Study of natural antioxidants in food systems and in active or edible packaging

In this doctoral thesis, the antioxidant capacity of certain aromatic plants was examined. The studied aromatic plants were thyme (Thymus capitatus), oregano (Origanum vulgare hirtum), savory (Satureja thymbra) and rosemary (Rosmarinus officinalis). Two chemotypes of oregano, rich in carvacrol and rich in carvacrol and thymol, were used to identify the differences between them. The extraction and identification of the antioxidant components of the plants were carried out with ethyl acetate and ethanol. In mixed chemotype of oregano and rosemary, their phenolic components were also extracted directly with ethanol in order to assess the possibility to obtain antioxidants directly with one extraction. The plants before extraction were dried at room temperature and subjected to hydro-distillation to remove essential oils. The aim was to exploit the deodorized plant mass, as it is a source of phenolic compounds but is a by-product of the essential oil manufacturing industry. The solvents used to extract the antioxidant components were chosen to be food grade and to separate polar and nonpolar plant components. The extracts were analyzed by liquid chromatography.Ethanol extracts are rich in polar phenolic components, such as rosmarinic acid and flavonoid glycosides. On the other hand, ethyl acetate extracts are rich in non polar phenolic compounds such as flavonoid aglycones. Rosmarinic acid was detected in all the tested extracts. Its content, however, varies significantly, with savory showing the highest concentration of rosmarinic acid in ethanol extract (129.11 mg/g dry extract), followed by the ethanol extract of carvacrol-rich oregano (116.17 mg/g dry extract). All the other extracts have lower rosmarinic acid values, with the lowest found in the ethyl acetate extract from thyme (12.98 mg/g dry extract). It is observed that ethanol extracts are richer in rosmarinic acid than ethyl acetate extracts for all the studied aromatic plants.Carvacrol and its isomer, thymol were detected in the studied aromatic plants. These compounds are components of the essential oils of aromatic plants, but the plant mass that remains as waste after the hydro-distillation of the essential oils, and was used for the preparation of the extracts, contains significant amount of these two substances. Specifically, carvacrol was detected in the ethyl acetate extracts, with thyme having the highest concentration (176.34 mg/g dry extract) and savory the lowest (16.99 mg/g dry extract). The total ethanolic extract of oregano of the mixed chemotype also showed a low value (25.45 mg/g of dry extract). On the other hand, oregano of mixed chemotype contains thymol.Flavonoids, such as taxifolin, dihydrokaempferol, eriodictyol and naringenin, were detected in ethyl acetate extracts of aromatic plants and identified with reference standards. Savory showed the highest content of all the abone mentioned flavonoids, while thyme showed the lowest.Rosmarinic acid was the major phenolic acid in all savory extracts, followed by salvianolic acid A and lithospermic acid. Salvianolic acid A was identified only in Satureja thymbra extracts. Caffeic acid was detected in small amounts in the aqueous and ethanolic extract of the plant.The extracts were incorporated in vegetable oils to investigate their ability to inhibit oxidation. The oils used were corn oil and palm oil. More specifically, all extracts of the mixed oregano chemotype (ethyl acetate, ethanolic and total ethanolic extract) were incorpotated in corn oil to compare each other. The oxidation of pure corn oil and enriched oils was carried out at 70 ⁰C. Peroxide value (PV) and conjugated dienes and trienes were measured. The primary oxidation products of all oil samples were observed over a period of 20 days. Ethanol extracts do not show antioxidant protection, as the oxidation rate of the samples enriched with them, coincides with that of pure corn oil, based on the measurement of the peroxide value. This behavior is probably due to the inability of ethanolic extracts to integrate well into the oil. In particular, their polar components do not dissolve in oils, and in general in high-fat food products, as the non-polar compounds.Ethyl acetate extract of oregano provided antioxidant activity and was able to prolong the incubation period of the enriched oil by 1.5 days compared to the pure oil. Linear regression of peroxide value of the oil samples in the accelerated oxidation period was estimated, following pseudo-zero-order kinetics. The constant rate (k) of peroxide value of the enriched oil samples do not show significant differences from the corresponding value of the pure