Makalah Dan Sumber Referensi PSM

MAKALAH PENENTUAN STRUKTUR MOLEKUL
Penentuan Struktur Molekul Apigenin, Luteolin, dan Myricetine

dengan Menggunakan Spektroskopi UV-Visible
Dosen Pengampu : Drs. Dede Sukandar, M.Si
Disusun Oleh:
Kelompok 2
Tiara Hadi Ashari 11180960000097
Seto Muhammad Adli 11180960000102
Risma Mutiara Pertiwi 11180960000105
PROGRAM STUDI KIMIA
FAKULTAS SAINS DAN TEKNOLOGI
UNIVERSITAS ISLAM NEGERI SYARIF HIDAYATULLAH

JAKARTA
2021
KATA PENGANTAR
Segala Puji dan Syukur penulis panjatkan atas kehadirat Allah S.W.T. yang
melimpahkan segala nikmat dan karunia Nya. Sehingga kami dapat menyelesaikan
tugas mata kuliah Penentuan Struktur Molekul ini dengan baik. Makalah ini dibuat
untuk memenuhi tugas mata kuliah Penentuan Struktur Molekul, dan pada kesempatan
ini pula penulis mengucapkan terima kasih kepada Bapak Drs. Dede Sukandar, M.Si
selaku Dosen Pengajar mata kuliah Penentuan Struktur Molekul.
Apabila di dalam penulisan laporan ini teradapat banyak kesalahan atau

kekurangan baik kelalaian maupun ketidaksengajaan, maka kami selaku penulis
memohon maaf atas berbagai hal tersebut. Penulis secara terbuka menerima segala saran
dan kritik yang membangun. Akhirnya dengan ini penulis berharap semoga laporan ini
dapat bermanfaat untuk penulis pada khususnya dan kalangan pembaca pada umumnya.
Tim Penulis
Ciputat, Maret 2021
ii
DAFTAR ISI
KATA PENGANTAR .......................................................................................................ii

DAFTAR ISI ...................................................................................................................... iii
BAB 1 PENDAHULUAN ..................................................................................................5
1.1 Latar Belakang ......................................................................................................5
1.2 Rumusan Masalah .................................................................................................6
1.3 Tujuan..................................................................................................................... 6
BAB II PEMBAHASAN ...................................................................................................7
2.1 Tanaman Sage (Savia Officinalis. L.) ....................................................................7
2.2 Senyawa Apigenin ...................................................................................................8
2.3 Senyawa Luteolin ....................................................................................................9
2.4 Senyawa Myricetine ................................................................................................ 10
2.5 Spektroskopi UV-Visible ........................................................................................ 11
BAB III METODOLOGI PERCOBAAN .......................................................................17
3.1 Alat dan Bahan........................................................................................................17
3.1.1 Alat .................................................................................................................17
3.1.2 Bahan .............................................................................................................17
3.2 Diagram Alir ...........................................................................................................17
3.2.1 Isolasi untuk Senyawa Apigenin dan Luteolin ...........................................17
3.2.2 Penentuan Struktur Senyawa Apigenin pada Zat I ..................................18
3.2.3 Penentuan Struktur Senyawa Luteolin pada Zat II ..................................18
3.2.4 Penentuan Struktur Senyawa Myricetine pada Zat III ............................ 19
3.3 Prosedur percobaan ................................................................................................ 19
3.3.1 Isolasi untuk Senyawa Apigenin dan Luteolin ...........................................19
3.3.2 Penentuan Struktur Senyawa Apigenin pada Zat I ..................................20
3.3.3 Penentuan Struktur Senyawa Luteolin pada Zat II ..................................20
3.3.4 Penentuan Struktur Senyawa Myricetine pada Zat III ............................ 20
BAB IV HASIL DAN PEMBAHASAN ...........................................................................22
4.1 Penentuan Struktur Molekul Apigenin (Đorđević, Cakić, and Amr,
2000) ........................................................................................................................ 25
iii
4.2 Penentuan Struktur Molekul Luteolin (Đorđević et al. 2000) ..................... 26
4.3 Penentuan Struktur Myricetine (Jomová et al., 2019) .................................27
BAB V PENUTUP .............................................................................................................30
5.1 Kesimpulan .........................................................................................................30
DAFTAR PUSTAKA ........................................................................................................31
iv
BAB I
PENDAHULAN
1.1 Latar Belakang

Spektrofotometri adalah suatu metode yang digunakan untuk mengukur
absorbansi dari suatu bahan terhadap spektrum sinar sebagai fungsi pada panjang
gelombang tertentu. Bila suatu zat disinari dengan elektromagnet, zat ini menyerap
panjang-panjang gelombang tertentu dari radiasi dan membiarkan
gelombanggelombang yang lain lewat, pola panjang gelombang yang diserap suatu zat
disebut spektrum absorpsi. Spektrum absopsi untuk senyawa molekul disebut
Spektrofotometri karena biasanya spektro itu terdiri dari daerah-daerah resapan yang
lebar. Salah satu alasan mengapa deteksi spektroskopi itu begitu berguna adalah bahwa
biasanya diperlukan hanya sedikit sekali sampel untuk pengujian zat tersebut
(Alamsyah, 2007).
Spektroskopi digunakan dalam banyak industri untuk menganalisis bahan baku
dan produk. Spektrofotometer Ultraviolet dan Visibel merupakan hasil transisi
elektronik yang disebabkan oleh foton berenergi. Foton dengan energi yang cukup
tinggi (lebih khas dalam daerah UV) dapat mengionkan atom dan molekul. Absorpsi
foton UV dan Vis tanpa pemutusan ikatan memberikan informasi mengenai susunan
dan struktur elektronik dari molekul-molekul. Spektrum didaerah sinar tampak (tampak
bagi mata manusia) sama dengan gelombang cahaya 400-800nm. Cahaya didaerah UV
mempunyai panjang gelombang yang lebih pendek yaitu 200-400nm (Departemen
Kesehatan Republik Indonesia, 1995).
Pada pengukuran serapan suatu larutan hampir selalu digunakan blanko yang
dipakai untuk mengatur spektrofotometer, hingga pada panjang gelombang pengukuran
mempunyai serapan nol. Maksud dari blanko tersebut adalah koreksi serapan yang
disebabkan oleh pelarut, pereaksi, sel atau pengukuran alat, blanko yang digunakan
adalah blanko untuk yang melarutkan zat terhadap larutan zat pembanding kimia yang
disiapkan dengan cara yang sama, dalam hal ini pengukuran serapan mula-mula
dilakukan terhadap pembanding kemudian terhadap larutan zat yang diperiksa (Day dan
Underwood, 1999).
1.2 Rumusan Masalah
1. Bagaimana cara menentukan struktur molekul pada senyawa apigenin
dengan menggunakan Spktroskopi UV-Visible?
2. Bagaimana cara menentukan struktur molekul pada senyawa luteolin
dengan menggunakan Spktroskopi UV-Visible?
3. Bagaimana cara menentukan struktur molekul pada senyawa myricetine
dengan menggunakan Spektroskopi UV-Visible?
1.3 Tujuan
1. Mampu mengetahui cara menentukan struktur molekul pada senyawa
apigenin dengan menggunakan Spktroskopi UV-Visible?
luteolin dengan menggunakan Spktroskopi UV-Visible?
myricetine dengan menggunakan Spktroskopi UV-Visible?
6
BAB II
TINJAUAN PUSTAKA
2.1 Tanaman Sage (Savia Officinalis. L)

Sage (Salvia Officinalis. L) merupakan tanaman herbal yang belum banyak
dikenal dan dimanfaatkan di Indonesia untuk alternatif pengobatan. Di Eropa dan Cina,
ekstrak dan minyak esensial tanaman sage telah banyak digunakan untuk berbagai
aplikasi seperti makanan, kosmetik, maupun keperluan industri farmasi (Stammati et
al., 1999). Secara tradisional, sage telah banyak digunakan untuk pengobatan seperti
penanganan pada kasus keradangan pada mulut dan tenggorokan (Baricevic et al.,
2001). Selain itu, sage juga memiliki kemapuan antimutagenik dan untuk penanganan
kanker (Craig, 1999; Simic et al., 2000; Knezevic-Vukcevic et al., 2001). Sage memiliki
efek antibakterial, fungisidal, virustatik dan astrigensia. Asam phenolic seperti salvin
dan salvin monometyl ether yang diisolasi dari Sage diduga memiliki aktivitas
antimikrobial khususnya dalam melawan infeksi Staphylococcus aureus (Dragana et
al., 2005).
Gambar 1. Daun Sage

Sage (Salvia officinalis L.) adalah tanaman obat yang bernilai tinggi dan banyak
digunakan dalam pengobatan tradisional karena aktivitas antioksidan, antibakteri,
antijamur, antiinflamasi, antiviral, dan antialergi (Dragan et al., 2007). Adanya
kandungan vitamin A, vitamin C, vitamin E, flavonoid, saponin, dan zink diduga
berperan sebagai senyawa yang mampu mempercepat proses penyembuhan luka.
Ekstrak etanol daun sage konsentrasi 50% bersifat antibakteri melalui mekanisme
penghambatan kerja enzim bakteri sehingga efektif untuk mencegah aktivitas
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kolagenolitik dari Porphyromonas gingivalis (Barnes et al., 2002). Menurut (BHMA,
1996) aplikasi topikal ekstrak 50% daun sage menunjukkan aktivitas antiinflamasi yang
kuat. (Zupko et al., 2001) menambahkan bahwa ekstrak 50% daun sage dapat
menghambat aktivitas enzim lipid peroksidase karena kandungan asam rosmarin yang
bersifat antioksidan.
2.2 Senyawa Apigenin

Apigenin merupakan flavonoid yang bersifat non-toksik yang banyak terdapat
pada tanaman sayur, terutama di peterseli, seledri, buah zaitun, dan rempah-rempah,
yang dapat memblokir ekspresi COX-2 (cyclooxygenase) pada peradangan. Apigenin
juga berfungsi sebagai anti-apoptosis terutama menghambat c-FLIP sebagai regulator
apoptosis yang berperan penting sebagai fungsi pemeliharaan dan kekebalan pada sel
peradangan dan juga autoimunitas. Apigenin bersifat vasorelaksan (penurun tekanan
darah pada pembuluh darah), anti-platelet (menghambat agregasi platelet), dan
antioksidan, yang dapat mengurangi risiko penyakit koroner dan meningkatkan fungsi
endotel (Kang et al., 2009). Adapaun bentuk struktur senyawa apigenin, sebagai berikut:
Gambar 2. Struktur Senyawa Apigenin

Apigenin berfungsi seperti beta blocker, yaitu memperlambat detak jantung dan
menurunkan kekuatan kontraksi jantung sehingga aliran darah yang terpompa lebih
sedikit dan tekanan darah menjadi berkurang. Manitol dan apiin, bersifat diuretik yaitu
membantu ginjal mengeluarkan kelebihan cairan dan garam dari dalam tubuh, sehingga
berkurangnya cairan dalam darah akan menurunkan tekanan darah (Smeltzer, 2008).
Apigenin yang terkandung dapat bermanfaat untuk mencegah penyempitan
pembuluh darah sehingga membuat pembuluh darah rileks. Kandungan itulah yang
mengatur aliran darah sehingga pembuluh darah membesar dan mengurangi tekanan
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darah (Goodman, 2008). Apigenin berkhasiat sebagai peluruh air seni, obat rematik,
serta penurun tekanan darah tinggi (Putra, 2013).
2.3 Senyawa Luteolin

Luteolin (3‟,4‟,5,7-tetrahidroksiflavon) merupakan senyawa polifenol golongan
flavon yang terdapat dalam jumlah yang berlimpah di dalam lada hijau, perilla, seledri,
dan teh chamomile. Penelitian sebelumnya telah menunjukkan efek kemopreventive
kanker dari luteolin.Luteolin dapat menginduksi terjadinya apoptosis pada banyak jenis
sel kanker. Luteolin juga mampu menghambat berbagai jenis rangsangan yang diinduksi
oleh ekspresi gen proinflamatori dan senyawa kmia penginduksi karsinogenesis
payudara, kolon dan kulit (Lin et al., 2008). Adapun bentuk struktur senyawa luteolin,
sebagai berikut:
Gambar 3. Struktur Senyawa Luteolin

Luteolin dalam bentuk glikosida dapat menghambat aktivitas mediasi
transcriptional Hh/GLI1 dengan IC50 pada konsentrasi 0,5 µM, senyawa tersebut juga
bersifat sitotoksik terhadap sel PANC1 (IC50 = 0,7 µM) dan sel DU145 (IC50 = 0,8
µM) (Rifai et al., 2011), Hh/GLI1 signaling pathway merupakan jalur signal yang
berperan dalam pertumbuhan dan perkembangan embrio serta pada pemeliharaan
jaringan dewasa.
Luteolin juga dapat menginduksi terjadinya apoptosis melalui penghambatan
enzim topoisomerase I dengan nilai IC50 = 5 µM (Chowdhury et al., 2002) dan dapat
menghambat jalur signal TNF–α pada konsentrasi 5 µM hingga 20 µM (Jong et al.,
2011), serta menghambat enzim 15-lipoksigenase dengan nilai IC50 = 0,6 µM (Sadik et
al., 2003).
9
Senyawa luteolin murni dapat diperoleh dengan cara ekstraksi dan isolasi senyawa
tersebut dari tanaman penghasilnya, seperti Vitex negundo, Tomentosa salvia dan
berbagai jenis tanaman lainnya (Chowdhury et al., 2002). Senyawa ini dapat bersifat
inhibitor signal Hedgehog pada konsentrasi < 0,5 µM (IC50) (DB et al., 2008).
Aktivitas ini perlu ditingkatkan menjadi skala nM untuk memperoleh efek terapi kanker
yang lebih baik lagi, oleh karena itu perlu dilakukan sintesis, senyawa derivat luteolin
yang memiliki aktivitas antikanker yang lebih baik daripada senyawa bahan alamnya.
2.4 Senyawa Myricetin

Senyawa fenolik ini sangat umum pada buah beri, sayuran, dan teh serta anggur
yang diproduksi dari berbagai tanaman. Myricetin ditemukan dalam bentuk bebas dan
terikat secara glikosidik, yang meliputi myricetin-3-O-(3"-acetyl)-α-L-
arabinopyranoside, myricetin-3-O-(4"-acetyl)-α-L-arabinopyranoside, myricetin-3-O-α-
L-rhamnopyranoside, myricetin-3-O-β-D-galactopyranoside, myricetin-3-O-(6"-
galloyl)-β-D-galactopyranoside, myricetin-3-O-β-D-xylopyranoside, myricetin 3-O-α-
L- arabinofuranoside (De Leo et al., 2006), myricetin-3-O-(2"-O-galloyl)-α-L-
rhamnoside, myricetin-3-O-(3"-O-galloyl)-α-L-rhamnoside, dan myricetin-3-O-α-L-
rhamnoside (Kong et al., 2014). Myricetin sulit larut dalam air, pada 16,6 µg / mL,
tetapi larut cepat ketika dideprotonasi dalam media berair dasar dan dalam beberapa
pelarut organik seperti dimetilformamida, dimetilasetamida, tetrahidrofuran dan aseton
(Yao et al., 2014). Selain itu, degradasi senyawa ini paling stabil pada pH 2, dan sangat
bergantung pada pH dan suhu. Adapun bentuk struktur senyawa myricetin, sebagai
berikut:
Gambar 4. Struktur Senyawa Myricetin
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Sejarah myricetin (Lau-cam et al., 1973) dimulai lebih dari seratus tahun.
Myricetin pertama kali diisolasi di akhir abad kedelapan belas dari kulit kayu Myrica
nagi Thunb. (Myricaceae), dipanen di India, sebagai cahaya kristal berwarna kuning
(Perkin et al., 1896). Isolasi terutama dipicu oleh minat pada properti pewarnaan dari
senyawa. Myricetin dicirikan dengan baik dalam studi lebih lanjut dari Perkin (Lin et
al., 2012), yang menetapkan peleburan titik 357 ᵒC dan disiapkan berbagai analog
bromo, metil, etil dan kalium. Laporan ini juga menjelaskan myricitrin (Jones et al.,
2011), sebuah myricetin glycoside (myricetin-3-O-rhamnoside), untuk pertama kalinya.
Dalam penelitian selanjutnya, (Perkin et al., 1896) menemukan bahwa myricetin
menghasilkan asam kloroglusinol dan galat pada hidrolisis, yang berfungsi untuk
memastikan struktur kimianya.
Myricetin secara struktural terkait dengan beberapa senyawa fenolik.
Nutraceuticals dan sifat anti-oksidan myricetin sangat dihargai, bukti ilmiah (Lin et al.,
2012) menggaris bawahi klaim tersebut saat senyawa tersebut menampilkan berbagai
aktivitas farmakologis, termasuk antiinflamasi, analgesik, aktivitas antitumor,
hepatoprotektif dan antidiabetes.
2.5 Spektrofotometer UV-Visible

Prinsip kerja Spektrofotometer UV-Vis, yaitu apabila cahaya monokromatik
melalui suatu media (larutan), maka sebagian cahaya tersebut diserap (I), sebagian
dipantulkan (lr), dan sebagian lagi dipancarkan (It) (Yanlinastuti and Fatimah, 2016).
Aplikasi rumus tersebut dalam pengukuran kuantitatif dilaksanakan dengan cara
komparatif menggunakan kurva kalibrasi dari hubungan konsentrasi deret larutan alat
untuk analisa suatu unsur yang berkadar rendah baik secara kuantitatif maupun secara
kualitatif. Pada penentuan secara kualitatif berdasarkan puncak-puncak yang dihasilkan
spektrum dari suatu unsur tertentu pada panjang gelombang tertentu.
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Gambar 5. Contoh Bagian Spektrum Absorpsi UV-Vis pada Uap Benzene
(Sumber: Underwood dan Day, 2017)
Sedangkan penentuan secara kuantitatif berdasarkan nilai absorbansi yang
dihasilkan dari spektrum dengan adanya senyawa pengompleks sesuai unsur yang
dianalisisnya. Adapun yang melandasi pengukuran spektrofotometer ini dalam
penggunaannya adalah hukum Lambert-Beer yaitu bila suatu cahaya monokromatis
dilewatkan melalui suatu media yang transparan, maka intensitas cahaya yang
ditransmisikan sebanding dengan tebal dan kepekaan media larutan yang digunakan
berdasarkan persamaan berikut:
Dimana,
( )
(Yanlinastuti dan Fatimah, 2016).

