Antioxidant, anti-inflammatory potential and chemical constituents of Origanum dubium Boiss., growing wild in Cyprus

Abstract

Origanum dubium Boiss. is a flavouring herb widely used in Cyprus. In this study, both lipophilic and polar extracts of the aerial parts of O. dubium were investigated for their chemical contents and their antioxidant potential. Overall, 20 constituents were isolated and identified, belonging mainly to three significant classes of compounds: terpenes, phenolic derivatives, such as hydroquinone glycosides and flavonoids and alicyclic derivatives. None of them was previously reported as constituent of O. dubium The inhibitory potencies of all total extracts and the isolated compounds on lipid peroxidation and their interaction with 1,1-diphenyl-picrylhydrazyl (DPPH) activity is discussed. The polar extract showed strong interaction with DPPH stable radical and significant inhibition of lipoxygenase and lipid peroxidation.

• 1,1-Diphenyl-picrylhydrazyl stable radical
• flavonoids
• hydroquinone glycosides
• lipid peroxidation
• lipoxygenase
• terpenes

Introduction

Antioxidants have been widely used as food additives to provide protection against oxidative degradation of foods. Due to the negative health effects, some of the most widely used commercial synthetic antioxidants [butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG) and tertiary butylhydroquinone (TBHQ)] have been restricted in the food industry1. Therefore, natural substances isolated from plants are considered as promising sources of potential antioxidants2–5. Among the plant species, aromatic herbs from Lamiaceae family, and especially spices belonging to the genus Origanum L. have emerged as effective agents to provide antioxidant activity6–8.

The genus Origanum L. is divided into 10 sections. In particularly nine taxa are restricted to Greece, South Balkans and East Mediterranean area9^,10. Origanum spp. have been used for thousands of years as spices and as local medicines in traditional medicine. Today, they are among the most common and widely used aromatic herbs in agriculture, pharmaceutical and cosmetic industries with important economic potential11–13. Phytochemical and biological studies on Origanum spp. confirm that the genus is a rich source of active principles with antibacterial, antifungal, insecticidal, antioxidant and anti-carcinogenic activities8^,14–19. Origanum dubium Boiss. is an endemic Mediterranean shrub. In Cyprus, it is known as “rigani” and extensively used to add a distinctive aroma and flavour to food instead of common oregano (O. vulgare L.). In the traditional Cyprus medicine, an infusion of the leaves, flowering stems and flowers is used as a digestive and carminative, while carvacrol-rich essential oil is used externally as an antirheumatic agent20. The essential oil of O. dubium from Cyprus has been previously investigated and proved to possess significant antimicrobial and antioxidant activities, mainly due to its high content on carvacrol, a known antimicrobial, antiseptic and antioxidant agent21. Apart from the chemical analysis of Greek common oregano14, the inhibitory effect of the polar oregano extracts on aldose reductase and soybean lypoxygenase have been evaluated15. Furthermore, the inhibition of both enzymes by some secondary metabolites isolated from oregano, as well as docking studies thereof, have been investigated16^,17. The aim of this study was to search for new natural antioxidant agents deriving from the genus Origanum L. Both lipophilic and polar extracts of O. dubium were subjected to detailed phytochemical analysis, isolation and structural elucidation of their secondary metabolites. The isolates were evaluated for their antioxidant activity through their interaction with 1,1-diphenyl-picrylhydrazyl (DPPH) free stable radical, their anti-lipid peroxidation, as well as their inhibitory activity on lipoxygenase.

Methods

Plant material

The aerial-parts of O. dubium Boiss. were collected in Cyprus (Amargheti, Paphos) in April 2004. The plant material was collected and identified by Dr. T. Vrahimi-Hadjilouca, and Dr. D. Droushiotis (Agricultural Research Institute, Ministry of Agriculture, Natural Resources and Environment, Nicosia, Cyprus). The plant voucher specimen (ARI 3215) was deposited at the Agricultural Research Institute, Cyprus.

