LAPORAN AWAL PRAKTIKUM

KIMIA BAHAN ALAM

OBJEK II
ISOLASI SENYAWA FLAVONOID DARI KULIT JERUK MANIS
(Citrus sinensis)

OLEH :

NAMA : INDRI ISTIQOMAH

NO.BP : 1811012014
SHIFT / KELOMPOK :5/1
REKAN KERJA : 1. INSAN KAMIL S 1811011036
2. RIZKYA ALIFA. A 1811012050
3. INTAN PERMATA. S 1811013022

LABORATORIUM KIMIA BAHAN ALAM

FAKULTAS FARMASI
UNIVERSITAS ANDALAS
PADANG
2020
 BAB I
PENDAHULUAN

1.1 Tujuan Percobaan

1. Mengetahui dan mempraktekkan cara mengisolasi flavonoid dari kulit
jeruk manis (Citrus sinensis).
2. Mengetahui cara mengidentifikasi flavonoid dari kulit jeruk manis (Citrus
sinensis).

1.2 Manfaat
1. Dapat diharapkan mengetahui adanya potensi senyawa aktif pada kulit
jeruk manis (Citrus sinensis) yang dapat memberikan manfaat sebagai
pengobatan.
2. Dapat memahami teori isolasi senyawa flavonoid dengan adanya
praktikum ini.
3. Diharapkan dapat memperoleh suatu metoda isolasi serta metoda ekstraksi
dari senyawa flavonoid dari kulit jeruk (Citrus sinensis).
 BAB II
TINJAUAN PUSTAKA

2.1 Tinjauan Botani

Gambar 1. Kulit buah jeruk

2.1.1 Klasifikasi
Kingdom : Plantae
Subkingdom : Tracheobionta
Super divisi : Spermatophyte
Divisi : Magnoliophyta
Kelas : Magnoliopsida
Subkelas : Rosidae
Ordo : Sapindales
Family : Rutaceae
Genus : Citrus
Spesies : Citurus sinensis L.1

2.1.2 Morfologi
Buah jeruk manis berbentuk bulat atau hampir bulat , berukuran agak
besar, bertangkai bulat, kulit buah berwarna hijau sampai kuning mengkilat. Kulit
buah sulit dilepaskan.3
Jeruk manis dicirikan dengan tangkai daun yang mempunyai sayap dan
bunganya berwarna puth. Morfologi tanaman jeruk nipis mempunyai batang yang
dapat mencapai ketinggian 6-10m, bercabang banyak, tajuk daun bundar dan
umumnya berbuah satu kali satu tahun. Ranting yang muda biasanya berduri,
bercabang rendah dan berbentuk tajuk bulat dengan kerimbunan sedang. Batang
tanaman jeruk beerkayu dan keras.3
 Secara umum, daun jeruk berwarna hijau tua sampai hijau cerah dan
terkesan tebal. Jika daun itu diperas akan menimbulkan aroma sesuai dengan jenis
jeruknya. Tulang daun berbentuk menyirip beraturan, tetapi ada juga
berselangseling. Tepian daun bergerigi dengan ukuran gerigi ada yang besar dan
ada yang kecil. Bentuk fisik daun oval, meruncing, tetapi ada juga oval tumpul
dan membulat. Lembaran daun (petiolus) kecil terletak dekat dengan tangkai
daun. Tetapi ada juga daun yang tidak memiliki petiolus. Permukaan daun sekilas
terlihat mengkilap, karena dilapisi oleh kutikula yang mengandung sedikit pektin
sehingga tetesan air hujan cepat meluncur.4
Bunga tumbuh pada ketiak daun, bau sangat harum, bila membuka penuh
garis tengahnya 2-3cm. Kelopak berbentuk mangkok bergaris tengah 0,4-0,5 m.
Mahkota bunga berjumlah 5 helai, warnanya putih atau kekuningan, bentuknya
bulat telur yang bagian bawah menyempit dan ujungnya tumbuh atau runcing
tidak berbulu. Tangkai benang sari berwarna putih tidak berbulu. Tangkai putik
panjang berwarna putih kehijauan.3

2.1.3 Sinonim
Database MMPDN memberikan sinonim berikut.Citrus aurantium L., Var
Sinensis L., Aurantium sinensis (L.) Mill., Citrus aurantium L.,subsp, sinensis
(L.) P. Fourn., Citrus aurantium L., Citrus macracantha Hassk.2