oil, which proves that the extracts were not able to inhibit the production of primary oxidation products. In particular, the pure corn oil shows oxidation rate equal to (7.50 ± 0.16) days-1, while the oil containing the ethyl acetate extract, which showed one of the lowest oxidation rates, shows an oxidation rate equal to (7.27 ± 0.04) days-1. The increase of conjugated dienes appears to be similar to that of peroxides, confirming the above mentioned conclusions.Ethyl acetate extracts from thyme and carvacrol-rich oregano were incorporated in corn oil, in order to study their effectiveness against oil oxidation. The rate constants (k) of the accelerated oxidation period of the enriched oil samples did not show significant differences from the pure corn oil, which proves that the extracts were not able to inhibit the production of primary oxidation products.Ethyl acetate extracts of the same plants (thyme and the two types of oregano) as well as Satureja thymbra (savory), were incorporated in palm oil in order to observe any differences or similarities of their antioxidant activity in a different vagetble oil. All extracts showed better antioxidant protection of palm oil, than in corn oil, both by prolonging its incubation period and by reducing the rate constant of peroxides, during the accelerated oxidation period. In order to compare the antioxidant activity of the extracts during the accelerated oxidation period, the Protection parameter (P) was determined. Protection parameter (P) expresses the % reduction of the hydroperoxide formation, in terms of the pure oil hydroperoxide formation. Comparing the Protection (P) values, it is concluded that the extracts of the Satureja thymbra and mixed chemotype of oregano showed the best protection against oxidation, while the other extracts showed satisfactory antioxidant protection, reducing the oxidation rate by ≥ 30%.Choosing palm oil, in which the best antioxidant activity of the extracts appeared and distinguishing savory (Satureja thymbra) that offered the best antioxiadant protection in the previous oxidation tests, the effect of its extracts on the oxidation of oil-in-water emulsions was studied. The studied extracts were ethyl acetate, ethanol and the aqueous extract obtained after hydro-distillation of the plant. Both aqueous and ethanol extracts of the plant reduced the increase of primary oxidation products in the emulsions compared to the non-enriched emulsions. The emulsion with ethyl acetate extract of the plant showed a similar increase of peroxide value as the emulsion without additive. The secondary oxidation products of the emulsions enriched with ethyl acetate and ethanol extracts of savory, as well as the emulsion without additive, remained low and only after 20 days of storage at 70 ⁰C showed an increasing trend, as determined by the p-anisidine method. The secondary oxidation products of th emulsion with the aqueous extract of the plant, increased significantly during storage. Sunflower oil is one of the most common oil widely consumed and used to prepare emulsions, such as mayonnaise and salad dressings. Ethyl acetate and ethanol extracts of savory were added to sunflower oil-in-water emulsions, while the aqueous extract of the plant was not incorporated, as it did not show good antioxidant activity in the previous oxidation tests of palm oil emulsions. Quercetin and rosmarinic acid were also added, in order to correlate the action of the extracts with their components. The aim of the present research was to investigate the antioxidant protection of the emulsions, to study the shelf life of quercetin, rosmarinic acid and the active compounds found in the extracts after encapsulation in the emulsions in order to ascertain whether the enrichment potentially leads to the preservation of the functional products. The oxidation of the emulsions was studied at three temperatures, 5, 25 and 40 ⁰C, in order to check their degradation at refrigerator, ambient temperature and under stress due to bad storage conditions. The dependence of oxidation rate on temperature was studied, through the Arrhenius equation.All antioxidant additives reduced the rate constant (k) of hydroperoxide formation, with the ethanol extract presenting the highest protection, followed by quercetin, ethyl acetate extract, and rosmarinic acid. No significant differences between the samples with different phenolic additives was observed at 5 ⁰C, while at higher temperatures the samples enriched with the ethanol extract or quercetin showed lower oxidation rates than those with the ethyl acetate extract or rosmarinic acid. Comparing the two pure compounds, quercetin presented significantly higher protection than rosmarinic acid, which can be