization mass spectroscopy (HRESIMS), and X-ray methods.
Compound 1 showed obvious inhibition activity of all tested cancer cell lines, and the content of
compound 1 in edible fruit flesh is 13.89 ± 0.57 µg/kg, suggesting that the fruit could be of potential
value for the prevention and treatment of cancer.

2. Results and Discussion

Repeated column chromatography of 95% ethanol crude extraction of G. mangostana led to the
isolation of one new compound (1), and 13 known compounds (Figure 1). The chemical structures of
known compounds mangostanin (2) [19], 8-deoxygartanin (3) [20], gartanin (4) [21], garcinone E (5) [22],
trapezifolixanthone (6) [23], padiaxanthone (7) [7], tovophyllin A (8) [24], 1,5,8-trihydroxy-3-methoxy-2
[3-methyl-2-butenyl]xanthone (9) [25], garcinone B (10) [6,26], 1,3,7-trihydroxy-2,8-di-(3-methylbut-
2-enyl)xanthone (11) [27], mangostenone D (12) [6], 2-geranyl-1,3,5-trihydroxyxanthone (mangostinone)
(13) [28], and 1,7-dihydroxy-2-(3-methylbut-2-enyl)-3-methoxyxanthone (14) [29] were identified by
comparing spectroscopic data with those of published values. The chemical structure of the new compound
was elucidated as follows.
7-O-Demethyl mangostanin (1) was obtained as a yellow solid. Its molecular formula was
determined to be C23 H22 O6 by the quasi-molecular ion peak [M + H]+ at m/z 395.1507 in HRESIMS.
The 1 H and 13 C-NMR spectra showed some characteristic signals of tetracyclic xanthone with
a 3-methylbut-2-enyl-group and a pyrano ring (Table 1). There is a chelated hydroxyl group at
δH 14.08 (1H, s, 1-OH), a chelated carbonyl at δC 181.7 (s, C-9), two characteristic proton and
carbon signals of the xanthone [δH 6.32 (1H, s, H-4), 6.76 (1H, s, H-5) and δC 93.6 (d, C-4), 100.1
(d, C-5)], two coupled aromatic protons [δH 6.61 (1H, d, 9.9, H-11) and 5.73 (1H, d, 9.9, H-12),
δC 114.8 (d, C-11), 128.0 (d, C-12)], and a 3-methylbut-2-enyl-group signal [δH 4.02 (2H, d, 6.8, H-16),
5.18 (1H, t, H-17), 1.61 (3H, s, H-19) and 1.78 (3H, s, H-20); δC 25.3 (t, C-16), 123.5 (d, C-17), 130.2
(s, C-18), 25.67 (t, C-19) and 18.3 (t, C-20)] in the NMR spectra. Comparing NMR data of 1 with
mangostanin (19), the chemical shift values at C-6 and C-7 was moved to up-field due to existence
a chelated hydroxyl group, and two significant broad hydroxyl signals at δH 11.2 (1H, OH-7) and
8.7 (1H, OH-8) were observed in the 1 H NMR. The assignments of protonated and quaternary
carbons were assigned from heteronuclear singular quantum correlation (HSQC) and heteronuclear
multiple bond correlation (HMBC) spectra (see Table 1). The connection of the 3-methylbut-2-enyl
moieties at C-8 was established on the basis of the HMBC correlations of H-16 to C-8 and C-7.
Two aromatic protons were assigned to positions C-4 and C-5 from the HMBC correlations of
H-4 at δH 6.32 (1H, s) with C-2 (δC 103.6), C-32 (δC 158.4), and C-9a (δC 102.9) and correlations
of H-5 at δH 6.32 (1H, s) with C-6 (δC 152.8), C-7 (δC 141.2), C-8a (δC 109.6), and C-10a (δC 152.0)
(see Figure 2A). The structure was further confirmed by X-ray diffraction (see Figure 2B). Compound 1
was therefore identified as 1,6,7-trihydroxy-8-isoprenyl-60 ,60 -dimethylpyrano (2,30 :3,2) xanthone,
named as 7-O-demethyl mangostanin.
Molecules 2017, 22, 683 3 of 10

Figure 1. Chemical structure of xanthones 1–14 isolated from Garcinia mangostana L.

Table 1. 1 H- and 13 C-NMR spectral data of compound 1 in DMSO-d6 .

Compound 1
Position
13 C 1H

1 157.1
2 103.6
3 158.4
4 93.6 6.32, s
4a 155.6
5 100.1 6.76, s
Molecules 2017, 22, 683 4 of 10

Table 1. Cont.

Compound 1
Position
13 C 1H

6 152.8
7 141.2
8 127.6
8a 109.6
9 181.7
9a 102.9
10a 152.0
11 114.8 6.61, d, 9.9
12 128.0 5.73, d, 9.9
13 77.9
14 27.4 1.43, s
15 27.4 1.43, s
16 25.3 4.02, d, 6.8
17 123.5 5.18, t, 6.8
18 130.2
19 25.7 1.61, s
20 18.3 1.78, s
DMSO: dimethyl sulfoxide; δ in ppm, J in Hz.

Figure 2. Identification of chemical structure of compound 1 from G. mangostana. (A) Selected

heteronuclear multiple bond correlation (HMBC) correlations (H → C) of 1 and (B) X-ray
crystallographic analysis of 1.

In the activity evaluation of xanthones from the pericarp of G. mangostana, compounds 1–14
were tested for their inhibition effect on two common cell lines using 3-(4,5-dimethyl-2-thiazolyl)-2,
5-diphenyl-2-H-tetrazolium bromide (MTT) method (see Table 2 and refer to Tables S1 and S2).
Six compounds 1, 2, 3, 4, 5, 11 inhibit PC12 (pheochromocytoma cell) and U87 (human malignant
glioma cell) cell proliferation in a dose-dependent fashion, but the six compounds had stronger
activities against U87 than PC12. By comparing their chemical structure–activity relationship,
the activity was reduced with pyrano ring at C-7 and C-8 like compounds 7, 8, 10, and 12.
The cytotoxicity was weaker with the methyl group at C-7 comparing 2 with 1. To intensively assess
the potential cytotoxic activity of the new compound 1, 1 was tested by seven cell lines CNE-1, CNE-2
(nasopharyngeal carcinoma cell line), A549, H460 (lung cancer cell line), SGC-7901 (gastric cancer cell
line), PC-3 (prostate cancer cell line), and U87, and the half maximal inhibitory concentration (IC50 )
values were 3.35, 4.01, 4.84, 7.84, 6.21, 8.09, and 6.39 µM, respectively (Table 2). Hirsutanol A was used
as positive control, and the IC50 were 9.52, 11.86, 12.00, 11.00, 11.25, and 15.00, 6.3 µM. Compound 1
was greater than positive control Hirsutanol A.
Molecules 2017, 22, 683 5 of 10

Table 2. Cytotoxic activities of compound 1.

Concentration Cell Lines

(µM) CNE-1 CNE-2 A549 H460 PC-3 SGC-7901 U87 PC12
0 0 0 0 0 0 0 0 0
0.40 0 5.4 10.6 0 7.27 1.94 0 /
0.81 4.35 12.87 11.11 0 10.08 3.37 14.26 /
1.58 47.42 15.96 23.81 1.15 12.27 17.98 16.25 /
3.17 63.68 51.92 49.6 20.04 32.93 47.7 26.55 2.19
6.34 69.4 71.24 62.54 50.72 58.68 49.17 49.15 11.33
12.69 78.42 80.01 63.18 58.45 64.59 51.74 68.24 4.66
25.3 83.9 83.89 73.98 84.86 72.64 54.91 72.56 23.82
50.7 / / / / / / / 40.57
101.5 / / / / / / / 58.21
6.39 >50
IC50 3.35 4.01 4.84 7.84 6.21 8.09
(2.51 µg/mL) (19.7 µg/mL)
IC50 : half maximal inhibitory concentration; /: no detection.

Programmed cell death mainly occurs through either type I cell death (apoptosis) or type II
cell death (autophagy). The present results showed that compound 1 can significantly induce the
late and early stage apoptosis of CNE-2 cell at 6.34 µM—40.3% and 5.5%, respectively (see Figure 3).
Side population cells are commonly thought to represent cancer stem cell populations, and inhibition
of the growth of cancer stem cell populations is an effective way to treat cancer. The inhibition effect of
compound 1 on the side population of CNE-2 cell was measured by flow cytometry (FCM). It showed
that compound 1 had very significant cytotoxic active against the growth of side population of CNE-2
cell from 1.26 to 5.07 µM (see Figure 4).

Figure 3. Compound 1 can promote CNE-2 cell apoptosis. Cells were labeled with Annexin-Fluorescein
isothiocyanate (FITC) and Propidium Iodide (PI), and analyzed by flow cytometry (FCM): (A) untreated;
(B) treated with 6.34 µM for 72 h; (C) Effects of compound 1 on apoptosis in CNE-2 cell. Q2 and Q4
stand for late stage and early stage, respectively. * p < 0.05, ** p < 0.01 compared with the control group.
Data are expressed as the mean ± standard deviation (SD). n = 3.

The content of bioactive compound 1 from fresh aril was 13.89 ± 0.57 µg/kg by ultra-performance
liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS)
analysis (Table S3). The new compound is not only isolated from pericarp, but is also distributed
in flesh aril. The bioactive compound 1 could be potential matter for cytotoxic activity from fresh
G. mangostana, suggesting that animal experiments and pharmacological analysis is worth carrying out
to support the opinion.
Molecules 2017, 22, 683 6 of 10

Figure 4. Compound 1 effect of side population growth CNE-2. (A) Compound 1 inhibited side
population growth in a concentration-dependent manner as determined by FCM analysis. (B) The
inhibition was visualized by adding Annexin-FITC and PI at different concentrations. * p < 0.05,
*** p < 0.001 compared with the control group. Data are expressed as the mean ± SD. n = 3.

3. Materials and Methods

3.1. General Experimental Procedures

NMR spectra were recorded on a Bruker advance 400 NMR spectrometer (Bruker Biospin, Zurich,
Switzerland). HRESIMS mass spectra were obtained on an ultra-performance liquid chromatography
(UPLC) 1290-6540B quadrupole-time of flight (Q-TOF) instrument (Waters. Ltd., Milford, MA, USA)
in positive ion mode after direct injection of the test solutions. ESIMS data were obtained using
a MDS SCIEX API 2000 LC/MS/MS system (Waters. Ltd., Milford, MA, USA). UPLC-MS was carried
out on a C18 column (ACQUITY UPLC BEH C18 2.1 × 50 mm i.d., 1.7 µm, Waters. Ltd., Milford,
MA, USA). UPLC-TQS-MS was operated using an Acquity UPLC system (Waters Corporation, Milford,
MA, USA) coupled with a MS (Xevo TQ-S, Waters MS Technologies, Manchester, UK), controlled by
MassLynx v4.1 software (Waters Corp., Milford, MA, USA). Open column chromatography (CC) was
carried out using silica gel (80–100 and 200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao,
China), and Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden). Thin layer
chromatography (TLC) was performed on HSGF254 TLC (Yantai Jiangyou silica gel Co. Ltd., Yantai,
China), and spots were visualized by heating the silica gel plates sprayed with 10% sulphuric acid in
ethanol (v/v). HPLC-MS-grade acetonitrile, water, and formic acid were purchased from J. T. Baker
(Philipsburg, NJ, USA). All analytical-grade reagents were purchased from the Tianjin Fuyu Fine
Chemical Industry Co. Ltd., Tianjin, China.

