Optimizacija Procesa Ekstrakcije Ulja Divljeg Nara (Punica Granatum L) Primenom Mikrotalasa i Ispitivanje Biološke Aktivnosti Dobijenog Ulja

Abstract

Cilj ove doktorske disertacije bio je ispitivanje potencijalne primene mikrotalasnog zračenja na povećanje prinosa ulja semena divljeg nara (Punica granatum L.) nakon ekstrakcije i ispitivanje biološke aktivnosti dobijenog ulja. Ispitan je uticaj operativnih parametara (snage i vremena) mikrotalasnog zračenja na prinos i sastav ulja dobijenog natkritičnom ekstrakcijom (NKE) sa ugljenik(IV)–oksidom (CO2) i ekstrakcijom heksanom u aparaturi po Soxhlet–u (SE). Seme divljeg nara je odabrano kao sirovina za ispitivanje zbog svoje vredne biološke aktivnosti (jakog antioksidativnog i potencijalnog citotoksičnog dejstva).

Za predtretman semena divljeg nara mikrotalasima odabrani su uslovi snage mikrotalasa 100, 250 i 600 W i vremena 2 i 6 minuta. Prikazane su kinetike NKE ulja iz semena divljeg nara tretirano mikrotalasima pri različitim uslovima, kao i rezultati SE. Dobijeni rezultati su upoređeni sa rezultatima ekstrakcije ulja iz semena divljeg nara koje nije tretirano mikrotalasima. Pokazano je da čak i pri najnižim vrednostima snage i vremena dejstva mikrotalasa (100 W i 2 minuta) dolazi do povećanja prinosa ulja (sa 27,7% na 34,0% za SE i sa 21,6% na 25,5% za NKE). Maksimalni prinos ulja za NKE (27,2%) ostvaren je nakon mikrotalasnog predtretmana semena divljeg nara od 250 W tokom 6 minuta, dok je za SE (36,6%) maksimalni prinos ostvaren nakon predtretmana od 600 W tokom 6 minuta.

Procesi NKE I SE semena divljeg nara omogućili su dobijanje ekstrakta bogatih masnim kiselinama, a u cilju njihove identifikacije, derivatizacijom su dobijeni metil- estri nezasićenih masnih kiselina. Za kvalitativni i kvantitativni sastav masnih kiselina. dobijenog ulja korišćene su gasna hromatografija uz plameno-jonizujuću detekciju. (GC/FID) i kombinacija gasne hromatografije i masene spektrometrije (GC/MS). Odabrane analitičke metode su pokazale da predtretman mikrotalasima ima zanemarljiv uticaj na količinu masnih kiselina, u poređenju sa značajnim uticajem na povećanje prinosa ulja. Analiza ulja pokazala je visok sadržaj polinezasićenih masnih kiselina (83,5%), dok je udeo mononezasićenih masnih kiselina (9,5%) i zasićenih masnih kiselina (7.0%) bio niži.

Lipofilni antioksidativni kapacitet dobijenog ulja semena divljeg nara određen je korišćenjem a-trolox ekvivalent antioksidativne aktivnosti (a-TEAC). Pokazano je da dobijeno ulje ispoljava visoku antioksidativnu aktivnost (410 + 89 µmol a-TE/100 g).

Citotoksična aktivnost dobijenog ulja semena divljeg nara je ispitana na sledećim malignim ćelijskim linijama: HeLa (adenokarcinom cerviksa), LS174 (adenokarcinom debelog creva), A549 (adenokarcinom pluća), EA.hy926 (humane endotelijalne ćelije umbilikalne vene), kao i na zdravim MRC-5 (fetalni fibroblasti pluća) ćelijama, korišćenjem kolorimetrijskog MTT testa. Ispitivano masno ulje nije pokazalo citotoksično dejstvo prema normalnim MRC-5 i endotelijalnim EA.hy926 ćelijama (IC50>200µg/mL), dok je prema malignim ćelijama HeLa, LS174 i A549 (IC50-49,51-91,54 µgmL) pokazalo umeren citotoksični efekat. Ispitivanja na endotelijalnim EA.hy926 ćelijama ukazuju na umereni antiangiogenetski efekat ulja semena divljeg nara, pa se stoga može smatrati kandidatom za inhibiciju angiogeneze.

Alternate abstract:

The aim of this PhD thesis was investigation of the potential application of microwave radiation, in order to increase the yield of wild growing pomegranate seed oil (Punica granatumL.) after exctraction and analysis of biological activity of the obtained oil. The influence of operating parameters (power and time) of microwave irradiation on yield and composition of the oil obtained by supercritical fluid extraction with carbon dioxide (SFE) and extraction with hexane in the Soxhlet apparatus (SE) was examined. Wild growing pomegranate seed was used as raw material for experiments, due to its valuable biological activity (strong antioxidant and potential cytotoxic effect).

For pretreatment of the seeds with microwaves, power of 100, 250 and 600 W and two irradiation times (2 and 6 min) were selected. Kinetics of oil extraction from wild growing pomegranate seed using SFE, treated with microwaves at different conditions, as well as, results of SE are also presented here. Obtained results were compared with the results of extraction of oil from non-treated seeds. Results showed that even the lowest values of applied power and time of microwave irradiation (100 W during 2 min) contributed to increase oil yield (from 27.7% to 34.0% and from 21.6% to 25.5% for SE and SFE, respectively). Maximal oil yield for SFE (27.2%) was obtained with microwave irradiation of 250 W during 6 minutes, while for SE maximal oil yield (36.3%) was achived for pretreatment of 600 W during 6 minutes.

Processes of SFE and SE from wild growing pomegranate seed enabled obtaining extracts rich in fatty acids. In order to identify them, fatty acids were derivatized to their methyl esters. The qualitative and quantitative composition of fatty acids of the obtained oil was performed by gas chromatography/flame ionization detection (GC/FID) and gas chromatography/mass spectrometry (GC/MS). Selected analytical methods showed that microvawe pretreatment of seeds has a negligible influence on the amount of fatty acids, compared with its significant influence on oil extraction yield. The oil analysis showed high content of polyunsaturated fatty acids (83.5%), while the content of monounsaturated fatty acid (9.5%) and saturated fatty acid (7.0%) were much lower.

The lipophilic antioxidant capacity of oil obtained from wild growing pomegranate seed was determined using a-tocopherol equivalent antioxidant capacity assay (a-TEAC). Results showed that obtained oil exhibited high antioxidant effects (410 + 89 µmoL a-TE/100g).

The cytotoxic activities of oil obtained from wild growing pomegranate seed was tested against selected human malignant cell lines: HeLa (cervix adenocarcinoma), LS174 (colon adenocarcinoma), A549 (lung adenocarcinoma), EA.hy926 (human umbilical vein endothelial) as well as against normal fetal lung fibroblast (MRC-5) cell lines, using MTT colorimetric assay. Cytotoxic activity of fatty oil on normal MRC-5 and EA.hy926 cells were not observed. The IC50 values in malignant HeLa, LS174 and A549 cells ranged from 49.51±0.57 µg/mL. to 91.54±1.32 µg/mL showing a moderate cytotoxic effect. The investigation of EA.hy926 cells indicated moderate anti-angiogenic effects of pomegranate seed oil. It can thus be considered as a candidate for the inhibition of angiogenesis.