THE EFFECT OF STORAGE ON QUALITY OF HERBS GENUS ORIGANUM

Herbs of Origanum genus are rich in essential oils and contain large amounts of phenols, lipids, fatty acids, flavonoids and anthocyanins. Antioxidant activity of these herbs depends on many factors, including the type herbs, post-harvest processing and subsequent processing. The aim of this study was therefore to confirm the hypothesis that the composition of oils of these two herbs of the Origanum genus depends on the post-harvest treatment of herbs and that the dried herb antioxidant activity is higher for fresh than that of frozen herbs. Lamiaceae family herbs: oregano ( Origanum vulgare L.) and Greek oregano ( Origanum heracleoticum L.) were planted and analyzed. Herb samples were extracted by hot demineralised water. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method was used for antioxidant activity assessment. The total phenolic content was determined spectrophotometrically by using Folin-Ciocalteu reagent. Steam distillation of essential oils was carried out via Clevenger Apparatus. The obtained essential oils were analysed by GC-MS technique. Results of tested fresh, dried and frozen herbs showed a considerable potential for quenching the free DPPH radical. Significantly higher antioxidant activity was found in dried herbs comparing to fresh and frozen, but only in case of values calculated per 100 g of the sample. However, the differences were not statistically significant after recalculation when expressed on dry matter content. There was no difference between fresh and frozen samples. The content of total phenols was significantly higher in dried than in frozen herbs in values recalculated per 100 g of sample. A strong correlation between the results of DPPH and TPC was found again only for values expressed per 100 g of the sample. Postharvest treatment of herbs affects the composition of their essential oils. The dominant essential oil component of Greek oregano is carvacrol with a proportion of 60% or more. On the contrary, there is no such dominating component in oregano essential oil but there are more components with a share of 10 to 20%.


INTRODUCTION
Herbs of the Origanum genus are found mainly in the Mediterranean region and Asia. This genus is rich in essential oils where the quantity and quality vary considerably (Asensio et al., 2015). Herbs of Origanum genus contain large amounts of phenols, lipids, fatty acids, flavonoids and anthocyanins (Kintzios, 2002).
Oregano is a perennial plant of Lamiaceae family groving in Asia, Europe and North Africa. This aromatic herb is widely used in many world cuisines ( Origanum vulgare L. has been used in Persian traditional medicine for its anti-inflamantory (Javadian et al., 2016) and other effects such as diuretics, stomachics, antineuralgics, antitussives and expectorans (Afsharypour et al., 1997).
Greek oregano is widely used in gastronomy. Its typical flavour is produced by the presence of essential oils Greek oregano such as rosmarinic acid and its derivatives, caffeic acid, protocatechuic acid and phenyl glucoside. In a report by Zheng et al., (2009), the essential oil of Greek oregano consists of 78.28% of phenolic compounds carvacrol and thymol followed by γ-terpinene (5.54%) and p-cymene (7.35%). The antioxidant activity of essential oil of Greek oregano and its use in the food industry was also discussed by Kulisic et al., (2004).
Antioxidant activity of herbs depends on many factors, including the type herbs, the methods and conditions of cultivation, harvest, post-harvest processing and subsequent processing. The extraction method, the manner of extraction and the type of solvent used also affect the level of antioxidant activity (Škrovánková et al., 2012). The effect of different growing conditions on the chemical composition and biological activity of the essential oil of oregano was studied De Falco et al., (2013). Kouřimská et al., (2014) investigated the antioxidant activity of plants of the Lamiaceae family grown under organic and conventional conditions. Ozkan et al., (2010) and Baranauskienè et al., (2013) focused on the impact of harvest on essential oil composition and antioxidant activity of oregano. They found the highest antioxidant activity was found in herbs harvested during their flowering.
The water content in fresh Lamiaceae herbs is typically in the range of 75 -80%. For the preservation of the herbs, it is necessary to reduce the amount of water to less than 15% (Diaz-Maroto et al., 2002). Lowering the water activity inactivates the enzymes, which in its active form may be cause degradation of antioxidant ingredients of fresh herbs (Hossain et al., 2010). Water activity may be reduced via different methods. The most commonly used method is drying. This method results in an increase in the content of some substances. Cell tissues of herbs are damaged during the drying process which leads to the release of phenolic compounds and increase of antioxidant activity. Changes in appearance and flavour are due to loss or development of volatile compounds because of oxidation and esterification reactions (Hossain et al., 2010).
Air drying is the simplest and cheapest method of drying. Low temperature prevents the degradation of the active ingredients of herbs during this type of conservation method, but the drying is relatively slow and thus the metabolic processes may continue and cause changes in quality of herbs (Keinänen and Julkunen-Tiitto, 1996). Slow loss of water can act as a stressor and the defensive mechanism of most plants is the production of phenolic compounds ( Although there is a lot of scientific literature focussed on the effects of drying or freezing on the antioxidant activity and composition of certain medicinal and aromatic herbs, there is still lack of a comprehensive study comparing these two conservation practices both at the same time in the case of oregano and Greek oregano. The aim of this study was therefore to confirm the hypothesis that the composition of oils of these two herbs of the Origanum genus depends on the post-harvest treatment of herbs and that the dried herb antioxidant activity is higher for fresh than that of frozen herbs.