Transisi elektronik yang terjadi merupakan perpindahan elektron dari orbital
ikatan atau nonikatan ke tingkat orbital antiikatan atau disebut dengan tingkat eksitasi.
12
Orbital ikatan atau nonikatan sering disebut dengan orbital dasar, sehingga transisi
elektron sering dinyatakan sebagai transisi elektron dari tingkat dasar ke tingkat
tereksitasi.Tingkat tereksitasi dari elektron molekul organik hanya ada duajenis, yaitu
phi bintang (π*) dan sigma bintang (σ*). Agar terjadi transisi elektronik ini diperlukan
energi yang besarnya sesuai dengan jenis elektron ikatan dan nonikatan yang ada dalam
molekul organik. Besarnya energi untuk transisi dapat dihitung dari persamaan Planck,
yaitu:
Dimana,
(Suharti, 2017),
Semua molekul dapat mengabsorpsi radiasi dalam daerah UV dan sinar tampak
karena mereka mengandung electron, baik sekutu maupun menyendiri, yang dapat
diesksitasikan ke tingkat yang lebih tinggi. Panjang gelombang dimana eksitasi itu
terjadi, bergantung pada kuat electron itu terikat dalam molekul itu. Electron dalam
suatu ikatan kovalen tunggal terikat dengan kuat, dan diperlukan radiasi berenergi tinggi
atau panjang gelombang pendek untuk eksitasinya (Underwood dan Day, 2017).
Gambar 6. Tipe Transisi Elektronik dalam Molekul Organik

(Sumber: Underwood dan Day, 2017)
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Jika suatu molekul mengandung sebuah atom klor yang mempunyai pasangan
pasangan electron bebas, sebuah electron tak terikat (nonbonding) dapat dieksitasikan
ke tingkat energy yang lebih tinggi. Karena electron nonbonding tak terikat terlalu kuat
seperti electron bonding-sigma, maka absorpsinya terjadi pada panjang gelombang yang
lebih panjang. Pada suatu transisi terjadi dalam CH3Cl pada 173 nm, dan transisi dalam
CH3Br dan CH3I terjadi pada panjang gelombang yang lebih panjang lagi. Hal ini
disebabkan oleh kurang kuatnya ikatan electron dalam brom dan iod. Transisi yang
dijelaskan disini diberi tanda n-σ* untuk menujukan bahwa sebuah electron nonbonding
dinaikkan ke orbital antibonding-σ (Underwood and Day, 2017).
Tabel 1. Transisi Elektronik dalam Molekul-molekul Organik
(Sumber: Underwood dan Day, 2017).
Electron dalam ikatan rangkap dua dan rangkap tiga agak mudah dieksitasikan ke
orbital yang lebih tinggi. Suatu transisi dilambangkan π-π* bila sebuah electron-π
ditingkatkan dari suatu orbital bonding-π ke suatu orbital antibonding-π. Dalam molekul
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terkonjugasi (yakni molekul yang memiliki suatu deret ikatan-ikatan rangkap yang
berselang seling) absorpsinya bergeser ke panjang gelombang yang lebih panjang,
seperti pada Tabel 1. Molekul-molekul semacam itu digambarkan dengan menulis
struktur-struktur resonans, dengan mengatakan bahwa elektronnya lebih “tak-
terlokalisir” daripada jika mereka dikungkung dalam suatu ikatan antara dua atom.
Geseran ke panjnag gelombang yang lebih panjang mencerminkan fakta bahwa electron
dalam suatu sistem terkonjugasi kurang kuat terikat dari pada dalam suatu sistem tak-
terkonjugasi (Underwood dan Day, 2017).
Telah dijumpai bahwa transisi π-π* dalam molekul-molekul yang mengandung
gugus-gugus tak jenuh sangan mirip terlepas dari jenis atom yang membentuk ikatan
rangkapnya. Pada Tabel 2.1 bahwa absorpsi asetilena dan hydrogen sianida, terjadi kira-
kira pada panjang gelombang yang sama. Demikian pula untuk sistem terkonjugasi, -
(CH=CH)2- dan CH3-CH=CH-CHO, yang keduanya memberikan puncak sekitar 217
nm. Bertahun-tahun yang lalu kaitan antara ketidakjenuhan dan absorpsi cahaya
dikenali oleh ahli kimia organic, dan dilontarkan istilah kromofor untuk
menggambarkan peranan gugus-gugus semacam C=C, C=O, dan N=N dalam
menggeser absorpsi cahaya kea rah daerah tampak (Underwood dan Day, 2017).
Kebanyakan penerapan spektrofotometri UV-Vis pada senyawa organic
didasarkan pada transisi n-π* atau π-π* dan karenanya memerlukan hadirnya gugus
kromofor dalam molekul itu. Transisi ini terjadi dalam daerah spectrum (sekitar 200
hingga 700 nm) yang praktis untuk digunakan dalam eksperimen. Spektrofotometer
UV-Vis yang komersial biasanya beroperasi dari sekitar 175 atau 200 hingga 1000 nm.
Identifikasi kualitataif senyawa organic dalam daerah ini jauh lebih terbatas daripada
dalam daerah inframerah. Hal ini karena pita absorpsi terlalu besar dan kurang terinci.
Tetapi, gugus-gugus fungsional tertentu sepert karbonil, nitro, dan sistem
terkonjugasi,benar-benar menunjukan puncak karakteristik, dan sering dapat diperoleh
informasi yang berguna mengenai ada tidaknya gugus semacam itu dalam molekul
tersebut (Underwood dan Day, 2017).
Bila cahaya putih yang berisi seluruh spectrum panjang gelombang, melewati
suatu medium seperti kacaatau suatu larutan kimia berwarna yang tembus cahaya bagi
panjang-panjang gelombang tertentu tetapi berwarna bagi pengamat. Karena hanya
15
gelombang yang diteruskan sampai ke mata, panjang-panjang gelomang merekalah
yang menentukan warna medium. Warna ini dikatakan komplementer pada warna yang
akan diinderai seandainya cahaya yang akan terserap itu dapat ditilik, karena cahaya
yang diteruskan dan cahaya yang diabsorpsi menyusun warna putih aslinya. Serupa pula
objek berwarna yang kedap cahaya menyerap beberapa panjang gelombang dan
memantulkan panjang-panjang gelombang yang lain, bila dicahayai dengan cahaya
putih (Underwood dan Day, 2017).
Tabel 2. Spektrum Cahaya Tampak dan Warna-warna Komplementer
(Sumber: Underwood dan Day, 2017).
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BAB III
METODOLOGI PERCOBAAN
3.1 Alat dan Bahan

3.1.1 Alat
Peralatan yang digunakan pada percobaan ini, berupa rotary evaporator,
Therapeutic Lifestyle Changes (TLC), Spektrofotometer UV-Visible, kertas
saring, corong, dan peralatan-peralatan gelas.
3.1.2 Bahan
Bahan yang digunakan pada percobaan ini, berupa etanol 98%, n-
butanol, aquadest, tersier butanol, asam asetat, etanol, pereaksi penggeser
(MeOH + NaOMe; AlCl2 + HCl; NaOAc + H3BO3), dan senyawa kompleks
Cu (II)-kompleks fenolik.
3.2 Diagram Alir

3.2.1 Isolasi untuk Senyawa Apigenin dan Luteolin
Sampel Daun Sage kering halus
 Diekstraksi dengan etanol 98% selama 20 jam

dengan pengadukan intermiten
 Diuapkan dengan rotary evaporator sampai kira kira
sepersepuluh dari aslinya
Ekstrak Etanol
 Dimurnikan dengan ekstraksi cair-cair dengan n-

butanol dan air
Lapisan ekstrak dengan n- Lapisan Ekstrak dengan

butanol Air
17
Lapisan ekstrak dengan n-butanol
 Dilakukan pemisahan dengan TLC (menggunakan

eluen Tersier butanol- Asam asetat-air (6:2:2))
Terlihat 2 bercak noda

hasil pemisahan
 Dimurnikan dengan ekstraksi cair-cair dengan n-butanol dan air

 Masing-masing bercak dikerok, dilarutkan masing-masing
dengan etanol, dan silica diendapkan, serta disaring
 Diuapkan larutannya sampai volume berkurang banyak dan
dikristalkan
 Disaring masing masing bercak noda yang telah dilarutkan dan
diuapkan
Senyawa Murni Zat I Senyawa Murni Zat II
3.2.2 Penentuan Struktur Senyawa Apigenin pada Zat I
Senyawa murni Zat I
 Diuji Spektrofotometri UV-Vis dengan penambahan

pereaksi penggeser (MeOH + NaOMe, AlCl3 + HCl,
NaOAc + H3BO3)
 Diuji pada panjang gelombang 269nm
Hasil
3.2.3 Penentuan Struktur Senyawa Luteolin pada Zat II
18
Senyawa murni Zat II

pereaksi penggeser (MeOH + NaOMe, AlCl3 + HCl,
NaOAc + H3BO3)
 Diuji pada panjang gelombang 366nm
Hasil
3.2.4 Penentuan Struktur Senyawa Myricetine pada Zat III
Ekstrak etanol
 Dimurnikan dengan ekstraksi cair-cair dengan n-butanol dan air

 Diuapkan larutannya sampai volume berkurang banyak
Senyawa Murni Zat III
Senyawa murni Zat III

senyawa kompleks Cu (II)-kompleks fenolik pada
panjang gelombang 266nm
Hasil
3.3 Prosedur Percobaan

3.3.1 Isolasi untuk Senyawa Apigenin dan Luteolin (Dordevic S, et al., 2000)
Sampel Daun Sage pertama tama dikeringkan, setelah kering sampel
kemudian dihaluskan. Setelah itu, sampel diekstraksi selama 20 jam dengan
pengadukan intermiten, Ektraksi dilakukan dengan metode ekstraksi maserasi
19
dengan pelarut etanol 98%. Setelah diekstraksi, sampel diuapkan dengan rotary
evaporator sampai kira kira sepersepuluh dari aslinya. Ekstrak etanol yang didapat
kemudian dimurnikan dengan ekstraksi cair-cair dengan n-butanol dan air.
Didapat ekstrak n-butanol dan ekstrak air.
Ekstrak n-butanol kemudian dipisahkan dengan TLC (menggunakan eluen
Tersier butanol- Asam asetat-air (6:2:2)). Terlihat 2 bercak noda hasil pemisahan
yang kemudian dimurnikan dengan ekstraksi cair-cair dengan n-butanol dan air,
masing-masing bercak dikerok, dilarutkan dengan etanol, dan silica diendapkan,
serta disaring. Diuapkan larutannya sampai volume berkurang banyak dan
dikristalkan. Disaring masing masing bercak noda yang telah dilarutkan dan
diuapkan, dan didapat 2 senyawa murni yang berbeda.
3.3.2 Penentuan Struktur Senyawa Apigenin pada Zat I (Dordevic S. et al.,

2000)
Senyawa apigenin pada Zat I Diuji Spektrofotometri UV-Vis dengan
penambahan pereaksi penggeser (MeOH + NaOMe, AlCl3 + HCl, NaOAc +
H3BO3). Larutan diukur absorbansinya menggunakan spektrofotometri UV-Vis
pada panjang gelombang 269 nm.
3.3.3 Penentuan Struktur Senyawa Luteolin pada Zat II (Dordevic S. et al.,

2000)
Senyawa luteolin pada Zat II Diuji Spektrofotometri UV-Vis dengan
penambahan pereaksi penggeser (MeOH + NaOMe, AlCl3 + HCl, NaOAc +
H3BO3). Larutan diukur absorbansinya menggunakan sinar UV dengan panjang
gelombang 366 nm.
3.3.4 Isolasi dan Penentuan Struktur Senyawa Myricetine pada Zat II

(Jomová et al., 2019)
Ekstrak etanol dari isolate sampel tumbuhan yang diyakini terdapat senyawa
myrecetin kemudian dimurnikan dengan ekstraksi cair-cair dengan n-butanol dan
air. Hasil pemurnian pelarut n-butanol diuapkan larutannya sampai volume
20
berkurang banyak. Hasil ekstraksi n-butanol di uji dengan spektrofotometer UV-
Vis pada panjang gelombang 266 nm. Sampel ditambahkan dengan senyawa
kompleks Cu (II)-kompleks fenolik.
21
BAB IV
HASIL DAN PEMBAHASAN
Spektrofotometer UV-Vis merupakan suatu metode identifikasi yang didasarkan

pada struktur elektronik molekul, yang dikenal sebagai spektroskopi elektronik
(Sastrohamidjojo, 1991).
Penyerapan sinar tampak dan ultraviolet oleh suatu molekul dapat menyebabkan
terjadinya eksitasi molekul dari tingkat energi dasar ke tingkat energi yang lebih tinggi.
Elektron-elektron yang menyerap radiasi ultraviolet dalam suatu moleku organik adalah
elektron-elektron yang terlibat langsung dari ikatan antara dua atom dan elektron-
elektron bebeas seperti pada atom oksigen, halogen, nitrogen dan belerang (Underwood
dan Day, 2002).
Transisi yang penting pada daerah ultraviolet dan tampak yaitu transisi n→π* dan
π→π* jarang terjadi (Fessenden and Fessenden, 1989). Transisi yang terjadi pada
flavonoid yaitu transisi π→π* akibat adanya ikatan rangkap terkonjugasi dan transisi
n→π* karena adanya elektron bebas. Flavonoid mengandung sistem aromatik yang
terkonjugasi, oleh karena itu menunjukkan pita serapan yang kuat pada daerah
ultraviolet dan tampak (Harborne, 1987). Senyawa dengan ikatan rangkap terkonjugasi
seperti flavonoid akan mengalami penyerapan radiasi pada panjang gelombang yang
lebih besar dari 217 nm (Sastrohamidjojo, 1991).
Spektrofotometer UV-vis dapat digunakan untuk mengidentifikasi jenis flavonoid
dan menentukan pola oksigenasi. Kedudukan gugus hdroksil fenol bebas pada inti
flavonoid dapat ditentukan dengan menambahan pereaksi geser ke dalam larutan
cuplikan dan mengalami pergeseran puncak serapan yang terjadi. Metode ini secara
tidak langsung juga berguna untuk menentukan kedudukan gula atau metal yang terikat
pada salah satu gugus hidroksil fenol. Pereaksi geser yang biasa digunakan adalah
NaOMe/NaOH, NaOAc/H3BO3, AlCl3 dan AlCl3/HCl (Markham, 1988).
Penambahan NaOMe/NaOH menyebabkan batokromik karena adanya ionisasi
dari gugus hidroksilnya peka basa. Pergeseran terjadi pada pita I sebesar 45-65 nm
untuk flavon dan flavonol (Markham 1988). Bathochrome effect (red shift), merupakan
pergeseran dari absorban yang maksimum ke panjang gelombang yang lebih besar,
22
disebabkan oleh perubahan medium atau adanya suatu auksokrom (Suhartati, 2017).
Reaksi yang terjadi, yaitu:
Gambar 7. Reaksi NaOH dengan Senyawa Flavonoid

(Sumber: Markham, 1988).
Penambahahan AlCl3 5 % dimaksudkan untuk mendeteksi adanya gugus o-
hidroksi (biasanya pada posisi 3’, 4’) dan gugus o-hidroksi keton (4-keto, 5-OH).
Karena AlCl3 dapat membentuk kompleks dengan gugus tersebut. Sedangkan spektrum
AlCl3/HCl hanya merupakan pengaruh kompleks hidroksi keto. Adapun reaksi
pembentukan kompleks tersebut adalah (Markham, 1988):
Gambar 8. Reaksi Pembentukan Kompleks Flavonoid dengan AlCl3 dan HCl

Penambahan natrium asetat digunakan terutama untuk mendeteksi adanya gugus
7-hidroksil bebas atau yang setara. Pergeseran terjadi pada pita II sekitar 5-20 nm untuk
flavon, flavonol dan isoflavon. Sedangkan pergeseran sebesar 35 nm untuk flavanon
dan 60 nm untuk dihidroksiflavonol (Markham, 1988). Adapun reaksi yang terjadi,
sebagai berikut:
23
Gambar 9. Reaksi NaOAc dengan Senyawa Flavonoid
Penambahan asam borat dimaksudkan untuk mendeteksi orto dihidroksi pada
cincin B. Hal ini menyebabkan, pergeseran batokromik pita I sebesar 12-36 nm untuk
flavon, flavonol, auron dan khalkon. Reaksi pembentukan kompleks asam borat dengan
flavonoid adalah (Markham, 1988):
Gambar 10. Reaksi Pembentukan Kompleks Asam Borat dengan Flavonoid

Menurut Markham (1988), bahwa spektrum flavonoid biasanya ditentukan dengan
pelarut metanol atau etanol. Spektrum khas terdiri dari dua maksima pada rentang 240-
285 nm (pita II) dan 300-350 (pita I). Kedudukan yang tepat dan kekuatan nisbi
maksimal tersebut memberikan informasi yang berharga mengenai sifat flavonoid dan
pola oksigenasinya.
24
Gambar 11. Spektrum hasil analisis spektrofotometer UV-Vis sampel tanpa
pereaksi penggeser (a) dan dengan pereaksi penggeser (b,c,d)
(Sumber: Đorđević et al., 2000)
4.1 Penentuan Struktur Molekul Apigenin (Đorđević, Cakić, and Amr, 2000)
Spectrum UV pada larutan methanol, senyawa apigenin memiliki dua
karakteristik pita, I pada λ = 336 nm dan II pada λ = 267 nm, yang menunjukan bahwa
senyawa termasuk dalam kelompok Flavonoid. Dari pergeseran batokromik dengan
MeOH+NaOMe (Gambar b) dan dengan AlCl3 (Gambar c). Gambar c dengan
pergeseran batokromik AlCl3, pada pita I bergeser dari λ = 336 nm, terbagi menjadi dua
pita dengan puncak pada λ = 384 nm dan λ = 348 nm, menunjukan adanya gugus OH
pada posisi 5. Pada penambahan NaOAc (Gambar d), band shif II sebesar 7 nm (274
nm-267nm), menunjukan keberadaan gugus OH diposisi 7. Pada penambahan
NaOAc+H3BO3 (Gambar d), pergeseran pita I sebesar 2 nm (338 nm – 336 nm)
menunjukan adanya gugus OH pada posisi 3’ atau 4’.
25
Gambar 12. Struktur Senyawa Apigenin
(Sumber: Đorđević et al., 2000).
4.2 Penentuan Struktur Molekul Luteolin (Đorđević et al. 2000)

Spektrum UV senyawa II memiliki dua pita karakteristik, I di λ = 349 nm, dan
band II pada λ = 253 nm, yang menunjukkan bahwa senyawa tersebut termasuk dalam
kelompok flavonoid (Gambar 4a). Dari pergeseran batokromik dengan AlCl3 (Gambar
4c), yaitu pita I bergeser dari λ = 349 nm, terbagi menjadi dua pita dengan puncak pada
λ = 426 nm dan λ = 328 nm, menunjukkan adanya gugus OH di posisi 5. Setelah
menambahkan agen NaOAc (Gambar 4d), band shift II sebesar 16 nm (269 nm - 253
nm), menunjukkan keberadaan dan gugus OH pada posisi 7. Saat agen NaOAc + H3BO3
ditambahkan (Gambar 4d), pergeseran pita I sebesar 21 nm (370 nm - 349 nm),
menunjukkan adanya gugus OH pada posisi 3 dan 4.
26
Gambar 13. Spektrum senyawa luteolin hasil analisis spektrofotometer UV-
Vis sampel tanpa pereaksi penggeser (a) dan dengan pereaksi penggeser (b,c,d)
Gambar 14. Struktur Senyawa Luteolin

4.3 Penentuan Struktur Myricetine (Jomová et al., 2019)

Molekul dari kelompok spesifik flavonol memiliki tiga lokasi utama di mana ion
logam transisi berpotensi terkoordinasi: (i) kelompok 3-hidroksil dan 4-karbonil dari
cincin C; (ii) kelompok 5-hidroksil dari cincin A dan kelompok 4-karbonil dari cincin
C; dan (iii) kelompok 3’-dan4’-hidroksil dari cincin B (Gambar 15) (Jomová et al.,
2019).
Gambar 15. Sistem Khelasi Logam (M) pada Flavonoid

(Sumber: Jomová et al., 2019).
Pada senyawa flavonoid myricetin memiliki gugus kromofor ikatan rangkap dua
terkonjugasi dan ikatan rangkap pada karbonil yang terkonjugasi dengan ikatan rangkap
dua, serta gugus auksokrom -OH yang berikatan dengan gugus kromofor (Gambar 15),
membuat senyawa myricetin dapat terbaca pada daerah UV-Vis saat diidentifikasi
dengan spektrofotometer UV-Vis. Gugus kromofor (Al-Gouti et al., 2004), gugus
senyawa radikal yang terdiri dari ikatan ganda terkonjugasi yang mengandung electron
terdelokalisasi. Gugus kromofor biasanya meliputi gugus azo (-N=N-), karbonil (-C=O-
), karbon (-C=C-), karbon-nitrogen (-C=NH- atau -CH=N-), nitroso (-NO atau N-OH),
27
nitro (-NO2 atau =NO-OH), dan sulfur (C=S). Kromogen adalah senyawa aromatis yang
biasanya mengandung cincin benzena, naftalena, atau antrasena merupakan bagian dari
struktur kromogen-kromofor pada auksokrom. Adanya gugus terionisasi yang dikenal
sebagai auksokrom memberikan peningkatan absorpsi dan kekuatan ikatan pada suatu
senyawa. Beberapa gugus auksokrom adalah –NH3, -COOH, -HSO3, -OH.
Gambar 16. Struktur Senyawa Myrecetine dengan Gugus Kromofor yang

Terkonjugasi
Spektrum UV-Vis dapat melihatkan interaksi antara ion logam dan molekul
organik. Interaksi senyawa fenolik dengan ion cu dievaluasi berdasarkan perubahan
pada Spektrum UV-Vis senyawa fenolik bebas, mengikuti penambahan ion logam Cu2+
ke fenolik rasio molar majemuk 1: 1 dan 1: 2. Spektrum direkam pada empat waktu
berbeda yaitu di awal reaksi (hingga 10 menit) dan setelah 1 jam, 3 jam, dan kemudian
6 jam.
Gambar 17. Spektrum Pengukuran Myrecetine