3.2. Plant Material

The fresh G. mangostana of Thailand was purchased from Guangzhou market in July 2015. A dry
voucher specimen (No.: 20160105GM) has been deposited in the herbarium of the College of Natural
Resources and Environment, South China Agricultural University, China.
Molecules 2017, 22, 683 7 of 10

3.3. Sample Preparation and Isolation

For the extraction and isolation of compounds from the pericarp of G. mangostana, the pericarps
were separated from fresh fruits and naturally dried, then milled with a grinder, and 1 kg of material
was extracted with 95% ethanol to obtain crude extract (188 g). Then, 150 g silica gel (80–100 mesh)
was used to mix with crude extract (128 g) and was directly filled into the silica gel column
chromatograph (70 × 920 mm, 200–300 mesh), eluted with petroleum ether, dichloromethane, and
dichloromethane:methanol (50:1, 20:1, 10:1, 5:1), respectively. The collected fractions were monitored
by TLC, and combined to yield five fractions (Fr.1–2). Fr.1 (52 g) was chromatographed on a silica gel
column washed with petroleum ether–dichloromethane (10:1, 5:1, and 1:1, v/v) to obtain sub-fractions
Fr.1.1, 1.2, and 1.3. Fr.1.1 was further repeatedly chromatographed on a silica column eluting
with petroleum ether–dichloromethane (15:1, 7:1, 3:1, v/v) to get compounds 2 (13 mg), 3 (9 mg),
4 (20 mg), 5 (10 mg), and 6 (7 mg). Fr.1.2 was subjected to a silica column eluting with petroleum
ether–dichloromethane (2:1 and 1:1, v/v) to yield compounds 2 (5 mg), 7 (7 mg), and 8 (11 mg). Fr.1.3
was subjected to a silica gel column eluting with gradient system chloroform–methanol (30:1, 20:1, and
10:1, v/v), and further purified by Sephadex LH-20 with eluting methanol to give 9 (20 mg), 10 (6 mg),
and 11 (13 mg). The Fr.2 was rechromatographed on a silica column with chloroform–methanol (70:1,
60:1, and 50:1, v/v) as gradient system to obtain 1 (10 mg), 11 (10 mg), 12 (18 mg), 13 (8 mg), and 14
(11 mg).
7-O-demethyl mangostanin (1): yellow solid, IR ν max cm−1 : 3500 (OH), 1650 (C=O), 1604 (Ar).
1 H- and 13 C-NMR (DMSO-d ) data, see Table 1; HRESIMS m/z 395.1507 [M + H]+ (C H O calcd
6 23 23 6
for 395.1416).
Crystallographic data for 1: C23 H22 O6 ·C3 H6 O, MW = 452.48, monoclinic, a = 6.1328(5) Å,
b = 19.7854(13) Å, c = 18.1183(13) Å, α = 90.00◦ , β = 90.00◦ , γ = 90.00◦ , V = 2198.5(3) Å3 , T = 100(2) K,
space group P21/c, Z = 4, µ (CuKα) = 0.816 mm−1 , 10,923 reflections measured, 3497 independent
reflections (Rint = 0.1292). The final R1 values were 0.1121 (I > 2σ (I)). The final Wr (F2 ) values were
0.2686 (I > 2σ (I)). The final R1 values were 0.1670 (all data). The final wR (F2 ) values were 0.3160
(all data). The goodness of fit on F2 was 0.982.
For the determination of bioactive constituents from mangosteen, the fresh aril segments were
carefully separated into arils (edible parts) and seeds by a stainless-steel knife. The fresh arils (each 4
g) were each stirred with 2 mL 95% ethanol followed by ultrasonic extraction for 30 min in a water
bath at room temperature. After membrane-filtration (0.25 µm), the extract solutions were directly
used for the quantification of bioactive compounds by UPLC-QTS-MS analysis.
UPLC-TQS-MS analysis: Quantitative analysis of samples was performed on an ACQUITY
UPLC system couple with a triple-quadrupole Xevo TQ-S mass spectrometer (Waters Corp., Milford,
MA, USA). An ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) (Waters Corp., Milford,
MA, USA) was employed, and the column temperature was maintained at 40 ◦ C. The gradient elution
with acetonitrile containing 0.1% formic acid (A) and water containing 0.1% formic acid (B) was
performed as follows: 0–0.5 min, 30–35% A; 0.5–2 min, 35–75% A; 2–3.5 min, 75–90% A; 3.5–4.5
min, 90–95% A; 4.5–4.8 min, 95–30% A; 4.8–6 min, 30% A. The flow rate was set at 0.4 mL/min.
The auto-sampler was conditioned at 25 ◦ C, and the injection volume of solution was 5 µL for analysis.
Mass spectrometric detection was performed on Xevo TQ-S equipped with an electrospray ionization
source (ESI). The capillary voltage was set to 3.5 kV, and the source temperature was maintained at
150 ◦ C. The collision gas was Ar, and N2 gas was used as desolvation at temperature of 400 ◦ C and
cone gas at a flow rate of 700 L/h, the cone gas set to 50 L/h. Compound 1 was optimized in multiple
reaction monitoring in positive mode. The cone voltage was 48 V, m/z 394.0 was selected as parent
ion, and m/z 323.8 and 339.0 were set as the daughter qualitative and quantitative ions, respectively,
with the corresponding collision energy settings at 38 and 28 V. The dwell time was 0.025 s. Targetlynx
(Waters Corp., Milford, MA, USA) software was used to analyze the data.
Molecules 2017, 22, 683 8 of 10

3.4. Cell Culture

All cell lines were cultured in dulbecco’s modified eagle's medium (DMEM) medium containing
10% fetal bovine serum, 5% horse serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. All cell
lines were placed in an incubator at 37 ◦ C with 5% carbon dioxide.

3.5. MTT Assay

Cells were seeded into 96-well plates (about 3000 cells/well), and were treated with seven
concentration gradients: 0, 0.79, 1.58, 3.17, 6.34, 12.59, 25.38, 50.76, and 101.56 µM, then the cells
were incubated for 48 h. Subsequently, MTT was added to the culture medium to yield a final MTT
concentration of 800 µg/mL, and cells were incubated with the MTT for 4 h in the incubator, then
collected and dissolved in dimethyl sulphoxide (DMSO). Colorimetric analysis was measured at
570 nm.

3.6. FCM Assay

Cells were treated with 6.34 µM compound 1, then incubated for 72 h, cells were collected,
and Annexin-FITC and PI were added; after dark reaction for 10 min, cell apoptosis was analyzed
using FCM for the treated group and the control group.

4. Conclusions
In summary, 14 compounds were isolated and identified from G. mangostana. The new compound
was tested by seven cancer cell lines and side population growth of CNE-2, showing that the compound
has potential anti-cancer properties. It is noteworthy that this bioactive constituent can also be detected
in mangosteen aril. There is some possibility that eating mangosteen is useful for the prevention and
treatment cancer.

Supplementary Materials: NMR data of 1, Growth (%) of tumor PC12 and U87 and linear regression data of 1
are available online.
Acknowledgments: This work was supported by science and technology planning project of Guangdong Province,
China (2016A010105018).
Author Contributions: R.Y., P.L. and L.S. performed the experiments; N.L., Q.Z., X.B., L.W. and Y.X. contributed
reagents/materials/analysis tools; J.Y. and Q.Y. conceived and designed the experiments, wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.

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Sample Availability: Samples of the compounds are available from the authors.

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
 Saudi Journal of Biological Sciences 25 (2018) 1090–1095

Contents lists available at ScienceDirect

Saudi Journal of Biological Sciences

journal homepage: www.sciencedirect.com

Original article

The naturally occurring xanthone a-mangostin induces ROS-mediated

cytotoxicity in non-small scale lung cancer cells
Chunyun Zhang a,⇑, Guifang Yu b, Yifeng Shen c
a
Department of Respiration, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510700, China
b
Department of Oncology, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou 510700, China
c
Guangzhou Wondfo Biotech Co., Ltd, Guangzhou 510663, China

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

Article history: Small cell lung cancer (NSCLC) accounts for 85% of total deaths globally, and recent studies indicate the
Received 6 January 2017 increasing risks of NSCLC in China and South Asian countries. Hence, development of new therapeutics
Revised 8 March 2017 against NSCLC has been a major concern. a-Mangostin, a naturally occurring xanthone, found abundantly
Accepted 12 March 2017
in pericarps of mangosteen fruit is well known for its medicinal importance. The anticancer properties of
Available online 14 March 2017
a-mangostin against several types of cancer are also well documented. But the mechanism of action of
a-mangostin against lung cancer is not well understood and requires further investigation. Therefore
Keywords:
in the present study, we explored the therapeutic potential of a-mangostin against A549 cells.
a-Mangostin
ROS
Treatment of A549 cells with a-mangostin resulted in a dose-dependent loss of cell viability, while the
NSCLC non-malignant cells such as hPBMC and WI-38 remained unaffected. Further we observed that the ROS
Apoptosis plays an important role in a-mangostin -induced apoptosis in A549 cells, and administration of
N-acetyl cysteine significantly abrogates a-mangostin -mediated cytotoxicity in lung cancer cells.
Overall, a-mangostin induces ROS-mediated cytotoxicity in NSCLC cells.
Ó 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction accelerated metastasis and high rate of tumor recurrence are

Cancer is a group of diseases involving abnormal cell growth
with the potential to invade or spread to other parts of the body.
Not all tumors are cancerous; benign tumors do not spread to other
parts of the body. Possible signs and symptoms include a lump,
abnormal bleeding, prolonged cough, unexplained weight loss
and a change in bowel movements. While these symptoms may
indicate cancer, they may have other causes. Over 100 types of can-
cers affect humans. Lung cancer is responsible for most number of
cancer- related deaths worldwide, and among the cancer affected
patients, non-small cell lung cancer (NSCLC) accounts for 85% of
the total death percentage, while small cell lung cancer (SCLC) is
responsible for only 15% of mortality (Jemal et al., 2005; Smith
et al., 2009; Yang et al., 2015). Mainly the poor prognosis,
⇑ Corresponding author.
E-mail address: chunyunzhang66@hotmail.com (C. Zhang).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
responsible for reduced survival rate and high lethality of NSCLC
(Chan et al., 2002). Although the frequency of lung cancer is not
very significant in the developed countries, recent epidemiological
studies indicate that incidence of NSCLC had significantly increased
in the Asian countries including China (Zhou, 2014; Fan et al.,
2015). According to National Central Cancer Registry (NCCR) in
2010, the estimated lung cancer incidence in China was 46.08
per 100,000 populations, and over 600,000 patients were newly
diagnosed. Hence lung cancer has become the major concern in
China, leading to a high mortality rate of 37.00 per 100,000 popu-
lations. Although the platinum-based chemotherapy, e.g. cisplatin,
is used for the treatment of advanced NSCLC patients (Gridelli and
Sacco, 2016), but the acquisition of chemoresistance has signifi-
cantly resulted in the poor survival rate of the patients (Jiang
et al., 2016). Hence development of novel therapeutic agents
against NSCLC is an urgent need for the improved treatment of
the disease.
Mangosteen Linn (Garcinia mangostana) is a type of fruit mainly
a grows in the Asian counties such as Thailand, Philippines, Sri
Lanka, Malaysia, India and Myanmar, which have a significant
medical importance, due to its usage in the treatment of trauma,
abdominal pain, skin infection, dysentery and also wound-