General experimental procedures

Optical rotation values were determined at 25 °C at 589 nm in CHCl₃ (Uvasol) on a Perkin-Elmer 341. UV spectra were recorded using Shimadzu UV-160A spectrophotometer, according to standard procedures22. IR spectra were obtained on a Perkin-Elmer Paragon 500 FT-IR spectrometer. The 1D and 2D NMR spectra were recorded using Bruker DRX 400 and Bruker AC 200. Vacuum-liquid chromatography (VLC) was carried out on silica gel (Merck; 43–63 µm) and column chromatography (CC) on silica gel 60H SDS (Merck; 40–63 µm) and on Sephadex LH-20 (Pharmacia). Medium Pressure Liquid Chromatography (MPLC) was performed on a Büchi 688 pump and with column RP-silica gel 60 (Merck, Art. 10167). Fractionations were monitored by TLC silica gel 60 F-254; Merck, Art. 5554; cellulose Merck, Art. 5716 with visualization under de UV light (absorbance: λ 254 and λ 365 nm) and with spray reagents, vanillin–H₂SO₄ on silica gel, Neu’s reagent on cellulose23.

Extraction and isolation

The air-dried powdered aerial parts (0.40 kg) were successively extracted at room temperature with dichloromethane (extract A; 9.9 g), methanol (extract B; 27.1 g) and methanol:water (5:1; extract C; 62.7 g) and evaporated to dryness in vacuo. Based on the ¹H-NMR spectra and the TLC analyses of all extracts, the non-polar dichloromethane extract and polar methanol–water were chosen for further fractionation.

The dichloromethane residue (extract A) was subjected to VLC over silica gel (8.0 × 6.5 cm) using as eluents mixtures of cyclohexane–EtΟAc (100:0–55:45) of increasing polarity. Sixteen groups (AA–AK) of 500 mL each, were collected. Group AΒ (cyclohexane–EtΟAc, 95:5; 2.4 g) was further purified by column chromatography on silica gel (11.0 × 3.0 cm; cyclohexane–CH₂Cl₂ 100:0 to 45:55) and yielded 12 (689.9 mg) and 13 (21.5 mg). Group AD (cyclohexane–EtΟAc, 85:15; 314.7 mg) was subjected by column chromatography on silica gel (13.0 × 2.0 cm; Pe–EtΟAc, 95:5 to70:30) and yielded 16 (6.1 mg). Group AI (cyclohexane–EtΟAc 55:45; 868.0 mg) was purified by several column chromatography on silica gel (15.0 × 3.0 cm; cyclohexane–CH₂Cl₂–MeOH 80:80:1 to 40:80:4) and yielded 17 (4.8 mg), 6 (12.5 mg), 18 (6.5 mg), 3 (5.5 mg) and 7 (6.5 mg).

The residue methanol–water 5:1 (extract C) was subjected to MPLC (10.0 × 5.0 cm) over reversed phase silica gel column (RP-18, Merck, Art. 10167) using as eluents mixtures of H₂O–MeOH of decreasing polarity. Eleven fractions (CA–CK) of 1 L were received. Fraction CB (1.4 g) was similarly subjected to RP–MPLC with H₂O–MeOH of decreasing polarity. Fifty five sub fractions were recombined in sixteen groups (DBA–DBR).

The group CBΒ (H₂O:MeOH, 100:0; 175.3 mg) was purified by CC over Sephadex LH-20 (25.0 × 3.0 cm; MeOH–Η₂Ο 19:3) and yielded compounds 8 (3.4 mg), 19 (5.2 mg) and 20 (18.5 mg). Based on TLC analyses, groups CBC and CBD (H₂O–MeOH, 95:5) were combined as fraction CBC′ (104.5 mg) and subjected to column chromatography on silica gel (8.0 × 2.0 cm; CH₂Cl₂–MeOH–H₂O, 100:0:0 to 0:0:100) and yielded 9 (3.8 mg). Group CBG (H₂O–MeOH 95:5–90:10, 72.0 mg) was further purified by column chromatography on silica gel (8.0 × 2.0 cm; CH₂Cl₂–MeOH–H₂O, 100:0:0 to 0:50:50) and afforded compounds 8 (1.3 mg) and 10 (1.6 mg). Group CBJ (H₂O–MeOH, 80:20; 389.4 mg) was similarly fractionated by column chromatography over Sephadex LH-20 (25.0 × 2.0 cm; MeOH–Η₂Ο 19:3) and afforded 5 (0.8 mg), a mixture of 14 and 15 (2.7 mg) and 11 (6.4 mg). Finally, group CBK (149.0 mg) was purified by column chromatography on silica gel (12.0 × 2.0 cm; CH₂Cl₂–MeOH–H₂O, 100:0:0 to 0:0:100) and yielded 4 (4.3 mg), 2 (0.8 mg) and 1 (11.6 mg) and 15 (1.7 mg).