2.1.4 Habitat dan Sebaran

Jeruk (Citrus sp) merupakan tanaman buah tahunan yang berasal dari Asia.
Cina dipercaya sebagai tempat pertama kali jeruk tumbuh. Jeruk merupakan
tanaman yang dapat tumbuh baik di daerah tropis dan subtropis. Jeruk manis
dapat beradaptasi dengan baik di daerah tropis pada ketinggian 900-1200 meter di
atas permukaan laut dan udara senantiasa lembab, serta mempunyai persyaratan
air tertentu.5
Jeruk memiliki beberapa jenis, seperti spesies jeruk kemprok (Citrus
nobilus), Spesies jeruk besar (Citrus maxima). Di Indonesia ditemukan banyak
kultivar jeruk baik yang tumbuh secara alami ataupun di budidayakan di
Indonesia. Tanaman jeruk mulai dibudidayakan sejak orang-orang Belanda berada
di Indonesia. 6
Genus Citrus dan genera (Fortunella, Poncirus, Eremocitrus dan
Microcitrus) termasuk dalam subfamili angiosperma Aurantioideae dari keluarga
Rutaceae, yang tersebar luas di wilayah monsun dari Pakistan barat ke Cina utara-
utara dan selatan melalui Kepulauan India Timur ke Nugini dan Kepulauan
Bismarck, Australia timur laut, Kaledonia Baru, Melanesia, dan pulau-pulau
Polinesia barat. Habitat asli jeruk dan genus terkait kira-kira meluas di seluruh
wilayah, meskipun asal geografis, waktu dan penyebaran spesies jeruk di Asia
Tenggara masih belum jelas.15
 2.2 Kandungan Kimia
Kandungan kimia yang terdapat dalam kulit jeruk sangat banyak
diantaranya hesperidin, naragin, kuarsetin dan pektin.7
Kandungan kulit jeruk tidak kalah banyak dibandingkan dengan
kandungan buah jeruknya sendiri. Zat bermanfaat yang terkandung dalam kulit
jeruk salah satunya adalah minyak atsiri. Kandungan kulit jeruk yang satu ini
banyak ber‐manfaat bagi manusia. Minyak atsiri adalah sejenis minyak nabati
yang dapat berubah mengental bila diletakkan pada suhu ruangan. Minyak ini
mengeluarkan aroma yang sangat khas dan biasa digunakan sebagai bahan
pembuat minyak gosok alami yang digunakan untuk pengobatan dan kosmetika.
Kulit jeruk mengandung atsiri yang terdiri dari berbagai komponen seperti tepen,
sesquiten, aldehida, ester dan sterol.8
Kulit buah jeruk Bali (Citrus maxima Merr.) mengandung senyawa
flavonoid yaitu naringin dan hesperidin. Hal ini dibuktikan berdasarkan penelitian
yang dilakukan Dianingati terhadap kandungan ekstrak etanol kulit jeruk Bali
dengan metode kromatografi lapis tipis, terlihat bahwa kandungan senyawa
flavonoid yang terkandung dalam ekstrak jeruk Bali yaitu rutin, naringin, dan
hesperidin.9
2.2.1 Struktur Kimia

Gambar 2. Hesperidin Gambar 3. Naragin

Gambar 4. Pektin Gambar 5. Nobiletin

 Gambar 6. Sitronelal, Sitronelol, dan Geraniol

2.3 Kegunaan Tradisional

Kandungan flavonoid dan bioflavonoid dalam jeruk manis bermanfaat
sebagai antiradang, anti alergi, antivirus, antikanker, dan antioksidan. Kandungan
flavonoid dan bioflavonoid juga bermanfaat untuk memperlambat proses penuaan,
menurunkan kolesterol darah serta mengobati artritis dan pengerasan arteri
(arterosklerosis). Menyembuhkan sariawan karena memiliki kandungan vit c yang
tinggi.10
Daging buah jeruk memiliki kandungan vitamin C yang tinggi yang
mampu menambah daya tahan tubuh. Selain daging jeruk, khasiat dan manfaat
buah jeruk juga banyak terkandung pada kulit jeruk. Kulit jeruk memiliki
kandungan manfaat yang tidak kalah banyak dibandingkan dengan kandungan
buah jeruknya sendiri. Kandungan kulit jeruk memiliki manfaat diantaranya mulai
dari penenang, penghalus kulit hingga obat anti nyamuk.8
Jeruk yang dijus bila menghilangkan rasa mual dan muntah-muntah pada
wanita hamil. Meringankan sakit pada tenggorokan, batuk, pilek, selesma, seta
asma. Kandungan limonene tertinggi terdapat dibagian agen anti kaner dan
mendorong sistem kekebalan tubuh.14

2.4 Uji Bioaktivitas

2.4.1 Ekstrak
Ekstrak kulit jeruk (OPE) mengandung banyak flavonoid termasuk flavon
polimtoksilasi (PMF). Flavon C atau o-glikosilasi, flavonon o-glikosilasi, flavonol
dan banyak asam fenolat lainnya, bersama dengan turunan terkait citrus flavonoid
memiliki berbagai bioaktivitas, termasuk aktivitas antioksidan, anti-inflamasi,
anti-karsinogenik, dan anti-aterosklerosis.11

2.4.2 Metabolit Sekunder

Semua flavonoid yang dijelaskan dalam citrus sp. Dapat diklasidikasikan ke
dalam kelompok-kelompok ini : flavon, dan flavonol. Serta spesies yang dicirikan
dengan gugus flavonon glikosida tertentu. Faktanya tanaman citrus mengandung
berbagai konstituen flavonoid, beberapa diantaranya merupakan karakteristik
mereka.13
Telah dibuktikan juga bahea nobiletin menunjukkan aktivitas antiprofetatif
dalam sel kanker ovarium dan payudara. Dengan sel kanker ovarium dan
payudara. Dengan demikian telah diakui bahwa nobiletin dapat membantu
mencegah kanker serviks dan berpotensi menjadi agen yang berguna untuk
pengobatan penyakit-penyakit tertentu yang sangat ganas.13