http://dx.doi.org/10.1016/j.sjbs.2017.03.005
1319-562X/Ó 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 C. Zhang et al. / Saudi Journal of Biological Sciences 25 (2018) 1090–1095
healing properties (Peres et al., 2000) a-mangostin is a naturally
occurring xanthone, found abundantly in pericarps of mangosteen
fruits, which has diverse biological importance such as anti-
inflammatory, antifungal, anti-tumor, antiparasitic, antioxidant,
cardio-protective, and antibacterial properties to name a few
(Jindarat, 2014). The anticancer properties of a-mangostin against
various carcinomas are well documented (Kumazaki et al., 2015;
Verma et al., 2016; Xia et al., 2016a, 2016b). But there are not
much reports on the effect of a-mangostin on lung cancer cells.
Hence in the present study we have investigated the effect of
a-mangostin on NSCLC cells A549 and also on the noncancerous
cells such as lung fibroblasts WI-38 cells and human peripheral
blood mononuclear cells (PBMC).
2. Materials and methods
2.1. Cell culture and maintenance
Human lung fibroblast cells WI-38 and non-small cell lung can-
cer cells A549 were cultured in DMEM complete medium supple-
mented with 10% FBS. Human Peripheral Blood mononuclear
cells (hPBMC) were harvested from the blood samples of the
healthy donors. hPBMC were isolated by density gradient centrifu-
gation technique, using Ficoll-Hypaque (1:1) (Histopaque 1077,
Sigma Aldrich-USA), following the published protocol
(Bhattacharya et al., 2013).
2.2. MTT assay
Cultured WI-38, A549 and hPBMC were seeded at a density of
1
104 cells/ml, grown to 80% confluency and treated with
a-mangostin (0–50 mM) for 24 h. After treatment, cellular viability
was monitored by MTT assay.
2.3. Determination of apoptosis by FACS
Induction of apoptosis in Α-MANGOSTIN -treated A549 cells
was monitored by AnnexinV/PI double staining, using BD
Fluorescence Activated Cell Sorter (ARIA II). Cultured A549 cells
(1
104 cells/ml) were treated with varying concentrations of
a-mangostin (0–10 lM) for 24 h. Induction of apoptosis was
determined by flow cytometry using Annexin V-FITC apoptosis
kit (Cayman Chemicals) following the protocol supplied by the
manufacturers.
2.4. Determination of migration by Boyden chamber assay
Cultured A549 cells (1
104 cells/ml) were treated with vary-
ing concentrations of a-mangostin (0–10 lM) for 24 h and approx-
imately 1
103 cells from each treatment groups were placed in
the upper portion of the Boyden chamber, containing serum free
medium. Cells, which invaded to the lower surface of the mem-
brane, containing FBS supplemented DMEM, were then stained
with crystal violet. The crystal violet complex thus formed, was
dissolved in 10% acetic acid, and the absorbance was measured
at 600 nm. The absorbance is directly proportional to the extent
of migration.
2.5. Determination of ROS by DCF-DA staining
Cultured A549 cells (1
104 cells/ml) treated with varying con-
centrations of a-mangostin (0–10 lM) for 24 h, incubated with
25 lM 20 ,70 - dichlorodihydrofluorescin diacetate (DCFH-DA) for
30 min at 37 °C and the images were taken by a confocal
microscope (Olympus FluoView-1000). Fluorescence intensities
1091
were calculated from approximately 1000 cells taken from
different fields.
2.6. Estimation of antioxidant enzymes
For these assays A549 cells (untreated and treated with
a-mangostin) were collected, resuspended in 200 ml of assay buf-
fers, sonicated and centrifuged at 15,000 rpm for 20 min at 4 °C.
The supernatant was collected and total protein was estimated
by Bradford method. Catalase activity was measured according to
protocol reported by Seizer& Beer in 1952 (Beers and Sizer,
1952). Briefly, 20 ml of the sample was added to 100 ml of assay buf-
fer [50 mM potassium phosphate buffer (pH-7)] in a quartz cuvette
to which 180 ml of 30 mM H2O2 was added. Decomposition of H2O2
was monitored spectrophotometrically at 240 nm for 4 min at
room temperature. For each samples the catalase activity was
measured by calculating mmol of H2O2 consumed per minute per
milligram of protein.
For determination of cellular glutathione peroxidase (GPx)
activity, 10 ml of sample was added to 170 ml of the assay buffer
[50 mM potassium phosphate buffer with 1 mM EDTA and 2 mM
sodium azide, pH-7], 30 ml of GSH (10 mM), 30 ml of glutathione
reductase (2.4 U/ml) and 30 ml of 1.5 mM NADPH (dissolved in
0.1% sodium bicarbonate) in a quartz cuvette. The whole mixture
was incubated at 37 °C for 10 min and the reaction is started by
adding 30 ml of 2 mM H2O2. The consumption of NADPH was mon-
itored spectrophotometrically at 340 nm at 37 °C for 10 min and
the specific activity was calculated from nanomoles of NADPH con-
sumed per minute per mg of protein (Das et al., 2012).
For the determination of total GSH, the cell lysates were mixed
with 10% TCA (1:1 volume) and centrifuged at 150,000 RPM at 4 °C
for 5 min, to eliminate the cellular proteins. This step was repeated
and the resultant supernatant was checked for the residual pro-
teins by Bradford method. Finally to the protein free supernatant,
1 mm of DTNB was added in a quartz cuvette Formation of TNB
was monitored spectrophotometrically at 412 nm for 10 min. A
standard curve for GSH was prepared by using known concentra-
tion of GSH (0.1–50 mM) and their respective absorbances at
412 nm, and the total GSH was calculated from the standard curve
(Forman et al., 2009).
2.7. Western blot analysis
Cultured A549 cells (1
104 cells/ml) were treated with vary-
ing concentrations of a-mangostin (0–10 lM) for 24 h and western
blots were performed for the detection of Bax and Bcl-2 proteins.
The primary antibodies e.g. mouse monoclonal anti- Bcl-2 antibody
(sc-7382) and rabbit polyclonal anti-Bax antibody (sc-493) were
purchased from Santa Cruz (USA).
2.8. Statistical analysis
Statistical analysis was performed using GraphPad Software. All
results are expressed as the mean ± SD. Statistical significance was
determined by one way Anova using Newman-Keuls Multiple
Comparison Test.
3. Results
3.1. a-Mangostin induces loss of cell viability in NSCLC cells
The cytotoxic effects of a-mangostin on NSCLC cells A549 and
nonmalignant WI-38 and hPBMC were determined by MTT assay.
a-Mangostin -treatment resulted in a dose-dependent loss of cell
viability in A549 cells after 24 h of treatment (IC50  10 lM)
1092 C. Zhang et al. / Saudi Journal of Biological Sciences 25 (2018) 1090–1095

Fig. 1. Effect of xanthone a-mangostin on cell viability in NSCLC cells.

(Fig. 1). But interestingly, negligible cytotoxicity were observed in the presence of 10 lM a-mangostin, 38% of the cells were found
normal lung fibroblasts WI-38 and also human PBMC, with the to be apoptotic (Fig. 2A). Moreover, we also observed that,
respective IC50 concentrations being observed at around 50 lM Bax/Bcl-2 ratio was also increased in a-mangostin –treated A549
a-mangostin, after 24 h of treatment. cells, confirming the involvement of apoptosis (Fig. 2B).

3.2. a-Mangostin -treatment induces apoptosis in NSCLC cells via 3.3. a-Mangostin -treatment inhibits the migratory activity of NSCLC
modulation of Bax/Bcl-2 ratio cells

a-Mangostin -treatment of A549 cells resulted in the significant The migratory properties of A549 cells in the absence and
induction of apoptosis in a dose-dependent fashion, as determined presence of a-mangostin was accessed by Boyden chamber assay.
by annexin V/PI staining. Treatment of A549 cells with 5 lM a-Mangostin -treatment of A549 cells resulted in the inhibition
a-mangostin, increased the apoptotic population to 18%, while in of migratory properties in a dose-dependent fashion (Fig. 2C).

Fig. 2. Different concentration of xanthone a-mangostin treatment induces apoptosis in NSCLC cells via modulation of Bax/Bcl-2 ratio.
 C. Zhang et al. / Saudi Journal of Biological Sciences 25 (2018) 1090–1095 1093

When treated with 5 lM a-mangostin, cellular migration was 3.5. a-Mangostin -treatment resulted in the down regulation of
found to be inhibited by almost 33%, and in the presence of antioxidant machinery of NSCLC cells
10 lM a-mangostin, migration was inhibited by 60%.
After confirming the involvement of ROS in a-mangostin -
mediated cytotoxicity in A549 cells we further monitored the sta-
3.4. a-Mangostin -treatment resulted in ROS-generation in NSCLC cells
tus of different antioxidant markers such as catalase, glutathione
peroxidase (GPx) and reduced glutathione (GSH). Thymoquinone
a-Mangostin was known to induce ROS generation in several
was reported to have the unique property of modulating both
cancer cells, but there was no report of a-mangostin -induced
pro-oxidant as well as antioxidant machinery in cancer cells. We
ROS generation in NSCLC cells. Hence we investigated whether
observed the activity levels of the antioxidant enzymes like cata-
ROS is involved in a-mangostin -mediated cytotoxicity in A549
lase and GPx were enhanced in the presence of low a-mangostin
cells. We observed that, DCF-fluorescence, an indicator of ROS
-doses, while at IC50 or near IC50 concentrations, the activity
accumulation was increased in a-mangostin -treated cells in a
levels of these enzymes as well as the total GSH-levels were signif-
dose-dependent fashion (Fig. 3). In the presence of 10 lM
icantly decreased (Fig. 4).
a-mangostin, ROS levels were increased by 6-folds compared to
the untreated cells (Fig. 3B).

Fig. 3. Effect of xanthone a-mangostin on the migratory activity of NSCLC cells.

Fig. 4. Effect of xanthone a-mangostin on ROS-generation in NSCLC cell.

1094 C. Zhang et al. / Saudi Journal of Biological Sciences 25 (2018) 1090–1095
Fig. 5. Effect of N-acetyl cysteine (NAC) attenuates ROS-mediated cytotoxicity.
3.6. Antioxidant N-acetyl cysteine (NAC) attenuates ROS-mediated
cytotoxicity in a-mangostin -treated NSCLC cells
Since a-mangostin -treatment resulted in significant ROS gen-
eration in A549 cells, we further investigated whether ROS is
responsible for a-mangostin -induced cell death A549 cells. Co-
treatment of a-mangostin -treated A549 cells with the antioxidant
NAC significantly diminished ROS generation as indicated by the
decrease in DCF-fluorescence (Fig. 5). Moreover, administration
of NAC significantly restored the cell viability in a-mangostin -
treated A549 cells (Fig. 5C). Thus we might conclude that ROS is
a key player in a-mangostin -mediated cytotoxicity in NSCLC cells.
4. Discussion
The naturally occurring xanthone a-mangostin is well known
for diverse biological benefits (Jindarat, 2014). The anticancer
properties of a-mangostin against various malignancies are also
well documented (Verma et al., 2016). It has been reported that
reactive oxygen species (ROS) are known to play important roles
in drug induced apoptosis in cancer cells (Forman et al., 2009;
Jeong and Joo, 2016; Liou and Storz, 2010). Hence in the present
study we have investigated whether oxidative stress plays any role
in a-mangostin induced cytotoxicity, in NSCLC cells.
Treatment of A549 cells with a-mangostin resulted in a dose-
dependent loss of cell viability with a limited toxicity towards
the noncancerous cells such as WI-38 and hPBMC cells. Further-
more, we observed that the mode of cell death by a-mangostin is
apoptosis, as confirmed by annexin V/PI staining and monitoring
Bax/Bcl-2 ratio. a-Mangostin -treatment also resulted in the signif-
icant inhibition of the migratory properties of A549 cells, as deter-
mined by Boyden camber assay. To determine the involvement of
ROS, we monitored ROS-generation in a-mangostin treated A549
cells by DCF-DA staining. We observed that, there is significant
increase in ROS-generation in a-mangostin treated A549 cells, in
a dose-dependent fashion. There has been several reports which
suggest that a-mangostin can modulate antioxidant enzymes (Lei
et al., 2014; Fang et al., 2016). Hence we estimated the enzymatic
activities of catalase and superoxide dismutase, and also the total
GSH levels in a-mangostin treated A549 cells. We observed that
a-mangostin imparts dual effect on the antioxidant markers. At
lower doses, a-mangostin acts as an antioxidant and enhances
the activities of these enzymes, while at near IC50 doses, the
pro-oxidant effect of a-mangostin was more prominent. In pres-
ence of 5 lM and 10 lM a-mangostin, the activities of catalase
and GPx, and also the levels of reduced glutathione was found to
be significantly reduced. Finally the involvement of ROS in
a-mangostin -mediated cytotoxicity was confirmed, when we
observed that administration of NAC significantly restored the via-
bility in a-mangostin treated cells. Thus we can conclude that ROS
plays an important role in a-mangostin -mediated cytotoxicity in
NSCLC cells.
Conflict of interest
The authors declare that they have no conflicts of interest.
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N U S AN T AR A BIO S C IEN C E ISSN: 2087-3948
Vol. 11, No. 1, pp. 49-55 E-ISSN: 2087-3956
May 2019 DOI: 10.13057/nusbiosci/n110109

