WELCOME TO MY PRESENTATION

November 26th 2011 Chemistry Department, Faculty of Science SYIAH KUALA UNIVERSITY

Antioksidant activity relationship of phenolic compounds in Hypericum perforatum L.

By Hijjatin Nur SP 1008103010031

Guaed by Muhammad Bahi,Ph.D

Chemistry Department, Faculty of Science SYIAH KUALA UNIVERSITY

Introduction

Hypericum perforatum L.

Clusiaceae

LC-MS

DPPH assay

FRAP

Superoxide anion test

N0 Radikal

Lipid peroxide

Material and Method

A. Hypericum perforatum L. Extraction
Hypericum perforatum L
Dried and powdered at 150 C dy steam heating Extracted using chlorofrom for 97 h Extraced with methanol for 72 h Extraced again whit Petroleum eter and Etil asetat

residu
Fractionated using sephadex LH-20

I/1

I/2

I/3

I/4

I/5

II/2p

II/2S

II/3

B. LC-MS Analysis
H. perforatum L fractions Dissolved in methanol (except I/4 fraction) Determined by rapid resolution liquid chromatography with mass selective detection

result

C. DPPH assay
Fraction H. Perforatum (10l)
Selected concentration (0.001-12 mg/ml)of H.perforatum fraction Mixde with 190 l methanol and 100 l methanolic solution containing DPPH radikal Incubated for 60 min at room temperatur Measured absorption at 515nm by microlate reader Calculated IC50

results

D. FRAP (Ferric reducing ability of plasma)

Fraction H. Perforatum (10l)
Selected concentration (0.001-12 mg/ml)of H.perforatum fraction Treated with 300 l of FRAP reagent Incubated for 6 min Measured absorbance at 593nm by microplate resder Calculated IC50

results

E. Superoxide anion test

Fraction H. Perforatum (10l)
Selected concentration (0.001-12 mg/ml)of H.perforatum fraction mixed with 40 l (144mol/L )NBT, 20 l (677mol/L)NADH, 20 l (60mol/L)PMS, 220 l(buffer Ph 8,3) Incubated for 6 min Measured absorbance at 560nm by microplate resder Calculated IC50

results

F. Inhibition of NO radical
Fraction H. Perforatum (10l)
Selected concentration (0.001-12 mg/ml)of H.perforatum fraction Mixed with 75L of SNP and 75L of phospate buffer (0.064 mol/L, pH=7.4 Incubated under a flurescent lam p for 1 h Added Griess reagen Measured absorbance at 546nm by microplate resder Calculated IC50

results

G. Lipid peroxidation
Fraction H. Perforatum (10l)
Prepared substrat linseed oil for lipid peroxidation Selected concentration (0.0001-1,2 mg/ml)of H.perforatum fraction Mixed 20 L FeSO4 1.875 mmol/L and 20 L ascorbate (15.4 g/Ml) Incubated at 37 C for 1 h Added 200l EDTA (0.1 mmol/L) and added 2mL TBA reagent Centrifuged for 15 min at 3700 rpm Measured absorbance at 532nm by microplate resder Calculated IC50

results
Note: RSC = ( A rata-rata - Acorr) x 100 A kontrol
IC50

RSC vs. Concentration curve

Results & Discussion

Table 1 Retention times and [M-H]- ions of identified peaks
Punca k 1 tR [min] 0.13 [M-H]-, m/z 353 463 Senyawa caffeoylquinic acid quercetin-3-O-b-Dgalactopyranoside (hyperoside) quercetin-3-O-rutinoside (rutin) quercetin-3-O-a-Lrhamnopyranoside (quercitrin) Quercetin I3, II8-biapigenin amentoflavone (I3, II8biapigenin) protopseudohypericin Pseudohypericin Protohypericin Hypericin Hyperfiri Adhyperfirin Hyperforin Adhyperforin

609 2 0.16 447

3 4 5 6 7 8 9 10 11 12 13

0.34 0.96 1.47 2.75 2.87 3.69 3.83 4.80 4.95 5.53 5.68

301 537 537 521 519 505 503 467 481 535 549

Figure 1 LC-MS-MS chromatograms (base peak chromatograms, BPC) of H. perforatum extract fractions: 1. quercetin-3-O-b-D-galactopyranoside (hyperoside) and quercetin-3-O-rutinoside (rutin), 2. quercetin-3-O-a-Lrhamnopyranoside (quercitrin), 3. quercetin, 4. biapigenin, 5. amentoflavone, 6. protopseudohypericin, 7. pseudohypericin, 8. protohypericin, 9. hypericin, 10. hyperfirin, 11. adhyperfirin, 12. hyperforin, 13. adhyperforin

Tabel 2. relativ abundancea of compound classes in analyzed fractions

fractions Total b I-1 I-2 I-3 I-4 I-5 II-2P II-2S II-3S
a

16.2 37.2 29.9 0.584 420 17.9 17.3 31.4

Puncak 0- 2,5 min Flavonoid Flavonoid As. Fenolik glycosida aglycones 0.430 2.72 4.41 1.30 6.49 20.3 nde 0.414 8.49 Nd Nd nd Nd Nd 11.7 0.866 5.77 7.67 0.743 5.83 6.70 0.257 4.50 7.75

biflavonoid 6.14 3.02 12.7 nd 263 0.496 14.0 14.2

Puncak 2.5- 4.7 min c 31.8 49.1 94.1 137 1000 36.9 41.5 11.9

Puncak 4.7 -6.5 min d 68.7 nd 610 968 nd nd nd 28.4

given as peak areas divided by injected mass, normalized to 1000 for convenience including phenolic acids, flavonoids, biflavonoids and unidentified peaks c Naftodiantrone d floroglusinol e not detected
b

Tabel 3. results of antioxidant activity assays

Fraksi
I/1 I/2 I/3 I/4 I/5 II/2s II/2p II/3 BHT BHA DPPH IC50(g/ml) 1.55 1.09 6.66 32.8 11.0 0.520 1.16 2.61 8.28 12.4 (g/umol)a 69.2 48.7 297 1464 491 23.2 51.8 117 369 553 SO IC50(g/ml) 11.1 1.86 11.7 20.6 32.4 7.10 5.70 8.80 n/ac n/a LP IC50(g/ml) 2.25 7.95 17.8 2.33 8.09 7.00 12.2 8.31 0.859 0.138 NO IC50(g/ml) 32.8 >>22 53.7 >120 53.7 83.9 30.2 6.11 n/a n/a FRAP (Mg Fe/b)b 80.0 104.0 10.0 7.00 12.0 25.0 44.0 17.0 25.3 n/a

mass of extract (in g) needed for 50% neutralization of 1 mol of DPPH of Fe3+ that can be reduced by 1 g of H. perforatum fraction. c 50% inhibition not reached
a b mass

Conclusion

LC-MS technique has been succsessfully applied for separation and identification of compounts in Hypericum perforatum L
Flavonoid and fenolic acids have expressed a very high antioxsidan activity when compared to synthetic antioxsidant