2.5 Metode Ekstraksi

Metode ekstraksi kulit jeruk mengikuti metode yang dilakukan oleh
Mehmood, yaitu kulit jeruk diiris tipis dan dikeringkan pada suhu 60oC
mengguakan cabinet dryer selama 24 jam. Kulit jeruk yang sudah kering
dihaluskan dengan menggunakan grinder kemudian disaring dengan ayakan 70
mesh.12
Ekstrak kulit jeruk didapatkan dengan metode maserasi. Sebanyak 30
gram bubuk kulit jeruk direndam dalam 300ml etanol anhydrous dengan
perbandingan 1:10, dilanjutkan dengan agitasi 500 rpm pada suhu ruang (28±2oC)
selama 2 jam. Kemudian, agitasi dihentikan dengan maserasi dilakukan hingga
mencapai 24 jam. Ekstrak yang didapat disaring dengan kertas saring lalu eluen
diuapkan dengan rotary evaporator dengan pengaturan rotasi 60 rpm, suhu 50oC,
dan tekanan 100 mbar selama 2 jam. Penguapan dihentikan ketika didapatkan
ekstrak kulit jeruk yang pekat (berwarna kuning kehijauan). Sebelum
diaplikasikan ke bahan pangan, ekstrak kulit jeruk lebih dulu dilarutkan ke dalam
akuades dengan konsentrasi 1% (b/v) dan dipanaskan pada suhu 70oC selama 30
menit.12
Pengujian dengan Pereaksi Kimia: Untuk identifikasi alkoloid Ekstrak
direaksikan dengan pereaksi Mayer, pereaksi Wagner, dan pereaksi Dragendorf.
Jika masing-masing pereaksi menghasilkan endapan jingga, berwarna coklat dan
berwana putih menunjukkan adanya alkaloid. Selanjutnya untuk identifikasi
flavonoid ekstrak ditambahkan dengan serbuk Magnesium 0,1 mg dan 2 tetes HCl
pekat. Jika menghasilkan warna merah menunjukkan adanya flavonoid. Untuk
identifikasi saponin ekstrak ditambahkan 2-3 tetes HCl 2N dan dikocok kuat. Jika
menghasilkan busa menunjukkan adanya saponin. Identifikasi terpenoid ekstrak
dilakukan dengan penambahan 2mL kloroform dan 2mL asetat anhidrida lalu
didinginkan dan ditambahkan H2SO4. Jika terjadi warna hijau atau biru
(triterpenoid), dan merah atau ungu (steroid). Terakhir, untuk identifikasi tanin
ekstrak ditambahkan FeCl3 1% sebanyak 2 - 3 tetes, jika menghasilkan warna
coklat kehijauan atau biru kehitaman menunjukkan adanya tannin.9
 BAB III
PROSEDUR KERJA
3.1 Alat dan Bahan
3.1.1 Alat
Seperangkat alat sokletasi, rotary evaporator, pipet tetes,
chamber, pipa kapiler, vial, corong, spatel.
3.1.2 Bahan
Kulit jeruk manis (Citrus sinensis) 200g, metanol, n-heksana, etil
asetat, kertas saring, benang jagung, plat KLT.

3.2 Cara Kerja

Kulit Jeruk

Dikeringkan

Kulit jeruk kering

Dihaluskan dan ditimbang 200 gram

Bubuk halus kulit jeruk kering

Direfkluks dengan 500 ml n-heksana,

selama 2 jam

Ekstrak n-heksana kulit jeruk

Saring, uapkan pelarut filtrat dengan

rotary evaporator

Ekstrak kental kulit jeruk

Rekristalisasi, ambil endapan yang

terbentuk
Kristal flavonoid

Pengujian menggunakan metode KLT

dengan mengamati fase diam
Nobiletin
pada sinar uv
 DAFTAR PUSTAKA

1. Novitasari, R. Studi Pembuatan Sirup Jeruk Manis Pasaman. Jurnal

Teknologi Pangan. 2018; 7 (2) :1-9.
2. Ravindran, PN. The Encyclopedia Of Herbs & Spices. London : Cabi ; 2017.
3. Aziz, MF. Distribusi Beberapa Jenis Kutu Sisik pada Perkebunan Jeruk
Manis (Citrus sinensis). Malang : Fakultas Sains dan Teknologi UIN Maulana
Malik Ibrahim Malang ; 2010.
4. Tuasamu, Yati. Karakteristik Morfologi Daun dan Anatomi Stomata pada
Beberapa Spesies Tanaman Jeruk (Citrus sp). Jurnal Agribisnis Perikanan.
2018; 11.(2) : 85-90.
5. Adelina, OS., Adelina, E., dan Hasriyanty. Identifikasi Morfologi dan
Anatomi Jeruk Lokal (Citrus sp) di Desa Doda dan Desa Lempe Kecamatan
Lore Tengah Kabupaten Poso. J. Agrotekbis. 2017; 5.(1): 58-65.
6. Rukmana, Rahmat. Jeruk Nipis: Proses Agribisnis, Budidaya, dan
Pascapanen. Yogyakarta : Kanisius; 2010.
7. Milind, P., & Dev., C. Orange: Range of benefits. Int. Res. J. Pharm. 2012. 3,
59–63
8. Friatna, ER., Rizqi, A., dan Hidayah, T. Uji Aktivitas Antioksidan pada Kulit
Jeruk Manis (Citrus sinensis ) sebagai Alternatif Bahan Pembuatan Masker
Wajah. UNY. 2011; 6.(2).
9. Suryanita, Aliyah., Djabir, YY. Et all. Identifikasi Senyawa Kimia dan Uji
Aktivitas Antioksidan Ekstrak Etanol Kulit Jeruk Bali (Citrus maxima Merr.).
MFF.2019; 23. (1): 16-20.
10. Rusllanti. Jus Ajaib Penumpas Aneka Penyakit. Jakarta : PT. Agromedia
Pustaka ; 2013.
11. Xiu Min Chen., Andrew, PT, David, DK. Flavonoid Composition Of Orange
Peel and its Association With Antioxidant and Antiinflammatory Activities.
2017; 2.(8): 15-21.
12. Dewi, ADR. Aktivitas Antioksidan dan Antibakteri Ekstrak Kulit Jeruk
Manis (Citrus sinensis) dan Aplikasinya Sebagai Pengawet Pangan. Jurnal
Teknologi dari Industri Pangan. 2019; 30(1); 83-90 .
13. I. Jabri Karoui., Brahim,M. Characterization Of Bioactive Compounds In
Tunisian Bitter Orange (Citrus aurantium L.) Peel and Juice and
Determination Or Their Antioxidant Activities. BioMed Research
International. 2013;1.(1):1-13.
14. Dr. AP. Bangun, MHA. Jus Buah dan Sayuran untuk Mengatasi Kanker.
Jakarta: Agromedia; 2009.
15. Wu, GA., Terol, J., Ibanez, V., et all. Genomics Of The Origin and Evolution
Of Citrus. Research Article. 2018; vol 554 : 311-316.
 REVIEW JURNAL