Ethanolic extract of mangosteen (Garcinia mangostana) pericarp as

sensitivity enhancer of doxorubicin on MCF-7 cells by inhibiting P-
glycoprotein

NI PUTU LINDA LAKSMIANI

Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Udayana. Bukit Jimbaran, Badung 80362, Bali, Indonesia
Tel.: +62-361-701954, email: lindalaksmiani@gmail.com

Manuscript received: 20 November 2018. Revision accepted: 23 February 2019.

Abstract. Laksmiani NPL. 2019. Ethanolic extract of mangosteen (Garcinia mangostana) pericarp as sensitivity enhancer of doxorubicin
on MCF-7 cells by inhibiting P-glycoprotein. Nusantara Bioscience 11: 49-55. Generally, doxorubicin is used in combination with other
anticancer agents. Reduction side effects tend to be better in combination use than the use of single doxorubicin. Therefore the
development of anticancer agents with low side effects and combination agents that decrease the side effects of doxorubicin still need to
be pursued. The use of doxorubicin also shows a phenomenon of resistance due to over-expression of P-glycoprotein (Pgp). The
research was conducted to evaluate the cytotoxic effect of lower dose doxorubicin combine with ethanolic extract of mangosteen
pericarp (EEMP) on MCF-7 cells and determine the mechanism of alpha ()-mangostin as the active compound in EEMP that
contribute increasing sensitivity of doxorubicin on MCF-7 through Pgp by in silico. The cytotoxic activity was observed using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Molecular docking with 4.2 autodock program was used to
evaluate the interaction of -mangostin with Pgp. EEMP has IC50 of 54 μg/mL on MCF-7 cells while for doxorubicin has IC50 of 1392
nM. Exposure EEMP enhanced cytotoxic effect of doxorubicin on MCF-7 cells. Solely doxorubicin 750 nM treatment gave cell viability
percentage 94.98%, but when combined with EEMP 22.5 μg/mL, the cell viability percentage was reduced to 16.91%. This shows that
EEMP potent to be used as a combination agent for doxorubicin chemotherapy. The in silico assay demonstrate that -mangostin has an
affinity for Pgp with binding energy of -6.13 kcal/mol.

Keywords: Combination, EEMP, MCF-7, doxorubicin, Pgp

INTRODUCTION major chemotherapeutic agents can be mediated by

Cancer cells maintain themselves to stay alive from
various radiation treatments and chemotherapy agents.
Cancer cells try to avoid cell cycle arrest or apoptosis
(Hassan et al. 2014; Liu et al. 2015). They unresponse to
antiproliferative signals and have their own growth factors
to proliferate continuously. In addition, cancer cells avoid
the apoptosis mechanism when abnormalities occur
(Hanahan et al. 2011). These two things become important
targets of chemotherapy agents. Even cancer cells that are
sensitive to chemotherapy agents with its mechanism
induce cell cycle arrest and apoptosis, cancer cells at some
point turn out to be resistant to these agents (Shah et al.
2001; Ricci et al. 2006; Pistritto et al. 2016). There is
molecular evidence that cancer cell resistance due to
chemotherapy by the increased expression of Bcl-2 and P-
glycoprotein (Pgp) (Bao et al. 2012; Housman et al. 2014;
Geretto et al. 2017). Bcl-2 gene has a function to increase
cell immunity against the detachment of apoptosis and Pgp
as a protein transporter to maintain low concentrations of
chemotherapeutic agents in cells by pumping drugs out of
the cell (Alfarouk et al. 2015; Li et al. 2016). One
alternative to overcome cancer cell resistance is
combination therapy with several drugs. However, cancer
cell resistance can also occur due to improper
chemotherapy agent combination regimens. Resistance to
differences in the target inhibition in the cell cycle phase.
Combination agents can actually weaken the main
chemotherapeutic agent by blocking its action in the cell
cycle so that the apoptosis mechanism is inhibited. In this
case, the optimization of drug combination is needed
including the method of giving each sole agent to the
patient.
Doxorubicin is an anthracycline class antibiotic that is
widely used for the treatment of various types of cancers
such as acute leukemia, breast cancer, bone and ovarian
cancer (Kumar et al. 2014). Generally, doxorubicin is used
in combination with other anticancer agents such as
cyclophosphamide, cisplatin, and 5-FU. The combination
of doxorubicin and anticancer agents or co-chemotherapy
agents from natural ingredients is expected to reduce the
side effects and phenomena of cancer cell resistance. The
doxorubicin resistance is caused by the over-expression of
Pgp so that doxorubicin to be pumped out of the cell
leading to the dropped concentration of doxorubicin in the
cell. Therefore another or combination agents are needed to
overcome the problem of doxorubicin resistance and to
reduce the side effects of using doxorubicin.
One plant that has the potential to be developed as a
companion agent for doxorubicin is mangosteen pericarp.
The mangosteen pericarp turns out to have a cytotoxic
effect and cell cycle modulation. Mangosteen pericarp has
50 N U S A N T A R A B I O S C I E N C E 11 (1): 49-55, May 2019
cytotoxic effects on MCF-7 cells (Setiawati et al. 2014),
SKBR3 cells (Moongkarndi et al. 2004) and T47D cells
(Rofida et al. 2017) with IC50 each of them were 45 µg/mL,
15.45 µg/mL and 8.96 µg/mL, respectively. α-mangostin is
an active ingredient isolated from mangosteen pericarp
which is cytotoxic to cancer cells. Akao et al. 2008
reported that α-mangostin is cytotoxic in DLD-1 cells of
human colon cancer with IC50 7.5 µM. Based on this,
mangosteen pericarp is a potential source of anticancer or
combination agent to reduce the cancer drug resistance
with specific molecular targets. Various types of
compounds have been shown to be able to inhibit the work
of Pgp, including alkaloid compounds, flavonoids, and
xanthones. The approach of the chemical structure of the α-
mangostin compound from xanthone is the basic compound
which can be potentially used as Pgp inhibitor. This study
was designed to evaluate the cytotoxic effect of
combination of EEMP and doxorubicin in MCF-7 cells and
to investigate the molecular mechanism of α-mangostin in
EEMP through inhibiting Pgp as target protein by in silico
to increase the sensitivity cytotoxic effect of lower-dose
usage of doxorubicin.
MATERIALS AND METHODS
Sample collection
The pericarp of mangosteen (Garcinia mangostana L.)
was collected from traditional market in Pupuan, Tabanan,
Bali, Indonesia.
Chemical materials
Ethanol 70% was purchased from Trio Brothers,
Surabaya, Indonesia, advanced Dulbecco`s modified
Eagle`s medium (DMEM) and Fetal Bovine Serum (FBS)
(Sigma-Aldrich USA), Doxorubicin, Penicillin, streptomycin,
fungizone, and MTT were obtained from Sigma Aldrich,
Germany, Trypsin-EDTA was supplied from Gibco,
Dimethylsulfoxide (DMSO) (Sigma), Sodium Dodecyl
Sulfate Solution (SDS) (Merck) and Phosphate Buffer
Saline (PBS) (Sigma).
Extraction of mangosteen pericarp
Dried mangosteen pericarp was ground into powder and
extracted by maceration with ethanol 70%. The ratio of
powder sample and solvent was 1:10 (for 48 h). The
ethanolic extract was pressured using rotary evaporator.
Cytotoxicity and Cell viability assay: 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay
Cells with concentration of 5x103 cells/well distributed
into the 96-well plate were incubated for 24 h prior to
addition of test compound. Afterward, the medium was
removed and the cells were washed with PBS. Various
series of test compounds (EEMP and doxorubicin) in the
culture medium were added both solely and in
combination. They were then incubated for 24 hours at 5%
CO2 incubator at temperature of 37°C. After 24 hours of
incubation, the medium was removed and the cells were
washed with 100 µL of PBS followed by adding to each
mixture 100 µl MTT reagent 0.5 mg/ml in DMEM High-
Glucose medium and incubated for 3-4 hours in 5% CO2
incubator with the temperature of 37 ° C. Living cells will
react with MTT to form purple color. The MTT reaction
was stopped with a reagent stopper (SDS 10% in 0.1 N
HCl), which was then incubated overnight at room
temperature and in a dark place (covered with aluminum
foil). The next day, the plate was shaken for 10 minutes,
and the absorption was read by an ELISA reader at a
wavelength of 595 nm.
Molecular docking
Target protein preparation
Protein preparation began by selecting the structure of
the protein in an active form which bound to native ligand
namely Pgp (GDP ID: 5KOY). The native ligand on the
target protein was then eliminated by the Chimera 1.10.1
program aiming to provide pocket/cavity so that the pocket
coordinates and binding site center as docking material
could be known. The ligands of each target protein were
also separated for method validation.
Alpha-mangostin optimization
The 3-dimensional structure of -mangostin compound
was optimized using the Hyperchem 8 program complete
with hydrogen atoms. The optimization of the 3-
dimensional structure of -mangostin was done using AM1
semi-empirical computational methods and single-point
calculations and geometry optimization. Single point
calculations were used to find out the total energy of the -
mangostin configurations, while geometry optimization
was used to find stable configuration values with lower
values than single point calculations.
Molecular docking method validation
Validation of the molecular docking method was done
by redocking the native ligand of each target protein to the
target protein previously removed by the native ligand.
Redocking was done using the Autodock 4.2 program. The
results of the docking validation analysis showed the
compound with conformation, which has the lowest bond
energy to bind to the target protein. Root mean square
distances (RMSD) compound docking results were then
compared with references. The method was valid if the
value of RMSD was ≤ 3 Å. Afterward, docking of the test
compound on the target protein could be done (Jain and
Nicholls 2008).
Docking -mangostin to Pgp as target protein
The optimization result of -mangostin was then
docking to the target protein, which has been prepared (the
native ligand was omitted). Docking was done using the
Autodock 4.2 program. The results of the analysis will
show the compound with confirmation with the lowest
bond energy to bind to the target protein.
 LAKSMIANI et al. – Mangosteen extract as enhancer of doxorubicin
Data analysis
Single compound cytotoxic test using MTT assay
In the cytotoxicity assay using MTT method, the
calculation of % cell death due to treatment was conducted
by comparing the difference in the absorbance of the
solution from the cell control with the absorbance of the
solution from the treated cell to the absorbance of the
solution from the cell control multiplied by 100%
(Equation (1). To determine whether or not there is a
cytotoxic effect, a computerized linear regression analysis
is performed to obtain the IC50 value.
Combination cytotoxic test using MTT assay (co-
chemotherapy application analysis)
The combined effect of -mangostin-doxorubicin could
be analyzed by evaluating the percentage of cell viability
from the treatment of each concentration used both singular
and a combination. Data was then analyzed by ANOVA
with a confidence level of 95%.
Molecular docking analysis
Molecular docking results are binding energy. Binding
energy values indicated the bond strength between compounds
and proteins. The lower binding energy value means that
the bond was stronger and more stable. If the binding
energy between α-mangostin and Pgp as target protein was
getting lower, then the interaction between α-mangostin
and Pgp was stronger so that α-mangostin in EEMP has the
potential as a Pgp inhibitor. Pgp, a reflux pump protein that
comes out drugs from the cell cause the drug concentration
in the cell decreases and will need a larger dose to get the
same effect. Pgp is one of protein that responds to
resistance event. Molecular docking result show EEMP
inhibit Pgp that occur to EEMP enhance the sensitivity of
chemotherapy agents and less resistance of drugs.
RESULTS AND DISCUSSION
Ethanolic extract of mangosteen pericarp performed
cytotoxic to MCF-7 cells
Cytotoxic assay was carried out to determine the cell
toxicity potential of the compound expressed in IC 50
A B
Figure 2. Morphology changing of MCF-7 cells after treated by ethanolic extract of mangosteen pericarp (EEMP). Cell control (a), after
24 hours 12.5 g/mL EEMP treatment (b), 25 g/mL EEMP treatment (c), 50 g/mL EEMP treatment (d). The cell morphology
changes showed by arrow ( )
51
parameters. The test was carried out with MTT assay,
where the absorbance of purple formazan salt was formed
due to the reduction of reagent MTT tetrazolium salt which
was yellow because of the succinate dehydrogenase
enzyme found in living cells (Bahuguna et al. 2017). The
intensity of purple crystal is measured at a wavelength of
595nm. Color intensity is proportional to the number of
living cells, the obtained absorbance could be converted to
the percentage of cell viability.
EEMP treatment with series content of 12.5; 25; 50;
62.5; 75 and 87.5 µg/mL for 24 hours applied in this study.
Results showed that higher treatment doses gave a decrease
in cell viability, indicating a correlation between the
concentration of the test solution and the cytotoxic effects
occurred. It also showed that a dose-dependent
phenomenon, cytotoxic effects increase with the increase of
concentration. The increase in cytotoxic effects was
indicated by the smaller percentage of living cells after it
was treated in higher concentrations of test compound or
extract. The greater concentration of extract, the
absorbance value is smaller, which means smaller number
of living cells. IC50 of EEMP to MCF-7 cell was 54 µg/mL
(Figure 1). The cell morphology of MCF-7 which died
because of the extract treatment could be observed in figure
2. Cell morphology changes significantly since the EEMP
concentration increased. EEMP caused changes in cell
morphology to become rounded, irregular and experience
condensation before it died.
Figure 1. Effect of ethanolic extract of mangosteen pericarp
(EEMP) on MCF-7 cell viability by using MTT assay
C D
52 N U S A N T A R A B I O S C I E N C E 11 (1): 49-55, May 2019