Spektrum UV-Vis myricetin (Gambar 17) memiliki dua puncak intens pada 253
nm (pita II) dan pada 370 nm (band I). Setelah penambahan ion cupric pita II
menunjukkan pergeseran kecil ke arah panjang gelombang yang lebih panjang (~ 9 nm),
tetapi pita I terpecah, menyebabkan penurunan intensitasnya dan penyerapan maksimum
28
baru diamati pada 422 nm, menunjukkan pergeseran merah (Batokromik) dari puncak
aslinya ~ 52 nm, yang menunjukkan partisipasi sistem cincin cinnamoyl dalam interaksi
myricetin dengan ion cupric melalui gugus 3-OH dan 4-CO (lihat skema atas pada
Gambar 17). Gugus -OH yang berdekatan dari cincin-B dalam molekul myricetin dapat
memberikan tempat alternatif untuk interaksi dengan ion cupric. Intensitas yang lebih
rendah dari pita yang baru terbentuk pada 442 nm menunjukkan bahwa beberapa ion
tembaga juga dapat berikatan dengan myricetin melalui gugus 3’-, 4’-, 5’-OH dari
bagian katekol (cincin B).
29
BAB V
PENUTUP
5.1 Kesimpulan
Berdasarkan penjelasan yang telah dibahas maka dapat disimpulkan, bahwa
senyawa metabolit sekunder Flavonoid dapat diidentifikasi mennggunakan
spektrofotometer UV-Vis pada Spektrum khas terdiri dari dua maksimal pada rentang
240-285 nm (pita II) dan 300-350 (pita I). Identifikasi dapat dilakukan pada isolate hasil
isolasi tumbuhan yang diyakini memiliki kandungan senyawa flavonoid. Syarat
senyawa organic untuk pengukuran menggunakan spektrofotometer UV-Vis, yaitu
memiliki gugus kromofor dan menggunakan pelarut yang tidak memiliki gugus
kromofor (alcohol). Untuk mendapatkan pembacaan pada panjang gelombang yang
lebih besar (mengalami pergeseran batokromik), maka dapat ditambahkan reagen
penggeser yang diharapkan akan terbentuk kompleks atau terjadi resonansi pada
senyawa flavonoid yang akan diidentifikasi sehingga mengalami pergeseran panjang
gelombang ke yang lebih besar.
30
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of myricetin: A natural antioxidant flavonoid. Pharmazie 2014, 69, 19–26.
Zupko I, Hohmann J, Redei D, Falkay G, Janicsak G, Mathe I. Antioxidant

Activity of Leaves of Salvia Species in Enzyme- Dependent and Enzyme-Independent
Systems of Lipid Peroxidation and their Phenolic Constituents, Planta Medica, 2001.
67: 366-368 (Abstr.).
34
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The extraction of apigenin and luteolin from the sage Salvia ofﬁcinalis L. from
Jordan
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UNIVERSITY OF NIŠ
The scientific journal FACTA UNIVERSITATIS
Series: Working and Living Environmental Protection Vol. 1, No 5, 2000, pp. 87 - 93
Editor of series: Ljiljana Rašković, e-mail: ral@junis.ni.ac.yu
Address: Univerzitetski trg 2, 18000 Niš,YU, Tel. +381 18 547-095, Fax: +381 18 547-950
http:// ni.ac.yu/Facta
THE EXTRACTION OF APIGENIN AND LUTEOLIN

FROM THE SAGE SALVIA OFFICINALIS L. FROM JORDAN
UDC 582.824
Siniša Đorđević1, Milorad Cakić1, Salameh Amr2

1
Faculty of Technology, Bulevar oslobodjenja 124, Leskovac, Yugoslavia
2
Royal Science Institute, Amman- Alqwesme, Box 38365, Jordan
Abstract. In the Kingdom of Jordan first class sage (maramia) (Salvia officinalis L.)
grows both in wilderness and cultivated on plantations, and it is the most important
plant species in the country.
Our aim is to investigate the chemical composition of the sage originating from
Jordan, and in this paper the original procedure is described for the extraction of
apigenin and luteolin.
The preparative chromatography on paper was used for the isolation of these
compounds from ethanol extract (1:10) of the plant (flower and top leaves) in the n-
butanol-water-acetic acid system 12:2:1 v/v/v.
Considering the Rf values of IR and UV spectra (with and without adding the specific
agents NaOMe, NaOAc, NaOAc+H3BO3, AlCl3 and AlCl3+Hcl) and compared with the
literature data it was determined that the isolated compounds were apigenin and luteolin.
Key words: sage, extraction, apigenin, luteolin
1. INTRODUCTION
The sage growing in Jordan both in wilderness and cultivated on plantations is of first
class quality. It is the compulsory ingredient in cooking recipes and for tea making and is
known as traditional medicine for numerous diseases. The popular names for this plant
are: žalfija (in towns in Serbia), plem (in Montenegro and Herzegovina), kadulja (in
middle Dalmatia), kuš (in the north coast), kalaver or džiger (in eastern Serbia), meramia
or marjamia (in Jordan). Beside the above, a great number of other names are also used.
The sage is one of the oldest medicinal herbs. It is mentioned by all the medical
writers in the ancient Rome. The Latin name Salvia comes from the Latin verb "salvare"
meaning to save, to heal. Officinalis in Latin means medicinal. The mere fact that both
Received March 23, 2001

88 S. ĐORĐEVIĆ, M. CAKIĆ, S. AMR
Latin names are referring to the medicinal properties, which cannot be said for any other
plant, shows how much the ancient Romans appreciated the sage two thousand years ago,
and used it for healing in various ways.
The sage is also important from the ecological point of view because it improves the
environment, the air around its habitat, thanks to the antiseptic effect of its ether oil.
The scope of this work is the isolation, by an original technological method, of the
pure bioactive components of the sage, apigenin and luteolin, and the determination of
their structure by modern instrumental methods.
2. EXPERIMENTAL CONDITIONS
The plant material (flowers and top leaves), gathered at the locality of Hfashijet Al
Dbajbe (The Kingdom of Jordan) in the phase of blooming, dried in dark, well ventilated
place, ground by electric grinder and screened through the 1.4 mm mesh sieve, was
analyzed according to the Jordanian Agricultural Products Quality Control Regulations,
1982. The extraction by the maceration method in 98% ethanol was carried out with the
drug to solvent ratio 1:10, for a period of 20 hours with intermittent stirring.
The extract obtained, evaporated under vacuum at the temperature of 50oC to
approximately one tenth of its original volume, was purified by liquid-liquid extraction
method with n-butanol and water. In a 2000 ml separation funnel 50 ml of ethanol extract
was mixed with 125 ml of n-butanol, and then 40 ml portions of water were added in 15
minutes' intervals (with manual stirring), while at the same time the lower water layer was
separated. The procedure was repeated 12 times. The residual upper layer (n-butanol) was
applied to Whatman 1 chromatographic band, 2 cm3 to band size 30 × 60 cm, and
chromatographed for 48 hours in n-butanol-water-acetic acid system 12:2:1 v/v/v. In the
chromatograms obtained two zones were clearly visible: Rf - 0.9 and Rf - 0.81, and these
were separated by cutting the paper. The procedure was repeated several times for
preparatory isolation. The substances were eluted from the chromatographic paper by
ethanol. The solutions were evaporated to small volume from which, when cooled,
crystallized the separated substances (substance I at Rf = 0.9 and substance II at 0,81).
The pure compounds isolated by this method were tested for their UV spectra, as well
as their respective shifts (bathochromic and hypsochromic), that occur due to addition of
specific agents: NaOMe, NaOAc, NaOAc + H3BO3, AlCl3, and AlCl3 + HCl. IR spectra
were also made. For the determination of Rf value in the TBA system (tertiary butanol-
acetic acid-water 6:2:2 v/v/v) Whatman 1 chromatographic band was used.
3. RESULTS AND DISCUSSION

In Table 1 the investigation results are shown of common quality parameters of
Jordanian sage.
Table 1. Quality parameters of sage (Salvia officinalis L.) originating from Jordan
Moisture % Ashes % Ashes not soluble in HCl % Ether oil % Plant drug scent
9.41 12.57 2.45 2.09 specific and pleasant
The Extraction of Apigenin and Luteolin from the Sage Salvia Officinalis L. from Jordan 89
All the quality parameters tested comply with the requirements of Jordanian Regulations.
Table 2 shows the Rf values of the isolated substances in various systems.
Table 2. Basic properties of chromatographically isolated compounds 1 and 2
No Property Compound 1 Compound 2
1. Rf value in TBA system 0.87 0.77
2. Rf value in n-butanol-water-acetic acid system 12:2:1 v/v/v 0.9 0.81
Some Rf values in the TBA system were identical to those of apigenin and luteolin
mentioned in literature, and even more in n-butanol-water-acetic acid system 12:2:1 v/v/v
due to polarity difference. Bearing in mind the limitations of the identification of sub-
stances by the chromatographic method, the two isolated substances were further tested
by UV and IR spectroscopy methods for identification.
Compound 1 structure determination

The UV spectrum of the methanol solution of compound 1 has characteristic bands: I
at λ = 336 nm, and band II at λ = 267 nm (Figures 1 and 2) which leads to the conclusion
that the compound belongs to the group of flavonoides.
Fig. 1. UV spectra of compound 1 (a) with, and (b, c, d)

without the addition of specific agents
From the bathochromic shift with AlCl3 (Figure 1c), i.e. the band I shift from λ = 336 nm,
splitting into two bands with peaks at λ = 384 nm and λ = 348 nm, the presence of an OH-
group was detected in position 5. After the addition of agent NaOAc (Figure 1d), the band shift
II by 7 nm (274 nm - 267 nm), indicated the presence of and OH-group in position 7.
When agent NaOAc+H3BO3 was added (Figure 1d) a slight shift of band I by 2 nm
(338 nm - 336 nm) indicated the absence of OH-group in position 3.
From the UV spectrum the presence of OH-group in positions 5, 7, and 4' was recorded.
However, the UV spectra do not furnish information whether the compound tested belongs
to the group of flavon glycosides, i.e. whether it contains more OH-groups involved in the
forming of glycoside bond with some sugar. That is why the IR spectrum was made, as
shown in Figure 2.
Fig. 2. IR spectrum of compound 1
It can be seen that there are no intensive bands in the 1100-1000 cm-1 span that are
characteristic of glycoside bond, nor band of any sugar type. For example, there are no
bands of νCH vibrations of CH2 group at approx. 2950 cm-1 and 2850 cm-1, or any δCH
vibrations at approx. 1470 cm-1 or sugar vibration band νOH (expected frequency about
3400 cm-1). In the spectrum of νOH vibrations two bands are found, one at approx. 3307 cm-
1
, and the other at approx. 3149 cm-1, that are most probably the result of νOH vibrations of
phenol OH groups. The intensive band at 1608 cm-1 is most probably the result of νC=O
vibrations of C=O group from the central heterocyclic ring, while the νC-O vibration should
occur at approx. 1162 cm-1. The above spectral characteristics indicate with high probability
that the compound is apigenin. These data were compared with data from the book by T.J.
Mabry and the identical UV spectra made with typical agents are proof that the isolated
compound 1, the structure of which is shown in Figure 3, is apigenin.
HO O
OH
OH O
Fig. 3. The structure of apigenin Compound 2 structure determination -
The UV spectrum of the methanol solution of the compound 2 has two characteristic
bands, I at λ = 349 nm, and band II at λ = 253 nm, which indicates that the compound
belongs to the group of flavonoides (Figures 4a). From the bathochromic shift with AlCl3
(Figure 4c), i.e. the band I shift from λ = 349 nm, splitting into two bands with peaks at
λ = 426 nm and λ = 328 nm, indicated the presence of an OH-group in position 5.
After adding the agent NaOAc (Figure 4d), the band shift II by 16 nm (269 nm -
253 nm), indicated the presence of and OH-group in position 7.
When agent NaOAc+H3BO3 was added (Figure 4d), the shift of band I by 21 nm
(370 nm - 349 nm), indicated the presence of OH-group in positions 3 and 4.
Fig. 4. UV spectra of compound 2 (a) with, and (b, c and d)

without the addition of specific agents
From the UV spectrum the presence of OH-group in positions 5, 7, 3' and 4' was
recorded. However, the UV spectra do not furnish information whether the compound
tested belongs to the group of flavon glycosides, i.e. whether it contains more OH-groups
involved in the forming of glycoside bond with some sugar. That is why the IR spectrum
was made, as shown in Figure 5.
Fig. 5. IR spectrum of compound 2
It can be seen that there are no intensive bands in the 1100-1000 cm-1 span that are
characteristic of glycoside bond, nor band of any sugar type. For example, there are no
bands of νCH vibrations of CH2 group at approx. 2950 cm-1 and 2850 cm-1, or any δCH
vibrations at approx. 1470 cm-1 or sugar vibration band νOH (expected frequency about
3400 cm-1). In the spectrum of νOH vibrations two a band is found, at approx. 3144 cm-1,
that is most probably the result of νOH vibrations of phenol OH groups. The intensive
band at 1616 cm-1 is most probably the result of νC=O vibrations of C=O group from the
central heterocyclic ring, while the νC-O vibration should occur at approx. 1172 cm-1. The
above spectral characteristics indicate with high probability that the compound is luteolin.
These data were compared with data from the book by y T.J. Mabry and the identical UV
spectra made with typical agents are proof that the isolated compound 2, the structure of
which is shown in Figure 6, is luteolin.
OH
HO O
OH
OH O
Fig. 6. The structure of luteolin
CONCLUSIONS
The sage Salvia officinalis L. originating from Jordan complies with the requirements
of the Jordanian Agricultural Products Quality Control Regulations with respect to
general quality parameters.
The original procedure with n-butanol-water-acetic acid system 12:2:1 v/v/v was used
to isolate two bioactive substances, that were identified, through PC, UV-Vis and IR
spectroscopy as compound 1, which is apigenin, and compound 2, which is luteolin.
REFERENCES
1. Medicinal Plants Encyclopedia, First Edition , Editor Al-Arabia Lidirasad, Jordan, 1993
2. Flora SR Srbije, Naučno delo, Beograd VI, 1974.
3. Pharmacopoea Jugoslavica III, Izdanje saveznog zavoda za zdravstvenu zaštitu, Beograd, 1972.
4. Harborne, J.B., Chromatographic Identification of Anthocyanin Pigments, Chromatogr. Rev. 1(1959).
5. Mabry, T. J., K. R. Markham and M. B. Thomas, The Systematic Identification of Flavonoides,
Springer Verlag, New York-Heidelberg-Berlin 1970.
6. Jordanian Regulations For The Quality Control Of Agricultural Products, 1982.
IZOLOVANJE APIGENINA I LUTEOLINA IZ ŽALFIJE

SALVIA OFFICINALIS L. POREKLOM IZ JORDANA
Siniša Đorđević, Milorad Cakić, Salameh Amr
U Kraljevini Jordan spontano raste, ali se i plantažno gaji žalfija (maramija) (Salvia
officinalis L.) izvanrednog kvaliteta i najvažnija je biljna vrsta u ovoj zemlji.
Naš cilj je istraživanje hemijskog sastava žalfije poreklom iz Jordana, a u ovom radu izložen je
originalni postupak za izolovanje apigenina i luteolina.
Za izolovanje ovih jedinjenja primenjena je preparativna hromatografija na papiru etanolnog
ekstrakta (1:10) biljke (cvet + gornji lisni deo) u sistemu n-butanol-voda-sirćetna kiselina 12:2:1
v/v/v.
Na osnovu Rf vrednosti, IR i UV spektra (sa i bez dodataka specifičnih reagensa NaOMe,
NaOAc, NaOAc+H3BO3, AlCl3 i AlCl3+HCl) kao i poredjenjem sa literaturnim podacima
utvrdjeno je da su izolovana jedinjenja apigenin i luteolin.
Ključne reči: žalfija, ekstrakcija, apigenin, luteolin
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molecules
Article
A Switch between Antioxidant and Prooxidant
Properties of the Phenolic Compounds Myricetin,
Morin, 30,40-Dihydroxyflavone, Taxifolin and
4-Hydroxy-Coumarin in the Presence of Copper(II)
Ions: A Spectroscopic, Absorption Titration and DNA
Damage Study
Klaudia Jomová 1 , Lenka Hudecova 1 , Peter Lauro 1 , Miriama Simunkova 2 , Saleh H. Alwasel 3 ,
Ibrahim M. Alhazza 3 and Marian Valko 2,3, *
1 Department of Chemistry, Constantine the Philosopher University in Nitra, 949 74 Nitra, Slovakia;
kjomova@ukf.sk (K.J.); lhudecova@ukf.sk (L.H.); plauro@ukf.sk (P.L.)
2 Faculty Chemical and Food Technology, Slovak University of Technology, 812 37 Bratislava, Slovakia;
miriama.simunkova@stuba.sk
3 King Saud University, Zoology Department, College of Science, Riyadh 11451, Saudi Arabia;
salwasel@ksu.edu.sa (S.H.A.); imalhazza@hotmail.com (I.M.A.)
* Correspondence: marian.valko@gmail.com

Received: 26 October 2019; Accepted: 25 November 2019; Published: 27 November 2019
Abstract: The beneficial effects of polyphenols, predominantly in the context of oxidative

stress-related diseases such as cancer, cardiovascular diseases and neurological conditions including
Alzheimer’s and Parkinson’s diseases, have been documented by a number of papers and reviews.
The antioxidant/prooxidant properties of phenolic compounds are related mainly to the number and
positions of hydroxyl groups and to their redox metal (Cu, Fe) chelating capacity. In this work we
studied structurally distinct phenolic molecules such as myricetin, morin, 30 ,40 -dihydroxy-flavone,
taxifolin and 4-hydroxycoumarin, either alone or as interacting with Cu2+ ions. EPR and UV-Vis
spectroscopy confirmed that the effective binding of cupric ions to phenolic compounds requires
the presence of the 3-OH and 4-CO groups on the flavonoid C ring and unsaturated C2-C3 bond
of the C-ring, which permits through-conjugation with the B-ring. An ABTS assay revealed that
radical scavenging activities of phenolic compounds are related to their number of hydroxyl groups,
planarity of the molecular skeleton, extent of delocalization and they decrease in the order: myricetin
> morin > 30 ,40 -dihydroxyflavone ~ 4-hydroxy coumarin > taxifolin. Absorption titrations indicate
that copper ions can modulate the DNA binding affinity of flavonoids via the formation of their
Cu-chelates. Gel electrophoresis measurements indicated that the protective effect of the phenolic
compounds decreases in the order: 30 ,40 -dihydroxyflavone > 4-OH coumarin > morin > taxifolin ~
myricetin. This can be explained by the fact that myricetin, taxifolin and morin form stable Cu(II)
complexes capable of causing DNA damage via interaction with DNA and ROS formation via the
Fenton reaction. Application of ROS scavengers revealed the formation of singlet oxygen, superoxide
and hydroxyl radicals and their concerted synergistic effect on the DNA. The overall results suggest
that the most pronounced DNA damage has been observed for flavonoids containing higher number
of hydroxyl groups (including 3-OH group of the C ring), such as myricetin (six hydroxyl groups),
morin and taxifolin (five hydroxyl groups) in the presence of Cu(II) ions. The proposed mechanism
of action by which Cu(II) complexes of myricetin, morin and taxifolin interact with DNA predispose
these substances to act as potential anticancer agents. The anticancer activity of phenolic compounds
can be explained by their moderate prooxidant properties, which can boost ROS formation and kill
cancer cells. Alternatively, slight prooxidant properties may activate antioxidant systems, including
Molecules 2019, 24, 4335; doi:10.3390/molecules24234335 www.mdpi.com/journal/molecules

Molecules 2019, 24, 4335 2 of 28
Molecules 2019, 24, x 2 of 28
properties may
antioxidant activate
enzymes and antioxidant systems,
low molecular including
antioxidants suchantioxidant enzymes
as glutathione and
and thus actlow molecular
as preventive
antioxidants
anticancer such as glutathione and thus act as preventive anticancer agents.
agents.
Keywords: flavonoids; hydroxyl