Judul : Fractionation of Phenolic and Flavonoid Compounds

from Kaffir Lime (Citrus hystrix) Peel Extract and
Evaluation of Antioxidant Activity.
Nama jurnal : Ejurnal undip
Volume dan halaman : Vol. 17 No.3 Hal 111-117
Tahun : 2017
Penulis : Yusak Adi Wijaya, Daniel Widyadinata, Wenny Irawaty,
and Aning Ayucitra
Reviewer : Indri Istiqomah (1811012014)
Tujuan Penelitian : untuk memisahkan fenolik dan senyawa flavonoid dari
ekstrak kasar kaffir kulit jeruk purut dengan pelarut yang
memiliki polaritas berbeda, khusus dengan pelarut heksana,
etil asetat, dan n-butanol.
Objek penelitian : Kulit Jeruk purut (Citrus hystrix)
Metode Penelitian :
• Persiapan sampel
Jeruk purut dibeli di pasar Keputran tanpa kerusakan mikroba yang diamati. Buah
jeruk purut dicuci dengan air, kulitnya dikupas dan dipotong kecil-kecil dengan
ukuran 0,5 cm. Kulit jeruk purut dikeringkan dalam oven 35oC dan disimpan
dalam wadah ketat.
• Ekstraksi dan fraksinasi
Kulit jeruk purut diektraksi dengan etanol menggunakan metode maserasi
dengan rasio 1:40 dalan 8 jam pada suhu kamar kondisi gelap. Bagian padat
kemudian dipisahkan dan disaring melalui whatman no 1 ketas saring. Filtrat
dipekatkan dengan rotary evaporator pada 45oC. ekstrak minyak mentah
digunakan untuk fraksinasi selanjutnya. Rasio volume ektrak kasar dan pelarut
1:1. Fraksi heksana, etil asetat, dan n-butanol dikumpulkan secara individual dan
dikeringkan dibawah vakum 45 oC, sedangkan residu disimpan tanpa perawatan
lebih lanjut.
• Penentuan total flavonoid
Metode kalorometri aluminium klorida yang dipilih unruk menentukan total
flavonoid. 0,4 ml sampel (ekstrak kasar, fraksi, atau residu) ditambahkan ke 1,8
ml metanol, 2 ml 0,01 M larutan aluminium klorida. Setelah 30 menit masa
inkubasi dalam kondisi gelap, adsorbansi diukur dengan 431 nm terhadap
metanol kosong menggunakan spektrofotometer. Total flavonoid dinyatakan
sebagai Rutin Ekuivalen (RE)/ mg sampel.
• Identifikasi senyawa fenolik dan flavonoid oleh HPLC
 Senyawa fenolik terdeteksi menggunakan jasco HPLC (UV-2077 Plus)
digabung dengan detektor UV. Identfikasi fenolik dan flavonoid dicapai dengan
membandingkan Rf dlm sampel standar 280 nm. Beberapa senyawa standar
untuk mengidentifikasi flavonoid yaitu naringin, hesperidin, hesperetin, dan
nobiletin yg umumnya ditemukan pada buah jeruk.

Hasil Penelitian :
Pada total fenolik, Urutan fenolat dlm fraksi adalah etil asetat>fraksi n-
butanol>residu>heksana. Pada total flavonoid, fraksi etil asetat jumlah flavonoid
tertinggi diikuti n-butanol dn heksan. Pada identifikasi fenolik oleh HPLC,
Senyawa polaritas rendah quercerin tidak terdeteksi dlm pelarut heksana dan etil
asetat, tapi terdeteksi dlm n-butanol. Pada identifikai flavonoid oleh HPLC
didapat 4 senyawa yang diidentifikasi sebagai hesperidin, naringenin, dan
nobiletin. Nobiletin dalam fraksi heksana dan etil asetat dan naringenin sebagian
kecil di etil asetat.

