Antioxidant activity of rosemary and oregano ethanol extracts in soybean oil under thermal oxidation

Ação antioxidante de extratos etanólicos de alecrim (Rosmarinus officinalis L.) e orégano (Origanum vulgare L.) em óleo de soja submetido à termoxidação

ANTIOXIDANT ACTIVITY OF ROSEMARY AND OREGANO ETHANOL EXTRACTS IN SOYBEAN OIL UNDER THERMAL OXIDATION¹

R.F. ALMEIDA-DORIA², Marisa A.B. REGITANO-D'ARCE²,^*

SUMMARY

Four experiments were conducted to measure the antioxidant activity of ethanol extracts of rosemary and oregano compared with synthetic antioxidants such as TBHQ and BHA/BHT. The antioxidant activity was determined and results differed from those of the Oven test at 63º C. Peroxide values and absorptivities at 232 nm of soybean oil under Oven test were lower in treatments with 25, 50, 75, 100 and 200 mg.Kg^-1 TBHQ than in treatments with 1000 mg.Kg^-1 oregano extract (O), 500 mg.Kg^-1 rosemary extract (R) and their mixture R+O. All the treatments were effective in controlling the thermal oxidation of oils; the natural extracts were as effective as BHA+BHT and less effective than TBHQ. The natural extracts were mixed with 25, 50, 75 and 100 mg.Kg^-1 TBHQ and then added to the oil. No improvement in antioxidative properties was observed. The best antioxidant concentration could be determined from polynomial regression and quadratic equation from the experimental data.

Keywords: natural antioxidants; soybean oil; oxidation; rosemary; oregano.

RESUMO

AÇÃO ANTIOXIDANTE DE EXTRATOS ETANÓLICOS DE ALECRIM (Rosmarinus officinalis L.) E ORÉGANO (Origanum vulgare L.) EM ÓLEO DE SOJA SUBMETIDO À TERMOXIDAÇÃO. Foram realizados quatro ensaios para verificação da atividade antioxidante de extratos etanólicos de alecrim (A) e orégano (O) comparados com os antioxidantes sintéticos TBHQ e BHA+BHT. Os resultados de atividade antioxidante do teste usando sistema modelo diferiram das respostas do teste acelerado em estufa. Nos ensaios em estufa os valores de peróxido e absortividade em 232nm dos óleos de soja, adicionados de 25, 50, 75, 100 e 200mg TBHQ. Kg^-1, foram menores do que os dos óleos adicionados dos extratos de orégano (O) (1000mg.Kg^-1), de alecrim (A) (500mg.Kg^-1) ou da mistura deles (A + O). Todos os tratamentos retardaram a oxidação do óleo, entretanto os extratos naturais não atingiram a eficiência do TBHQ, mas foram tão efetivos quanto a mistura BHA+BHT. A adição dos extratos naturais a doses reduzidas de TBHQ não melhorou a eficiência em retardar a oxidação.

Palavras-chave: antioxidantes naturais; óleo de soja; oxidação; alecrim; orégano.

1 — INTRODUCTION

World edible vegetable oil has been increasing gradually each year. In Brazil, the major oil is soybean with a production that may reach 4,085 thousand metric tons in 1998/1999 [16].

Soybean oil is known for its instability due to the high content of unsaturated fatty acids [34]. Lipids are oxidized by light, temperature and metal ions, generating hydroperoxides that decompose through secondary reaction to aldehydes, ketones, acids and alcohols [39, 42]. Lipid oxidation is one of the main causes of food deterioration and results in loss of quality, turning products unacceptable for consumption. Oxidative reactions, besides affecting organoleptic characteristics (color, flavor and texture) and decreasing nutritional value due to degradation of fat soluble vitamins (A, D, E and K) and essential fatty acids (linolenic and linoleic acids), are also responsible for the formation of potentially toxic polymeric compounds [33, 40].

In order to prevent or retard oxidative deterioration, antioxidants have been widely used. They may act as free radical quenchers, reducing compounds, singlet oxygen scavengers and as pro-oxidant metals suppressors [11, 21, 39, 40].