attributed to the polarity of the acid, according to the polar paradox. Rosmarinic acid, as a polar compound, is distributed in the aqueous phase and cannot protect the oil from oxidation reactions, which take place mainly at the emulsion interface. Quercetin can be located in both aqueous and oily phases of the emulsion as well as at the droplet interface. Ethyl acetate extract is rich in non-polar compounds, such as flavonoids and flavonoid aglycones, which are soluble in the emulsion oily phase. Therefore, it is possible that these compounds are located inside the droplets of the dispersed phase of the emulsions and not at their interface. Free radicals are generated at the interface of the emulsion droplets and therefore the phenolic components of the ethyl acetate extract are not able to protect the emulsion oil from oxidation to the same extent as the ethanol extract. In contrast, ethanol extract of the plant, which is rich in polar phenolic components, such as phenolic acids and glycosides of flavonoids, located at the emulsion interface, was effective in reducing the increasing rate of primary oxidation products.Temperature affect the oxidation rate of sunflower oil-in-water emulsions with pure rosmarinic acid and with ethyl acetate extract. The lowest effect of temperature was observed for the samples with quercetin and ethanol extract. All the tested additives protect the emulsions at 5 ⁰C. Protection factors (P) decreased as the temperature increased, especially in the emulsions with rosmarinic acid. Protection (P) of 50% was achieved with the addition of ethanol extract and quercetin even at 40 ⁰C.Measurement of total phenolic components (TPC) of the emulsions indicate the fast loss of phenolic compounds in the emulsion treated with the ethyl acetate extract, which coincides with the higher oxidation rates of this emulsion. Furthermore, the effect of temperature on the reduction of total phenolic content was more pronounced in the emulsions enriched with ethyl acetate extract compared to those with ethanol extract, and the calculated activation energy values, according to the Arrhenius equation, were 27.20 kJ/mol and 19.30 kJ/mol, respectively. Rosmarinic acid was the main component reduced in emulsions containing both extracts, as determined by liquid chromatography, following first-order kinetics and the rate of consumption was concentration dependent. The reduction rate reduction of rosmarinic acid is higher in the emulsion containing ethanol extract compared to the emulsion containing ethyl acetate extract. The reduction rate of quercetin in the emulsions was also examined by liquid chromatography. The results showed that quercetin concentrstion reduced significantly compared to other flavonoids of Satureja thymbra. The decrease of quercetin concentration followed first order kinetics and the reduction rates were 0.006, 0.010 and 0.014 days-1 at 5, 25 and 40 ⁰C, respectively. The reduction rates of quercetin are lower than those of rosmarinic acid. In fact quercetin is one of the most active flavonoid antioxidants, due to its structure, and consequently part of it is oxidized during storage, offering high antioxidant protection.In the next experiment, natural antioxidants were added to edible films for coating sea bream fillets. Sea bream has high commercial value and high content of polyunsaturated fatty acids. Extracts of Satureja thymbra (ethanol and ethyl acetate) were used as sources of natural antioxidants, in order to investigate their effectiveness in the coating technology and to study their interaction with a complex food. Essential oils of aromatic plants play an important role in the inhibition of microbial growth and it is interesting to investigate the possible inhibition of oxidation with the use of essential oil of Satureja thymbra in the coating solution. Specifically, 3 series of samples were prepared, in which the carboxy-methyl-cellulose (CMC) coating membrane contained one of the above fractions at a concentration of 500 mg GAE/L coating solution. Also, another 2 series of samples were prepared, one of which contained a combination of essential oil (2% v/v) and ethanol extract and the other a combination of essential oil (2% v/v) and ethyl acetate extract. All samples were stored at 0 ⁰C, to monitor their oxidation course. The peroxide values of CMC-coated samples without antioxidant additive were lower than those of the uncoated samples, indicating that edible coating itself provide protection against oxidation. Ethyl acetate extract of Satureja thymbra did not show antioxidant activity, while ethanol extract was effective in retarding the production of primary oxidation products. The incorporation of essential oil in edible coating