Kesimpulan :
Polaritas pelarut yg digunakan fraksinasi telah memisahkan fenolik dan
senyawa flavonoid dalam kulit jeruk purut. Kandungan fenolik dan flavonoid
tinggi tidak menjamin aktivitas pembersihan yang lebih tinggi terhadap senyawa
radikal bebas yang sama. Berdasarkan jumlah total fenolik dan flavonoid yang
bisa diekstraksi dari ekstrak kulit jeruk purut dengan etil asetat sebagai pelarut
terbaik.
   p-ISSN 0852 – 0798
e-ISSN 2407 – 5973

Terakreditasi: SK No.: 60/E/KPT/2017

Website : http://ejournal.undip.ac.id/index.php/reaktor/

Reaktor, Vol. 17 No. 3, September Tahun 2017, Hal. 111-117

Fractionation of Phenolic and Flavonoid Compounds from Kaffir

Lime (Citrus hystrix) Peel Extract and Evaluation of Antioxidant
Activity

Yusak Adi Wijaya, Daniel Widyadinata, Wenny Irawaty*), and Aning Ayucitra
Department of Chemical Engineering, Faculty of Engineering, Universitas Katolik Widya Mandala, Surabaya
Jl. Kalijudan 37 Surabaya, Indonesia 60114
Telp./Fax. (031)3891264 / (031)3891267
*)
Coresponding author: wenny_i_s@ukwms.ac.id

Abstract

The observation of side effects during drug consumption results a numerous research to search natural
antioxidant such as kaffir lime which has not been utilized. Fruit peel has been reported to exhibit
higher antioxidant content than the edible part and therefore, kaffir lime peel was selected in this study.
This work was aimed to investigate the effect of solvent polarity (hexane, ethyl acetate and n-butanol)
employed during fractionation of ethanolic crude extract and assess its antioxidative activity to
neutralize DPPH (2,2-diphenyl-1-picrylhydrazyl) radical. The results show the employment of solvents
possessing different polarity resulted fractions of hexane, ethyl acetate and n-butanol with different
phenolic and flavonoid amount in each fraction. Semi polar solvent of ethyl acetate performed as the
best solvent with total phenolic and flavonoid content was detected 0.1157 mg Gallic Acid
Equivalent/mg and 0.1147 mg Rutin Equivalent/mg, respectively. Accordingly, each fraction exhibited
different antioxidant activity against DPPH. N-butanol fraction demonstrated the strongest antioxidant
activity. Phenolics and flavonoids identification by HPLC (High Performance Liquid Chromatography)
indicate the presence different phenolics and flavonoid compounds in each fraction that contribute to
antioxidant activity to different extents.

Keywords: antioxidant; fractionation; flavonoid; kaffir lime (Citrus hystrix); peel; phenolic

Abstrak

FRAKSINASI SENYAWA FENOLIK DAN FLAVONOID DARI EKSTRAK KULIT JERUK

PURUT (Citrus hystrix) DAN EVALUASI AKTIVITAS ANTIOKSIDAN. Efek negatif dari
konsumsi obat-obatan menyebabkan banyaknya penelitian untuk mencari antioksidan alami seperti
jeruk purut yang selama ini belum dimanfaatkan secara maksimal. Kandungan antioksidan kulit buah
dilaporkan lebih tinggi daripada bagian buah sehingga kulit jeruk purut dipilih pada penelitian ini.
Tujuan penelitian ini adalah mempelajari pengaruh polaritas pelarut (heksana, etil asetat dan n-
butanol) pada proses fraksinasi ekstrak kulit jeruk purut terhadap perolehan total fenolik dan flavonoid
serta mempelajari kemampuan antioksidannya dalam menetralkan radikal bebas DPPH (2,2-diphenyl-
1-picrylhydrazyl). Hasil penelitian menunjukkan bahwa penggunaan pelarut dengan polaritas berbeda
menghasilkan fraksi heksana, etil asetat dan n-butanol dengan kandungan fenolik dan flavonoid
tertentu di setiap fraksinya. Pelarut dengan polaritas sedang yaitu etil asetat merupakan pelarut terbaik
dimana kandungan total fenolik dan flavonoidnya masing-masing 0,1157 mg Gallic Acid Equivalent/mg
dan 0,1147 mg Rutin Equivalent/mg. Akibatnya, tiap fraksi mempunyai aktivitas antioksidan yang
berbeda di dalam menetralkan DPPH. Fraksi n-butanol mempunyai aktivitas antioksidan tertinggi.

111
Fractionation of Phenolic and Flavonoid ... (Wijaya et al.)

Identifikasi senyawa fenolik dan flavonoid dengan menggunakan HPLC (High Performance Liquid
Chromatography) menunjukkan bahwa di dalam masing-masing fraksi terdapat senyawa fenolik dan
flavonoid yang berbeda yang menyebabkan kemampuan antioksidan dari masing-masing fraksi juga
berbeda.

Kata kunci: antioksidan; fraksinasi; flavonoid; jeruk purut (Citrus hystrix); kulit; fenolik

How to Cite This Article: Wijaya, Y.A., Widyadinata, D., Irawaty, W., and Ayucitra, A., (2017), Fractionation of
Phenolic and Flavonoid Compounds from Kaffir Lime (Citrus hystrix) Peel Extract and Evaluation of Antioxidant
Activity, Reaktor, 17(3), 111-117, http://dx.doi.org/10.14710/reaktor.17.3.111-117