The most used synthetic antioxidants are butylated hydroxytoluene (BHT), butylated hydroxyanisol (BHA) and tertiary butylhydroquinone (TBHQ) [11, 15]. These compounds have been reported as health risks and their use is restricted in several countries. Many laboratory studies demonstrated possible health damage in animals. BHA may induce hepatic activity, liver enlargement, growth reduction and carcinoma formation. BHT was toxic to the liver, kidney and lungs, and reduced weight gain, among other effects observed in animals. Hemoglobin level reduction and basal cells hyperplasy were attributed to TBHQ use [26, 41]. In Brazil the use of antioxidants is regulated by the Health Ministry, resolution number 22/77 of the law decree number 986, that allows 0.02 g.100g^-1 oil of BHA and TBHQ and 0.01 g.100g^-1 oil of BHT as maximum antioxidant concentration [5].

There is an increasing number of research for development and utilization of antioxidants from natural sources. Since pre-historical times, seasonings and herbs have been used not just as food flavorings, but also for its medicinal and antiseptical properties. Preservative effects have suggested the presence of antimicrobial and antioxidant constituents [12, 30, 44].

Many spices have been studied and it has been observed that rosemary, oregano and sage, from Labiatae family, possessed strong antioxidant activity [4, 6, 7, 8, 9, 13]. Several antioxidant phenolic compounds have been isolated from rosemary (carnosol, rosmanol, rosmaridiphenol and rosmariquinone) [20, 22, 23, 31, 37, 49] and from oregano (glucosides, phenolic acids and terpene derivatives) [25, 32, 48]. Phenolic antioxidants donate hydrogen to the free radicals, yielding low energy stable radicals. These antioxidants phenolic structure make them stable through resonance inside the ring [10, 14].

The oxidative stability of oils and fats with added antioxidants can be determined during storage under normal ambient conditions and packing. However, this information is usually needed beforehand and at a much shorter time, so accelerated or aging tests are conducted. Tests like Active Oxygen Method (AOM) and the Schaal Oven Test are two widely used procedures adopted in order to have an idea of both oil and the antioxidant type and concentration behavior. Besides these methods, there are others that require complex instrumentation like Oxidograph, Rancimat and OSI (Oil Stability Instrument) [15, 19]. During lipid oxidation, primary changes can be detected by loss of unsaturated fatty acids, weight gain by oxygen incorporation or by the formation of hydroperoxides and conjugated dienes. Secondary changes can be monitored by the detection of the presence of carbonyl compounds, malonaldehyde and other aldehydes, hydrocarbons and fluorescent products formed [17, 38].

CHIPAULT et al [9] studied the antioxidant effect of 32 spices added to lard as petroleum ether and ethanol extracts. Rosemary, sage and oregano presented the highest antioxidant power in both extracts. In a subsequent study, CHIPAULT et al [7] observed that rosemary and oregano were better antioxidants in an oil in water emulsion than sage. Stability was monitored by the oxygen absorption method.

CHANG et al [6] studied the antioxidant properties of rosemary and sage extracts in a wider variety of solvents (hexane, benzene, ethyl ether, chloroform, dichlorethylene, dioxane and methanol) applied to lard stored at 60ºC in the dark. This time, peroxide value was determined and rosemary extracts in dichlorethylene, ethyl ether and methanol showed the lowest results. WU et al [50] confirmed the antioxidant efficiency of the rosemary (0.02%) methanolic extract in lard stored in the dark for 6, 14, 21, 28 and 36 days through peroxide value determination. Rosemary extract efficiency was comparable to BHT and superior to BHA under the same concentration.

The antioxidant activity of carnosinic acid, purified from rosemary extract was evaluated with the Rancimat method. In most experimental times, it was more potent than BHA and BHT, but less efficient than TBHQ in soybean oil.

Oregano methanol extract was fractionated and their ethyl ether solubles presented strong antioxidant activity on linoleic acid when ferric tiocyanate method was used [25]. VEKIARI et al [47] also fractionated oregano extract with several solvents (petroleum ether, ethyl ether, ethyl acetate and butanol). Ethyl ether soluble fraction was more effective in protecting lard (in the dark at 65ºC) against oxidation and its activity was equivalent to BHT. The same was observed in refined vegetable oils stored in the dark at 35ºC and 65ºC and under frying operation.