Keywords: hydroxyl groups;
groups; copper;
copper; antioxidant;
antioxidant; prooxidant;
prooxidant; DNA
DNAdamage
damage
1.
1. Introduction
Owing
Owing to to their
their potential
potential beneficial
beneficial health
health effects,
effects, polyphenolic
polyphenolic compounds
compounds have have attracted
attracted
significant
significant attention
attention during
during the the past
past twotwo decades
decades [1].[1]. The most plentiful group group of of polyphenol
polyphenol
compounds
compounds that that occurs
occurs in in Nature
Nature is is the
the flavonoids.
flavonoids. Flavonoid
Flavonoid biosynthesis
biosynthesis is is stimulated
stimulated by by light
light and
and
therefore these compounds accumulate mainly in the peel and bark of plants,
therefore these compounds accumulate mainly in the peel and bark of plants, but also in their seeds but also in their seeds
and
and flowers.
flowers. In plants, the primary function of flavonoids is to to provide
provide protection
protection against
against reactive
reactive
oxygen
oxygen species
species (ROS)
(ROS) thatthat are
are produced
producedduringduringthe theprocess
processof ofphotosynthesis
photosynthesis[2]. [2].
The
The beneficial
beneficial health
health effects
effects ofof flavonoids
flavonoids have have been
been associated
associated withwith their
their ability
ability toto prevent
prevent
oxidative damage to
oxidative damage toliving
livingorganisms,
organisms,causedcaused bybyROS ROS and/or
and/or reactive
reactive nitrogen
nitrogen species
species (RNS) (RNS) [3],
[3], and
and it is believed that one of the most important mechanisms associated with
it is believed that one of the most important mechanisms associated with the beneficial effects of these the beneficial effects
of thesepigments
natural natural pigments and the prevention
and the prevention of variety of of diseases
variety of diseases
is their is their
ability abilityastoantioxidants.
to function function as
antioxidants.
The beneficialThe beneficial
effects effects of polyphenols
of polyphenols have been documented
have been documented predominantly predominantly
in the context in theof
context of oxidative stress-related diseases such as cancer, and also neurological
oxidative stress-related diseases such as cancer, and also neurological conditions including conditions including
Alzheimer’s
Alzheimer’s and and Parkinson’s
Parkinson’sdiseases
diseases[4].[4].In In addition
addition to their
to their antioxidant
antioxidant properties,
properties, flavonoids
flavonoids may
may
confer confer further
further beneficial
beneficial consequences,
consequences, including
including digestive
digestive stimulation,
stimulation, and anti-inflammatory,
and anti-inflammatory, anti-
anti-microbial, anti-viral
microbial, anti-viral and and anti-mutagenic
anti-mutagenic effects
effects [5–7].[5–7].
Although
Althoughmore morethan than4000 flavonoids
4000 flavonoidsare currently knownknown
are currently [8], it is[8],
quercetin, kaempferol,
it is quercetin, myricetin,
kaempferol,
apigenin and luteolin that have been reported as important components
myricetin, apigenin and luteolin that have been reported as important components in human in human diets (as present
diets
in
(asfruits,
presentvegetables, cereals) [9].cereals)
in fruits, vegetables, The characteristic feature of feature
[9]. The characteristic each flavonoid molecule molecule
of each flavonoid is a skeletonis a
composed of 15 carbon
skeleton composed of 15atoms
carbonwhich
atomscontains two aromatic
which contains rings, A
two aromatic and AB,and
rings, linked together
B, linked by a
together
three-carbon
by a three-carbon bridge that isthat
bridge partisof a heterocyclic,
part pyranpyran
of a heterocyclic, ring Cring(Figure 1). 1).
C (Figure
Figure 1. Basic
Figure 1. Basic skeleton
skeleton of
of flavonoids.
flavonoids.
Due
Due toto their
their great
great variety,
variety, flavonoids
flavonoids are are categorized
categorized according
according toto structural
structural characteristics
characteristics
(oxidation
(oxidation level, substituents) into multiple classes [10]. They may also exist as
level, substituents) into multiple classes [10]. They may also exist as glycosides,
glycosides, which,
which, inin
contrast
contrast to
to aglycones,
aglycones, possess
possess bound
bound carbohydrate
carbohydrate groupsgroups at at positions
positions 33 or
or 7,7, or
or they
they maymay contain
contain
methyl 0 , 30 , 40 and 50 , which
methyl groups.
groups. TheThe hydroxyl
hydroxyl groups
groups ofof flavonoids
flavonoids cancan bebein
inpositions
positions3,3,5,5,7,7,22′, 3′, 4′ and 5′, which
give
give these
these compounds
compounds the the properties
properties of of weak
weak acids,
acids, which
which cancan deprotonate
deprotonate to to form
form anionic
anionic species,
species,
depending both on their pKa, and the pH of the local
depending both on their pKa, and the pH of the local environment. environment.
Flavonoids
Flavonoids play
play an
an important
important role
role in
in the
the bioavailability
bioavailability of of metal
metal ions
ions which
which are are present
present inin trace
trace
amounts
amounts in the body, and can also detoxify heavy metals such as chromium or lead: the latter being
in the body, and can also detoxify heavy metals such as chromium or lead: the latter being
due
due to
to the
the ability
ability of
of their hydroxyl groups
their hydroxyl groups to to chelate
chelate free
free redox
redox metal ions and
metal ions and form
form stable
stable complexes,
complexes,
which
which are subsequently eliminated from the organism [11]. Molecules of the specific flavonols
are subsequently eliminated from the organism [11]. Molecules of the specific group of group of
have threehave
flavonols mainthree
sitesmain
where transition
sites metal ionsmetal
where transition can potentially be coordinated:
ions can potentially (i) the 3-hydroxyl
be coordinated: (i) the 3-
hydroxyl and the 4-carbonyl groups in the C ring, (ii) the 5-hydroxyl group of the A ring and the 4-
carbonyl group in the C ring and (iii) the 3′- and 4′-hydroxyl groups of the B ring (Figure 2) [7,12–14].
Molecules 2019, 24, 4335 3 of 28
and the 4-carbonyl groups in the C ring, (ii) the 5-hydroxyl group of the A ring and the 4-carbonyl
Molecules
group in2019,
the 24, x
C ring and (iii) the 30 - and 40 -hydroxyl groups of the B ring (Figure 2) [7,12–14]. 3 of 28
3'
2' 4'
8 B
1'
2 5'
7
6'
A C
3
6
5 4
Figure 2. Typical metal (M) chelation sites in flavonoids.

Figure 2. Typical metal (M) chelation sites in flavonoids.
The position and number of particular functional hydroxyl groups in flavonoids and their
The position and number of particular functional hydroxyl groups in flavonoids and their
interaction with redox ions such as iron or copper have several important consequences:
interaction with redox ions such as iron or copper have several important consequences:
(i) A flavonoid molecule that contains multiple hydroxyl groups typically exhibits a greater
(i) A flavonoid molecule that contains multiple hydroxyl groups typically exhibits a greater
antioxidant (radical scavenging) capacity than a flavonoid with a smaller number of hydroxyl
antioxidant (radical scavenging) capacity than a flavonoid with a smaller number of hydroxyl groups
groups [15,16]. (ii) Transition metal ions such as copper or iron catalyze the decomposition of hydrogen
[15,16]. (ii) Transition metal ions such as copper or iron catalyze the decomposition of hydrogen
peroxide, resulting in the formation of damaging hydroxyl radicals, via the Fenton reaction [17]:
peroxide, resulting in the formation of damaging hydroxyl radicals, via the Fenton reaction [17]:
+
Fe2+Fe
/Cu
2+/Cu++ H
+H2O2 2→
2O →Fe
3+
Fe3+/Cu
2+
OH−−+ +•OH
/Cu2+ ++OH
•
OH (1)
(1)
Metal ions
Metal ions coordinated/chelated
coordinated/chelated by by flavonoids
flavonoids [18–22]
[18–22] usually
usually exhibit
exhibit aa suppressed
suppressed catalytic
catalytic
activity in the Fenton reaction and hence a reduced formation of hydroxyl
activity in the Fenton reaction and hence a reduced formation of hydroxyl radicals. However, many radicals. However, many
flavonoids also
flavonoids also have
have reducing
reducing properties
properties and and when
when coordinated
coordinated to to copper(II)
copper(II) they they may
may (partially)
(partially)
reduce the metal ion (i.e., Cu 2+ → Cu+
2+ +) which causes them to be more active as catalysts in the Fenton
reduce the metal ion (i.e., Cu → Cu ) which causes them to be more active as catalysts in the Fenton
reaction [15].
reaction [15]. There
There are
are thus
thus two
two competing
competing effects
effects in
in that
that interaction
interaction of of metal
metal ionsions with
with flavonoids:
flavonoids:
metal-chelation effect
metal-chelation effect ofof flavonoids
flavonoids suppresses
suppresses catalytic
catalytic activity
activity ofof metal
metal ions
ions inin the
the Fenton
Fenton reaction,
reaction,
while alternatively, reduction of metal ions to lower oxidation states, due
while alternatively, reduction of metal ions to lower oxidation states, due to the reducing capacity of to the reducing capacity of
flavonoids, results
flavonoids, results in
in the
the augmentation
augmentation of of hydroxyl
hydroxylradical
radicalformation.
formation. (iii)(iii) Complexes
Complexes of of transition
transition
metal ions with flavonoids are effective SOD mimetic compounds and
metal ions with flavonoids are effective SOD mimetic compounds and are thus capable of affecting are thus capable of affecting
the
the redox state of cells [20,21]. (iv) Redox metals coordinated with flavonoids
redox state of cells [20,21]. (iv) Redox metals coordinated with flavonoids may exert redox cycling may exert redox cycling
mechanismresulting
mechanism resultingin in
thethe formation
formation of ROSof (prooxidant
ROS (prooxidant behaviour)
behaviour) [18]. (v)[18]. (v) The of
The majority majority
flavonoid of
flavonoid molecules have essentially flat structures with rigid rings
molecules have essentially flat structures with rigid rings that contain a system of conjugated double that contain a system of
conjugated
bonds double
and those bonds and
flavonoids those flavonoids
coordinated to redox coordinated to redox ions
ions may intercalate intomayDNA intercalate
and causeinto DNA
damage
and cause damage
(prooxidant (prooxidant
behaviour) [18]. behaviour) [18].
From the above discussion, itit follows
From the above discussion, follows thatthat in
in the
the presence
presence of of redox
redox ions,
ions, flavonoids
flavonoids maymay behave
behave
either as
either asantioxidants
antioxidantsororasasprooxidants.
prooxidants. Since
Since thethe results
results obtained
obtained to date
to date are fully
are not not fully consistent,
consistent, due
due to the complexity of the systems investigated, and the various
to the complexity of the systems investigated, and the various experimental conditions employed, the experimental conditions
employed,
aim the aimwork
of the present of the present
is to study work is to study
structurally structurally
distinct distinct phenolic
phenolic molecules such asmolecules
myricetin,such as
morin,
3myricetin,
0 0 morin, 3′,4′-dihydroxyflavone,
,4 -dihydroxyflavone, taxifolin and 4-hydroxycoumarin
taxifolin and 4-hydroxycoumarin (Figure 3), either(Figure alone 3),
or either alone or
as interacting
as interacting
2+ with Cu 2+ ions. The flavonoids were selected with the aim to determine and contrast
with Cu ions. The flavonoids were selected with the aim to determine and contrast the effect of the
the effectand
number of the number
positions ofand
the positions
hydroxyl of the hydroxyl
groups groupschelating
on the copper on the copper chelating
efficiency efficiency and
and antioxidant vs
antioxidant vs prooxidant activity, paying particular attention to elucidating
prooxidant activity, paying particular attention to elucidating those structural characteristics of the those structural
characteristics
compounds of thewhich
studied compoundsinfluencestudied
a switchwhich
frominfluence
antioxidanta switch from antioxidant
to prooxidant behaviourto[23].
prooxidant
behaviour [23].
Copper(II) ions were chosen for interaction studies with flavonoids on the basis of the biological
importance of copper as an integral element of metalloenzymes, in the synthesis of neurotransmitters,
in the origin of oxidative stress in Alzheimer’s and Parkinson’s diseases, cancer diseases and in other
conditions [4]. Those pathological states of an organism that are characterized by free (unbound)
copper, which can catalyze formation of ROS via the Fenton reaction and consequently cause damage
to all important biomolecules including DNA are of great clinical importance [15]. Thus, we see that
in Alzheimer’s disease, which is characterized by a significantly raised content of copper in amyloid
copper(II)-flavonoid interactions not only from a molecular structural point of view but also in a
search for prospective flavonoid chelation therapies.
The nature of copper(II)-flavonoid interactions were studied using both EPR and UV-Vis
spectroscopy, while the biological activity of flavonoids, both as free compounds, and as copper(II)-
flavonoid chelates, was studied through their interactions with DNA, as determined using absorption
Molecules 2019, 24, 4335 4 of 28
titrations and gel electrophoresis.
Figure 3. Structure of phenolic compounds used in the study based on their categorization into classes.
Figure 3. Structure of phenolic compounds used in the study based on their categorization into
The conjugation of A/C-rings to a polyhydroxyphenyl moiety at the C-2 position is marked in yellow.
classes. The conjugation of A/C-rings to a polyhydroxyphenyl moiety at the C-2 position is marked
in yellow. ions were chosen for interaction studies with flavonoids on the basis of the biological
Copper(II)
importance of copper as an integral element of metalloenzymes, in the synthesis of neurotransmitters,
2. Results and Discussion
in the origin of oxidative stress in Alzheimer’s and Parkinson’s diseases, cancer diseases and in other
conditions [4]. Thoseofpathological
The interaction each of the states
phenolicof an organism studied
compounds that are with
characterized
cupric ions bywas
free determined
(unbound)
copper,
using awhich can catalyze
combination formation
of EPR and of ROS via
UV-Vis the Fenton reaction
spectroscopic and Additionally,
methods. consequently cause damage
the effect of
to all important biomolecules including DNA are of great clinical importance
coordination with copper(II) ions on the antioxidant properties of the phenolic compounds was [15]. Thus, we see that
in Alzheimer’s
studied disease,
using ABTS which
assays; theisinteraction
characterized byphenolic
of the a significantly raisedand
compounds content of copper
of their copperin amyloid
complexes
plaques,
with DNA, therapeutic
along with approaches based onrole
DNA protective a dietary supplementation
was studied by means with
of a flavonoids
combination that
of are capable
absorption
of chelating
titrations andfree
gelcopper, resulted in an ameliorated progression of those symptoms that are typical for
electrophoresis.
the condition. From this and other examples of pathological states of an organism that are characterized
2.1.a Chelation
by of Cu(II)copper
malfunctioning by Phenolic Compounds
metabolism, it follows that it is worthwhile to study copper(II)-flavonoid
interactions not only from a molecular structural point of view but also in a search for prospective
The interaction of cupric ions with phenolic compounds was studied in DMSO solution at 77 K
flavonoid chelation therapies.
using EPR spectroscopy and at room temperature by UV-Vis spectroscopy.
The nature of copper(II)-flavonoid interactions were studied using both EPR and UV-Vis
spectroscopy, while theof biological
2.1.1. EPR Spectroscopy activity of
Copper(II)-Phenolic flavonoids, both as free compounds, and as
Complexes
copper(II)-flavonoid chelates, was studied through their interactions with DNA, as determined
usingEPR spectroscopy
absorption titrationsisand
a sensitive physical technique that is used for the detection and
gel electrophoresis.
determination of paramagnetic species in various coordination environments and because cupric ions
(d9Results
2. configuration) contain one unpaired electron they can be studied using EPR [24]. EPR spectra of
and Discussion
the CuCl2 reference system, along with selected Cu(II)-flavonoid complexes, all measured in their
The interaction of each of the phenolic compounds studied with cupric ions was determined
DMSO solutions at 77 K are presented in Figure 4. It is immediately clear that the EPR spectrum of
using a combination of EPR and UV-Vis spectroscopic methods. Additionally, the effect of coordination
Cu(II)-complex with myricetin is different from those recorded for Cu(II) interaction with morin,
with copper(II) ions on the antioxidant properties of the phenolic compounds was studied using
ABTS assays; the interaction of the phenolic compounds and of their copper complexes with DNA,
along with DNA protective role was studied by means of a combination of absorption titrations and
gel electrophoresis.
2.1. Chelation of Cu(II) by Phenolic Compounds

The interaction of cupric ions with phenolic compounds was studied in DMSO solution at 77 K
using EPR spectroscopy and at room temperature by UV-Vis spectroscopy.
Molecules 2019, 24, 4335 5 of 28
2.1.1. EPR Spectroscopy of Copper(II)-Phenolic Complexes

EPR spectroscopy is a sensitive physical technique that is used for the detection and determination
of paramagnetic species in various coordination environments and because cupric ions (d9 configuration)
contain one unpaired electron they can be studied using EPR [24]. EPR spectra of the CuCl2 reference
system, along with selected Cu(II)-flavonoid complexes, all measured in their DMSO solutions at
77 K are2019,
Molecules presented
24, x in Figure 4. It is immediately clear that the EPR spectrum of Cu(II)-complex 5 of 28
with myricetin is different from those recorded for Cu(II) interaction with morin, dihydroxyflavone,
dihydroxyflavone,
taxifolin taxifolin and 4-hydroxycoumarin.
and 4-hydroxycoumarin. The spectra
The difference in the EPR difference in theinteracting
of Cu(II) EPR spectra
with ofmorin,
Cu(II)
interacting with morin,
dihydroxyflavone, dihydroxyflavone,
taxifolin taxifolin
and hydroxycoumarin is and hydroxycoumarin
substantiated is substantiated
by the presence by the
of an overshoot
presence of an overshoot peak at about 3500–3600 Gauss, originating probably from
peak at about 3500–3600 Gauss, originating probably from an existence of aggregated Cu(II) speciesan existence of
aggregated
in Cu(II) species
DMSO solution in DMSO
upon cooling solution
to 77 upon cooling
K. It appears to 77
that with theK.exception
It appears ofthat with theflavonoids
myricetin, exception
of myricetin, flavonoids with lower number of hydroxyl groups and reduced
with lower number of hydroxyl groups and reduced Cu(II) chelating capacity have greater tendency Cu(II) chelating
capacity have greater tendency to aggregate upon cooling in DMSO solution.
to aggregate upon cooling in DMSO solution. Considerably more informative were UV-Vis spectra Considerably more
informative
discussed were
in the UV-Vis
next spectra discussed in the next section.
section.
CuCl2 Myricetin Morin