Ethanolic extract of mangosteen pericarp enhanced
cytotoxic activity of doxorubicin on MCF-7 cells
Combination tests were carried out to analyze the
effects of EEMP and doxorubicin on MCF7-7 cells. The
efficacy of those combinations is synergistic, additive, or
antagonistic. Synergistic combinations of them can
increase the efficacy of doxorubicin resulting in a decrease
in the treatment dose to obtain the same level of cytotoxic
effect. The interaction of EEMP and doxorubicin on MCF-
7 cells was observed by the combination of cytotoxic test
using lower concentrations than the IC50 single treatment of
each test compound, which was then analyzed their
percentage of cell viability.
The combination of EEMP and doxorubicin with
concentration of 22.5 μg/mL EEMP and 750 nM
doxorubicin produced percentage of MCF-7 cell viability is
16.91 % (Figure 3). The percentage of cell viability is still
high at 94.89 % due to treatment with single doxorubicin
was 750 nM. It means that the EEMP increased the
cytotoxic effect of doxorubicin. This also shows that
EEMP has the potential to be used as a companion (co-
chemotherapy) agent for doxorubicin chemotherapy. The
lower doses of doxorubicin could reduce its side effects,
interestingly the activity of this agent could still be
maintained, which could be seen from the decreased
percentage of viability of MCF-7 cells caused by the
EEMP exposure. Figure 4 demonstrates the changing in
cell morphology occurring because of combination
treatments. The cell nucleus appeared to shrink, and some
cells underwent membrane blebbing, while the boundary
between cells was very thin, and the cell indicated in
apoptosis process.

Figure 3. The cytotoxic effect of combination treatment of ethanolic extract of mangosteen pericarp (EEMP) and doxorubicin (Dox).
Results are shown as mean  SD (n=3)

A B C D

Figure 4. Effect of combination of ethanolic extract of mangosteen pericarp (EEMP) and doxorubicin on MCF-7 cells undergo
morphological changes. Cell control (A); 22.5 μg/mL EEMP exposure (B); Doxorubicin 750 nM exposure (C); combination 22.5 μg/mL
EEMP and 750 nM doxorubicin exposure (D)
 LAKSMIANI et al. – Mangosteen extract as enhancer of doxorubicin 53

Ethanolic extract of mangosteen pericarp (EEMP) with
-mangostin as active compound potentially increases
the cytotoxic effect of doxorubicin by inhibiting the
target protein Pgp
Molecular docking is designed to determine the
molecular mechanisms mediating EEMP cytotoxic effects
in silico. Molecular docking test results using the Autodock
4.2 program were listed in Table 1, while the interactions
between α-mangostin and Pgp could be seen in Table II. In
the validation process, 10 native ligand bond conformations
to the target protein were obtained. From the 10
conformations, one conformation was chosen with the
lowest RMSD (Root Mean Square Deviation) value.
RMSD is a measurement representing the deviation of the
obtained native ligand bond position experimentally
compared to the actual one. If RMSD value was greater,
the deviation of the bond position was also greater.
Therefore, the possibility of bond prediction errors of the
test compound was also getting bigger when docking was
complete (Bordogna et al. 2011; Kufareva and Abagyan
2012). The RMSD value obtained in this study was 2.74 Å
(Table I). A molecular docking method is valid if it has a
value of RMSD ≤ 3 Å.
Discussion
This study aims to explore the natural ingredients of
mangosteen pericarp which has the potential as a co-
chemotherapeutic agent in the treatment of breast cancer.
Doxorubicin as one of the widely used chemotherapeutic
agents also affects normal tissues, immune system
suppression, and resistance. Chemotherapy is a cancer
therapy strategy by combining a compound with
chemotherapy agents. Combinations with phytochemical
compounds can be used as a choice. Therefore, the effect of
mangosteen pericarp usage both as substituents and
complement, in the treatment of breast cancer was studied.
The first study was carried out to the single EEMP
cytotoxic potency. The results of this study with the MTT
assay method showed that EEMP was able to inhibit the
growth of MCF-7 cells with an IC50 value of 54 µg/mL.
This value shows a potent cytotoxic effect, which was
under 100 µg/mL (Ueda et al. 2002).

Table 1. RMSD value of molecular docking validation method and binding energy of α-mangostin with Pgp using molecular docking

Binding energy (Ligand-protein)

No. Target proteins RMSD (Å) Ligand Hydrogen bond
(kcal/mol) group
1 Pgp (5KOY) 2.74 Native ligand -7.01 GLY 428 O3-HN
SER 430 O1G-HN
ASP 160 HN61-OD2
LYS 429 O2G-HZ2
LYS 429 O2G-HN

α-mangostin -6.13 ASP 160 H-OD2

THR 431 O-HN
ARG 901 O-HH21

Table 2. Interaction between of α-mangostin with target protein Pgp with amino acid residue on target protein

No. Target proteins Interaction between brazilein and amino acid residue of target proteins

1 Pgp
54 N U S A N T A R A B I O S C I E N C E 11 (1): 49-55, May 2019
When the dose of extract treatment increases, which is
in line with the decrease in cell viability, it means that the
inhibitory effect is dose-dependent. The growth inhibition
by this extract could also be caused by the content of active
substance namely α-mangostin. α-mangostin which is the
major component of EEMP is capable to induce the cell
death in oral squamous cell carcinoma cell (Kwak et al.
2016) and to induce the apoptosis in human osteosarcoma
cell line MG63 (Park et al. 2018). α-mangostin has been
shown to inhibit the proliferation pathway through p38
mitogen-activated protein kinase (MAPK) and to induce
the apoptosis mechanism by increasing the levels of Bax,
followed by the activation of the caspase 3 and caspase 9
(Lee et al. 2016). Those protein are involved in the
regulation of cell death, cell cycle development, and cell
division so that the mechanism mediating EEMP inhibition
is the induction of apoptosis or the inhibition of the cell
cycle in certain phases. The potent cytotoxic activity shows
that EEMP could be developed specifically as a
combination with doxorubicin breast cancer chemotherapy
agents to improve its efficacy. Doxorubicin has a cytotoxic
effect on MCF-7 with IC50 1392 nM. However, subsequent
exposure to doxorubicin will trigger resistance. Resistance
mechanism of doxorubicin in MCF-7 cells is the
overexpression of antiapoptotic proteins Bcl-2 and Pgp.
Therefore, to increase the cytotoxic effect of doxorubicin
on MCF-7 cells, it was combined with EEMP.
EEMP and doxorubicin interactions in MCF-7 cells
were observed by combination cytotoxic tests using low
concentrations of the IC50 single treatment of each test
compound followed by the percentage of cell viability
analysis. The combination treatment of EEMP and
doxorubicin with each used concentration showed that a
higher inhibitory has an effect on the growth of MCF-7
cells compared to a single treatment of each compound and
was able to inhibit cell growth up to 50% of the population.
From the results of the combination of cytotoxicity test
between EEMP and doxorubicin, it showed that EEMP can
be developed into a co-chemotherapy agent so that the
concentration of doxorubicin producing the cell inhibition
up to 50% of the population could be reduced. Decreasing
the concentration of doxorubicin correlates directly with
the decrease of side effects and toxicity as well as cases of
resistance caused by doxorubicin. Figure 3 demonstrates
that EEMP enhanced the cytotoxicity effect of doxorubicin.
Single doxorubicin application with concentration of 750
nM exposed to MCF-7 cells still resulted in the percentage
of cell viability about 94.89%, but when it was combined
with EEMP 22.5 μg/mL, the percentage of cell viability
become 16.91%.
The strong activity of EEMP can occur through a
variety of mechanisms, including induction of apoptosis
and cell cycle modulation. In the EEMP and doxorubicin
combination test, there was a change in cell morphology
when it was compared with the single treatment of each test
compound (Figure 4). Almost all cells experienced cell
shrinkage and morphological changes in these cells led to
indications of apoptosis. Further research is needed to
determine the mechanism for increasing cytotoxic activity
from a combination of EEMP and doxorubicin against
MCF-7 cells. Therefore, in silico assay was conducted to
trace and provide an overview of the molecular mechanism
of α-mangostin with Pgp used as target protein, which
caused EEMP to increase the sensitivity of cytotoxic effect
of doxorubicin (Toffoli et al. 1996). Tracking the molecular
mechanism of α-mangostin was carried out using molecular
docking by in silico on Pgp protein.
Molecular docking analysis of α-mangostin was
evaluated from the binding site of Pgp after the preparation
of the target protein, to determine the affinity of α-
mangostin in Pgp and the inhibition of the target protein.
These obstacles will disrupt Pgp activities because there
was no energy used in the active transport process by Pgp
(Aller et al. 2009). From the results of molecular docking
in tables I and II, interactions α-mangostin was weaker than
native ligand because it had higher binding energy value, -
6.13 kcal/mol compared to native ligand -7.01 kcal/mol
(Table I). Nevertheless, the interaction of α-mangostin at
Pgp binding site is quite strong, as evidenced by the
presence of 3 hydrogen bonds between α-mangostin and
amino acid residues in Pgp, which is similar to amino acid
residues (ASP 160). This could be seen from the interaction
between α-mangostin at Pgp binding site when it was
compared with the interaction between native ligands in the
same pocket from Pgp. However, further research to find
out α-mangostin in EEMP inhibiting Pgp activity in vitro
should be done.
It can be concluded that EEMP cytotoxic on MCF-7
cells and its combination with doxorubicin increases the
cytotoxic activity of doxorubicin chemotherapy agent,
which may be related to MCF-7 cell apoptosis induction
and the inhibition of Pgp expression. The results of this
study are expected to give scientific data regarding the use
of EEMP, specifically as a co-chemotherapy agent for
breast cancer with doxorubicin.
ACKNOWLEDGEMENTS
The author would like to thank Udayana University for
funding this research (PUPS 2018) and Cancer
Chemoprevention Research Center (CCRC) for supporting
both material and non-material of the implementation of
this research.
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 INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2155-2165, 2016