Ethanol was used in this study because among all the solvents found in the literature used in the preparation of herb extracts, it is the safest healthwise.

The objective of this work was to evaluate the antioxidant activity of the rosemary and oregano ethanolic extracts in refined soybean oil, compared to TBHQ, BHA and BHT, by means of the accelerated procedure Schaal Oven Test, adopted in so many studies like this [18, 28, 46] and the determination of the antioxidant activity under a model system [27], adopted by others [3, 28, 35, 36].

2 — MATERIAL AND METHODS

Refined, bleached and deodorized (RBD) soybean oil, free from any antioxidant addition was furnished by a Brazilian vegetable oil refinery.

Rosemary and oregano extracts were prepared from 5g of dried leaves, ground in 95% ethanol, in a domestic homogenizer (Arno multiprocessor) for 2 minutes at potency 2. After filtration in a filter paper, extracts were concentrated in a rotary evaporator under vacuum at 50°C to a final volume of 20ml or a concentration of 0.25g dried material per ml ethanol. Based on this stock concentration, the extract was added to the soybean oil in different concentrations according to preliminary tests results.

Sigma BHA and BHT and Eastman TBHQ were added to the oil in different concentrations. BHA and BHT were always added together under the proportion of 1:1. All antioxidants were added directly to the oil at room temperature and dissolution was obtained by manual homogenization for about five minutes.

Four trials were conducted to compare natural and synthetic antioxidants in soybean oil.

Assay 1 - The antioxidant activity of the extract was determined in a model system according to Marco's procedure [27], which was modified by Miller [29] and adapted by Araújo [2]. The antioxidant is evaluated comparatively according to its ability to prevent the oxidation of the emulsion b-carotene - linoleic acid, monitored by the loss of color at 470nm for one to two hours. Ethanolic solutions of 100 and 200mg TBHQ.kg^-1, 200mg BHA+BHT.kg^-1, 500 and 1000mg rosemary and oregano. kg^-1 were compared to a blank system (control, no antioxidants added). The experiment was conducted twice with replicates for all treatments.

Assays 2, 3 and 4 consisted of evaluation of the antioxidants according to our adapted Schaal Oven Test procedures, conducted in a regular laboratory oven adjusted to 63°C ± 1°C for different periods of days. Fifty ml beakers without any lid, containing 20g of oil were placed on the same shelf and withdrawn, in triplicate, for analysis. A control (blank, no antioxidant added) sample was always evaluated in triplicate.

Assay 2 - 200 mg TBHQ.kg^-1, 200mg BHA+BHT.kg^-1, 500mg rosemary ethanolic extract.kg^-1 (R) and 1000mg oregano ethanolic extract. kg^-1 (O) were added to the soybean oil to be stored in the oven for 1.5, 3, 5 and 7 days.

Assay 3 - The mixture R + O [500mg rosemary ethanolic extract and 1000 mg oregano extract. kg^-1] added or not of 25, 50, 75 and 100 mg TBHQ . kg^-1 was evaluated on a 7-day oven storage essay.

Assay 4 - 25, 50, 75 and 100 mg TBHQ . kg^-1 were added to soybean oil to evaluate the effect of the addition of natural antioxidants mixture performed in assay 3. The experiment lasted 7 days.

Determination of the absorptivity (ratio of the absorbance reading and the oil concentration in isoctane solution) at 232 and 270nm (NGD C-40) [45], UV-absorbance scanning between 220 and 320nm (II.D.23) [24] and peroxide value (Cd 8-53) [1] were performed in all oil samples from assays 2, 3 and 4. A Shimadzu spectrophotometer UV 1203 provided with software PC-1201 was employed for absorbance readings and scannings. The determinations were conducted in triplicate and analyzed statistically in a factorial model, randomly casualized, considering the factors antioxidant and time. A Tukey test was applied to the means of antioxidant, time and interaction antioxidant x time, considering a level of 5% significance.