1800 2400 3000 3600 4200 1800 2400 3000 3600 4200 1800 2400 3000 3600 4200
Magnetic field / Gauss Magnetic field / Gauss Magnetic field / Gauss
Taxifolin
Dihydroxyflavone 4-hydroxycoumarin
1800 2400 3000 3600 4200 1800 2400 3000 3600 4200 1800 2400 3000 3600 4200
Magnetic field / Gauss Magnetic field / Gauss Magnetic field / Gauss
Figure
Figure4. 4.EPR spectra
EPR of CuCl
spectra of2 and Cu(II)
CuCl complexes (1:2) with myricetin, morin, 30 ,40 -dihydroxyflavone,
2 and Cu(II) complexes (1:2) with myricetin, morin, 3′,4′-
taxifolin and 4-hydroxycoumarin
dihydroxyflavone, (1:2), recorded in DMSO
taxifolin and 4-hydroxycoumarin solution at
(1:2), recorded in77 K. EPR
DMSO data: CuCl
solution ⊥ =
(gEPR
at 772K.
2.083, gk = 2.414,
data: CuCl Ak = 118
2 (g⊥ = 2.083, g|| =Gauss); Myricetin
2.414, A (g⊥ = Myricetin
|| = 118 Gauss);
2.084, gk =(g2.413, Ak =g||115
⊥ = 2.084,
Gauss);
= 2.413, A|| =Morin (g⊥ =
115 Gauss);
2.085, k = ⊥ = gk = 2.397, A k =
Morin (g⊥ = 2.085, g|| = 2.521); Dihydroxyflavone (g⊥ = 2.085, g|| = 2.397, A|| = 115 Gauss); Taxifolin g
g 2.521); Dihydroxyflavone (g 2.085, 115 Gauss); Taxifolin (g ⊥ = 2.081, (gk⊥==
2.393, A k = 120 Gauss); 4-hydroxycoumarin (g⊥ = 2.084, g k = 2.451, A k = 115 Gauss).
2.081, g|| = 2.393, A|| = 120 Gauss); 4-hydroxycoumarin (g⊥ = 2.084, g|| = 2.451, A|| = 115 Gauss).
2.1.2. UV-Vis Spectroscopy of Cu(II)-Phenolic Complexes
2.1.2. UV-Vis Spectroscopy of Cu(II)-Phenolic Complexes
UV-Vis spectra can sensitively reflect interactions between the metal ions and organic molecules.
UV-Vis spectra can sensitively reflect interactions between the metal ions and organic molecules.
The interaction of the phenolic compounds with cupric ions was evaluated on the basis of changes in the
The interaction of the phenolic compounds with cupric ions was evaluated on the basis of changes in
UV-Vis spectra of the free phenolic compounds, following addition of cupric ions at metal-to-phenolic
the UV-Vis spectra of the free phenolic compounds, following addition of cupric ions at metal-to-
compound molar ratios of 1:1 and 1:2. Spectra were recorded at four different times: at the beginning
phenolic compound molar ratios of 1:1 and 1:2. Spectra were recorded at four different times: at the
of the reaction (up to 10 min) and after 1 h, 3 h and then 6 h. Figure 5 shows the UV-Vis spectra of
beginning of the reaction (up to 10 min) and after 1 h, 3 h and then 6 h. Figure 5 shows the UV-Vis
myricetin, morin and 30 ,40 -dihydroxyflavone and their Cu(II) complexes. Figure 6 shows the UV-Vis
spectra of myricetin, morin and 3′,4′-dihydroxyflavone and their Cu(II) complexes. Figure 6 shows
spectra of taxifolin and 4-hydroxycoumarin and their Cu(II) complexes.
the UV-Vis spectra of taxifolin and 4-hydroxycoumarin and their Cu(II) complexes.
Generally, the UV-Vis spectra of both flavones and flavonols exhibit two characteristic absorption
Generally, the UV-Vis spectra of both flavones and flavonols exhibit two characteristic
maxima related to the π → π* transitions localized within the A ring and ring C (~240–290 nm,
absorption maxima related to the π → π* transitions localized within the A ring and ring C (~240–
referred to as band II, benzoyl system) and within the B-ring conjugated with the carbonyl of ring C
290 nm, referred to as band II, benzoyl system) and within the B-ring conjugated with the carbonyl
(~300–415 nm, referred to as band I, cinnamoyl system) [25,26]. Complex formation between a phenolic
of ring C (~300–415 nm, referred to as band I, cinnamoyl system) [25,26]. Complex formation between
a phenolic compound (organic ligand) and metal ions (cupric ions) causes a marked bathochromic
(red) shift of band I (up to 115 nm) toward the longer wavelengths due to the enhanced conjugative
effect as a new ring is formed [27]. Following interaction of phenolic compounds with cupric ions,
band II undergoes a mild red shift (up to 30 nm). Larger shifts of band I are linked with the
participation of the cinnamoyl system in the complex formation, and larger shifts of band II reflect
Molecules 2019, 24, 4335 6 of 28
The UV-Vis spectrum of myricetin (Figure 5, upper part) has two intense peaks at 253 nm (band
II) and
compound at 370 nm (band
(organic I). Following
ligand) and metaladdition of cupric
ions (cupric ions)ions, theaband
causes markedII exhibits a small (red)
bathochromic shift toward
shift of
the
band longer
I (up wavelength (~ 9 nm),
to 115 nm) toward thebut the wavelengths
longer band I is split, causing
due to the aenhanced
decreaseconjugative
in its intensity and
effect as aa new
new
absorption maximum is observed at 422 nm: this represents a red shift from the
ring is formed [27]. Following interaction of phenolic compounds with cupric ions, band II undergoesoriginal peak of ~ 52
nm, which suggests participation of cinnamoyl ring system in the interaction of myricetin
a mild red shift (up to 30 nm). Larger shifts of band I are linked with the participation of the cinnamoyl with cupric
ions
systemviainitsthe
3-OH and formation,
complex 4-CO groups and(see upper
larger scheme
shifts of band inIIFigure
reflect 5). Adjacent -OH
involvement of thegroups
benzoyl ofsystem
the B-
ring
in the formation of complexes between the phenolic compounds and the metal ions. To simulateThe
in the myricetin molecule can provide an alternative site for the interaction with cupric ions. the
lower intensity
conditions closeof
tothe
thenewly formed
biological band at 442
environment, thenm suggests
UV-Vis thatof
spectra some copperinteracting
flavonoids ions may alsowithbind to
Cu(II)
myricetin through the 3′-,4′-,5′-OH groups of
ions were measured in DMSO-phosphate buffer (5% v/v). the catechol moiety (ring B).
0.7
MYR 0.h
253 nm 370 nm MYR 1.h
0.6 MYR 3.h
MYR 6.h
262 nm 1 MYR : 1 Cu 0.h
0.5 1 MYR : 1 Cu 1.h
Absorbance
1 MYR : 1 Cu 3.h
0.4 1 MYR : 1 Cu 6.h
2 MYR : 1 Cu 0.h
422 nm 2 MYR : 1 Cu 1.h
0.3 2 MYR : 1 Cu 3.h
2 MYR : 1 Cu 6.h
0.2
0.1
0.0
250 300 350 400 450 500 550 600
Wavelength (nm)
0.9
261 nm MOR 0.h
0.8 273 nm MOR 1.h
MOR 3.h
0.7 MOR 6.h
1 MOR : 1 Cu 0.h
0.6 371 nm 1 MOR : 1 Cu 1.h
Absorbance
1 MOR : 1 Cu 3.h
0.5 1 MOR : 1 Cu 6.h
2 MOR : 1 Cu 0.h
0.4 390 nm 2 MOR : 1 Cu 6.h
2 MOR : 1 Cu 3.h
0.3 2 MOR : 1 Cu 6.h
0.2
0.1
0.0
250 300 350 400 450 500 550 600
Wavelength (nm)
1.4 237 nm
340 nm DHF 0.h
244 nm DHF 1.h
1.2 396 nm DHF 3.h
DHF 6.h
342 nm
1.0 314 nm 1 DHF : 1 Cu 0.h
1 DHF : 1 Cu 1.h
Absorbance
1 DHF : 1 Cu 3.h
0.8 1 DHF : 1 Cu 6.h
2 DHF : 1 Cu 0.h
0.6 2 DHF : 1 Cu 1.h
2 DHF : 1 Cu 3.h
2 DHF : 1 Cu 6.h
0.4
0.2
0.0
250 300 350 400 450 500 550
Wavelength (nm)
UV-Vis spectra 0 ,40 -dihydroxyflavone

Figure 5.
Figure 5. UV-Vis spectra of
of flavonols
flavonols myricetin
myricetin (MYR),
(MYR), morin
morin (MOR)
(MOR) andand 33′,4′-dihydroxyflavone
(DHF) and
(DHF) and their
their Cu(II)
Cu(II) complexes
complexes in
in DMSO-phosphate
DMSO-phosphate buffer (5% v/v)
buffer (5% v/v) at
at molar
molar ratios Cu:flavonols =
ratios Cu:flavonols =
1:1 a 1:2 recorded at the beginning of the reaction (up to 10 min), then after 1, 3 and 6 h. Potential
1:1 a 1:2 recorded at the beginning of the reaction (up to 10 min), then after 1, 3 and 6 h. Potential Cu(II)
chelating sites of myricetin, morin and 30 ,40 -dihydroxyflavone are shown on the right panel.
Cu(II) chelating sites of myricetin, morin and 3′,4′-dihydroxyflavone are shown on the right panel.
changes can be attributed to the weak Cu(II)-binding to the 3′,4′-dihydroxy groups of the catechol
moiety
Molecules (new
2019, band at 390 nm).
24, 4335 7 of 28
Figure 6. UV-Vis spectra of flavonols taxifolin (TAX) and simple phenol 4-hydroxycoumarin (CUM)
andFigure
their Cu(II) complexes
6. UV-Vis spectra in
of DMSO-phosphate buffer and
flavonols taxifolin (TAX) v/v) at molar
(5% simple phenolratios Cu: phenolic compound
4-hydroxycoumarin (CUM)
= 1:1
anda 1:2 recorded
their at the beginning
Cu(II) complexes of the reaction buffer
in DMSO-phosphate (up to 10
(5%min),
v/v) then after 1,
at molar 3 and
ratios 6 h.
Cu: at molar
phenolic
ratios Cu: phenolic compound = 1:1 a 1:2 recorded at the beginning of the reaction
compound = 1:1 a 1:2 recorded at the beginning of the reaction (up to 10 min), then after 1, 3 and (up to 106min),
h.
after 1, 3 and
at molar 6 h. Cu:
ratios Potential Cu(II)
phenolic chelating
compound sites
= 1:1 of taxifolin
a 1:2 recorded and 4-hydroxycoumarin
at the are shown
beginning of the reaction (up to on10the
right panel.
min), after 1, 3 and 6 h. Potential Cu(II) chelating sites of taxifolin and 4-hydroxycoumarin are shown
on the right panel.
The UV-Vis spectrum of myricetin (Figure 5, upper part) has two intense peaks at 253 nm (band
II) and atThe UV-Vis
370 spectrum
nm (band of the coumarin
I). Following additionderivative
of cupric shows
ions, major peaks
the band at 286 nm,
II exhibits 298 nmshift
a small andtoward
~345
thenm (Figure
longer 6, lower panel).
wavelength (~9 nm), However, following
but the band its interaction
I is split, causing a with cupric
decrease ions
in its the firstand
intensity peak is
a new
slightly skewed to the lower wavelengths and the band at ~345 disappeared.
absorption maximum is observed at 422 nm: this represents a red shift from the original peak of Such negligible changes
~52innm,
the which
UV-Vissuggests
spectrum, following the
participation of interaction
cinnamoyl of ringcoumarin
system with
in theCu(II) are in of
interaction agreement
myricetin with
with
previous
cupric reports
ions via and and
its 3-OH can be4-COexplained
groups by(seethe isolated
upper schemeketo-inor hydroxyl-
Figure groups -OH
5). Adjacent in itsgroups
molecular
of the
structure,
B-ring in thewhich cannot
myricetin stronglycan
molecule bindprovide
metal ions [31,33].
an alternative site for the interaction with cupric ions.
The lower intensity of the newly formed band at 442phenolic
In summary, to effectively bind cupric ions to nm suggestscompounds
that some requires
copper the presence
ions may alsoof the
bind
3-OH and 4-CO groups of0 the 0 flavonoid
0 C ring, which is documented by the formation of a new band
to myricetin through the 3 -,4 -,5 -OH groups of the catechol moiety (ring B).
in the UV-Vis spectrum; however, adjacent -OH groups in ring B can also serve as donor groups to
The UV-Vis spectrum of morin (Figure 5 middle part) shows peaks centered at 261 nm (band II)
interact with cupric ions. The results further indicate that the tight interaction between phenolic
and 371 nm (band I). When this flavonol is mixed with cupric ions, a slight shift occurs for the band II
compounds and cupric ions via the 3-OH and 4-CO groups is only achieved if the unsaturated C2-
(~12 nm), but in contrast with the behaviour of myricetin, band I disappears and a new absorption
C3 bond of the C-ring is present, which permits through-conjugation with the B-ring. From a
band is observed
spectroscopic at 390
point nm (red
of view, shift about
the more intense19 nm).
and moreBoth hydroxyl
greatly shiftedgroups
towardon ringwavelengths
longer B are separatedis
fromthe newly-present band (>400 nm) in the UV-Vis spectra, the stronger is the interaction ofshift
one other and therefore cannot act together to coordinate with cupric ions. The of band
cupric ions I
results
with from a change of
the polyphenol, andthethis
electronic charge density
occurs preferentially in theitscinnamoyl
through 3-OH and system containing
4-CO groups. the 3-OH
Coordination
group, which suggests that complex formation with Cu(II) has occurred through
of the copper(II) ions to the 3′-OH and 4′-OH groups of the polyphenol [33] is signified in the UV-Vis the 3-OH and 4-CO
groups
spectra[27].
by the appearance of a less intense band at wavelengths greater than 400 nm. According to
This type of interaction is supported by the greater proton acidity of the 3-OH group [28],
as compared with the 5-OH group. The dissociation constants of phenolic -OH groups are influenced
by the presence of the 4-CO group, which induces electronic density shifts via resonance effects [29].
The first complexation introduces a steric barrier that will hinder further complexation via the 4-CO and
5-OH groups [30]. Nevertheless, the comparable mild shifts of the band II towards higher wavelengths
Molecules 2019, 24, 4335 8 of 28
in the spectra of both flavonols (9 nm for myricetin and 12 nm for morin) indicate that an interaction of
cupric ions through the 4-CO and 5-OH cannot be excluded.
Although the shift of band I is an indicator of interaction through the 3-OH and 4-CO groups for
both myricetin and morin, the differences in the intensities of the new peaks observed at 422 nm for
myricetin and at 390 nm for morin correlate with differences in their molecular structures. In the morin
molecule, copper cannot bind to the OH groups of the B ring, so the 3-OH-4-CO groups most likely
provide the only available site for interaction with cupric ions on the cinnamoyl part of the molecule:
this is in agreement with the observation of a new absorption maximum at 390 nm along with the
disappearance of the original band at 371 nm observed for the interaction of Cu(II) with morin.
The 30 ,40 -dihydroxyflavone molecule possesses an isolated 4-CO group and also contains two
-OH groups in the B-ring through which it can interact/complex with cupric ions. In the electronic
absorption spectrum of 30 ,40 -dihydroxyflavone (Figure 5, lower part), two peaks appear, at 244 nm
(associated with the benzoyl skeleton) and at 340 nm (corresponding to the B-ring, cinnamoyl system).
As it can be seen from Figure 5, the molar ratio of copper ions to this flavone significantly affects the
shape of the spectrum. In the case of equimolar ratio of cupric ions and 30 ,40 -dihydroxyflavone (green
line in the spectrum), the first peak is slightly skewed to the lower wavelength (~7 nm) and the second,
intense absorption maximum (340 nm) is split, causing a decrease in its intensity and a new absorption
maximum is observed at 396 nm: this represents a red shift from the original peak of ~56 nm. Due to
the absence of functional (-OH) groups in the C ring to be involved in binding copper ions, the only
Cu(II) binding site in 30 ,40 -dihydroxyflavone is characterised by the 30 -OH and 40 -OH groups in the B
ring (see lower scheme in Figure 5).
Figure 5 also shows the change in band intensity over time. While in the spectrum recorded
after 10 min (indicated as 0. hour) the intensity of the new band at 396 nm (indicating copper-flavone
interaction) is weak, after 1 h its intensity increases significantly with a concomitant disappearing of
main absorption band of the free 30 ,40 -dihydroxyflavone.
If 30 ,40 -dihydroxyflavone is in excess to copper ions (blue line), the spectrum shows only a small
decrease in intensity of band at 340 nm, with the concomitant appearance of a low intensity flat band
at 396 nm; band at 244 nm follows the spectrum of free 30 ,40 -dihydroxyflavone.
In the UV-Vis spectrum of taxifolin, the absorption maximum is observed at 289 nm with a
shoulder centered at around 326 nm (Figure 6). Inspection of the taxifolin structure indicates two
potential binding sites for the cupric ions: the 4-CO/5-OH groups (A/C rings) and the 30 ,40 -dihydroxy
groups of the catechol moiety (B-ring) [31,32]. The presence of the saturated C2-C3 bond in the C-ring
interrupts the delocalization of the π-system between the A/C fused rings and the B-ring which (unlike
flavonols) become deconjugated and independent of one other. Consequently, the interaction of cupric
ions through the 3-OH/4-CO groups is much less favourable, because the interaction is preferentially
directed towards the formation of a more stable, conjugated planar system. UV-Vis spectra (Figure 6)
recorded in the presence of cupric ions exhibit loss of the flat band at 326 nm and simultaneous
appearance of a new, very low in intensity band at 390 nm. The observed spectral changes can be
attributed to the weak Cu(II)-binding to the 30 ,40 -dihydroxy groups of the catechol moiety (new band
at 390 nm).
The UV-Vis spectrum of the coumarin derivative shows major peaks at 286 nm, 298 nm and
~345 nm (Figure 6, lower panel). However, following its interaction with cupric ions the first peak is
slightly skewed to the lower wavelengths and the band at ~345 disappeared. Such negligible changes in
the UV-Vis spectrum, following the interaction of coumarin with Cu(II) are in agreement with previous
reports and can be explained by the isolated keto- or hydroxyl- groups in its molecular structure, which
cannot strongly bind metal ions [31,33].
In summary, to effectively bind cupric ions to phenolic compounds requires the presence of the
3-OH and 4-CO groups of the flavonoid C ring, which is documented by the formation of a new band in
the UV-Vis spectrum; however, adjacent -OH groups in ring B can also serve as donor groups to interact
with cupric ions. The results further indicate that the tight interaction between phenolic compounds
Molecules 2019, 24, 4335 9 of 28
and cupric ions via the 3-OH and 4-CO groups is only achieved if the unsaturated C2-C3 bond of the
C-ring is present, which permits through-conjugation with the B-ring. From a spectroscopic point of
view, the more intense and more greatly shifted toward longer wavelengths is the newly-present band
(>400 nm) in the UV-Vis spectra, the stronger is the interaction of cupric ions with the polyphenol, and
this occurs preferentially through its 3-OH and 4-CO groups. Coordination of the copper(II) ions to the
30 -OH and 40 -OH groups of the polyphenol [33] is signified in the UV-Vis spectra by the appearance of
a less intense band at wavelengths greater than 400 nm. According to the UV-Vis data, the phenolic
compound most tightly coordinated to Cu(II) ions, within the series of compounds studied is myricetin
and morin.
2.2. Antioxidant Properties of Phenolic Compounds and Their Cu(II) Complexes

The antioxidant (radical scavenging) activity of phenolic compounds and their complexes with
cupric ions (prepared in situ using metal-to-ligand ratios of 1:1 and 1:2) was monitored using the ABTS
assay, which, briefly, is a simple non-enzymatic method commonly used for the in vitro evaluation of the
antioxidant activity of H-donor substances. Formation of the blue-green coloured ABTS radical cations
(ABTS•+ ) is initiated chemically and their decay, following addition of the compound being studied
is monitored using UV-Vis spectroscopic measurements of the ABTS•+ absorption band at 734 nm.
The reaction between ABTS•+ and the phenolic compound is accompanied by the decolorization of the
ABTS•+ as it is reduced back to its colourless neutral form. Radical scavenging activity is one of the
criteria that a substance must meet to be described as an antioxidant, and so those molecules with better
radical scavenging activity are generally considered to have better antioxidant properties. One should
bear in mind, however, that the antioxidant activities of phenolic compounds also indirectly depend
on their metal-chelating abilities, as discussed above.
The results of the ABTS assay are summarized in Table 1, which clearly show there are significant
differences in radical scavenging activities among the individual phenolic compounds.
Table 1. Inhibition [%] of radical cation of ABTS (ABTS•+ ) by phenolic compounds (PhC).
% Inhibition
MYR MOR DHF TAX CUM
PhC 98.10 ± 1.42 74.05 ± 0.41 36.56 ± 0.77 21.79 ± 0.71 35.67 ± 0.77
Cu:PhC (1:1) 81.50 ± 1.48 69.22 ± 1.36 38.02 ± 1.12 23.71 ± 0.34 36.62 ± 1.20
Cu:PhC (1:2) 79.66 ± 1.76 60.59 ± 1.66 38.82 ± 1.32 29.19 ± 0.45 36.28 ± 1.13
PhC = Phenolic compound; MYR = Myricetin; MOR = Morin; DHF = 30 ,40 -dihydroxyflavone; TAX = Taxifolin;
CUM = 4-hydroxycoumarin. Concentration of phenolic compounds = 8.33 µmol.
The observed differences in radical scavenging activities of phenolic compounds are related to
their molecular structures and decrease in the order: MYR > MOR > DHF ~ CUM > TAX. Generally,
there are two main mechanisms through which phenolic compounds can exert their scavenging activity:
the hydrogen transfer mechanism and electron donation mechanism. In the ABTS assay, the ABTS•+
reacts with hydrogen donor molecules and therefore we can expect a positive correlation between
the radical scavenging activity of phenolic compounds and the number of their hydroxyl groups [34].
Myricetin, with six hydroxyl groups, exerts the highest antioxidant activity, followed by morin which
possesses five hydroxyl groups. In the presence of Cu(II) ions, the antioxidant activity of both myricetin
and morin decreases because the coordinated cupric ions replace hydrogen atoms and thus the ability
of phenolic molecule to provide hydrogen atoms for reaction with ABTS radical is reduced.
30 ,40 -Dihydroxyflavone with two hydroxyl groups achieves about 50% of the scavenging activity
of morin. A common structural characteristic of myricetin, morin and 30 ,40 -dihydroxyflavone is
the presence of the planar 2-phenyl-benzo-γ-pyrone skeleton. A significantly lower activity of the
pure flavone compounds, as compared with the flavonols, correlates with their smaller number of
hydroxyl substituents.
Molecules 2019, 24, 4335 10 of 28
Although the molecule of 30 ,40 -dihydroxyflavone has only one site for copper chelation (30 -OH
and 40 -OH), the radical scavenging activity is about the same for the pure flavone, or the flavone
in the presence of cupric ions in either molar ratio, which can be attributed to a weaker chelation
ability observed in fresh prepared solution (after 10 min), as is indicated by the UV-Vis spectrum of
this flavone (Figure 5). The higher scavenging activity of the 30 ,40 -dihydroxyflavone, (with just two
hydroxyl groups) compared to that of taxifolin (which has five hydroxyl groups), can be explained in
terms of the radical stability associated with the above mentioned, planar, 2-phenyl-benzo-γ-pyrone
skeleton. While the entire 30 ,40 -dihydroxyflavone molecule possesses an entire conjugated system,
in which the radical is extensively stabilized by resonance, in the taxifolin molecule, the conjugation
of the system is disrupted by the absence of a C2-C3 double bond and the resulting radical will be,
accordingly, less stable.
The antioxidant activity of 4-hydroxycoumarin is comparable with 30 ,40 -dihydroxyflavone (one
hydroxyl group vs. two hydroxyl groups); however, the separation between the hydroxyl and
carbonyl groups of 4-hydroxycoumarin does not allow them to provide a site for copper chelation (as
documented by the UV-Vis spectra) and thus the activity of 4-hydroxycoumarin in the presence of
Cu(II) ions is almost identical to the free compound.
In conclusion, when overviewing the reasons for the observed differences in antioxidant/radical
scavenging activities of the phenolic compounds studied, there are three main factors that must be
considered: (i) the number of hydroxyl groups (i.e., the number of potentially donatable hydrogen
atoms) (ii) the overall planarity of the molecular skeleton and (iii) the extent of the electronically
delocalized system. Molecules with more extended conjugated systems form resonance-stabilized free
radicals, which have more markedly increased half-lives.
It should be noted that the ABTS assay experiments express the relative rate of antioxidant activity,
as determined under in vitro conditions, which is significantly different from the physiological state
in the cells and biological fluids of living organisms [35]. In order to draw conclusions that are most
relevant to living systems, a more comprehensive assessment under different experimental conditions
is needed.
2.3. DNA Interaction Studies Using Absorption Titrations