Antiproliferative and apoptosis induction of

α-mangostin in T47D breast cancer cells
SOMCHAI KRITSANAWONG1, SUKANDA INNAJAK1, MASAYA IMOTO2 and RAMIDA WATANAPOKASIN1

1
Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand;
2
Department of Bioscience and Information, Faculty of Science and Technology, Keio University, Yokohama, Japan

Received November 5, 2015; Accepted February 8, 2016

DOI: 10.3892/ijo.2016.3399

Abstract. α-Mangostin extracted from mangosteen, Garcinia
mangostana Linn. is known as ‘queen of fruits’. The anti-
cancer activity of α-mangostin through apoptosis induction
and related signaling pathways in human breast cancer T47D
cells was investigated. Human epidermal growth factor
receptor 2 (HER2) and mitogen-activated protein kinase
(MAPK) signaling have been shown to play important roles in
apoptosis. The results showed that α-mangostin induced cell
proliferation inhibition, DNA fragmentation, nuclear conden-
sation, increased cleaved caspase-3 and cleaved caspase-9,
but decreased Bcl-2 and Mcl-1 expression. Mitochondrial
dysfunction and cytochrome c release were also detected.
In addition, phosphorylation of ERα, HER2, PI3K, Akt and
ERK1/2 were downregulated whereas p-JNK1/2 and p-p38
were upregulated. These results indicated that α-mangostin
induced apoptosis associated with HER2/PI3K/Akt and
MAPK signaling pathways suggesting that α-mangostin
may be used as food supplement or a potential therapeutic
compound for breast cancer.
Introduction
Breast cancer is the most common type of cancer in women
and is the second leading cause of cancer death following lung
cancer, resulting from coordinated actions between estrogen
receptor (ER) and growth factor receptor especially for HER2
and various survival signaling pathways (1). Increasing efforts
are conducted on identifying not only agents that selectively
target cancer cells but also signaling pathways that promote
or inhibit cancer progression. Targeting a specific pathway is
critical to successful treatment of breast cancer, as cancer cells
reflect the balance between cell death and survival (2). ERs are
Correspondence to: Professor Ramida Watanapokasin, Department
of Biochemistry, Faculty of Medicine, Srinakharinwirot University,
Bangkok 10110, Thailand
E-mail: ramidawa@yahoo.com
Key words: α-mangostin, breast cancer, apoptosis, PI3K, Akt,
MAPK
a group of nuclear receptor (NR) transcription factors, which
could be activated by estrogen hormone. There are two clas-
sical nuclear ER subtypes, ERα and ERβ. Approximately 70%
of breast cancer cases are ER-positive (3). Signaling of ERα is
involved in normal breast development, as well as in growth
and progression of breast cancer. HER2, an orphan receptor
with intrinsic tyrosine kinase activity, regulates cell growth,
differentiation and survival (4,5). Excess HER2 signaling leads
to numerous oncogenic processes including cell proliferation,
survival and carcinogenesis (6) via the RAS/Raf1/MEK/ERK
and the PI3K/Akt pathways. Moreover, crosstalk between ERα
and HER2 signaling pathways fundamentally contribute to the
development and aggressiveness of cancer (7).
Apoptosis is a form of programmed cell death which is
precisely regulated and plays important roles during embryo-
genesis and immunology (8-11). Apoptosis is characterized
by a number of well-defined features including cellular
morphological changes, chromatin condensation, DNA frag-
mentation and caspase activation. Apoptosis (program cell
death) proceeds through two main pathways, the extrinsic and
intrinsic pathways. The extrinsic pathway is triggered through
the binding of death ligands such as tumor necrosis factor-α
(TNF- α), cluster of differentiation 95 ligand (CD95L)/Fas
ligand (FasL) or TNF-related apoptosis-inducing ligand
(TRAIL) to death receptors on the cell surface resulting in
formation of death-inducing signaling complexes (DISC) and
activation of initiator caspases-8 (12). On the contrary, the
intrinsic or mitochondrial pathway is initiated by cellular or
DNA damage characterized by depolarization of mitochon-
drial membrane e controlled by the Bcl-2 family proteins
leading to cytochrome c release into cytoplasm and caspase-9
activation (13). Both pathways promote cleaved caspase-3
expression then PARP degradation, DNA fragmentation and
apoptotic cell death. Apoptosis is regulated by a variety of
signaling pathways including the PI3K/Akt pathway. Protein
kinase B (Akt) has been shown to regulate apoptosis related
proteins such as B cell lymphoma-2 (Bcl-2), Bcl-2 associated
X protein (Bax) and cysteine aspartic acid specific protease
(caspase-3) and is crucially involved in anticancer drug
induced apoptosis of cancer cells (14-17).
MAPK signaling, targeted for cancer prevention and
treatment, includes a three-tiered kinase core, where MAP3K
activates MAP2K that activates MAPKs including ERK1/2
which promotes cell growth, cell survival and differentia-
2156 Kritsanawong et al: Apoptosis induction of α-mangostin in T47D breast cancer cells
tion whereas JNK1/2 and p38 promote cell apoptosis (18,19).
Moreover, α-mangostin also showed decreased inactive-
MAPK(s) by dephosphorylation of c-Raf at Ser259. Therefore,
downregulation of phosphorylated-ERK1/2 may be an appro-
priate alternative therapy for breast cancer patients (20).
Various signaling pathways were activated upon the
binding of estrogen to ERα including Ras-Raf-MAP kinase
and PI3K/Akt signaling pathways (21). ERα phosphorylation
is involved in tamoxifen resistance including Ser104/106 and
Ser118 in breast cancer patients (22). These regions of ERα were
phosphorylated by ERK1/2 and MEK1/2 (23,24). ERK/MAPK
and PI3K/Akt pathways rapidly activated by the ERα-estrogen
complex also have a critical role in estrogen action as a survival
agent. Furthermore, p38 phosphorylated-ERα (at Ser294) in
coordination with S-phase kinase-associated protein 2 (Skp2)
which stimulated phosphorylation of ERα at Ser64 led to
proteosomal degradation of ERα. In addition, inhibition of
p38 and Skp2 affected the ERα function (25). Indeed, these
pathways enhanced Bcl-2 expression, blocked p38 activation
and reduced caspase-3 activation (26,27).
Mangosteen (Garcinia mangostana Linn.) family
Guttiferae has been used for hundreds of years around the
world, mainly in Southeast Asia. Mangosteen is round, dark
purple or reddish fruit with white juicy pulp possessing slightly
acidic and sweet flavor, known as the ‘queen of fruits’ (28).
The juicy pulp contains essential nutrients such as carbohy-
drates, fat, protein, vitamins and trace metals. The fruit hulls
were used as a traditional herbal medicine for the treatment of
abdominal pain, dysentery, wound infections, eczema, suppu-
ration and chronic ulcer (29-31). Xanthones, the most abundant
polyphenolic compounds present in mangosteen pericarp, have
long been reported to possess multiple health-promoting prop-
erties (32). The dominant xanthone extracted from the fruit
hulls of G. mangostana, α-mangostin, has been demonstrated
to possess antioxidant (33-35), antibacterial (36,37), antifungal
(38), anti-inflammatory (39,40), renoprotective activities (41).
Effects of α-mangostin have been reported in numerous pre-
clinical tumor models such as colon cancer (42), colorectal
(43), leukemia (44), chondrosarcoma (45).
In the present study, we investigated the hypothesis that
α-mangostin could inhibit cell proliferation and induce apop-
tosis associated with HER2/PI3K/Akt and MAPK signaling
pathway in human breast carcinoma T47D cells.
Materials and methods
Chemical. Hoechst 33342, fetal bovine serum (FBS), crystal
violet, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), phenylmethylsulphonylfluoride (PMSF),
2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulphonic acid
(HEPES), ethylenediaminetetraacetic acid (EDTA), Ethylene
glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid
(EGTA) and phenol:chloroform:isoamyl alcohol (25:24:1)
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Dulbecco's modified Eagle's medium (DMEM) and Roswell
Park Memorial Institute (RPMI)-1640 medium were
purchased from Gibco (Grand Island, NY, USA). Propidium
iodide (PI), Hoechst 33342 (2,5'-Bi-1H-benzimidazole,
2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-23491-52-3),
JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbo-
cyanine iodide) and DNase free RNase A were purchased
from Thermo Fisher Scientific Inc. (Pittsburgh, PA, USA).
Dimethylsulfoxide (DMSO) was purchased from Merck
calbiochem (San Diego, CA, USA). α-Mangostin was isolated
and purified by A. Krajarng according to our previous
report (45). The structure of G. mangostana is shown in
Fig. 1A (46).
Cell culture. Breast cancer cell lines MDA-MB-468, AU565,
SKBR3 and T47D were obtained from the American
Type Culture Collection (ATCC; Manassas, VA, USA).
MDA-MB‑468 was maintained as a monolayer in DMEM.
AU565, SKBR3 and T47D were maintained as a monolayer
in RPMI-1640. The media were supplemented with 10% fetal
bovine serum (FBS) 100 U/ml penicillin, 100 µg/ml strep-
tomycin (PAA Laboratories, Pasching, Austria). Cells were
cultured in 5% CO2 at 37˚C. The medium was refreshed every
2-3 days.
Evaluation of proliferation inhibitory potential (MTT assay).
The cytotoxicity of α-mangostin was determined by MTT
assay. MDA-MB-468, AU565, SKBR3 and T47D cells were
seeded at a density of 1x10 4 cells/well in a 96-well plate
and allowed to grow for 24 h. Cells were then treated with
α-mangostin at 7.5, 15 and 30 µM, whereas the control group
was treated with 0.03% DMSO. After incubation for 4, 8,
12, 16, 20 and 24 h, MTT solution (0.5 mg/ml) was added to
each well and was further incubated for 3 h at 37˚C. DMSO
was added to each well to solubilize water insoluble purple
formazan crystals. The absorbance at 570 nm was measured
using a microplate reader (Multiskan EX; Thermo electron
corp., Vantaa, Finland). Survival percentage (%) was calcu-
lated relative to the control.
The IC 50 of T47D cells was 7.5 µM, which was less
than Vero cells (14.26 µM), whereas those of SKBR3,
MDA-MB‑468 and AU565 were 23.88, 22.23 and 43.63 µM,
respectively. Thus, T47D cells were chosen for further study.
The reason for choosing different times depended on which
step of apoptosis occurs at each period, as each protein will
be expressed at different times. The survival proteins were
reduced with increasing of time.
Colony formation assay. T47D cell suspensions were seeded
at a density of 4x10 4 cells/well in a 35-mm tissue culture
dish and allowed to grow for 24 h at 37˚C. Cells were treated
with 15 and 30 µM α-mangostin for 3 and 6 h. After 14 days
colonies were fixed with 100% methanol and stained with 2%
(w/v) crystal violet (Suvchem Laboratory Chemicals, Mumbai,
India). The number of colonies ≥50 cells was counted and
colony formation efficiency was calculated.
Nuclear morphology staining with Hoechst 33342. T47D
cells were seeded at a density of 5x104 cells/dish for 24 h then
treated with 30 µM α-mangostin for 3, 6, 9 and 12 h at 37˚C.
The cells were then stained with 10 µM Hoechst 33342 and
examined under fluorescence microscope (IX73; Olympus,
Tokyo, Japan).
DNA fragmentation analysis. T47D cells at 2x106 cells/well
were seeded in 60-mm tissue culture dish and treated with 0,
 INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2155-2165, 2016
15 and 30 µM α-mangostin for 24 h. For DNA fragmentation
assay, low molecular weight DNA was isolated (47). Briefly,
T47D cells treated with α-mangostin were collected, washed
and resuspended in TE (10 mM Tris-HCl, pH 8.0 and 1 mM
EDTA) buffer. Then lysis buffer (5 mM Tris-HCl, pH 8.0
and 20 mM EDTA and 0.5% (v/v) Triton X-100) was added,
resuspended and kept at -20˚C overnight. The mixture was
centrifuged at 14,000 x g for 15 min. The supernatant was
then collected and extracted with phenol:chloroform:isoamyl
alcohol (25:24:1) followed by butanol and precipitated with
absolute ethanol. The pellet was dried and dissolved in TE
buffer, incubated with 100 µg/ml DNase free RNase A for 2 h
at 37˚C and incubated with 200 µg/ml proteinase K (Vivantis
Inc., Oceanside, CA, USA) at 60˚C. The sample DNA was
analyzed using 1.8% agarose gel (Vivantis) and visualized by
ethidium bromide staining.
Cell cycle analysis. T47D cells were seeded at 6x105 cells/well
in a 6-well plate then treated with 0, 15 and 30 µM α-mangostin
for 12 h, washed with ice cold PBS, fixed with 70% ethanol
and stored at -20˚C then resuspended in DNA staining solution
(50 µg/ml propidium iodide, 200 µg/ml DNase free RNase A,
0.1% Triton X-100, 0.1% sodium citrate). Cells were washed
and incubated at 4˚C overnight in the dark and analyzed for
DNA content on a BD Accuri C6 flow cytometer (Accuri
Cytometers, Inc., Ann Arbor, MI, USA) at 488 nm excitation.
The forward scatter and red fluorescence above 600 nm were
measured.
Measurement of mitochondrial membrane potential (∆Ψm).
The changes in mitochondrial membrane potential (∆Ψm)
were determined using JC-1, a lipophilic fluorescent cation
that incorporates into the mitochondrial membrane. Intact
living cells stained with JC-1 exhibits pronounced red
fluorescence, whereas break-down of the mitochondrial
membrane potential in apoptotic cells shows decreased red
fluorescence and reveals green fluorescence. Cells were
treated with 30 µM α-mangostin and the control cells were
treated with 0.03% DMSO, and then were stained with
10 µg/ml JC-1.
Detection of cytochrome c release. Upon treatment of T47D
cells in the absence or presence of 30 µM α-mangostin, cells
were resuspended in S-100 lysis buffer (20 mM HEPES,
pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM
EDTA and 250 mM sucrose) and homogenized for 30-40
strokes. Cell were centrifuged at 500 x g for 5 min at 4˚C
(to eliminate nuclei and unbroken cells). The supernatant
was taken to a new tube, then centrifuged at 10,000 x g for
30 min at 4˚C. Pellet contained mitochondria and supernatant
contained cytosolic fraction. The protein was separated by
12% sodium dodecyl sulphate polyacrylamide gel electropho-
resis (SDS-PAGE), transferred onto polyvinylidene fluoride
(PVDF) membranes (GE Healthcare, Buckinghamshire, UK)
and subjected to immunodetection of cytochrome c using a
rabbit polyclonal antibody against human cytochrome c (Cell
Signaling Technology, Beverly, MA, USA). The peroxidase
activity of bound secondary antibodies on the blots was
detected by enhanced chemiluminescence reagent (ECL; GE
Healthcare) and detected under chemiluminescent imaging
2157
system (GeneGnome gel documentation; Synoptics Ltd.,
Cambridge, UK).
Western blot analysis. T47D cells were seeded at 6x105
cells/well in a 35-mm culture dish, exposed with 30 µM
α-mangostin and harvested at designated time-points. Cells
were lysed with RIPA (radioimmunoprecipitation assay) buffer
(50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 250 mM NaCl, 0.5%
Triton X-100) supplemented with complete mini-protease
inhibitor cocktail (Roche Diagnostics GmbH, Mannheim,
Germany). The protein content was determined using Bio-Rad
protein assay kit (Bio-Rad Laboratories, Hercules, CA,
USA), separated by 10-12% SDS-PAGE and transferred onto
PVDF membrane using a Mini Trans-Blot Cell® (Bio-Rad
Laboratories). The blots were incubated overnight at 4˚C with
appropriate primary antibodies (Cell signaling Technology).
The membranes were washed and incubated with appropriate
secondary antibodies conjugated with horseradish peroxidase
(Cell signaling Technology) for 1 h at room temperature.
Immunoreactive protein bands were detected by chemilumi-
nescence using ECL.
Statistical analysis. All data presented were obtained from
at least three independent experiments and are presented
as mean ± standard deviation (SD). Statistical significance
was assessed by one-way analysis of variance (ANOVA).
Statistical analysis was performed using the SPSS statistical
software package (version 11.5) as well as GraphPad Prism
3.03 (GraphPad Software, Inc., San Diego, CA, USA). The
protein band intensity was quantified by ImageJ densitometer.
Asterisks in the figures indicate that the experimental values
are significantly different from those of the control (*p<0.05,
**
p<0.01).
Results
α-Mangostin decreases cell viability in T47D human breast
cancer cells. The effect of α-mangostin on cell viability in
T47D cells treated with α-mangostin at various concentrations
and times was determined and the results showed that the IC50
value was 7.5±0.5 µM at 24 h (Fig. 1A), and cell viability was
dose- and time-dependent comparing to the control cells.
α-Mangostin inhibits colony formation and proliferation of
breast cancer cells. The results indicated that α-mangostin
inhibited colony formation in T47D cells in a time- and dose-
dependent manner (Fig. 1B). T47D cells treated with 30 µM
α-mangostin for 6 h grew slightly, but less than at 15 µM
α-mangostin. The quantitative data of colony formation assay
were calculated and compared with untreated control cells in
three independent experiments (Fig. 1C and D).
Morphological and nuclei changes. Treatment of T47D cells
with 30 µM α-mangostin for 3, 6, 9 and 12 h showed evident
morphological changes including vacuolization, cell shrinkage
and rounding (Fig. 2A). The result also showed chromatin
condensation, a characteristic of apoptotic cells (Fig. 2B).
Cell cycle distribution and chromosomal DNA fragmenta-
tion. DNA fragmentation, a ladder-like pattern, typical DNA
2158 Kritsanawong et al: Apoptosis induction of α-mangostin in T47D breast cancer cells