3 — RESULTS AND DISCUSSION

The antioxidant power evaluated comparatively in assay 1 revealed, as expected, decrease of absorbance at 470nm for most treatments, except for BHA+BHT. According to this method, the mixture (1:1) 200mg BHA+BHT.kg^-1 was the most efficient in preventing the loss of the color of the emulsion of b-carotene-linoleic acid, followed by 200 and 100mg TBHQ. kg^-1, 1000mg rosemary extract.kg^-1 which was equivalent to 500mg of the same extract and by 1000 and 500mg oregano extract.kg^-1. Most efficient antioxidant concentration from each type was assayed again and the results are presented in Figure 1.

The natural extracts were efficient in retarding oxidation, but the synthetic antioxidants were better. Melo [28], employing the same method by Marco [27], found BHT to be a better antioxidant than cashew nut methanolic extract. However, palm and Brazil nut methanolic extracts were even better than BHT in retarding the oxidation. PASQUEL & BABBITT [35] found, using the same method, that Tenox IV, a synthetic antioxidant was more efficient than shrimp extracts in different solvents. Among the shrimp extracts, the ethanolic extract was the best.

In assays 2, 3, and 4, there was an increase in peroxide value and in the absorptivity at 232 and 270nm with time of storage in the oven, indicating the formation of primary and secondary oxidation products of the lipids. Oxidation was always stronger in the control than in the treatment samples.

Figures 2, ⁴ and ⁶ present the mean peroxide values of oils withdrawn from the oven at each time and Figures 3, ⁵, and ⁷ present the mean absorptivity at 232nm for the same samples. The small letters on the top of each column indicate similarity or not among the treatments in that period of the experiment.

After 1.5 and 3 days in the oven, no significant difference was observed among peroxide or absorptivity at 232nm (Figures 2 and 3). Antioxidants demonstrated to be statistically different in performance after 5 to 7 days in the oven. Best treatment was TBHQ, however rosemary had a protective action comparable to that of the mixture BHA+BHT and oregano after 5 days. CHANG et al [6] had already observed that rosemary extract was as efficient in retarding oxidation of animal fat as the mixture of BHA, BHT, propyl gallate and citric acid. In vegetable oil rosemary was even better than the synthetic mixture. WU et al [50] also found that the rosemary extract (0.02%) had a performance comparable to that of BHT in lard stored in the dark at 60ºC, but was superior to BHA, in the same concentrations.

Peroxide value and absorptivity at 232nm showed the same tendency, indicating a very strong correlation between these two methods. GUTIERREZ, REGITANO-D'ARCE & RAUEN-MIGUEL [18], working with crude Brazil nut oil submitted to the Oven test and Siqueira [43], working with refined canola, corn and soybean oils submitted to light induced oxidation observed a similar behavior.

Absorptivities at 270nm did not present any significant difference at a 5% level among the treatments. UV absorbance scannings at 220-320nm of the oils of all treatments after 7 days in the oven is presented in Figure 8. The same tendency observed for 232nm and 270nm can be visualized in these scannings.

Assay 3 was conducted in order to test the efficiency of the mixture R + O and the possibility of increasing its efficiency by adding increasing concentrations of the most efficient and also the most expensive synthetic antioxidant. Oils were kept in the oven for 7 days, because that was the time necessary to visualize any antioxidant effect in assay 2. The results are shown in ^{Figures 4} and ⁵. All treatments demonstrated some effect in retarding oxidation when compared to the control, howe-

ver the presence of TBHQ in any concentration was determinant of a better oxidation protection. There was no statistical difference among peroxide values and absorptivities at 232nm for any TBHQ concentration added to the natural extract mixture. There was no difference among the absorptivities at 270nm and again a good correlation was observed for peroxide values and absorptivity at 232nm.

Assay 4 was conducted in order to quantify the effect of the addition of natural antioxidants to increasing TBHQ concentrations. Another Oven Test was conducted for seven days, this time without any natural extract. Results are shown in ^{Figures 6} and ⁷. Peroxide values and absorptivities at 232nm for oils with added synthetic antioxidant did not differ at a 5% level of significance. The addition of the natural antioxidant mixture did not improve TBHQ antioxidant efficiency as can be observed from the similarity of results both in peroxide values (^{Figures 4} and ⁶) and absorptivities at 232nm (^{Figures 5} and ⁷).