solution retart the increase of peroxide value.Rosmarinus officinalis (rosemary), which is known for its antioxidant properties and is recognized by the European Union as a natural antioxidant, was studed as source of natural antioxidants. Extracts with different concentrations of rosemary ethanol extract were incorporated in edible coating films to protect smoked eel fillets from oxidation. The essential oil of Rosmarinus officinalis (0.2% v/v) was also icorporetad in edible coating solutions to investigate its antioxidant protection. The incorporation of savory ethyl acetate extract in the CMC edible coating solution did not show antioxidant activity, so only rosemary ethanol extract was studed in these smoked fish oxidation tests. Three series of experiment were prepared in which the coating film contained ethanol extract of rosemary at different concentrations of 200, 500 and 800 mg GAE / L coating solution, in order to find the optimal concentration for the antioxidant protection of the fish. In addition, two other sets of experiments were prepared, one of which contained a combination of the essential oil with the ethanol extract and the other contained only the essential oil. As the concentration of ethanol extract increased, the accumulation of hydroperoxides and conjugated dienes decreased and the best antioxidant protection found in the coating with the highest concentration, 800 mg GAE / L coating solution. The addition of rosemary essential oil to edible coating solution, its combination with the ethanol extract inhibit the formation of primary oxidation products. Both of the addition of essential oil and the combination of essential oil and ethanol extract protect smoked fish fillets against the formation of secondary oxidation products to a greater extent than the addition of ethanol extract at the lowest concentration.The effect of savory extracts on the oxidative stability of fried potato chips, when added to the frying oil, on the fried product or the packaging material, was studied. The frying oil was palm oil and 5 series of experiments were prepared. The first series involved potatoes, which were fried in palm oil enriched with ethyl acetate extract from savory. The same extract as well as the ethanol extract were sprayed at the surface of chips fried in pure palm oil or coated the packaging material of the chips. The addition of the extracts to the surface of the fried chips and to the frying oil reduced the formation of hydroperoxides compared to the samples without additive. Ethyl acetate extract added to the frying oil showed antioxidant activity better than the same extract when it was sprayed on the surface of the food, but similar to the ethanol extract sprayed at the product surface. Coating the packaging material with the natural antioxidants resulted in slower increase of primary oxidation products and therefore higher oxidation protection.Since the addition of natural antioxidants to the surface of the packaging material provided the best antioxidant protection, the active packaging of potato chips was further studied using the natural extracts of the dried Satureja thymbra as antioxidants. Three different concentrations of ethyl acetate and ethanol extracts were incorporated on the packaging material, 100, 200 and 300 mg GAE/m2. All concentrations of ethanol extract active packaging protect against oxidation the fried potato. The best antioxidant protection found at the concentration of 300 mg GAE/m2, which increased the incubation period 20 days and reduced the peroxide formation rate by 78.4%. The concentrations of 100 and 200 mg GAE/m2 of ethyl acetate extracts protect chips from oxidation, while the concentration of 300 mg GAE/m2 showed a pro-oxidant activity increasing the formation of peroxides by 7.44% in the accelerated oxidation period and reducing the incubation period by almost 10 days. The pro-oxidant activity of 300 mg GAE/m2 for the ethyl acetate extract can be attributed to pro-oxidant properties of the flavonoids which appear to be concentration-dependent. Results showed that migration of the antioxidants to the chips occurred mainly from the ethyl acetate extracts, which is attributed to the non-polar nature of both the extract and the food. After 55 days of storage, the sum of total phenolic content of the packaging and chips showed more than 50% consumption of antioxidant compounds for all the treatments.In conclusion, aromatic plants of Lamiaceae family are sources of natural antioxidants and can extend the shelf life of food products, preventing their oxidation. Ethyl acetate extracts of savory, thyme and two chemotypes of oregano were effective in protecting palm oil from oxidation. Their activity was not proportional