INTRODUCTION conditions which in turn influence secondary

Drugs consumption during disease treatment
may promote side effects and therefore, the
requirement to design alternative treatment from
natural product such as herbal medicine or botanicals is
essential. Plants contain phenolic compounds that
experimentally exhibited properties of anti
inflammatory (Menichini et al., 2011), anti microbial
(Yi et al., 2008), anti adipogenesis (Kim et al., 2012a),
and anti diabetic (Kim et al., 2009). All parts of plant
such as fruit, seed, leave and root contain phenolic
compounds to different extents (Kondo et al., 2002;
Olabinri et al., 2009). In fruit itself, part of fruit such as
skin, flesh and seed have been reported to have
different amount of phenolic compounds. Accordingly,
they have different antioxidant activity.
Citrus has been well known for health benefits
since the fruit contains a lot of vitamin, antioxidant
compounds and others. Accordingly, citrus is potential
to be further developed as natural antioxidant,
especially for degenerative diseases which has been
focused in recent years. Kaffir lime (Citrus hystrix) is a
type of citrus fruits that possesses high content of
phenolic compounds. In kaffir lime tree, only leaves
have been used as spiced-citrus flavor. In several
studies, fruit peel has been reported to exhibit higher
antioxidant activity compared to the edible part
(Gorinstein et al., 2002). Thus, the investigation of peel
as natural antioxidant is another challenge for treating
degenerative diseases (Garg et al., 2001; Irawaty et al.,
2014).
Even though investigations on kaffir lime peel
has been performed previously to obtain optimum
extraction conditions (Chan et al., 2009; Irawaty et al.,
2014) and the extract has been explored for several
applications (Irawaty et al., 2014; Jamilah et al., 2007;
Putri et al., 2013), but it is still limited to the usage of
crude extract. Further treatment on crude extract such
as fractionation step will provide deep insight of the
ability of fractions to neutralize free radicals which the
latter is responsible for degenerative diseases (diabetes,
organ inflammation, etc.) and this has not been reported
previously. Solvents possess different polarity will be
employed to separate phenolic compounds based on
like dissolves like principle. As a result, each fraction
will exhibit radical antioxidative activity that allows its
application to treat certain disease. The antioxidant
capability of extract from peel/leaves/seed/flesh is
different due to various factors, namely climate,
variety, soil, degree of maturation and environment
metabolites and phenolic compounds (Baniasadi et al.,
2014; Gebruers et al., 2010; Shewry et al., 2010).
Hence, the present study attempts to separate phenolic
and flavonoid compounds from crude extract of kaffir
lime peel with solvents possess different polarity,
specifically hexane, ethyl acetate and n-butanol with
relative polarity values of 0.009, 0.228, and 0.586,
respectively (Reichardt, 2003). Fractions were
prepared using successive solvents of varying polarity,
followed by assessment of antioxidant activity towards
the neutralization of free radical compound.
MATERIALS AND METHODS
Sample Preparation
Kaffir lime was purchased at Keputran market
(Surabaya, Indonesia) with no obvious physical or
microbial damage was observed. Kaffir lime fruit was
washed with tap water, peeled off sharply to collect the
peel which further cut into small pieces with a size of
approximately 0.5 cm. The kaffir lime peel were dried
in an oven at 35oC and kept in tight container for future
use.
Extraction and Fractionation
Kaffir lime peel was extracted with ethanol
using maceration method with a ratio of 1:40 (w/v) for 8
h at room temperature in dark condition. The solid part
was then separated and filtered through a Whatman No.
1 filter paper. The filtrate was concentrated with a
rotary evaporator (IKA RV 10) at 45°C. The crude
extract was further used in fractionation step. The crude
extract obtained was fractionated with solvents as
shown in Figure 1. Volume ratio of crude extract and
solvent was 1:1. The fractions of hexane, ethyl acetate,
and n-butanol were collected individually and dried
under vacuum below 45°C, while the residue was kept
without any further treatment. The three fractions and
residue were subjected for analysis to determine total
phenolic content, total flavonoid content, antioxidant
activity and identification of phenolic and flavonoid
compounds using HLPC (High Performance Liquid
Chromatography).
Determination of Total Phenolic Content
The amount of total phenolics was determined
according to the Folin-Ciocalteu colorimetric method
(Anagnostopoulou et al., 2006) with slight
modification. Briefly, 0.2 mL sample (crude extract,
fraction, or residue) was mixed with 1 mL Folin-