Considering all the peroxide values of oils with increasing TBHQ concentrations, 0, 25, 50, 75, 100 and 200 mg.kg^-1 after seven days in the oven at 63ºC, it was possible to determine the best antioxidant dose, by means of a polynomial regression and the quadratic equation obtained:

y = 0,00466201 x² + 1,2014066 x + 61,227352

where:

y = peroxide value (meq O₂ . kg^-1 oil)

x = antioxidant dose (mg . kg^-1)

By deriving this equation, one can obtain maximum function, or best oil quality, dy/dx = 0 and best TBHQ concentration is 128.85mg.kg^-1. Economic dose can also be calculated, based on RBD soybean oil price (US$ 736.9/t) and TBHQ price US$ 28.66/Kg. From that quadratic equation, economic dose (ED) can be defined as:

E. D. = [ (oil price/TBHQ price) + 1.2014066] / 0.00932402

The economical dose is calculated to be 126.09mg.kg^-1.

VIEIRA & REGITANO-D'ARCE [49] obtained 84.95mg TBHQ . kg^-1 as the best dose for Brazil crude nut oil from concentrations ranging from 0 to 100mg TBHQ . kg^-1 in a 120 hour Oven Test. Recommended doses vary according to the oil quality and range of antioxidant concentration tested.

When comparing results from assay 1 and 2, the sequence of antioxidant efficacy is not the same. In assay 1 the mixture BHT+BHA was the best, while in assay 2, TBHQ was the most efficient. Natural extracts, however presented the same performance, rosemary being more effective than oregano. MELO [28] could also not compare results from the model system with those from the Oven Test, by means of the peroxide value.

Analyzing data from assays 2, 3 and 4, after 7 days in the Oven Test, the efficiency sequence based on increasing peroxide values is : 200mg de TBHQ . kg^-1, 100 and 75mg TBHQ . kg^-1 did not differ from each other, 50 and 25mg TBHQ . kg^-1 were not statistically different from 50, 75 and 100mg TBHQ . kg^-1 with added natural antioxidant mixture R + O, while 25mg TBHQ . kg^-1 with added R + O was similar statistically to the last four treatments with the mixture R + O. Addition of 500mg rosemary ethanolic extract . kg^-1 was comparably efficient as 200 mg BHA+BHT . kg^-1 and more efficient than 1000 mg oregano ethanolic extract . kg^-1 oil sample.

UV absorbance scannings between 220 and 320nm of samples from assays 2, 3 and 4 after 7 days in Oven Test are presented in Figure 8. Their sequence of presentation in the graph is the same as that observed for peroxide values.

Rosemary and oregano ethanolic extracts are mixtures of compounds that may or not possess antioxidant activity, therefore, when compared to the synthetic antioxidants, that are pure substances, their efficiency is lower depending on the antioxidant compound concentration.

4 — CONCLUSIONS

Results of the antioxidant activity from the model system did not correspond to those from the Oven Test. It is still necessary to determine an accelerated aging method that resembles the real oxidation conditions while storing or using vegetable oils. The Oven Test was the most recommended for our conditions. 200mg TBHQ . kg^-1 was the best antioxidant in oven test, however it was possible to determine the best dose as 128.85mg . kg^-1 and 126.09 as the economical dose. Oregano and rosemary ethanolic extracts and their mixture can be used as natural antioxidants and presented the same efficiency as the synthetic antioxidants mixture BHT +BHA. The addition of natural antioxidants to TBHQ does not result in a better antioxidant effect. Natural antioxidants as ethanolic extracts can be an alternative in the preservation of oils.

5 — REFERENCES

² ESALQ-USP, Departamento de Agroindústria, Alimentos e Nutrição - Caixa Postal 09, CEP 13.418-900 , Piracicaba, SP. e-mail: [e-mail]

* A que a correspondência deve ser enviada.

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1

Recebido para publicação em 28/10/99. Aceito para publicação em 04/07/00.

Publication Dates

• Publication in this collection
  05 June 2001
• Date of issue
  Aug 2000

History

• Accepted
  04 July 2000
• Received
  28 Oct 1999