Electronic absorption spectroscopy is a sensitive method for determining the binding characteristics
of small organic molecules and/or their metal complexes with a DNA molecule. Changes observed
in the electronic absorption spectrum reflect the interaction between the molecules of a particular
compound/complex and DNA, and inferences may be drawn regarding the mechanism of this interaction
from wavelength shifts of the corresponding absorption bands. Furthermore, by determining the
internal binding constant Kb , a more quantitative basis may be established for the process [36–39].
In summary, we recall that small organic molecules and transition metal complexes can interact
with DNA either by covalent bonding and/or non-covalent interactions, for example by (i) intercalation
into DNA, (ii) electrostatic interactions involving phosphate groups that are present on the DNA chains
and (iii) interaction with major or minor grooves in the DNA [40]. The mechanism of interaction depends
on the structure of the molecule and thus, planar molecules with conjugated bonds interact with DNA
most frequently by intercalation, so creating “stacking” interactions of the aromatic chromophore with
DNA bases [41]. Generally, if the molecule interacts with DNA via intercalation, both hypochromism
and bathochromism (red shift) are observed in the absorption spectrum. Bathochromism can be
explained on the basis of the coupling of an antibonding π* orbital of the intercalating ligand and the
π-orbital of the DNA base pairs, which thus lowers the π → π* transition energy, and in turn results in
a shift to longer wavelengths. Given that the coupled π orbital is not fully filled with electrons, there is
a decrease in the values of the spectral transition probabilities and this is reflected in the spectrum
as a decrease in absorbance (hypochromism) [42–44]. Thus, the binding of a molecule to DNA via
intercalation involves a stacking interaction between the aromatic moiety of the molecule and the base
pairs of DNA, which is characterized by both a hypochromic shift and a red shift (bathochromism) in
Molecules 2019, 24, 4335 11 of 28
the electronic absorption spectra. The strength of binding of the complexes with DNA is determined
quantitatively by evaluating the binding constant Kb . Electrostatic interactions or hydrogen bonding
between the substance and DNA result in destabilization of the latter, which is manifested in the
spectrum by a hyperchromic shift [45].
In our experiments, we investigated the effect of adding of CT-DNA to a solution of phenolic
compounds or copper-phenolic complexes (Cu:phenolic compound = 1:2). Electronic transitions at
characteristic wavelengths can be observed in the absorption spectra of organic compounds, whose
molecules exhibit σ-σ* transitions in the range of 150–250 nm; π-π* transitions in systems with
conjugated bonds typically occur in the range of 200–400 nm; while n-π* transitions (with the lowest
energy of all) are observed at wavelengths longer than 400 nm (400–700 nm).
Figure 7 shows the absorption spectra of myricetin, morin, 30 ,40 -dihydroxyflavone, taxifolin and
4-hydroxycoumarin (both as the free phenolic compounds and as their Cu:phenolic (1:2) chelates) after
addition of an aliquot of CT-DNA solution: the occurrence of spectral shifts observed for flavonoids
in the presence of cupric ions relative to free flavonoids confirms the existence of Cu(II)-flavonoid
chelates in solution.
Please note that slight shifts in the absorption maxima of free phenolic compounds observed in
the DNA interaction studies (this Section) and UV-Vis spectroscopy study (Section 2.1.2) are caused by
the different compositions of the buffers used.
The electronic absorption spectrum of free myricetin (MYR) is more complex than that of the other
phenolic compounds, with significantly intense peaks at 267 nm and 378 nm. It was found that the
intensities of both peaks decreased as the concentration of added DNA increased (Figure 7, Table 2),
and for the band at 267 nm, a hypochromic shift of about 45.95% was observed, accompanied by a
small bathochromic shift (of about 8 nm).
Table 2. Overview of wavelength values of absorption peaks for free phenolic compounds and their
chelates with copper ions, percentages of hypochromic (↓) and hyperchromic (↑) shifts. Bathochromic
and hypsochromic shifts are denoted (→) and (←), respectively and are expressed in nm.
Phenolic ↓↑ % ↓↑ % ↓↑ %
Band 1 Band 2 Band 3
Compound →/← nm →/← nm →/← nm
↓ 45.95 ↑ 23.33 ↓ 72.97
MYR 267 nm 322 nm 378 nm
→ 8 nm → 10 nm ← 5 nm
↓ 36.51 ↑ 23.33 ↓ 66.67
Cu-MYR 267 nm 346 nm 441 nm
← 5 nm ← 21 nm → 2 nm
↓ 22.86 ↓ 21.64
MOR 269 nm 393 nm
- -
↓ 42.25 ↑ 21.62 ↓ 55.17
Cu-MOR 269 nm 330 nm 398 nm
→ 7 nm - ← 2 nm
↓ 17.86 ↓ 22.83
DHF 244 nm 342 nm
← 2 nm -
↓ 26.83 ↓ 28.00 ↓ 31.75
Cu-DHF 281 nm 308 nm 397 nm
- - -
↓ 25.37
TAX 326 nm
-
↓ 27.59
Cu-TAX 326 nm
-
↓ 20.59
CUM 287 nm
-
energy of all) are observed at wavelengths longer than 400 nm (400–700 nm).
Figure 7 shows the absorption spectra of myricetin, morin, 3′,4′-dihydroxyflavone, taxifolin and
4-hydroxycoumarin (both as the free phenolic compounds and as their Cu:phenolic (1:2) chelates)
after addition of an aliquot of CT-DNA solution: the occurrence of spectral shifts observed for
flavonoids
Molecules 2019,in24,
the presence of cupric ions relative to free flavonoids confirms the existence of Cu(II)-
4335 12 of 28
flavonoid chelates in solution.
Absorption spectra
Figure 7. Absorption
Figure spectra obtained
obtained by
by titration
titration of
of free
free phenolic
phenolic compound
compound (20 (20 µM) and phenolic
µM) and phenolic
compound in
compound in the
the presence
presence of
of Cu(II)
Cu(II) (Cu:phenolic compound == 1:2)
(Cu:phenolic compound 1:2) with
with CT-DNA
CT-DNA (0–50(0–50 µM)
µM) inin aa
solution containing
solution containing 55 mM mM Tris-HCl,
Tris-HCl, 50
50 mM
mM NaCl,
NaCl, pH
pH 7.2
7.2 in
in the
the absence
absence of
of CT-DNA
CT-DNA (red(red line)
line) and
and in
in
the presence
the presenceofofananincreasing concentration
increasing concentrationof CT-DNA
of CT-DNA (0–50(0–50
µM). The
µM).direction of the arrow
The direction indicates
of the arrow
the shift in
indicates thethe absorbance
shift after addition
in the absorbance of the CT-DNA
after addition solution.solution.
of the CT-DNA Abbreviations: myricetin
Abbreviations: (MYR),
myricetin
morin (MOR), 30 ,40 -dihydroxyflavone (DHF), taxifolin (TAX), 4-hydroxycoumarin (CUM).
(MYR), morin (MOR), 3′,4′-dihydroxyflavone (DHF), taxifolin (TAX), 4-hydroxycoumarin (CUM).
The band at 378 nm was found to decrease in intensity by up to 72.97% with a hypsochromic shift
(of about 5 nm). The myricetin spectrum also contains a less intense peak at 322 nm, but which gains
intensity as the concentration of DNA increases showing a hyperchromic shift (of about 23.33%) and
also exhibits a bathochromic shift (of about 10 nm). There are three isosbestic points in the spectrum at
284 nm, 347 nm and 447 nm, which indicate the existence of different binding mechanisms of MYR to
DNA. The binding constants of the phenolic compounds and their Cu(II) complexes with DNA are
Molecules 2019, 24, 4335 13 of 28
measures of the strength of the interaction and are summarized in Table 3; the calculated value of
binding constant for the interaction of free myricetin with DNA is 2.07 × 104 .
Table 3. Binding constants (Kb ) of free phenolic compounds (FL) with DNA, and in the presence of
cupric ions (Cu:FL = 1:2).
Flavonoid MYR MOR DHF TAX CUM

FL 2.07 × 104 3.23 ×104 2.43 × 104 1.44 × 104 6.61 × 104
Cu:FL (1:2) 4.02 × 104 8.08 × 103 1.23 × 105 - -
The electronic absorption spectrum of the Cu:MYR (1:2) complex has features similar to those
for free myricetin, with the exception that the band at 267 nm is flat and poorly resolved; the band at
441 nm is more intense and rather broad. Both bands show a hypochromic shift with increased DNA
concentration: for the band centered at 267 nm the decrease in intensity is 36.51% and is associated
with a hypsochromic shift of about 5 nm; the intensity of the band at 441 nm is reduced by 66.67%, and
is accompanied by a small bathochromic shift of about 2 nm. Similarly to free myricetin, addition of
DNA to the solution of the Cu:MYR (1:2) complex results in hyperchromic shift of the band at 346 nm
(23.33%) accompanied by a hypsochromic shift (about 21 nm); the isosbestic points are at 292 nm and
350 nm. The calculated value of the binding constant for the interaction between the Cu-MYR (1:2)
complex and DNA is 4.02 × 104 , which indicates a slightly higher strength of the intercalation process
than occurs for free myricetin (Table 3). The observed hypochromism confirms that intercalation
has taken place between myricetin, and its Cu-complex and DNA. In addition, the observations of
isosbestic points and hyperchromism indicate that, in addition to intercalation, other mechanisms of
interaction, such as electrostatic and covalent interactions have occurred. Disruption of the secondary
DNA structure may occur, as a result of covalent interactions between the Cu-MYR complex and the
negatively charged DNA phosphate backbone with subsequent cleavage.
The absorption spectrum of free morin (MOR) has two major bands at 269 nm and 393 nm, and
the gradual addition of CT-DNA into a solution containing morin resulted in the hypochromic shifts of
both bands: approximately 22.86% at 269 nm and 21.64% at 393 nm (Table 2). Based on this observed
hypochromism, we can assume that an intercalation mechanism occurs between free morin and DNA,
most probably mediated by the benzoyl moiety as is confirmed by the estimated value of the binding
constant, 3.23 × 104 .
Similar hypochromic shifts as that observed for the Cu-MYR (1:2) complex were also noted for the
Cu-MOR (1:2) system after the addition of CT-DNA. Two absorption maxima, one centered at 269 nm
and another at 398 nm, exhibited pronounced hypochromic shifts of 42.25% and 55.17%, respectively.
The band at 269 nm also showed a significant bathochromic shift of about 7 nm, while an almost
negligible hypsochromic (blue) shift (~2 nm) was observed for the 398 nm band; isosbestic points were
observed at 297 nm and 348 nm. The value of DNA binding constant for Cu-MOR (1:2) complex was
found to be 8.08 × 103 , which suggests that this complex intercalates less tightly into the DNA than
was observed either for free morin (3.23 × 104 ) or for MYR and the Cu-MYR (1:2) complex (Table 3).
The existence of a hyperchromic shift in the UV-Vis spectrum suggests that other types of interactions,
for example of electrostatic and covalent kinds, also occurred between the Cu-complex and DNA.
The spectroscopic data discussed above indicate that the interaction of free morin or myricetin
and their Cu-complexes with DNA is more than a simple intercalation process, and that other types of
interaction with the DNA molecule also occur. While hypochromism, accompanied by batochromism,
is typically attributed to intercalation mechanisms, the hyperchromic effect may be the result of
electrostatic interactions between the positively charged flavonoid molecule, or its copper chelate, with
the negatively charged phosphate groups of DNA.
30 ,40 -Dihydroxyflavone (DHF) exhibits absorption maxima at 244 nm and 342 nm (Figure 7)
and following the addition of the CT-DNA solution, a hypochromic shift in the spectrum occurred,
of approximately 17.86% and 22.83%, as is indicated for the corresponding peaks (Table 2). The
Molecules 2019, 24, 4335 14 of 28
spectrum recorded from the system containing 30 ,40 -dihydroxyflavone and Cu(II) ions (Cu-DHF =
1:2) shows two intense peaks at 308 nm and 397 nm and a lower intensity peak at 281 nm. After
addition of DNA, hypochromic shifts were measured of 26.83% (at 281 nm), 28% (at 308 nm) and
31.75% (at 397 nm) (Table 2). The absorption spectra of both dihydroxyflavone and its copper(II)
chelates show a hypochromic shift (no bathochromic shift was observed) which identifies that the
intercalation mechanism with DNA had occurred. In principle, hypochromism without any significant
shift to longer wavelengths (bathochromism) might be due to the loss of chromophore molecules into
the solution that have been taken up by the DNA molecule during the course of the titration [42].
From a consideration of the molecular structure of 30 ,40 -dihydroxyflavone, it seems probable that the
molecule will intercalate into DNA via its benzoyl moiety. The binding constants determined for the
interaction of free 30 ,40 -dihydroxyflavone and the Cu-DHF (1:2) complex with DNA are 2.43 × 104 and
1.23 × 105 , respectively (Table 3).
While the hypochromism and value of the binding constant for free dihydroxyflavone confirms
its interaction with DNA by the intercalation mechanism (~104 ), the binding constant of the Cu-DHF
complex with DNA is higher by one order of magnitude (~105 ) which confirms that the interaction
between this complex and DNA is much stronger.
The absorption spectrum obtained by titrating a solution of free taxifolin (TAX) and taxifolin
in the presence of cupric ions (Cu:TAX = 1:2) with DNA is shown in Figure 7, which shows an
intense absorption band at 326 nm, whose intensity is reduced by titration with CT-DNA solution.
This hypochromic shift (25.37%) is the result of the interaction of taxifolin with DNA by the intercalation
mechanism (Table 2); a binding constant value of 1.44 × 104 was determined which confirms the
function of a mild intercalation mechanism (Table 3). The absorption spectrum of the complex Cu-TAX
(1:2) has a maximum at the same wavelength as the free taxifolin, indicating that copper(II) is chelated
by taxifolin either only very weakly or not at all. The observed hypochromic shift (27.59%) is the
result of the interaction of taxifolin, and not the Cu-TAX (1:2) complex with CT-DNA; a weak copper
chelating abilities of taxifolin are also evident from the UV-Vis spectra (Figure 6).
As indicated above, the UV-Vis spectra (Figure 6) demonstrate that 4-hydroxycoumarin does not
interact with cupric ions, and hence electronic absorption titrations were performed only for the free
phenolic compound. The effect of adding an aliquot of CT-DNA solution to a constant concentration of
free 4-hydroxycoumarin is shown in Figure 7, the spectrum showing an intense maximum at 287 nm.
The hypochromic shift is evident from the spectrum (20.59% at 287 nm) and indicates the interaction of
coumarin with the DNA by weak intercalation, for which the binding constant is 6.61 × 104 (Table 3).
According to their binding constant values, we may order the free phenolic substances in order of
decreasing binding strength as follows: CUM > MOR ~ DHF ~ MYR > TAX, and very interestingly, the
highest interaction strength was observed for the smallest molecule, 4-hydroxycoumarin. For systems
containing copper ions, we noted an increase in the binding constant for Cu(II)-dihydroxyflavone, and
for Cu(II)-myricetin complexes over that for the free phenolic compounds, which indicates a potentially
important role of cupric ions as a transport vehicle for their intercalation into DNA. Conversely, for the
Cu-MOR complex, a decreased strength of interaction with DNA was observed, as compared with that
for free morin.
From the above, we can conclude that myricetin can interact with Cu(II) via donor atoms in the
C ring (3-OH and 4-CO groups) and the B ring (30 -OH and 40 -OH groups) and that it exhibits an
interaction with DNA of intermediate strength (Kb ~ 104 ); morin, which interacts exclusively via donor
atoms on the C ring (3-OH and 4-CO groups) exhibits a mild interaction strength with DNA (Kb ~ 103 ),
while 30 ,40 -dihydroxyflavone, which can interact exclusively via the 30 -OH and 40 -OH groups on the
ring B, exhibits a strong interaction with DNA (Kb ~ 105 ). The overall results indicate that copper ions
can modulate the DNA binding affinity of flavonoids via the formation of their Cu-chelates.
Mechanisms by which myricetin, morin and 30 ,40 -dihydroxyflavone and their Cu(II) complexes can
interact with DNA predispose these substances to act as potential anticancer agents. Weak prooxidant
Molecules 2019, 24, 4335 15 of 28
properties of phenolic compounds under study make them promising substances to boost cellular
antioxidant systems including antioxidant enzymes and low molecular antioxidants such as glutathione.
2.4. Protective Effect of Phenolic Compounds against DNA Damage