MATERIAL AND METHODOLOGY Herbs
Lamiaceae family herbs: oregano (Origanum vulgare L.) and Greek oregano (Origanum heracleoticum L.) were planted and analyzed. The seeds were sown on 20th April to the sunny, unfertilized plot of sandy loam medium soil in the Jirny locality (50° 6ʹ 56ʹʹ N, 14° 41ʹ 57ʹʹ E, district Prague-East). The seeds were purchased from Kiepenkerl company. Plant parts were harvested before flowering on 30th July and divided into three parts. One part was spread on papers in the laboratory and dried at 25 °C for one week. Another part was placed in plastic bags and frozen at -18 °C. The last third was analysed immediately.

Determination of dry matter content
Balances with infrared dryer, Precisa HA 300 (Precisa Instruments, Swirzerland) were used for dry mater content determination. Samples of herbs (1 g) were ground and spread on aluminium foil and dried at a maximum temperature 105 °C to the constant weight (the weight difference less than 2 mg for 30 s). All samples were measured in triplicate and the average was calculated. No. 1/2016

Herb extraction
Fresh herbs (6 g) or the equivalent amount of dried herbs (calculated from total dry matter of individual herbs) were taken for the preparation of water extracts. Herb samples were extracted twice by 50 mL of hot demineralised water in the ultrasonic bath for 10 min. Samples were then filtered into 100 mL volumetric flasks and filled up to the mark after cooling. The extracts were analysed on the same day.

DPPH
(2,2-diphenyl-1-picrylhydrazyl) radical scavenging method taken from Adámková et al., (2015) and Chrpová et al., (2010) was used for antioxidant activity assessment. The intensity of the violet DPPH radical solution was measured at 522 nm. As the reaction equilibrium is usually reached after two hours for most compounds, the absorbance of samples was measured after 1, 2 and 3 hours and its minimum was used for the antioxidant activity calculation. The method was calibrated with ascorbic acid and the results were expressed as equivalents of ascorbic acid per unit mass of sample.

Steam distillation of essential oils
Steam distillation of essential oils was carried out via Clevenger Apparatus (Wilmad-LabGlass, USA) for 4 hours (Memarzadeh et al., 2015). The apparatus was also used for the determination of extracted volume of essential oil.

GC analysis of essential oils
The obtained essential oils (10 µL) were placed into the vials with 500 µL of n-hexane (with a few crystals of anhydrous sodium sulphate) and analysed by GC-MS using Agilent 7890A GC coupled to Agilent 5975C singlequadrupole mass detector equipped with a HP-5MS column (30 m × 0.25 mm ID, 0.25 µm film). The sample volume of 1 µL was injected in split mode (ratio 12:1) into the injector heated to 250 °C. The initial oven temperature was 60 °C (hold 3 min), ramp to 250 °C at 3 °C . min -1 (hold 10 min). Helium was used as carrier gas with the flow rate of 1 mL.min -1 . The MS analysis was carried out in full scan mode, the electron ionization energy was set at 70 eV. The analytes were identified according to their relative retention times and by the comparison of their mass spectra with the National Institute of Standards and Technology Library (NIST, USA). The results were calculated by area normalisation method.

Statistical evaluation
The data obtained were analysed using statistical software Statistica 12.0 (StatSoft Inc.). Analysis of variance (one-way ANOVA) was performed and the significant differences in the means were separated using the Scheffé's test. The data were expressed as an average of triplicates ± standard deviation. For all statistical tests, a 5% level of significance was used.

