LOREDANA PLANTULE

Grau referinta

160
140

Absorbanta (mAU)

120
100
80
60
40
20
0
-20
0

10

20

30

40

50

60

70

Timp de retentie (min)

Fasole pamant

800

Absorbanta (mAU)

600

400

200

-200
20

40

60

80

100

Timp de retentie (min)

Fasole vata sap

140000
120000

Absorbanta (mAU)

100000
80000
60000
40000
20000
0
-20000
0

20

40

60

80

100

Timp de retentie (min)

Metoda carotenoide
Materialul vegetal cntrit a fost triturat i extras repetat cu aceton pn cnd reziduul a fost incolor. Extracia s-a fcut sub agitare
continu, n lumin diminuat, n prezen de BHT (butylhydroxytoluene) ca i antioxidant i de bicarbonat de sodiu (adugat n scopul prevenirii
rearanjrilor epoxidice ce pot avea loc n mediu acid) [1]. Extractele reunite, dup filtrare, au fost concentrate la rotavapor, la temperatur de 35
C i presiune sczut.
2. Saponificarea. Extractele ce conin pigmeni carotenoidici sunt de obicei supuse saponificrii n scopul eliberrii carotenoidelor aflate
sub form de esteri, a ndeprtrii clorofilelor i a ndeprtrii lipidelor saponificabile. Tinnd cont de cantitatea mic de prob disponibil s-a
3

utilizat o metod de saponificare modificat. Extractele aduse la un volum de 25 ml au fost tratate cu 5 g rin bazic Ambersep 900 OH, sub
agitare la ntuneric circa 8 ore [2]. Extractul obinut a fost trecut ntr-o plnie de separare cu eter etilic i splate cu soluie 5 % NaCl pn cnd
apele de splare au avut un pH neutru (fa de fenolftalein). Faza superioar eteric, coninnd pigmenii carotenoidici, a fost separat, evaporat
la sec i pstrat la 20 C pn la realizarea separrilor cromatografice. naintea separrii cromatografice probele au fost dizolvate ntr-un volum
cunoscut de faz mobil.
3. Determinarea cantitativ a carotenoidelor totale
Cantitatea de carotenoide totale din probe a fost calculat pe baza urmtoarei relaii de calcul.
X (mg carotenoide) = (A x V x 1000)/(2500 x l x 100)
n care:
A = absorbana probei citit la max = 450 nm
V = volumul probei (ml)
2500 = coeficient procentual de absorbie pentru carotenoide = A1 %1cm
l = 1 cm lungimea cuvei spectrofotometrului (drumul optic)
nregistrarea spectrului de absorbie i citirea absorbanei la 450 nm s-a fcut cu un spectrofotometru UV-VIS Jasco V-530.

4. Analiza HPLC a carotenoidelor

Analiza HPLC s-a efectuat cu un sistem Shimadzu LC20 AT, cu detector cu ir de fotodiode SPD-M20A i coloan YMC C30 (25
cm x 4.6 mm; 5 m).
Faza mobil:
Solventul A: metanol:tert-butil-metil-eter:apa (81:15:4)
Soventul B: tert-butil-metil-eter:metanol:apa (90:7:3)
4

Programul de gradient:
Minutul 0 1 % solvent B in A
Minutul 50 55 % solvent B in A
Minutul 51 60 % solvent B in A
Minutul 52 - 60 % solvent B in A
Minutul 54 - 1 % solvent B in A
Echilibrare cu 1 % solvent B in A pentru 15 minute

Tabel 1. Concentratia carotenoidelor in plantulele analizate (exprimata in mg/100g, proba proaspata)

Carotenoide totale
(mg/100 g)

Plantula
Sistem 1

Sistem 1

-caroten
(mg/100 g)

19.121.23

3.330.2

5011

20.131.36

4.360.3

6813

20.891.25

4.230.34

6115

22.322.23

5.630.12

7313

92.21 12
85 9

Linte
Sistem 2

Zeaxantina
(mg/100 g)

72 10

Rucola
Sistem 2

Luteina
(mg/100 g)

100.32 16
5

Sistem 1

28.312.69

7.130.21

519

312.58

8.910.54

6210

34.123.21

9.120.62

5712

414.12

10.120.87

698

252.36

5.210.32

517.6

293.36

6.320.5

565.6

86 8

Fasole
Sistem 2
Sistem 1

102.36 22
100.3 21

Gru
Sistem 2
Sistem 1

120.32 28
81.69 12

Mutar
Sistem 2

91.23 36

Metoda activitate antioxidanta (te rog tradu)

The antioxidant activity of the plant extracts and the standard was assessed on the basis of the radical scavenging effect of the stable 1, 1diphenyl-2-picrylhydrazyl (DPPH)-free radical activity by modified method (13). The diluted working solutions of the test extracts were
prepared in methanol. Ascorbic acid was used as standard in 1-100 g/ml solution. 0.002% of DPPH was prepared in methanol and 1 ml of this
solution was mixed with 1 ml of sample solution and standard solution separately. These solution mixtures were kept in dark for 30 min and
optical density was measured at 517 nm using Cecil-Elect Spectrophotometer. Methanol (1 ml) with DPPH solution (0.002%, 1 ml) was used as
blank. The optical density was recorded and % inhibition was calculated using the formula given below (14): A B Percent (%) inhibition of
DPPH activity = 100 A Where A = optical density of the blank and B = optical density of the sample.
Tabel 2. Activitatea antioxidanta a plantulelor analizate

Activitate antioxidanta
IC50 (g/ml)

Plantula
Sistem 1

1.32 0.2

Sistem 2

1.65 0.3

Sistem 1

2.31 0.9

Sistem 2

2.650.6

Sistem 1

1.96 0.5

Sistem 2

2.15 0.35

Rucola

Linte

Fasole

Sistem 1

2.1 0.49

Sistem 2

2.6 0.58

Sistem 1

1.1 0.2

Sistem 2

1.2 0.3

Gru

Mutar