Determination of total phenols and flavonoids

The total phenols were estimated according to the Folin-Ciocalteu method24. Extract was diluted to the concentration of 1 mg/mL, and aliquots of 0.5 mL were mixed with 2.5 mL of Folin–Ciocalteu reagent (previously diluted 10-fold with distilled water) and 2 mL of NaHCO₃ (7.5%). After 15 min of staying in the 45 °C, the absorbance was measured at 765 nm on spectrophotometer versus blank sample. Total phenols were determined as gallic acid equivalents (mg GA/g extract), and the values are presented as means of triplicate analyses. The total flavonoid content was determined according to Brighente et al.25. A 0.5 mL of 2% aluminium chloride (AlCl₃) in methanol was mixed with the same volume of the methanol solution of plant extract. After 1 h of staying at room temperature, the absorbance was measured at 415 nm on a spectrophotometer versus blank sample. Total flavonoids were determined as rutin equivalents (mg RU/g dry extract), and the values are presented as means of triplicate analyses.

Biological assays

In vitro assays

A Perkin Elmer Lambda 20 UV–Vis spectrophotometer has been used for the radical scavenging activity experiments. Each in vitro experiment was performed at least in triplicate and the standard deviation of absorbance was less than 10% of the mean.

All the chemicals used were of analytical grade and commercially available by Merck. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), trolox and nordihydroguairetic acid (NDGA) were purchased from the Aldrich Chemical Co. Milwaukee, WI. Soybean Lipoxygenase, linoleic acid sodium salt and 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) were obtained from Sigma Chemical, Co. (St. Louis, MO).

Evaluation of antioxidant and anti-inflammatory activity

The stock solution of the tested samples contains 5 mg/mL in ethanol. Interaction of the tested compounds with DPPH stable free radical was performed, as previously described26. About 20 μL from the stock solution of the sample were diluted in absolute ethanol to a final volume of 1 mL and then added to 1 mL DPPH (0.1 mM, in absolute ethanol). The reaction mixture was allowed at room temperature for 20 and 60 min. The optical density (OD) of the solution was measured and the percent reduction was calculated with the following equation at 517 nm. The optical densities of the samples without the presence of DPPH were recorded and subtracted from the corresponding OD with DPPH.

Inhibition of linoleic acid lipid peroxidation

It was performed according to a previously described procedure26. 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. About 10 mL of the 16 mM linoleic acid sodium salt solution was added to the UV cuvette containing 0.93 mL of 0.05 M phosphate buffer, pH 7.4 pre-thermostated at 37 °C. The oxidation reaction was initiated at 37 °C under air by the addition of 50 mL of 40 mM AAPH solution. Oxidation was carried out in the presence of aliquots (10 μL) of the examined samples. In the assay without antioxidant, lipid oxidation was measured in the presence of the same level of DMSO. The rate of oxidation at 37 °C was monitored by recording the increase in absorption at 234 nm caused by conjugated diene hydroperoxides (Table 2). Trolox was used as a standard.

Soybean lipoxygenase inhibition activity

It was evaluated as reported previously26. The tested samples 10 μL (3 mg/mL stock solution in DMSO) were incubated at room temperature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme solution (1/9 × 10⁴ w/v in saline) at a final volume of 1 mL. The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded and compared with the appropriate standard inhibitor (nor-dihydroguaiaretic acid 0.1 mM 83.7% inhibition).

Statistical analysis

Results were expressed as the mean ± standard deviation (SD). IBM SPSS, version 11.0 statistical program (Armonk, NY) used for data analysis (Pearson’s correlation coefficient).

Results

Phytochemical analysis

The chemical investigation of the dichloromethane and the methanol/water (5:1) extracts gave evidence to twenty different constituents (Figure 1).

Figure 1. Structures of substances isolated from the extracts of O. dubium.