RESULTS AND DISCUSSION Comparison of dry matter content, antioxidant activity and phenolics content
Dry matter content results of analysed herbs are presented in Table 1 Results of antioxidant activity of tested herbs analysed by DPPH method expressed in mg of ascorbic acid (AA) per 100 g of sample or per 100 g of dry matter are shown in  There can be seen, only a slight difference between fresh, dried and frozen samples of oregano expressed on dry matter content. When comparing oregano and Greek oregano there was always higher antioxidant activity in case of oregano samples. Table 3 shows the total phenolics content of tested herbs analysed by Folin-Ciocalteu reagent and expressed in mg of Gallic acid (GA) per 100 g of sample or per 100 g of dry matter. It can be observed that fresh, dried and frozen herbs contain reasonable amounts of phenols, which also corresponds to the results of Ivanova et al., (2005) and Skotti et al., (2014). The highest amount of phenols per 100 g of the plant was determined on the dried herbs, which correlates with the highest antioxidant activity. After recalculating the results based on dry matter content, fresh oregano samples had the highest phenol content. The lowest amount of total phenols showed frozen herbs even after recalculating to dry matter content. Higher phenol content in dried herbs corresponds with the conclusion of Ahmad-Qasem et al., (2013), who found lower levels of phenols in the frozen sample of olive-tree than in the dried sample. The authors explained this lower content of phenols in a frozen sample as a result of the temporary inactivation of the enzymes during freezing.
Statistical comparison of the results of the fresh, dried and frozen herbs expressed per dry matter content did not show any significant differences in antioxidant activity including the content of total phenols (pDPPH = 0.6509, pTPC = 0.0731). However, significant differences were found comparing the values per 100 g of herbs for both antioxidant activity (p = 0.0339) and in the content of total phenols (p = 0.0266). A more detailed analysis using Scheffé test showed a statistically significant difference in antioxidant activity between fresh and dried herbs (p = 0.0498) and between frozen and dried herbs (p = 0.0493). In the case of TPC, there was only a statistically significant difference between frozen and dried herbs (p = 0.0290). A strong correlation (r = 0.83) was observed between the values of DPPH and TPC expressed per 100 g of herbs.
Higher antioxidant activity by DPPH method in the dried sample may be due to stress of plants accompanied by the formation of phenolic compounds. Also the disruption of tissues during drying could result in the release of phenolics and increasing their content in the extraction process. This is partly confirmed by the results of total phenols, where a strong correlation between the values of TPC and DPPH was found, but only for the results expressed per 100 g of herbs. A significant correlation between the total content of phenols and antioxidant Higher antioxidant activity of frozen herbs (values expressed on dry matter) was found compared to fresh herbs. The reason could be damage of herb tissues due to the formation of crystals during freezing and subsequently increased release of secondary metabolites into the solvent

Comparison of extracted volume of essential oil and its composition
Extraction of the essential oils from the fresh, dried and frozen samples gave significantly higher yields from samples of Greek oregano (6.3, 14.8 and 8.2 mL.kg -1 respectively) than for oregano (0.2, 3.2 and 1.7 mL.kg -1 resp.). The lowest yield was in case of fresh samples. Baranauskienè et al., (2013) also found a higher yield at the Greek oregano compared with oregano and all samples that they harvested in different periods.
The main components of fresh oregano essential oil analysed by GC-MS were β-cubebene, caryophyllene, γ-terpinene and germacene D (Table 4) . Cubebol, sabinene and α-terpinene were found only in the essential oils of dried and frozen herbs, while βcubebene and β-phellandrene were only in the samples of fresh oregano.
Carvacrol was the main component of Greek oregano essential oil samples (Table 5) Najafian (2014) observed different representation of ingredients of lemon balm essential oil as a result of the storage of herbs in the freezer, refrigerator and room temperature.
The smallest changes in the composition of essential oils were found during the storage of herbs in the freezer and refrigerator compared to storage at room temperature. The highest level of carvacrol was found in the essential oil of fresh oregano Greek, while the lowest amount was in the frozen sample. Reduced proportion of carvacrol was always associated with increased content of its precursor γterpinene. This phenomenon was also highlighted by Novák et al., (2011). High concentration of carvacrol in the essential oil of Greek oregano is responsible for its antioxidant activity.

CONCLUSION
The antioxidant activity and the composition of essential oils of selected plants of the genus Origanum were affected by post-harvest treatment of plants. All fresh, dried and frozen herbs showed a considerable potential for quenching the free DPPH radical. Significantly higher antioxidant activity was found in dried herbs comparing to fresh and frozen, but only in case of values calculated per 100 g of the sample. However, the differences were not statistically significant after recalculation when expressed on dry matter content. There was no difference between fresh and frozen samples. The content of total phenols was significantly higher in dried than in frozen herbs in values recalculated per 100 g of sample. A strong correlation between the results of DPPH and TPC was found again only for values expressed per 100 g of the sample. Postharvest treatment of herbs affects the composition of their essential oils. The dominant essential oil component of Greek oregano is carvacrol with a proportion of 60% or more. On the contrary, there is no such dominating component in oregano essential oil but there are more components with a share of 10 to 20%.