Figure 1. Effects of α-mangostin on cell viability and colony formation in T47D cells. (A) Structure of α-mangostin isolated from G. mangostana fruit hull.
(B) Time- and dose-dependent effect of α-mangostin on T47D cells treated with different concentrations of α-mangostin at various time-points. Results are
mean values ± SD of three independent experiments performed in triplicate (n=3). (C) Representative dishes of colony formation assay. (D) The quantitative
data of colony formation assay are presented as the mean ± SD of relative increase as compared to the untreated control cells in three independent experiments.
Significant values were defined as *p<0.05 and **p<0.01 as compared to the control.

Figure 2. Effects of α-mangostin on cell morphology and nuclear condensation of T47D cells. (A) Morphological changes of control cells (0.03% DMSO),
30 µM α-mangostin for 3, 6, 9 and 12 h. (B) T47D cells stained with Hoechst 33342 and examined under a fluorescent microscope (magnification, x40).

cleavage between nucleosome was visible dose-dependently
after incubation with α-mangostin, whereas, not in the control
(Fig. 3A). The effect of α-mangostin on cell cycle distribution
showing two major peaks represented the G0/G1 and G2/M
phases, the sub G1 peak with reduced DNA content represented
apoptotic cells. The mean apoptotic population of T47D cells
was 3.3% under the control condition as compared to 12.6 and
21.0% upon treatment with 15 and 30 µM α-mangostin for
12 h, respectively (Fig. 3B and C).
Effects of α -mangostin on the mitochondrial membrane
potential (ΔΨm). Bax oligomers increased mitochondrial
 INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2155-2165, 2016 2159

Figure 3. DNA fragmentation and cell cycle distribution of T47D cells treated with α-mangostin. (A) DNA fragmentation of T47D cells treated with 0, 15 and
30 µM α-mangostin for 24 h (M, DNA size marker). (B) The sub G1 peak formed with reduced DNA content represent the presence of apoptotic cells. The two
major peaks represent G0/G1 and G2/M phases of the cell cycle. Mean apoptotic population of T47D cells was 3.3% for control cells, while it was increased to
12.6 and 21.0% after treatment with 15 and 30 µM α-mangostin for 12 h, respectively. (C) The percentage of cell population in sub G1, G1, S and G2/M phases.
Three independent experiments were performed and the results are expressed as mean ± SD. *p<0.05 compared to the control.