The dichloromethane extract yielded two phenolic monoterpenes: carvacrol 12, carvacrol acetate 1327, one methylated flavone, xanthomicrol 3 and two flavanones: naringenin 6, eriodictyol 728^,29, one oxygenated sesquiterpene, namely, spathulenol 1630, two triterpenic acids: oleanolic acid 17 and ursolic acid 1831 (Figure 1).

The polar extract methanol/water (5:1) afforded two phenolic monoterpene glucosides, namely thymoquinol-2-O-β-glucopyranoside 14 and thymoquinol-5-O-β-glucopyranoside 1527, three hydroquinone glycosides: arbutoside 832, seguinoside-B 933, osmantolide 1034, one phenolic acid: p-coumaric acid 1135, four flavonoids: apigenin 1, luteolin 2, apigenin 7-O-β-D-glucopyranoside 4 (=cosmoside), 6-hydroxykaempferol-3-methylether-6-O-β-D-glucopyranoside 528^,29 and two alicyclic derivatives: 12-hydroxyjasmonic acid 19, 12-hydroxyjasmonic acid 12-O-β-glucopyranoside 2036 (Figure 1). All structures were identified using spectroscopic methods 1D and 2D NMR, UV, GC-MS.

It is noteworthy that the chemical profile of O. dubium growing wild in Naxos island (Greece) has been revealed different, since four methylated flavonoids, namely 4′-methyl ether apigenin, 3,6,7-trimethyl ether kaempferol, 3,6-dimethylether quercetin and 3,6,7-trimethylether quercetin, have been isolated37. Although O. dubium is used in Cyprus as the common oregano (O. vulgare L.), the chemical profiles of the two plants are partially different. So far, our group has investigated two medicinal taxa from the genus Origanum L., used also in Greek cuisine as condiments: common oregano14, dittany38, as well as the endemic Greek species O. scabrum Boiss39.

The alicyclic compounds: 12-hydroxyjasmonic acid, 12-hydroxyjasmonic acid 12-O-β-glucopyranoside and the monoterpene glycosides thymoquinol-2-O-β-glucopyranoside and thymoquinol-5-O-β-glucopyranoside are present in all investigated taxa from Greece. Differences are observed concerning the content of the volatile monoterpene carvacrol in the extracts. While it is present in the dichloromethane extracts of O. dubium, it was not isolated during our previous study on common oregano, despite of its well-documented presence in high amounts in common oregano essential oils40^,41. These differences are expected, since the extraction scheme is not aiming at the volatile constituents. The extraction protocols involve powdered plant material (instead of finely cut aerial parts) and classic extraction (instead of hydrodistillation). Carvacrol and other volatile terpenes like carvacrol acetate and spathulenol can sometimes be extracted and detected in along with non volatile constituents. However, once carvacrol is present in the extracts, it might exert pharmacological activity. In addition, O. vulgare ssp. hirtum14 revealed the presence of p-menth-3-ene-1,2-diol 1-O-β-glucopyranoside and thymoquinol 2,5-O-β-diglucopyranoside and O. dictamnus L.38 of ursolic aldehyde and thymoquinone, none of them found in O. dubium.

When polar extracts are considered, phenolics are the marker/characteristic constituents. From this point of view, the most important differences between O. dubium in one hand and O. vulgare, O. dictamnus and O. scabrum on the other, is the total absence of depsides in O. dubium. All other taxa are characterised by the presence of rosmarinic acid and its derivatives (depsides): dimers (rosmarinic acid and methylesters), trimers (10-epi-lithospermic, salvianolic P acids) and tetramers (lithospermic acid B, epi-lithospermic acid B) of caffeic acid. Namely, O. vulgare ssp. hirtum14 revealed the presence of caffeic acid and its derivatives, rosmarinic acid, lithospermic acid B, 10-epi-lithospermic acid, epi-lithospermic acid; O. dictamnus38 yielded rosmarinic acid, rosmarinic acid methylester, salvianolic acid P; O. scabrum39 afforded rosmarinic acid, rosmarinic acid methyl ester and 3-O-methyl rosmarinic acid. As to their flavonoid contents, some similarities are observed since apigenin, luteolin, eriodictyol, narigenin, cosmoside are also present in O. dictamnus38^,42^,43 in addition apigenin, luteolin, cosmoside and eriodictyol are also found in O. vulgare L. ssp. hirtum14, while these two latter species also contain kaempferol, quercetin, taxifolin, luteolin 7-O-glucoside, eriodictyol 7-O-glucoside, vitexin, isovitexin, orientin and isoorientin; chrysoeriol, diosmetin, quercetin and vicenin-2, respectively, not found in O. dubium.