112
 Reaktor 17(3) 2017: 111-117
Ciocalteu’s reagent (1:1) and 3 mL Na2CO3 solution
(7.5%). The mixture was incubated at room
temperature for 30 min in dark condition. The
absorbance was subsequently measured at 730 nm
using a spectrophotometer (Shimadzu, UVmini-1240).
Total phenolic content was expressed as Gallic Acid
Equivalent (GAE)/mg sample, which is a common
reference compound.
Dry
peels
Crude
extract
Hexane
Hexane Ethyl acetate
fraction
Ethyl acetate
n-Butanol
fraction
n-Butanol
Residue
fraction
Figure 1. Schematic diagram for fractionation of crude
extract
Determination of Total Flavonoid Content
Aluminum chloride colorimetric method was
selected for determining total flavonoid content (Wu et
al., 2009). 0.4 mL of sample (crude extract, fraction, or
residue) was added to 1.8 mL methanol, 2 mL 0.01 M
aluminum chloride solution and the mixture was well
mixed. After 30 min of incubation period in dark
condition, the absorbance was measured at 431 nm
against a blank of methanol using a spectrophotometer
(Shimadzu, UVmini-1240). Total flavonoid content
was expressed as Rutin Equivalent (RE)/ mg sample.
Antioxidant Activity by DPPH Method
Radical scavenging activity of sample to
neutralize DPPH was measured according to (Liu et al.,
2011) with modification. Basically, 1.25 mL DPPH
solution and 1 mL sample were mixed and incubated at
room temperature for 30 min. Afterward, the
absorbance of the mixture was measured using a
spectrophotometer at 510 nm. For control, the
procedure was repeated by using DPPH solution only
without any sample. The antioxidant activity of the
sample was measured as a decrease in the absorbance
and calculated by using equation (1).
 A 
Radical s cavenging activity (%) =  1 - 1  x100% (1)
 A 
 0
where A1: absorbance of sample; A0: absorbance of
control.
In this study, antioxidant activity was expressed
as IC50 which is defined as the concentration of sample
required to scavenge the DPPH free radicals by 50%.
IC50 was obtained by linear regression analysis of dose-
response curve plotting between % inhibition and
concentrations.
Phenolics and Flavonoids Identification by HPLC
The presence of phenolic compounds were
detected using a Jasco HPLC (UV-2077 Plus) coupled
with a UV detector. Phenolics separation was achieved
on a C18 column and a mixture of acetic acid solution
(3%) and methanol was employed as the mobile phase
to facilitate the separation with total flow rate was 1 mL
min–1. By using the same column, the mobile phase for
flavonoids separation were acetonitrile, water and
acetic run with a rate of 1 mL.min–1. Identification of
phenolics and flavonoids was accomplished by
comparing the retention times of peaks in samples to
those of standards at 280 nm. Several standard
compounds employed to identify phenolic compounds
were gallic acid, catechin, caffeic acid, epicatechin, p-
coumaric acid, ferulic acid, rutin and quercetin.
Naringin, hesperidin, naringenin, hesperetin and
nobiletin were used for flavonoids identification. Those
compounds are generally found in citrus fruits.
RESULTS AND DISCUSSION
Total Phenolic Content
Phenolics are a major class of bioactive
compounds in plants that has been known as ideal
chemistry for free radical scavenging activity due to the
presence of high activity as hydrogen or electron donor.
Total Phenolic Content (TPC) in crude extract,
fractions (hexane, ethyl acetate, n-butanol) and residue
are displayed in Figure 2.
0.4
0.318
TPC (mg GAE/mg)
0.3
0.2
0.1157
0.0808
0.1
0.0372
0.015
0
Figure 2. Total phenolic content of crude extract,
fractions and residue
As seen, phenolic content detected in each
sample was varied according to type of solvent
113
Fractionation of Phenolic and Flavonoid ...
employed during fractionation. Whilst hexane fraction
possessed the least amount of phenolics with TPC value
is 0.015 mg GAE/mg, ethyl acetate exhibited the
highest fraction with TPC value was observed 0.12 mg
GAE/mg. N-butanol solvent gave lower TPC with
approximately 0.08 mg GAE was detected in each mg
of N-butanol fraction. Residue contains phenolic
compounds which were not taken by hexane, ethyl
acetate and n-butanol with TPC value was observed
approximately 0.4 mg GAE/mg. Crude extract,
however, shown the highest amount of TPC (0.32 mg
GAE/mg) since the phenolic compounds have not been
fractioned by solvents.
The results (Figure 2) show how the solvent
polarity influences type of phenolic compounds can be
extracted based on ‘like and dislike property’ (Patel et
al., 2011; Widyawati et al., 2014). The sequence of the
fractions and residue based on TPC detected are ethyl
asetate fraction > n-butanol fraction > residue > hexane
fraction. This indicates that phenolic compounds in
crude extract prefer to be extracted with solvent
possessing moderate polarity degree (semi-polar) such
as ethyl acetate and n-butanol than solvents with strong
polarity (water) or low polarity (hexane) (Zhao and
Hall, 2008). The highest TPC value observed in the
fraction of ethyl acetate indicates that major phenolic
compounds in kaffir lime peel were semi polar
compounds. The capability of ethyl acetate to extract
more phenolics compared to other solvents was also
reported by other group (Anagnostopoulou et al.,
2006). Complex formation among phenolics may
facilitate the extraction of the phenolic compounds in
ethyl acetate medium has been suggested in literature
(Zhu et al., 2011).
Total Flavonoid Content
Flavonoids are a noticeable group of secondary
metabolites in citrus fruits that may have beneficial
effects on human health (Kim et al., 2012b; Lee et al.,
2007; Yao et al., 2004). Total flavonoid contents of
crude extract, fractions and residue are shown in Figure
3. The results show the total flavonoid contents varied
from 0.0011 to 0.206 mg RE/mg. Among the fractions,
ethyl acetate contained the highest amount of flavonoid
0.25
0.206
TFC (mg RE/mg)
0.2
0.15 0.1147
0.1
0.05 0.0174
0.0011
0 0
Figure 3. Total flavonoid content of crude extract,
fractions and residue
114
(Wijaya et al.)
content, followed by n-butanol and hexane fractions.
This finding indicated that higher flavonoid content is
associated with higher total phenolic content of the
fraction.
Free Radical Scavenging Activity
In this study, scavenging activity of crude
extract and fractions (hexane, ethyl acetate, n-butanol
and residue) were tested on free radical DPPH. The
ability of the sample to neutralize DPPH was expressed
as IC50. IC50 is the sample concentration required to
inhibit the DPPH neutralization by 50%. Accordingly,
the lower IC50 value, the stronger ability of the sample
to quench DPPH. Figure 4 shows the antioxidant
activity of crude extract, fractions and residue
performed on DPPH.
As seen in Figure 4, crude extract exhibited the
lowest IC50 that around 0.09 mg of crude extract is
necessary to inhibit DPPH neutralization by 50%.
Following the crude extract, the n-butanol fraction
showed the second lowest position to neutralize the
same DPPH solution with IC50 is reported around 0.44
mg/mL. Ethyl acetate fraction, residue, and hexane
fraction have IC50 values of 1.5443, 5.3644 and
53.8386 mg/mL, respectively. Ascorbic acid was
selected as positive control. Since sample with lower
IC50 value means that it has higher antioxidant activity
to neutralize DPPH, thus the sequences of the four
fractions based on their antioxidant activity are as
follows: n-butanol fraction > ethyl acetate > residue >
hexane. Looking at the TPC values of ethyl acetate and
hexane fractions (Figure 2), higher TPC value exhibited
by ethyl acetate fraction may promise lower IC50 since
more phenolic compounds are available to neutralize
DPPH. Our study, however, observed higher IC50 in the
fraction of ethyl acetate. This finding leads to the
assumption that whilst ethyl acetate was able to extract
more phenolic compounds, not all compounds
performed well against DPPH. A reason to explain this
because each phenolic compound will exhibit particular
activity or selectivity to react with specific target (Islam
et al., 2016; Lau et al., 2015; Liu et al., 2017). Further
identification of phenolic compounds by instruments
such as HPLC may explain this.
60 53.8386
50
IC50 (mg/mL)
40
30
20
10 5.3644
0.0061 1.5443 0.4424
0.0904 0.1700
Figure 4. IC50 of crude extract, fractions and residue to
neutralize DPPH. Ascorbic acid was selected as
positive control
 Reaktor 17(3) 2017: 111-117