membrane permeability resulting in cytochrome c release
to the cytosol. JC-1 staining was used to determine ΔΨm of
T47D cells. At higher membrane potentials or healthy cells,
JC-1 forms a complex known as J-aggregates with intense
red fluorescence. In contrast, in apoptotic or unhealthy
cells with low ΔΨm, JC-1 remains in the monomeric form
with intense green fluorescence. The increased intensity
ratio of green to red fluorescence indicated mitochondrial
depolarization. The increased green/red ratio was detected
in T47D cells treated with 30 µM α -mangostin for 3, 6
and 9 h, while the control cells showed red fluorescence
(Fig. 4) indicating that α-mangostin induced loss of ΔΨm
in T47D cells.
Effects of α-mangostin on the expression of Bcl-2 family
proteins and induction of cytochrome c release. The Bcl-2
family is a group of proteins regulating cell survival and cell
death. The insertion of Bax into mitochondrial membrane
induces the opening of the mitochondrial voltage-dependent
anion channel (VDAC). The results showed that α-mangostin
decreased myeloid cell leukemia (Mcl-1) expression but not
B-cell lymphoma-extra large (Bcl-xL) and Bcl-2-associated
death promoter (Bad) expression (Fig. 5A). The Bax/Bcl-2 ratio
was increased significantly in each α-mangostin treated group
comparing to the control (Fig. 5B). Bax oligomers increased
mitochondrial membrane permeability and cytochrome c
release from mitochondria to cytosol in a time-dependent
pattern (Fig. 5C).
Effects of α-mangostin on caspase-3, caspase-9 and cleaved
PARP activation. The expression of active forms of caspases
was determined by western blot analysis. As shown in Fig. 5C,
cleaved caspase-3 and cleaved caspase-9 were induced upon
exposure to α-mangostin. Thus, α-mangostin-induced apop-
tosis was mediated by caspase-3 and may be associated with
the activation of intrinsic pathway (via caspase-9). In addition,
apoptosis induction by α-mangostin was accompanied by
expression of the apoptosis marker, cleaved PARP (Fig. 5C).
Modulation of MAPK signaling during apoptosis induction by
α-mangostin. MAPK are important signaling components that
control cellular proliferation, differentiation, motility, survival
and apoptosis. The results showed that α-mangostin induced
phosphorylated-p38 expression at 30 min and the highest level
was detected at 2 h. In addition, phosphorylated-JNK1/2 was
induced at 15 min and the highest level was at 6 h. Furthermore,
decreased phosphorylated-ERK1/2 at 15 min was detected.
Phosphorylated-c-Raf was significantly decreased at 15 and
30 min (Fig. 6A). Once activated, phosphorylated-c-Raf
activated the dual specific protein kinases mitogen-activated
protein kinase kinase 1 (MEK1) and MEK2, which in turn
activated the serine/threonine-specific protein kinase ERK1/2.
In summary α-mangostin modulated the MAPK(s) pathway
resulting in apoptosis induction.
Evaluation of transcription factor and downstream oncogenic
products of PI3K/Akt and MAPK. α-Mangostin enhanced
2160 Kritsanawong et al: Apoptosis induction of α-mangostin in T47D breast cancer cells

Figure 4. Effect of α-mangostin on mitochondrial membrane potential in T47D cells. Cells were treated with 30 µM α-mangostin for 3, 6 and 9 h and stained
with JC-1. The mitochondrial depolarization indicated by an increased green/red fluorescence intensity ratio (magnification, x40).

Iκ B kinases α (IKKα) and Src expression whereas decreased
nuclear transcription factor kappa B (NF-κ B p65) and c-Rel
expression (Fig. 6B). This could result in cell proliferation
inhibition and inflammation in cancer cells as well as increased
sensitivity to antitumor agent and ultimately apoptotic cell
death (48,49). Moreover, α-mangostin also activated C/EBP
homologous protein (CHOP) and c-Jun expression, while
suppressed c-Myc expression (Fig. 6B).
α -Mangostin inhibits HER2 activation and downstream
PI3K/Akt signaling pathway. Activation of the HER2 network
results in autophosphorylation of C-terminal tyrosine leading
to the recruitment of cytoplasmic signal transducer that
regulates cellular processes such as proliferation, apoptosis
inhibition and transformation. Therefore, we examined
whether α-mangostin could activate dephosphorylation of
phosphorylated-HER2 (p-HER2). Treatment of T47D cells
with 30 µM α-mangostin resulted in substantially decreased
phosphorylated-HER2 Tyr1221/1222 at 30 min (Fig. 7A),
which led to inactivation of RAS/Raf1/MEK/ERK and
PI3K/Akt transduction cascade. HER2 stimulates tumor cell
growth and renders cellular chemo-resistance involved with
the HER2 tyrosine kinase domain.
PI3K/Akt signaling is the major anti-apoptosis pathway that
confers survival advantage and drug resistance of cancer
cells. Our data clearly indicated the inhibition of PI3K/Akt
signaling cascade by α-mangostin in T47D cells (Fig. 7B).
In addition, Akt phosphorylation at Ser473 and Thr308 was
highly suppressed by α-mangostin resulting in inhibition of
cell proliferation and apoptosis induction.
Effect of α-mangostin on inhibition of ERα activation. ERα is
a member of NR family transcription factors that is involved
 INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2155-2165, 2016 2161

Figure 5. Effect of α-mangostin on caspase and Bcl-2 protein family expression in T47D cells. (A) Cells were treated with 30 µM α-mangostin and the expres-
sion of Bcl-2 family protein was examined by western blot analysis. (B) The increased Bax/Bcl-2 ratio upon treatment with α-mangostin. (C) Expression of
cleaved caspase-3, cleaved caspase-9 and cleaved PARP in T47D cells. Cytochrome c expression was determined both in cytosol and mitochondrial fraction.
(D) Relative protein expression of cytochrome c, caspase family and cleaved PARP. β-actin was used as the internal control. The quantitative data are presented
as the mean ± SD of the percent increase relative to untreated control cells in three independent experiments. Significant values were defined as *p<0.05 and
**
p<0.01 as compared to the control.

Figure 6. Effect of α-mangostin on phosphorylation of c-Raf, MAPK(s) and transcription factors in T47D cells. Cells were treated with 30 µM α-mangostin and
examined by western blot analysis. (A) Expression of phosphorylated-c-Raf, phosphorylated-ERK1/2, phosphorylated-p38, phosphorylated-JNK1/2 in T47D
cells. (B) Expression of transcription factors downstream of MAPK signaling. β-actin was used as the internal control. The quantitative data are presented
as the mean ± SD of the percent increase relative to untreated control cells in three independent experiments. Significant values were defined as *p<0.05 as
compared to the control.

in cell proliferation and survival. Once activated by estrogen, specific DNA response elements, known as estrogen response
the ERα is able to translocate into the nucleus and bind to elements (EREs), to regulate different gene activities. The
2162 Kritsanawong et al: Apoptosis induction of α-mangostin in T47D breast cancer cells

Figure 7. Inhibitory effect of α-mangostin on phosphorylation of HER2, PI3K/Akt and ERα signaling molecules in T47D cells upon treatment with
30 µM α-mangostin by western blot analysis. (A) Expression of total HER2 and phosphorylated-HER2. (B) Relative phosphorylated-HER2 expression.
(C) Phosphorylated-PI3K, phosphorylated-Akt at Ser473, Thr308 and total protein expression. (D) Relative phosphorylated-PI3K and phosphorylated-AKT
expression. (E) Expression of ERα (Ser104/106) and ERα (Ser118). (F) Relative phosphorylated-ERα expression. β-actin was used as the internal control. The
quantitative data are presented as the mean ± SD of the percent increase relative to untreated control cells in three independent experiments. *p<0.05 compared
with control.

results showed the decreased phosphorylation of Ser104/106
and Ser118 of ERα upon treatment with 30 µM α-mangostin
at 6 h (Fig. 7C) implying that α-mangostin could inhibit cell
proliferation and cell survival through the suppression of ERα
phosphorylation in T47D cells.
Discussion and Conclusion
The goal of the present study was to search for a natural
bioactive compound from a plant as anti-breast cancer agent
with high specificity to ER and progesterone receptor (PR)
positive T47D breast cancer cells. Approximately 75% of
all breast cancers are ER-positive and 65% are PR-positive
cells. Mammary ductal carcinoma is the most common type
of breast cancer in women with poor prognosis, resistant to
chemotherapy and radiation treatment. Apoptosis-inducing
agents are being investigated as a tool for the management of
cancer treatment. α-Mangostin, one of the xanthones, is the
secondary metabolite from mangosteen pericarp possessing
various biological activities (35). For this reason, we investi-
gated the potential of α-mangostin for human breast carcinoma
cell treatment. The results showed that α-mangostin could
inhibit cell growth in a time- and dose-dependent manner with
an IC50 value of 7.5±0.5 µM.
The present study showed that α-mangostin decreased
Mcl-1 expression (Fig. 5A) as well as increased Bax/Bcl-2
ratio (Fig. 5B). The cytosolic cytochrome c level was increased
corresponding to the loss of ΔΨm (Fig. 5C). Cytosolic cyto-
chrome c activated procaspase-9 by binding to apoptotic
protease activating factor 1 (Apaf1) and activated downstream
effector caspase (including caspase-3) then triggered apoptosis
induction through the intrinsic pathway.
 INTERNATIONAL JOURNAL OF ONCOLOGY 48: 2155-2165, 2016 2163

Figure 8. Potential mechanisms of apoptosis induction in T47D human breast cancer cells by α-mangostin. Inhibition of ERK1/2 and PI3K/Akt associated
with downregulation of phosphorylated-HER2 and ERα, which are upstream molecules of survival signaling pathways. α-Mangostin induced the activation of
p-p38 and p-JNK1/2, promoting apoptosis via modulation of Bcl-2 family proteins leading to cytochrome c release to the cytosol resulting in caspase activation
and apoptosis induction of cancer cells.

Our results illustrated that α -mangostin modulated
MAPK(s) (Fig. 6A) and inactivated ERK1/2 resulting in
decreased NF-κ Bp 65, c-Rel and c-Myc but increased IKKα
expression, whereas, activation of p38 and JNK1/2 led to CHOP
and c-Jun expression, respectively. A limitation of the study
at the present stage, is that further experiments with inhibi-
tors are required. There are many related studies including
phosphorylation of c-Jun leading to activator protein 1 (AP-1)
formation which involved pro-apoptotic protein transcrip-
tion and apoptosis induction (50). The JNK1/2 played a key
role in CHOP induction and cell death by ER stress in rat
hepatocytes (51). Moreover, CHOP/death receptor 5 (DR5)
upregulation was mediated via JNK1/2 phosphorylation
in α-TEA induced apoptosis in MDA-MB-231 and MCF-7
cells (52). Our results showed rapid cell growth inhibition and
apoptosis induction in T47D cells by α-mangostin at 15 min.
α-Mangostin also decreased p-HER2 at Tyr1221/1222 as
compared with proform-HER2 expression (Fig. 7A) leading
to downregulation of Ras-Raf-MAP kinase and PI3K/Akt
transduction pathways (53). PI3K/Akt signaling pathway is
essential for cell survival and the expression of a constitu-
tively active PI3K/Akt pathway induces multidrug resistance
and prevents apoptosis in a variety of cell types. Our results
showed that α-mangostin could decrease the phosphorylation
of PI3K and Akt at Ser473 and Thr308 (Fig. 7B). Interestingly,
we found that p-Akt at both sites were decreased significantly
suggesting that Akt may be a target of α-mangostin in breast
cancer. Previous studies showed that inhibition of the PI3K/Akt
pathway with doxorubicin, trastuzumab, etoposide, tamoxifen
increased apoptosis induction implying that measuring
Akt activity would directly be beneficial to breast cancer
patients (54,55). Thus, our results implied that inhibition of
the PI3K/Akt pathway by α-mangostin may be of great benefit
when combining with traditional cytotoxic chemotherapy,
immune therapy and endocrine based therapy.
In addition, estrogen is also involved in normal breast
development as well as in breast cancer growth and progres-
sion. The biological actions of estrogen are mediated by
binding to nuclear ERα and ERα. Treatment with ER antago-
nists is the current hormone therapy of choice for ERα-positive
breast cancer treatment. In the present study, we found that
α-mangostin could inhibit ERα phosphorylation in T47D cells
at 6 h.
In conclusion, α-mangostin inhibited cell proliferation
and induced apoptosis associated with HER2/PI3K/Akt and
MAPK signaling pathway in human breast carcinoma T47D
cells (Fig. 8). Therefore, α-mangostin may provide anticancer
action in clinical application in human breast cancer treatment.
Acknowledgements
We would like to thank the Royal Golden Jubilee Ph.D.
Program, Thailand Research Fund (grant no. PHD/0312/2550),
the Strategic Wisdom and Research Institute, Srinakharinwirot
University and the Research Division, Faculty of Medicine,
Srinakharinwirot University. We would also like thank
Dr Aungkana Krajarng for her help in α-mangostin preparation.
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