In conclusion, the chemical profile of O. dubium is distinct due to the presence of hydroquinone glycosides. So far, hydroquinone glycosides have been detected in O. majorana and O. dubium extracts44–46.

Previously quantification studies in common oregano have shown high amounts of rosmarinic acid. Shan et al.47 have reported 25.6 mg/g of plant, whereas Kivilompolo & Hyötyläinen48 after sonication assisted extraction reported 5.98 mg/g of rosmarinic acid in dry plant material. Unfortunately, in none of these studies, derivatives of rosmarinic acid or other depsides are reported. According to our studies, common Greek oregano (O. vulgare ssp. hirtum) contains significant amount of rosmarinic acid (at least 12.8 mg/g in dry plant material). Preliminary results (data not shown) in O. dictamnus showed the presence mainly of rosmarinic acid (4.9 mg/g of dry plant material) and salvianolic acid P (2.5 mg/g of dry plant), but these results largely depend on the extraction applied (solvent, time, temperature and DER)14^,37.

Total phenol and flavonoid content

Most of the therapeutic properties of medicinal plants are attributed to their phenolic content. Phenolic compounds are widely distributed and can be considered as the most abundant plant secondary metabolites with highly diversified structures. Their powerful antioxidant activity is highly related to the presence of phenol rings and hydroxyl groups, that act as electron traps to scavenge peroxy-, superoxide-anions and hydroxyl radicals49^,50. Positive correlation between total phenolic content of polar plant extracts and related antioxidant activity has been reported on Origanum species51^,52. For this reason, both total phenol and flavonoid contents of O. dubium were determined in order to have a general overview of the phenolic load of the extracts. In parallel, detailed phytochemical analyses were carried out in order to isolate molecules responsible for the antioxidant activity. A vast variety of chemical constituents were identified belonging to the groups of terpenes, some of them aromatic and phenols (simple phenolics and flavonoids).

The phenolic contents of the different extracts of O. dubium were high, as shown in Table 1. The methanol/water extract possessed the highest level of total phenolics (730.66 ± 6.82 mg GAEs/g dry weight) followed by the methanol extract (530.6 ± 12.7 mg GAEs/g dry weight) while the lowest level of phenols was observed in the dichloromethane extract (230.96 ± 4.29 GAEs/g). This evidence was further supported by the highest flavonoid content obtained from the most polar extract (70.15 ± 0.40) (Table 1). The phytochemical analysis is in complete agreement with present data, since the most polar extract (methanol:water 5:1) contains mainly phenolic compounds, while the dichloromethane extract was found to be rich in aromatic terpenes, such as carvacrol. Previous studies also reported positive correlation between phenolic and flavonoid content and DPPH radical scavenging activity of the plant extracts53.

Table 1. Total phenolic and flavonoid, content of O. dubium extracts.

Extract	Total phenolic content (mg GA g^−1)	Flavonoid content (mg RU g^−1)
A	230.96 ± 4.29	18.61 ± 0.29
B	650.79 ± 7.06	56.44 ± 0.63
C	730.66 ± 6.82	70.15 ± 0.40

Results are represented as mean values ±SD from three experiments. A: Dichloromethane extract; B: Methanol extract; C: Methanol–water (5:1) extract.

Bioactivity

All extracts and most of the isolated compounds after being submitted to three biological assays exhibited different levels of bioactivity (Table 2). Compounds 6-hydroxykaempferol-3-methylether-6-O-β-D-glucopyranoside, carvacrol and thymo-quinol-2-O-β-glucopyranoside and have been excluded from the present bioassays, as carvacrol has been previously tested21; the quantity of 6-hydroxykaempferol-3-methylether-6-O-β-d-glucopyranoside, was insufficient, while thymoquinol-2-O-β-glucopyranoside has been isolated only in mixture with thymoquinol-5-O-β-glucopyranoside. Naringenin interacted immediately. Taking in consideration that carvacrol is active⁽21, it is suggested that naringenin, isolated from the dichloromethane extract, acts synergistically with carvacrol for the antioxidant activity of this extract. Moreover, both polar extracts, characterised by high content of phenolic compounds, interacted with DPPH after 5 min. p-Coumaric acid and the methanol/water (5:1) extract (C) showed the highest inhibition of lipid peroxidation. Also, p-coumaric acid proved to be a very strong LOX inhibitor, while all flavonoids and hydroquinone glycosides, as well as the alicyclic compounds 12-hydroxyjasmonic acid, 12-hydroxyjasmonic acid 12-O-β-glucopyranoside have been proved totally inactive or interacted weakly.