DNA damage is associated with several diseases, as well as premature aging. Protection of DNA
from damage is important because of the important role of this molecule in the transfer of genetic
information and cell division. It is believed that phenolic compounds are suitable/promising natural
antioxidants that could prevent unwanted damage to DNA and in order to evaluate their potential
in this respect, gel electrophoresis was used with plasmid DNA (pDNA) under both normal and
Fenton conditions.
Gel electrophoresis represents a relatively simple way to evaluate radical induced DNA damage
(in the presence of Fenton like agents) and/or damage caused by intercalating molecules. If oxidative
cleavage to the covalent DNA bonds occurs, new conformations of pDNA can arise: cleavage in one
strand of the native superspiralized (SC) form of pDNA results in an open circular conformation (OC),
while cleavage in both strands results in a linear form (LIN). Both these DNA conformations differ in
their mobility in the gel and are made visible by staining with ethidium bromide [46]. From the band
intensities of the DNA conformational states, their relative abundances may be determined. In our
experiments, the reaction mixture contained cupric ions and a phenolic compound (with a constant
copper concentration and an increasing concentration of the phenolic compound). Experiments were
performed both in the absence, and presence of hydrogen peroxide (Fenton reaction (1)). The addition
of hydrogen peroxide (50 µM) followed by the DNA addition initiates the reaction with the copper
ions and the subsequent Cu(II)/Cu(I) redox cycle, which results in the formation of ROS. The degree
of DNA damage was correlated with the antioxidant (suppression of DNA damage) or prooxidant
(increase of DNA damage) effect of the given phenolic compound.
A protective/antioxidant effect of phenolic compounds (especially polyphenols) is generally
believed to be due to a combination of: (i) their metal (e.g., copper) chelating properties, resulting in
suppressed catalytic activity of the metal ions in the Fenton reaction, and/or ii) a direct scavenging
of free radicals [47]. In other words, the copper chelation by flavonoids reduces the ability of Cu(II)
ions to enter the redox cycling mechanism, initiated by hydrogen peroxide, which would otherwise
result in DNA damage. The protective effect of copper chelation by flavonoids is enhanced when the
Cu-flavonoid complex is formed before hydrogen peroxide has been added. Generally, an increase in
the intensity of the SC form of pDNA (preserved SC DNA) with a concomitant decrease of the OC
and/or LIN form intensities, when the phenolic compound is present, is considered to signify that the
substance being studied has exerted a protective effect. Generally, it is expected that increasing the
concentration of phenolic compounds enhances the DNA protective effect predominantly via a radical
scavenging mechanism.
Figure 8 shows the electrophoregram of the interaction of free phenolic compounds and their
Cu(II)-complexes with plasmid DNA in the absence of hydrogen peroxide (non-Fenton experiment).
In the absence of Cu(II) (see left panel, Figure 8), the most profound concentration dependent DNA
damage is noted for myricetin. A slight degree of DNA damage has also been observed for the
interaction of taxifolin with DNA. A similar trend as it was observed for free phenolic compounds
interacting with DNA was observed for their Cu(II) complexes.
Figure 8 shows the electrophoregram of the interaction of free phenolic compounds and their
Cu(II)-complexes with plasmid DNA in the absence of hydrogen peroxide (non-Fenton experiment).
In the absence of Cu(II) (see left panel, Figure 8), the most profound concentration dependent DNA
damage is noted for myricetin. A slight degree of DNA damage has also been observed for the
interaction of taxifolin with DNA. A similar trend as it was observed for free phenolic compounds
Molecules 2019, 24, 4335 16 of 28
interacting with DNA was observed for their Cu(II) complexes.
Figure 8. Gel electrophoregram of the interaction of phenolic compounds (PhC) and their Cu(II)
complexes with plasmid DNA in the absence of hydrogen peroxide (non-Fenton system). Electrophoretic
profile of agarose gel (0.8%) of the reaction mixture of 20 µM pBSK+ DNA, free phenolic compounds
(lanes 1–5) and Cu(II) complexes with phenolic compounds (lanes 6–10), incubated at RT for 30 min.
The order of the columns in the gel: (p) control pDNA; (1–5) pDNA + 5–500/5000 µM PhC (MYR,
MOR, DHF/TAX, CUM), (6–10) pDNA + 5–500/5000 µM PhC (MYR, MOR, DHF/TAX, CUM) + 5 µM
Cu(II). Experimental conditions of all reactions were the same, so the observed differences in gels are
related to structural differences of phenolic compounds. Due to the different solubility of phenolic
compounds, individual phenolic compounds were used under different concentrations. (PhC =
Phenolic compound; MYR = Myricetin; MOR = Morin; DHF = 30 ,40 -dihydroxyflavone; TAX = Taxifolin;
CUM = 4-hydroxycoumarin).
Interestingly, the Cu-MYR complex exhibit similar degree of DNA damage as it was observed for
free myricetin, which roughly correlates with the similar binding constants for MYR and the Cu-MYR
complex with DNA (~104 ). From the electrophoretic profiles obtained, it may be deduced that the
most severe DNA damage is caused by the myricetin, which contains a 3-OH group on ring C and
three adjacent -OH groups on ring B. Through these functional groups, myricetin can interact with
cupric ions. A weakly damaging effect of taxifolin and its Cu(II) complex accords with the presence
of a 3-OH group on ring C and two adjacent -OH groups on ring B; however, the C2-C3 bond of
ring C is saturated, which interrupts the extent of conjugation and coplanarity that are important for
the interaction with cupric ions. Coplanarity of the molecule is also important for the intercalation
mechanism of action with DNA.
Figure 9 shows the gel electrophoregram of the interaction of phenolic compounds (PhC) and
their Cu(II) complexes with plasmid DNA in the presence of hydrogen peroxide (Fenton-like system).
Generally, damage observed for a Fenton-like system is greater than for a non-Fenton system, due to
the ROS formed in the course of the Fenton reaction. Similarly to the non-Fenton system, the studied
Molecules 2019, 24, 4335 17 of 28
flavonoids, myricetin, morin, and 30 ,40 -dihydroxyflavone were tested within the concentration range
5–500 µM, and the flavonoids TAX and CUM allowed their testing up to the 5000 µM concentrations
(lanes 1–8). Each electrophoretic profile contains a control pDNA (lane p) indicating the quality of
the pDNA used in each reaction and control reaction with Fenton agents (cupric ions, H2 O2 ) (lane C)
showing new conformational states of pDNA in the absence of phenolic compounds. Thus the lane
C shows the DNA damage caused exclusively by ROS formed in the course of the copper-catalyzed
Fenton reaction
Molecules 2019, 24,(of
x which the main ROS is the hydroxyl radical). 17 of 28
Figure 9. Gel electrophoregram of the interaction of phenolic compounds (PhC) and plasmid DNA in
Figure
the 9. Gel
presence of electrophoregram
hydrogen peroxideof(Fenton
the interaction
system).ofElectrophoretic
phenolic compounds (PhC)agarose
profile (0.8% and plasmid
gel) ofDNA
DNAin
the presence of hydrogen peroxide (Fenton system). Electrophoretic profile (0.8% agarose
protection against oxidative damage in the reaction mixture (20 µL) containing Cu(II) ions, phenolic gel) of DNA
protection against oxidative damage in the reaction mixture (20 µL) containing Cu(II)
compounds, pBSK+ DNA, hydrogen peroxide and incubated at RT for 30 min. The order of the samples ions, phenolic
incompounds, pBSK+pDNA;
the gel: (p) control DNA, (C)hydrogen
Fenton peroxide andcontaining
like reaction incubatedpDNA
at RT+for
5 µM30 CuCl
min. 2The
+ 50order
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2 O2 ;
samples
(1–8) Fentonin like
the gel: (p) control
reaction pDNA;
+ phenolic (C) Fenton
compound like reaction containing
at concentrations of 5–500 µM pDNA
(MYR,+ 5MOR,
µM CuCl
DHF) 2 +or50
µM HµM
5–5000 2O2; (TAX,
(1–8) Fenton
CUM);like(L) reaction
1 kb DNA + phenolic
standardcompound at concentrations
(LIN) linearized pDNA. of 5–500 µM (MYR, MOR,
DHF) or 5–5000 µM (TAX, CUM); (L) 1 kb DNA standard (LIN) linearized pDNA.
As can be seen from the electrophoretic profiles, the flavonoids myricetin, morin, and taxifolin
As can be seen
show pro-oxidant from the
behaviour electrophoretic
being documentedprofiles, the
in gels by theflavonoids
complete myricetin, morin, suppression
loss or significant and taxifolin
ofshow
bandspro-oxidant
correspondingbehaviour being (Figure
to SC pDNA documented in gels by
9, MYR—lanes 1–5;the complete loss
MOR—lanes 1–3; or significant
—lanes 1–2)
accompanied by the enhanced band intensities corresponding to the cleaved plasmid DNA (OC and—
suppression of bands corresponding to SC pDNA (Figure 9, MYR—lanes 1–5; MOR—lanes 1–3;
lanesIn1–2)
LIN). accompanied
cases of morin and bytaxifolin
the enhanced band
and also intensities
partly corresponding
in the case of myricetin to the DNA
cleaved plasmid
damage DNA
can be
(OC and for
accounted LIN). In cases
by ROS of morin
products and taxifolin
originating and also
from Fenton partly in the case of myricetin the DNA
reaction.
damage
A weakcanprotective
be accounted forofby
effect ROS products
myricetin originating
was observed at afrom Fenton reaction.
concentration of 400 µM, which is well
above A theweak protective
copper effect of(Figure
concentration myricetin was observed
9, MYR—lane at ainconcentration
7), but comparison with of 400
theµM, which
control is well
variant
above the copper concentration (Figure 9, MYR—lane 7), but in comparison with the
(lane C), the loss of SC DNA is still significantly greater because of the interaction of Cu-myricetin control variant
(lane C),with
complex the DNA
loss of SCFigure
(see DNA 8). is still
Even significantly greater
at the highest because tested
concentration of the (500
interaction
µM, laneof 8),
Cu-myricetin
myricetin
complex with DNA (see Figure 8). Even at the highest concentration tested (500 µM, lane 8), myricetin
did not achieve a protective effect comparable to the effect of morin at 200 µM concentration (MOR—
lane 5), in agreement with the non-Fenton experiments (see Figure 8). In the case of morin, a gradual
increase in the band intensity of the SC form over a concentration range of 100–500 µM (MOR, lanes
4–8) suggests a stronger protective effect, as compared with myricetin. Considering the similarity in
the structures of myricetin and morin (differing by just one -OH group), this finding is rather
Molecules 2019, 24, 4335 18 of 28
did not achieve a protective effect comparable to the effect of morin at 200 µM concentration (MOR—lane
5), in agreement with the non-Fenton experiments (see Figure 8). In the case of morin, a gradual
increase in the band intensity of the SC form over a concentration range of 100–500 µM (MOR, lanes
4–8) suggests a stronger protective effect, as compared with myricetin. Considering the similarity in the
structures of myricetin and morin (differing by just one -OH group), this finding is rather unexpected,
because the protective effect of flavonoids (or generally antioxidants) against ROS is usually related to
their number of hydroxyl substituents.
Thus, the strong prooxidant behaviour of myricetin under the conditions of the Cu-Fenton reaction
must be related to a different mechanism of action. We propose that the formation of dimeric or even
polymeric species of Cu:myricetin complex, as documented by the EPR spectroscopic measurements
(see above) and hyperchromism observed in the absorption titrations (see above), point to the complex
mechanism of action of the Cu(myricetin)2 complex causing significant DNA damage. In addition,
dimeric/polymeric Cu complexes with chelated myricetin have a less tightly saturated coordination
sphere around the copper ion than do monomeric Cu complexes, so the catalytic activity of the
Cu-complex in the Fenton reaction is more profound and results in the formation of more ROS.
The prooxidant behaviour of myricetin in the presence of cupric ions indicates its possible use as
an anticancer agent [48]. From the results obtained, it follows that the DNA protective effect of
flavonoids against ROS, in the presence of redox metals such as copper, may not necessarily be related
to the number of hydroxyl groups in the molecular structure of the flavonoid. The presence of 3-OH
group on ring C, which is involved in the interaction with cupric ions, and planarity of the flavonoid,
are important factors that contribute to its prooxidant activity, and also the presence of adjacent
-OH groups on ring B, which can also assist in its interaction with cupric ions. Thus, the combined
interaction via donor atoms on ring C and ring B results in the formation of stable Cu-flavonoid
complexes that are capable of causing severe DNA damage. Thus, to have a high number of hydroxyl
groups, which otherwise is a beneficial structural feature of flavonoids, may become counterproductive
in the presence of metal ions (e.g., Cu2+ ), especially when a 3-OH group is present on ring C, and there
are adjacent -OH groups on ring B which can all participate in the interaction between the flavonoid
and cupric ions.
As illustrated in Figure 9, taxifolin shows a weaker, but similar, trend to myricetin. The presence
of a weak band from the SC form at a concentration of 100 µM (TAX—lane 3) indicates a somewhat
weaker pro-oxidant effect as compared with the same concentration of myricetin (MYR—lane 4).
However, a certain level of prooxidant activity for taxifolin is observed even at its highest (5000 µM)
concentration (TAX—lane 8). The prooxidant effect of taxifolin, in the presence of cupric ions, can be
explained on the basis of ROS-induced damage and to some extent by DNA intercalation mechanism
of action (Figure 8). Taxifolin also shows a strong tendency to form dimeric species when coordinated
to Cu(II) ions.
In contrast, 30 , 40 -dihydroxyflavone, significantly, and 4-hydroxycoumarin (no 3-OH group), to
some extent, both exhibit a protective effect within the entire concentration range tested, as is illustrated
by the degree of preservation of the SC form of plasmid DNA in gels (Figure 9, DHF—lanes 1–8,
CUM—lanes 1–8). A more pronounced concentration dependence is observed in the presence of
DHF, as seen in the gel by a gradual significant loss of OC pDNA. A very weak prooxidant effect of
dihydroxyflavone can be explained on the basis of its ROS-scavenging activity and by the existence
of only one potential copper chelating site (two hydroxyl groups on ring B) so creating a more labile
copper complex that cannot cause significant damage to DNA.
4-Hydroxycoumarin has no potential site to chelate cupric ions and therefore any potential DNA
damage caused by intercalation of its copper complex can be excluded (see also Figure 8). The overall
damage observed is due to the Cu-catalyzed Fenton reaction suppressed by the radical scavenging
activity of free (uncoordinated) 4-hydroxycoumarin.
Molecules 2019, 24, 4335 19 of 28
Quantification of band intensities was performed by densitometric analysis. The relative

concentration proportions of the individual conformational forms of plasmid DNA are shown in
Figure 10.
Figure 10. Relative densitometric quantification of band intensities of, at least, 3 agarose gels as
Figure 10. Relative densitometric quantification of band intensities of, at least, 3 agarose gels as
illustrated in Figure 9. Green colour corresponds to the SC form of plasmid DNA, red colour to the LIN
illustrated in Figure 9. Green colour corresponds to the SC form of plasmid DNA, red colour to the
form and orange colour to the OC form. Taxifolin and coumarin were tested up to a concentration of
LIN form and orange colour to the OC form. Taxifolin and coumarin were tested up to a concentration
5000 µM. p—SC form of pDNA, C—proportion of pDNA conformations formed in Fenton like reaction,
of 5000 µM. p—SC form of pDNA, C—proportion of pDNA conformations formed in Fenton like
1–8—proportion of pDNA conformations formed in Fenton like reaction in the presence of phenolic
reaction, 1–8—proportion of pDNA conformations formed in Fenton like reaction in the presence of
compound. Standard deviations are expressed as error bars.
phenolic compound. Standard deviations are expressed as error bars.
Based on the electrophoretic profiles, the protective effect of the phenolic compounds studied,
Based
against on the
plasmid electrophoretic
DNA damage under profiles, the protective
the experimental effect of the
conditions phenolic
described, compounds
decreases in the studied,
order:
against plasmid DNA damage under the experimental conditions
DHF > CUM > MOR > TAX ~ MYR. The results clearly show that more pronounced DNA damage described, decreases in the order:
DHF > CUM > MOR > TAX ~ MYR. The results clearly show that more pronounced
occurs in the presence of those flavonoids with more hydroxyl groups such as MYR (with 6-OH groups, DNA damage
occurs
and in the
where presence
a 3-OH group of isthose flavonoids
present), MOR (withwith more
5-OHhydroxyl
groups, and groups suchgroup
a 3-OH as MYR (with 6-OH
is present) and
groups, and where a 3-OH group is present), MOR (with 5-OH groups, and
TAX (with 5-OH groups and where a 3-OH group is present) all form stable copper(II) complexes, a 3-OH group is present)
and are
that TAX (withof5-OH
capable causing groups
DNAand damagewhere viaainteraction
3-OH group withisDNA
present) all form
and ROS stablevia
formation copper(II)
Fenton
complexes,
reaction [49].that are capable of causing DNA damage via interaction with DNA and ROS formation
via Fenton reaction [49].
2.5. Qualitative Analysis of ROS Formation
2.5. Qualitative Analysis of ROS Formation
During the course of the Fenton reaction, ROS such as singlet oxygen (1 O2 ), hydroxyl radical
•
( OH),During the course
and superoxide of the
anion Fenton
radical (O2reaction, ROS such
•− ) are formed. as singlet
In order oxygen
to identify these(1species,
O2), hydroxyl radical
the following
• •−
( OH), scavengers,
radical and superoxide anion radical
L-histidine (detection(O2 of) are formed.
singlet In order
oxygen), DMSOto identify these
(detection species, the
of hydroxyl following
radical) and
radical scavengers, L-histidine (detection of singlet oxygen), DMSO
SOD enzyme (detection of superoxide radical) were incorporated into the reaction system. (detection of hydroxyl radical)
and Since
SOD enzyme
the amount(detection
of ROS of formed
superoxide is notradical)
known,were incorporated
scavengers wereinto the reaction
applied system. high
in a relatively
Since the The
concentration. amount of ROS were
experiments formed is not
carried outknown,
using twoscavengers were applied
stoichiometric in aof
molar ratios relatively
Cu: phenolic high
compound. The molar ratio Cu(II):PhC = 1:2 was used (Figure 11—lanes K1 and 1–3). In a second
concentration. The experiments were carried out using two stoichiometric molar ratios of Cu:
phenolic compound. The molar ratio Cu(II):PhC = 1:2 was used (Figure 11—lanes K1 and 1–3). In a
second variant the phenolic compound was in a large excess (Cu:MYR/MOR/DHF = 1:40;
Cu:TAX/CUM = 1:1000) (Figure 11—lanes K2 and 4–6).
Molecules 2019, 24, 4335 20 of 28
variant the phenolic compound was in a large excess (Cu:MYR/MOR/DHF = 1:40; Cu:TAX/CUM =
Molecules
1:1000) 2019, 24,11—lanes
(Figure x K2 and 4–6). 20 of 28
Figure 11. Evidence of ROS formation in the Fenton-like system. Electrophoretic profile of agarose
Figure
gel 11.of
(0.8%) Evidence of ROS
the reaction formation
mixture of 20inµM
the Fenton-like
pBSK+, Cu(II) system.
ions,Electrophoretic
hydrogen peroxideprofileand
of agarose gel
phenolic
(0.8%) of the
compounds reaction
incubated mixture
at room of 20 µM
temperature forpBSK+,
30 min. Cu(II) ions,of hydrogen
The order the samplesperoxide and
in the gel: (p)phenolic
control
pDNA; (K1) Fenton like reaction containing pDNA + 5 µM CuCl2 + 50 µM H2 O2 and 10 µM of(p)
compounds incubated at room temperature for 30 min. The order of the samples in the gel: control
phenolic
pDNA; (K1) Fenton like reaction containing pDNA + 5 µM CuCl 2 + 50 µM H2O2 and 10 µM of phenolic
compound; lanes 1–3: Fenton like -reaction and 10 µM of phenolic compounds in the presence of
compound;(lane
l-Histidine lanes1),1–3:
DMSOFenton like
(lane 2) -reaction
and SOD and (lane103);µM of Fenton-like
(K2) phenolic compounds
reaction andin the
200 presence of L-
µM of MYR,
Histidine
MOR, DHF(lane 1), µM
or 5000 DMSO (laneand
of TAX 2) and
CUM. SOD (lane
lanes 4–6:3);Fenton
(K2) Fenton-like
like reaction reaction
and 200andµM200 µM ofMOR,
of MYR, MYR,
MOR, DHF or 5000 µM of TAX and CUM. lanes 4–6: Fenton like reaction and 200
DHF or 5000 µM of TAX, CUM in the presence of l-Histidine (lane 4), DMSO (lane 5) and SOD (lane 6). µM of MYR, MOR,
DHFscavengers
ROS or 5000 µM of TAX,
were added CUM in the presence
in following amounts: -Histidine (lane
of Ll-Histidine 20 mM, 4), DMSO
DMSO 6(lane 5) and
µL, SOD SOD (lane
15U.
6). ROS scavengers were added in following amounts: L-Histidine 20 mM, DMSO 6 µL, SOD 15U.
Figure 11 documents the formation of singlet oxygen, hydroxyl radicals and superoxide radical
anionsFigure
in the11 documents
course the formation
of the Cu-Fenton of singlet
reaction in theoxygen,
presencehydroxyl radicals
of plasmid DNA.andFrom
superoxide radical
the observed
anions
band in the course
intensities we canofconclude
the Cu-Fenton
that thereaction
additioninofthe presence(scavenger
L-histidine 1 O ) or
of plasmidofDNA. From
SOD the observed
(scavenger
2
O2 •−intensities
ofband we can
) significantly concludeSC
preserved that the addition
DNA. of L-histidine
Interestingly, addition(scavenger of •(scavenger
of 1O2) or SOD
of DMSO (scavenger OH) only
•−
of O2 ) significantly
partially preserved SCpreserved
DNA (seeSC DNA.intense
rather Interestingly,
bands ofaddition
OC DNA). of DMSO (scavenger
This can of •OH)
be explained by only
the
partially preserved
transformation SC DNA
of hydroxyl (see rather
radical to the intense
methyl bands
radicalof(• CH
OC3DNA).
) whichThis
can can
causebeDNA
explained by the
damage to
•
transformation
some extent, as a of hydroxylradical,
secondary radicalaccording
to the methyl
to theradical
reaction( CH 3) which can cause DNA damage to
[50]:
some extent, as a secondary radical, according to the reaction [50]:
(CH3 )SO + • OH → • CH3 + CH3 SO2 H (2)
(CH3)SO + •OH → •CH3 + CH3SO2H (2)
ItItmay
maybe
bespeculated
speculatedthat
thatthe
thepreservation
preservationofofSCSCDNA
DNAafter
afteraddition
additionofofeither
eitherL-histidine
L-histidineororSOD
SOD
(scavengers
(scavengersofofsuperoxide
superoxideradical or or
radical singlet oxygen,
singlet oxygen,respectively)
respectively)to the reaction
to the system
reaction is achieved
system by
is achieved
the
byscavenging of additional
the scavenging ROS ROS
of additional generated in theinsystem
generated at theatexpense
the system of theofscavenging
the expense activity
the scavenging of a
activity
given phenolic
of a given compound.
phenolic compound.
From the electrophoretic profiles also follows that scavenging of one of the formed ROS results
in significant preservation of SC DNA. This points to fact, that concerted action of ROS of various
origin has synergistic damaging effect on DNA damage [51].
3. Materials and Methods