Table 1. Phenolic compounds detected in crude extract, fractions and residue

Gallic Caffeic p-Coumaric Ferulic
Sample Catechin Epicatechin Rutin Quercetin
acid acid acid acid
Crude extract + + + – – + + –
Hexane fraction – – – – – – – +
Ethyl acetate fraction – – – – – – + +
N-butanol fraction – – + + + + – –
Residue + – + + + + + +
+ detected, – not detected

Table 2. Flavonoid compounds detected in crude extract, fractions and residue

Sample Naringin Hesperidin Naringenin Hesperetin Nobiletin
Crude extract – + + + +
Hexane fraction – – – – +
Ethyl acetate fraction – – + – +
N-butanol fraction + – – – –
Residue – – – – –
+ detected, – not detected

Phenolics Identification by HPLC identified as hesperidin, naringenin, hesperetin and

Different phenolic contents in each sample
(crude extract and fractions) resulted different total
phenolic content observed in this study as well as its
ability to quench DPPH as shown in Figures 2 and 3.
Therefore, crude extract and the four fractions have
been subjected for HPLC analysis to identify the
phenolic compounds and the result is shown in Table 1.
It is shown in Table 1 that phenolic compounds
distributed in crude extract and each fraction with
different extents. The polarity of solvent used in the
fractionation step influences the distribution of
phenolic compounds (Irawaty et al., 2014). Phenolic
compound with relative low polarity degree such as
quercetin was detected in low-medium solvents, i.e.
hexane and ethyl acetate. However, the same
compound was not detected in crude extract. Similar
observation was also noted for epicatechin and p-
coumaric acid that the two compounds were not
detected in the crude extract but presented in n-butanol
fraction and residue (Table 1). In addition to quercetin,
rutin was detected in the fraction of n-butanol but not
in ethyl acetate fraction. The presence of rutin in the n-
butanol fraction may facilitate the quenching process of
DPPH rather than quercetin since we observed that n-
butanol fraction exhibited higher activity to neutralize
DPPH. Without quantification analysis performed on
the sample, however, this cannot be further explaining
our observation. The stability of phenolic compounds
against oxidation process may also contribute to the
observation phenolic compounds in the four fractions
as detected in this study. For example, catechin was
initially detected in the crude extract and after
fractionation, none of the fractions contained this
compound. The poor oxidative stability of catechin
reported in literature may explain this observation
(Gadkari and Balaraman, 2015).
Flavonoids Identification by HPLC
The presence of flavonoid compounds was
detected by HPLC and the results are tabulated in Table
2. Among five flavonoid standards employed, crude
extract was found to have four compounds which were
nobiletin. Fractionation step performed on the crude
extract has distributed flavonoids into the four fractions
with different extents. For example, nobiletin was
detected in the fractions of hexane and ethyl acetate.
Naringenin was only found in the fraction of ethyl
acetate. On the other hand, both hesperidin and
hesperetin were not noticed in the fractions which the
compounds were originally present in the crude extract.
CONCLUSION
Solvent polarity employed during fractionation
stage has facilitated the separation of phenolic and
flavonoid compounds present in kaffir lime peel.
Different classes of phenolic and/or flavonoid
compounds are capable to supply scavenging activity
against DPPH to different extents. Although higher
total phenolic and flavonoid contents were detected in
the fraction of ethyl acetate than n-butanol, our results
showed, unexpectedly, the fraction of n-butanol
exhibited higher scavenging activity toward DPPH.
This inconsistency may be due that certain phenolic
compound has the ability to react with particular radical
compound (DPPH was selected in this study) and/or the
concentration of each phenolic and flavonoid
compound in the fractions will also important which the
latter was not determined in this study. Accordingly,
higher total phenolic and flavonoid content in a sample
did not guarantee higher scavenging activity against the
same radical compound. Further investigation is
necessary to confirm this. Based on the amount of total
phenolic and flavonoid compounds which can be
extracted from crude extract of kaffir lime peel, ethyl
acetate was observed as the best solvent.
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