Analisa Kadar Flavonoid dalam Plasma

(Metode Yuliatmoko 2007)

1. Persiapan Sampel.
Proses persiapan sampel mengikuti prosedur Crespy et al 2001.
Sampel plasma disimpan pada suhu -200 C sampai sampel dianalisis
lebih lanjut:
Sampel plasma diasamkan hingga mencapai pH 4.9 dengan 0,1
volume asam asetat 0,58 ml/L dan diinkubasi pada suhu 37 o C
selama 2 jam dengan penambahan -glucoronidase/sulfatase
untuk

mengubah

flavonoid

terkonjugasi

menjadi

aglikon

flavonoid bebas.
Protein plasma diprepisitasikan dengan cara penambahan 500 L
methanol/200 mmol/L HCL dan ekstraknya disentrifugasi selama

50 menit pada 14000 x g.

Setelah ekstraksi tersebut 20 L dari supernatant diinjeksikan
dan dianalisis dengan menggunakan HPLC. Injeksi Sampel ke

dalam HPLC.
2. Proses injeksi sampel ke dalam HPLC mengikuti prosedur Miean &
Mohamed 2001. Analisis HPLC dilakukan pada kondisi sebagai
berikut: kolom C18 (3,9 x 150 mm, 4 m); detektor UV pada panjang
gelombang 280 nm; panjang gelombang 280 nm merupakan
panjang gelombang optimal penyerapam flavonoid secara umum
(Lee 2000); fase bergerak menggunakan metanol/air (50:50 v/v, pH
2.5 menggunakan asam asetat) dan laju aliran 1 mL/menit. Menurut
Lee (2000), sejumlah kecil asam asetat (2-5%), asam fosfat, asam
trifluoroasetat
mencegah

(0,1%)

ionisasi

ditambahkan
komponen

ke

fenolik

dalam

pelarut

maupun

untuk

karbosiklik,

meningkatkan resolusi dan reprodusibilitas analisa. Kuantifikasi

komponen

flavonoid

dilakukan

dengan

cara

membandingkan

kromatogram sampel dengan kromatogram standar yaitu katekin

Quercetin, but not Its Glycosides, Is Absorbed fromthe Rat
Stomach (Crespy 2002)
Sample Treatment
Plasma, bile, and content of stomach samples were spiked with
diosmetin as an internal standard (5 M)

Samples were acidified with 0.1 vol of acetic acid 0.58 mol/L, except for
stomach content, and then incubated for 30 min at 37 C in the
absence (unconjugated forms) or in the presence (total forms) of 5
10 6 units/L Beta-glucuronidase and 2.5 10 5 units/L sulfatase.
Proteins were eliminated by adding 2.85 vol of methanol/HCl 200
mmol/L, and centrifugation was performed for 4 min at 14 000g.
The supernatant (20 L) was injected and analyzed by HPLC

Chromatographic Conditions
The HPLC system used consisted of an UV detector (set at 370 nm for
flavonols). The system was fitted with a 5-m C-18 Hypersil BDS
analytical column (150 4.6 mm; Life Sciences International, Cergy,
France).
The mobile phase consisted of H2O/H3PO4 (99.5:0.5) (solvent A) and
acetonitrile (solvent B)
Bile and plasma samples were analyzed in isocratic conditions (73% of
solvent A and 27% of solvent B) with a flow rate set at 1.5 mL/min
during 15 min.
The identification of the compounds present in samples was made by
comparison of the HPLC profile with that of standards according to their
respective retention times.
Human Metabolism of Dietary Flavonoids: Identification of Plasma
Metabolites of Quercetin (Day et. al 2001)
Extraction of Flavonols from Plasma
Baseline and 1.5 h plasma samples were treated in the same way.
Apigenin (100 mcrl, 60 mcrM) was added to plasma (10 ml) as an
internal standard.
For all samples, plasma was acidified to pH 5 with acetic acid (0.65
mM; 0.1 vol.).
Ascorbic acid was added to help stabilize the samples during
processing (final concentration, l mM).

Acetonitrile (2.5vol.) was used to precipitate plasma proteins and

extract flavonol metabolites for all plasma samples.
The samples were vortexed for 30s every 2min over a 10min period,
before centrifuging the precipitate (13,600g, 4C, 10min).
The supernatants were then taken to approximately 100 mcrl by
rotary evaporation under vacuum (50C).
Samples were made up to 500 mcrl with water followed by a further
500 mcrl methanol.
Samples were centrifuged (13,600g, 4C, 2 min), and filtered directly
into HPLC vials.
Aliquots (30 mcrl) were injected on to the HPLC column for analysis.

External standards of quercetin and

analysed approximately every five runs.

quercetin-3-glucoside

were

Indirect Determination of Conjugated Metabolites

Combined plasma samples were taken to dryness by rotary
evaporation and redissolved in buffer (1 ml, 0.1 M potassium
phosphate, pH 7).
A portion (200 mcrl) was incubated (37C, 30 min) with (a) Betaglucuronidase (5 U, 25 mcrl) and buffer (25 mcrl), (b) sulfatase
(0.4U, 25 mcrl) and buffer (25 mcrl), (c) Beta-glucuronidase and
sulfatase (25 mcrl of each), or (d) buffer (50 mcrl).
For Beta-glucuronidase one unit is 1 mcrmol of 4-nitrophenolreleased from 4-nitrophenyl-Beta-D-glucuronide per min at pH 7,
37C. For sulfatase one unit is 1 mcrmol of 4-nitrophenyl-sulfate
hydrolyzed per min at pH 7.1, 37C.
Reactions were stopped by addition of methanol (200 mcrl)
containing ascorbic acid (1 mM)
Samples were centrifuged (13,600g, 4C, 5 min), and then filtered
for analysis by HPLC
HPLC Analysis
Solvents A (water: tetrahydrofuran: trifluoroacetic acid, 98:2:0.1, v:v:v)
and
Solvents B (acetonitrile), were run at a flow rate of 1 ml/min, using a
gradient of 17% B (2min), increasing to 25% B ( 5 min), 35% B (8 min),
50% B (5 min) and then to 100% B (5 min).
A column clean-up stage maintained B at 100% (5 min) followed by a
re-equilibration at 17% B (15 min).
The column was packed with Prodigy 5 mcrm ODS3 reversed-phase
silica, 250ram by 4.6mm id (Phenomenex, Macclesfield, UK).
Diode array detection was at 270 and 370 nm.

Plasma concentrations of the flavonoids hesperetin, naringenin

and quercetin in human subjects following their habitual diets,
and diets high or low in fruit and vegetables
(Erlund et. al 2002)