Molecules 2019, 24, 4335 21 of 28
From the electrophoretic profiles also follows that scavenging of one of the formed ROS results in
significant preservation of SC DNA. This points to fact, that concerted action of ROS of various origin
has synergistic damaging effect on DNA damage [51].
3. Materials and Methods
3.1. Materials
Phenolic compounds myricetin (MYR), taxifolin (TAX), 4-hydroxycoumarin (CUM) were
purchased from Sigma Aldrich (Steinheim, Germany), 30 ,40 -dihydroxyflavone (DHF) and morin (MOR)
were obtained from Alfa Aesar (Ward Hill, MA USA). Myricetin, morin and 30 ,40 -dihydroxyflavone
contain a flavone structure (2-phenylbenzo-γ-pyrone) in which, because of presence of a double bond
between carbons 2 and 3 of C ring, all three rings form a planar delocalized system. The molecule
of 30 ,40 -dihydroxyflavone contains a 30 ,40 -dihydroxyphenyl group on the B-ring (catechol moiety).
Myricetin and morin belong to the class of flavonols, and their structures represent isomeric forms
of quercetin having a resorcinol moiety (A-ring) and a 3-hydroxy-4-carbonyl group on the C-ring,
but they differ in the hydroxylation pattern of the B-ring. While myricetin possesses a pyrogallol
moiety (30 ,40 ,50 -trihydroxyl), morin has two hydroxyl groups in a meta-20 ,40 -position. The presence
of a 4-carbonyl group on the C-ring and the hydroxylation pattern of taxifolin also corresponds with
the quercetin structure, but the lack of a C2-C3 double bond disrupts planarity and conjugation of
the rings. The 4-hydroxycoumarin is a coumarin (2H-chromen-2-one) derivative and represents the
simplest molecular structure included in the study.
Other chemicals and reagents were procured from different commercial companies:
disodium salt of calf thymus DNA (CT DNA), NaH2 PO4 , Na2 HPO4 , CuCl2 ·2H2 O
(cupric chloride dihydrate), H2 O2 (30%), DMSO, DPPH (diphenyl-picrylhydrazyl), ABTS
(2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) from Sigma Aldrich, absolute ethanol, TRIS
base, agarose, ethidium bromide from Serva (Heidelberg, Germany), glycerol from Merck (Darmstadt,
Germany), the 10×TBE (Trisborate-EDTA) and bromphenol blue from Applichem (Darmstadt,
Germany). The 1 kb DNA ladder was purchased from Solis BioDyne (Tartu, Estonia) and EcoRI
restriction enzyme from Thermo Fisher Scientific (Vienna, Austria). All chemicals were of analytical
grade purity and used without any purification. The solutions used in DNA experiments were prepared
using purified water (Simplicity Ultrapure Water System, Millipore, Burlington, MA, USA).
3.2. Experimental Solutions

Solutions of phenolic compounds: Stock solutions of phenolic compounds were prepared in
DMSO (3 mM) for both spectroscopic studies and DNA experiments. The solutions were always
prepared fresh and kept in the dark to avoid possible light induced changes.
Solution of CT DNA: Sodium CT DNA salt was gradually dissolved under continuous stirring in
Tris buffer Saline (TBS) solution (5 mM TRIS, 50 mM NaCl, pH 7.2) for 48 h and then stored at 4 ◦ C in
the dark.
Solution of plasmid DNA: Plasmid DNA (pBSK+) was isolated from Escherichia coli DH5α bacterial
cells, cultured for 16 h in LB medium (0.5% yeast extract, 1% trypton, % NaCl) under continual stirring at
150 rpm, temperature 37 ◦ C (Heidolph Unimax 1010 shaker, Heidolph Unimax, Schwabach, Germany).
Ampicilin (50 mg.cm−3 ) was used as a selection agent. Plasmid DNA was isolated by using commercial
isolation kit Fast-n-Easy Plasmid Mini-Prep Kit (Jena Bioscience, Jena, Germany). The pDNA obtained
in the last step of the isolation procedure was dissolved in 50 mM phosphate buffer, pH 7.2.
The concentration of DNA was determined spectrophotometrically at 260 nm on a NanoDrop
spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA). Each solution of DNA
was found to be of sufficient purity as evident from the absorbance ratio values A260 /A230 in the range
2.0–2.2 and A260 /A280 ≥ 1.8.
Molecules 2019, 24, 4335 22 of 28
3.3. UV-Vis Spectroscopic Studies

Complex formation between cupric ions and phenolic compounds was studied at molar ratios of
1:1 and 1:2 in DMSO-phosphate buffer (5% v/v). Phenolic compound (0.1 mM) was added to a solution
containing 0.1 mM (1:1) or 0.05 mM CuCl2 .2H2 O (Metal-to-ligand ratio 1:2). The final total volume in
the test tube was adjusted to 3 mL using DMSO and the solution was left to stand for 60 s. Subsequently,
the absorption spectra of the phenolic compounds (50 µM) and the changes in their respective spectra
in the presence of cupric ions were recorded in the wavelength range of 200–600 nm. The UV-Vis
absorbance measurements were performed on a Cary 60 spectrophotometer (Agilent Technologies,
Santa Clara, CA, USA) at room temperature (25 ◦ C) using matched quartz cells of 1.0 cm path length.
3.4. EPR Spectroscopy

EPR spectra of copper-flavonoid (metal-to-ligand ratio = 1:2) DMSO solutions (1 mM) were
measured using an EMX EPR spectrometer (Bruker, Karlsruhe, Germany) at 77 K at X-band (cca
9.4 GHz) using cylindrical quartz tubes [52].
3.5. ABTS Radical Scavenging Activity

The time dependent ABTS radical reduction activity was monitored from the decay of the
absorption band of the ABTS radical cation (ABTS•+ ) in the sample at a fixed wavelength
of 734 nm [53]. Firstly, the ABTS solution was prepared by dissolving 17.2 mg of ABTS
(2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 3.3 mg of potassium persulfate in 5 mL of
deionized water. To achieve complete oxidation of the ABTS salt to ABTS•+ the solution was left at
room temperature for 24 h. Working solution of ABTS•+ were prepared by diluting 1 mL of solution
containing oxidized ABTS•+ with 60 mL of deionized water. Subsequently, 2.8 mL of ABTS•+ solution
was added rapidly to the test tube containing 0.2 mL of sample. The sample consisted of free phenolic
compound or Cu(II)-phenolic complex in molar ratios 1:1 and 1:2 in DMSO. The kinetic of the reaction
of ABTS•+ in the presence of system studied was recorded for 900 s until a steady state level was
reached, which was observed as a plateau of the spectral curve. The ABTS radical cation inhibition
percentage was calculated according to the reaction: Inhibition % = 100 × (A0 − A)/A, where A0
indicates the absorbance of the blank and A indicates the absorbance of the sample (free phenolic
compound or phenolic compound in the presence of Cu(II) ions).
3.6. Electronic Absorption Titrations

CT DNA binding studies were performed by titrating a fixed concentration (0.02 mM) of
compounds (free phenolic compound and Cu(II)-phenolic compound system at molar ratio of 1:2)
while increasing the CT DNA concentration (10–50 µM) in TBS buffer (pH 7.2). The sample containing
DNA was allowed to incubate for 10 min at room temperature before the absorption spectra were
recorded. Absorption spectra for all free phenolic compounds and their mixtures with Cu(II) ions were
recorded in the range 200–600 nm. A correction was made for the absorbance of the DNA itself.
The intrinsic binding constant Kb (in M−1 ) of the systems studied, containing CT DNA reflects the
binding strength of the mutual interaction, and was calculated from the equation [54]:
[DNA] [DNA] 1
= + (3)
εa − ε f εb − ε f Kb εb − ε f
where [DNA] is the concentration of CT DNA in base pairs, the coefficients εa , εf , and εb are the
apparent absorption coefficients and correspond to Aobsd /[studied system], the extinction coefficient
of free studied system for each addition to DNA and the extinction coefficient for the bound studied
system, respectively. The data obtained were plotted to give a straight line, and the Kb value was
calculated from the ratio of slope to the intercept.
Molecules 2019, 24, 4335 23 of 28
3.7. Gel Electrophoresis

The reaction mixture was prepared in a total volume of 15 µL. Solutions of CuCl2 and of the
phenolic compounds were freshly prepared and used immediately. The 5 µM CuCl2 and respective
concentrations of phenolic compounds in DMSO (5–500 µM MYR/MOR/DHF; 5–5000 µM TAX/CUM)
were mixed at room temperature. The reaction mixture was allowed to stand for 10 min prior to addition
of 20 µM plasmid DNA in 50 mM sodium phosphate buffer (pH 7.2). After 5 min, 50 µM hydrogen
peroxide was added and the mixture was incubated for 30 min. Along with these experiments, variants
were performed under the same conditions with the selected concentrations of phenolic compounds,
both alone and with cupric ions, but without the addition of hydrogen peroxide. Following the 30-min
incubation, 6 µL of the quench buffer (0.25% bromphenol blue, 30% glycerol) was added and samples
were immediately loaded onto 0.8% agarose gel in TBE (89 mM Tris-borate acid, 2 mM EDTA, pH = 8.0)
containing 0.5 µg/mL of ethidium bromide. Electrophoresis was completed in TBE buffer for 1.5 h at
80 V. The 1 kb DNA ladder (Solis BioDyne) was used as a standard. The reference plasmid sample
was linearized with EcoRI endonuclease (Thermo Fisher Scientific, Vienna, Austria) and used as a
control for double strand breaks. The gel was imaged under UV light (Kodak Gel Logic 200, Eastman
Kodak Company, Rochester, NY, USA). Each experiment was performed in triplicate, under the same
conditions. Quantification of band intensities was performed by densitometric analysis using Kodak
Gel Logic 200 software. The relative intensities of bands of the respective conformation states were
normalized for each lane so that % SC + % OC + % LIN = 100% [55].
3.8. Identification of ROS

Radical scavenging agents, such as L-histidine (20 mM), DMSO (6 µL) and superoxide dismutase
(SOD) enzyme (15U) were added to the reaction mixture prior addition of hydrogen peroxide to
identify assumed formation of singlet oxygen (1 O2 ), hydroxyl radical (• OH) and a superoxide radical
anion (O2 •− ), respectively. Quantities of the scavengers are related to the final volume of 15 µL.
Electrophoretic conditions were identical, as described above.
4. Conclusions
The structural characteristics of flavonoids, particularly the number and position of hydroxyl
groups, planarity of the molecular skeleton, extent of delocalization and ability to chelate redox metal
ions are key features affecting their antioxidant or prooxidant properties. The aim of this work was to
ascertain the structural characteristics of a series of structurally different flavonoids responsible for
their antioxidant and/or prooxidant properties in the presence of Cu(II) ions. The selected phenolic
compounds were myricetin, morin, 30 ,40 -dihydroxyflavone, taxifolin and 4-hydroxycoumarin. These
substances differ in the number and positions of hydroxyl groups, the presence or absence of conjugation
in the system and their ability to chelate Cu(II) ions and other structural features.
The radical scavenging activity of flavonoid molecules (ABTS assay) decreases in the order
myricetin > morin > 30 ,40 -dihydroxyflavone ~ coumarin > taxifolin, in agreement with the deceasing
number of hydroxyl groups of flavonoids, planarity of the molecular skeleton and extent of electron
delocalization. The B ring of the flavonoid skeleton is the most biologically significant molecular
functionality in scavenging of ROS because it donates hydrogen and/or an electron to a variety of ROS
including hydroxyl and peroxyl radicals, stabilizing them and giving rise to formation of a relatively
stable flavonoid radicals. This is in agreement with the general rule that the molecules with more
extended conjugated systems form resonance-stabilized free radicals with prolonged life-times and
exhibit more profound radical scavenging activity.
A variety of diseases are characterized by disturbed metabolism of redox active metals such
as Cu(II), thus studies of their interaction with flavonoids have pharmacological importance.
Copper-chelating activity of studied flavonoids revealed that a tight interaction between phenolic
Molecules 2019, 24, 4335 24 of 28
compounds and Cu2+ ions is achieved via the 3-OH and 4-CO groups only if the unsaturated C2-C3
bond is present in the C-ring, which further permits through-conjugation with the B-ring.
The major mechanisms by which flavonoids can exert their biological effects involve the activation
of apoptotic proteins, increased formation of ROS and induction of DNA damage. Several flavonoids
including myricetin are known to inhibit DNA synthesis in bacteria [56]. It has been proposed that the
B ring of the flavonoids may intercalate or participate in the formation of hydrogen bonds with the
stacking of nucleic acid bases which in turn lead to inhibition of DNA synthesis. Interaction of phenolic
compounds and their Cu(II) complexes with DNA was studied by means of absorption titrations.
The results showed that myricetin can interact with Cu(II) via donor atoms in the C ring (3-OH and
4-CO groups) and the B ring (30 -OH and 40 -OH groups) and it exhibits an interaction with DNA of
intermediate strength. Morin interacts with Cu(II) exclusively via donor atoms on the C ring (3-OH and
4-CO groups) and exhibits a mild interaction strength with DNA, while 30 ,40 -dihydroxyflavone, which
can interact exclusively via the 30 -OH and 40 -OH groups on the ring B, exhibits a strong interaction
with DNA. The overall results indicate that copper ions can modulate the DNA binding affinity of
flavonoids via the formation of their Cu-chelates.
From a DNA damage/protection study, by means of gel electrophoresis, it was concluded that the
protective effect of the phenolic compounds studied decreases in the order: 30 ,40 -dihydroxyflavone >
coumarin > morin > taxifolin ~ myricetin. The results clearly show that more pronounced DNA damage
occurs in the presence of those flavonoids containing higher number of hydroxyl groups including
3-OH group and involve myricetin (six hydroxyl groups), morin and taxifolin (five hydroxyl groups).
All these flavonoids form stable Cu(II) chelates capable of causing DNA damage via their interaction
with DNA and the formation of ROS via the Fenton reaction. An application of ROS scavengers
documented formation of singlet oxygen, superoxide radical anion and hydroxyl radicals. An analysis
of electrophoretic profiles revealed that the concerted action of a variety of ROS synergistically enhances
DNA damage.
Based on the obtained results we may conclude that the flavonoids may behave as both antioxidants
or prooxidants, depending on the conditions such as presence of redox active Cu(II) ions. Recently
reported cytotoxicity studies have revealed that anticancer activity of flavonoids may be related to
their prooxidant mechanism of action [57]. Cancer cells are well adapted to higher level of oxidative
stress compared to normal cells, rendering malignant cells more vulnerable to being killed by drugs
such as flavonoids alone or their metal- (Cu-) flavonoid complexes that boost ROS levels in cancer
cells [51]. Whether a flavonoid will act as an antioxidant or a prooxidant depends on its dose, stability
of metal-flavonoid complex and number of hydroxyl groups. The sensitivities of cancer cells against
flavonoids or their metal-complexes may differ depending on the type of tissue, indicating that the
flavonoid-induced cytotoxicity might be related to certain types of cancers [57].
Overall, these results suggest that the higher number of hydroxyl groups including 3-OH group
of the C ring of phenolic compounds is critical for the induction of DNA damage in the presence of
cupric ions. There is the further indication that the mechanism by which myricetin, morin and taxifolin
and their Cu(II) complexes can interact with DNA, predisposes these substances to act as potential
anticancer agents [48]. The potential anticancer properties of phenolic compounds are related to their
moderate prooxidant activity, which can boost formation of ROS and kill cancer cells. Alternatively, as
a prevention strategy, slight prooxidant activity of flavonoids may induce cellular antioxidant systems,
including antioxidant enzymes and synthesis of low molecular antioxidants such as glutathione and
thus act as preventive anticancer therapeutic agents [58–64].
Author Contributions: K.J. and M.V. (Implementation of project, experiments, supervision, writing); L.H. and P.L.
(UV-Vis spectroscopy, absorption titrations, gel electrophoresis); M.S. (UV-Vis spectroscopy, EPR spectroscopy,
ABTS assay); S.H.A. and I.M.A. (Conceptualization, writing-review and editing).
Funding: This work was supported by Scientific Grant Agency (VEGA Project 1/0686/17) and Research and
Development Support Agency (APVV-15-0079). S. Alwasel would like extend his sincere appreciation to the
Researchers Supporting Project (RSP-2019/59), King Saud University, Riyadh, Saudi Arabia, for support.
Molecules 2019, 24, 4335 25 of 28
Conflicts of Interest: The authors declare no conflict of interest.
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MAKALAH PENENTUAN STRUKTUR MOLEKUL

Penentuan Struktur Molekul Apigenin, Luteolin, dan Myricetine

Dosen Pengampu : Drs. Dede Sukandar, M.Si

Tiara Hadi Ashari 11180960000097

Seto Muhammad Adli 11180960000102

Risma Mutiara Pertiwi 11180960000105

PROGRAM STUDI KIMIA

FAKULTAS SAINS DAN TEKNOLOGI

UNIVERSITAS ISLAM NEGERI SYARIF HIDAYATULLAH

Apabila di dalam penulisan laporan ini teradapat banyak kesalahan atau

Ciputat, Maret 2021

KATA PENGANTAR .......................................................................................................ii

1.1 Latar Belakang

2.1 Tanaman Sage (Savia Officinalis. L)

Gambar 1. Daun Sage

2.2 Senyawa Apigenin

Gambar 2. Struktur Senyawa Apigenin

2.3 Senyawa Luteolin

Gambar 3. Struktur Senyawa Luteolin

2.4 Senyawa Myricetin

Gambar 4. Struktur Senyawa Myricetin

2.5 Spektrofotometer UV-Visible

(Yanlinastuti dan Fatimah, 2016).

Gambar 6. Tipe Transisi Elektronik dalam Molekul Organik

3.1 Alat dan Bahan

3.2 Diagram Alir

Sampel Daun Sage kering halus

 Diekstraksi dengan etanol 98% selama 20 jam

 Dimurnikan dengan ekstraksi cair-cair dengan n-

Lapisan ekstrak dengan n- Lapisan Ekstrak dengan

 Dilakukan pemisahan dengan TLC (menggunakan

Terlihat 2 bercak noda

 Dimurnikan dengan ekstraksi cair-cair dengan n-butanol dan air

Senyawa Murni Zat I Senyawa Murni Zat II

3.2.2 Penentuan Struktur Senyawa Apigenin pada Zat I

Senyawa murni Zat I

 Diuji Spektrofotometri UV-Vis dengan penambahan

3.2.3 Penentuan Struktur Senyawa Luteolin pada Zat II

 Diuji Spektrofotometri UV-Vis dengan penambahan

3.2.4 Penentuan Struktur Senyawa Myricetine pada Zat III

 Dimurnikan dengan ekstraksi cair-cair dengan n-butanol dan air

Senyawa Murni Zat III

Senyawa murni Zat III

 Diuji Spektrofotometri UV-Vis dengan penambahan

3.3 Prosedur Percobaan

3.3.2 Penentuan Struktur Senyawa Apigenin pada Zat I (Dordevic S. et al.,

3.3.3 Penentuan Struktur Senyawa Luteolin pada Zat II (Dordevic S. et al.,

3.3.4 Isolasi dan Penentuan Struktur Senyawa Myricetine pada Zat II

Spektrofotometer UV-Vis merupakan suatu metode identifikasi yang didasarkan

Gambar 7. Reaksi NaOH dengan Senyawa Flavonoid

Gambar 8. Reaksi Pembentukan Kompleks Flavonoid dengan AlCl3 dan HCl

Gambar 10. Reaksi Pembentukan Kompleks Asam Borat dengan Flavonoid

4.2 Penentuan Struktur Molekul Luteolin (Đorđević et al. 2000)

Gambar 14. Struktur Senyawa Luteolin

4.3 Penentuan Struktur Myricetine (Jomová et al., 2019)

Gambar 15. Sistem Khelasi Logam (M) pada Flavonoid

Gambar 16. Struktur Senyawa Myrecetine dengan Gugus Kromofor yang

Gambar 17. Spektrum Pengukuran Myrecetine

Al-Gouti, M. .., M. A. .. Khraisheh, S. .. Allen, and M. .. Ahmad. 2004. “The

Chowdhury, Arnab Roy. Shalini sharma, Suparna mandal, Anindya goswami,

Craig, J.W. (1999). Health-promoting properties of common herbs. Am. J. Clin.

DB Lubahn. Anna slusarz, Nader shenouda, Mary S. Sakla. Phytoesterogen as

Goodman G. The pharmacological basis and therapeutics. Bandung: Penerbit