to their total phenolic content. It depended on their phenolic composition. As the content of phenolic acids and other flavonoids increased, the antioxidant capacity of the extracts improved. High values of monophenols, and mainly carvacrol, had the opposite results. Incorporation of ethyl acetate and ethanol extracts of aromatic plants in corn oil did not provide antioxidant protection.Extracts of Satureja thymbra can be incorpotated in o/w emulsions and upgrade their nutritional profile. Total phenolic components of the emulsions decreased during storage, mainly due to the decrease of rosmarinic acid. In addition to enriching the emulsions in phenolic compounds, The extracts of savory offer protection against oxidation to the emulsion. At low temperatures (5 ⁰C) the extracts reduce the oxidation rate by 75-80%. Higher storage temperatures accelerate oxidation of the emulsions and resulted in lower protection factors and greater loss of phenolic compounds.Edible films and coatings enriched with natural antioxidants can protect fish and fish products from lipid oxidation. CMC coating present lower peroxide values of the product than without it. The incorporation of savorys’ ethanol extract to CMC coating solution reduce food oxidation. Ethyl acetate extract of savory did not offer antioxidant protection on fish. The essential oil of savory proved to be a strong antioxidant, providing similar antioxidant protection to that of ethanol extract.Primary (peroxide value, PV) and secondary oxidation products (p-anisidine value, p-AV) of fish, stored at 4 ºC and coated with CMC enriched with ethanol extract of rosemary, showed that natural antioxiadants can offer protection to food and food products from oxidation. CMC films with essential oil and without additive did not protect smoked eel filets from oxidation phenomena. As the concentration of ethanol extract increased in the coating solution, an increase in antioxidant protection was observed.Incorporation of Satureja thymbra extracts in the packaging material creating active packaging films, can delay oxidative degradation of fried potato chips to a greater extent, compared to the addition of the same extracts in the frying oil or directly on the fried product. The antioxidant properties of the extracts depend on their concentration in the packaging material. Pro-oxidant phenomena can be observed when the concentration of flavonoids exceeds certain levels.Στην παρούσα διδακτορική διατριβή εξετάσθηκε η αντιοξειδωτική ικανότητα ορισμένων αρωματικών φυτών. Τα αρωματικά φυτά που μελετήθηκαν ήταν το θυμάρι (Τhymus capitatus), η ρίγανη (Origanum vulgare hirtum), το θρούμπι (Satureja thymbra) και το δεντρολίβανο (Rosmarinus officinalis). Ιδιαίτερα για τη ρίγανη, χρησιμοποιήθηκαν δύο χημειότυποι, ένας πλούσιος σε καρβακρόλη και ένας μεικτός με καρβακρόλη και θυμόλη, ώστε να εντοπιστούν τυχόν διαφ

Immunosuppressive regimens based on Cyclophospamide or Calcineurin inhibitors: Comparison of their effect in the long term outcome of Primary Membranous Nephropathy

Introduction Management of the Primary Membranous Nephropathy (PMN) usually involves administration of immunosuppressives. Cyclophosphamide (Cyclo) and Calcineurin Inhibitors (CNIs) are both widely used but only limited data exist to compare their efficacy in long term followup. Aim The aim of the present study was to estimate and compare long term effects of Cyclo and CNIs in patients with PMN. Patients-methods Clinical data, histologic findings and long term outcome were retrospectively studied. The response to treatment and rate of relapse was compared between patients treated with CNIs or Cyclo based immunosuppressive regimens. Results Twenty three centers participated in the study, with 752 PMN patients (Mean age 53.4(14- 87) yrs, M/F 467/285), followed for 10.1±5.7 years. All patients were initially treated with Renin Angiotensin Aldosterone System inhibitors (RAASi) for at least 6 months. Based on their response and tolerance to initial treatment, patients were divided into 3 groups, group I with spontaneous remission, who had no further treatment, group II, continued on RAASi only, and group III on RAASi+immunosuppression. Immunosuppressive regimes were mainly based on CNIs or Cyclo. Frequent relapses and failure to treatment were more common between patients who had started on CNIs (n = 381) compared to those initially treated with Cyclo (n = 110), relapse rate: 25.2% vs. 6.4%, p<0.0001, and no response rate: 22.5% vs. 13.6%, p = 0.04, respectively. Conclusions Long term follow up showed that administration of Cyclo in PMN is followed by better preservation of renal function, increased response rate and less frequent relapses, compared to CNIs. © 2019 Stangou et al