Table 2. Interaction % with 1,1-diphenyl-2-picrylhydrazyl (DPPH) at 0.1 mM, Inhibition of lipid peroxidation at 100 mM and % Inhibitory effect on soybean lipoxygenase of O. dubium extracts and metabolites.

Samples	RA% 20 μl at 20/60 min	AAPH % 10 μl	LOX % 10 μl
A	57 (20 min)/83	8	48
3	3/4	no	no
6	92 (immediately)/100	71	24
7	8/8	38	no
13	14/15	no	26
15	24/25	89	no
16	26/27	no	no
17	1 (20 min)/39 (60 min)	9	30
18	9/9	16	no
B	91 (5 min)/100	no	30
C	88 (5 min)/100	61	22
1	no	3	no
2	0 (20 min)/43(60 min)	29	no
4	No/no	44	no
8	15/16	31	no
9	29/30	37	no
10	63/65	77	1
11	16/15	100	100
19	37/38	13	no
20	36/38	16	no

A: Dichloromethane extract; B: Methanol extract; C: Methanol–water (5:1) extract.

Discussion and conclusions

Naringenin, although bearing only one hydroxy group at C-4′, was proved very active in contrast to eriodictyol, bearing an ortho-dihydroxy system, which revealed a weak activity. p-Coumaric acid, derived from the methanol:water (5:1) extract (C), also bearing only one hydroxy group proved to be a very strong LOX inhibitor. It is noteworthy that the p-coumaric acid anti-lipid peroxidation activity is significantly better than trolox (reference compound). Among the hydroquinone glycosides, osmantolide bearing a second oxygen function revealed the better interaction with DPPH and the highest inhibition of lipid peroxidation than arbutoside, seguinoside-B.

Concerning the inhibition of soybean lipoxygenase, it was observed that the absence of the sugar moiety in compound 12-hydroxyjasmonic acid did not increase its activity compared to its glucoside. Polar extracts of O. dubium containing mainly the above-mentioned constituents along with free sugars presented a weak inhibitory activity on LOX. In contrast, the dichloromethane extract of O. dubium gave the best result (48%) on soybean lipoxygenase inhibition at the same concentration. This inhibitory effect could be attributed to the extremely high content of carvacrol in the dichloromethane extract. It is noteworthy that the phytochemical analyses described in this study afforded almost 700 mg of pure carvacrol, much higher amount when compared to other terpenes present in the extract (carvacrol acetate, spathulenol, oleanolic acid, ursolic acid) or other phenolic compounds (naringenin, eriodictyol and xanthomicrol).

The aqueous methanolic extract of common oregano previously investigated17, proved to possess significant inhibitory activity (40.4%) on soybean lipoxygenase at the concentration of 10 μg/ml and in a dose dependent manner; this extract is rich in depsides, especially in trimers (10-epi-lithospermic acid) and tetramers (lithospermic acid B and epi-lithospermic acid B) of caffeic acid. These constituents contain several ortho-hydroxyl groups and are probably responsible for the inhibitory effect of the O. vulgare ssp. hirtum on soyabean lipoxygenase. Concerning the pure isolates, it was found that lithospermic acid B was the most potent compound. Furthermore, the docking results significantly supported the in vitro biological data17.

According to the literature, the antioxidant activity of oregano extracts is due to at least to two different anti-oxidative mechanisms54. The activity might be due both to non-phenolic compounds and to phenolic derivatives. The group of non-phenolic compounds act as scavengers of free radicals and are effective in early stages of oxidation. The group of phenolic compounds is effective in interrupting the chain processes responsible for oxygen consumption by a mechanism similar to that for tocopherols. In general, authors confirm the protective role of different Origanum taxa (O. vulgare, O. compactum, O. majorana) against the auto oxidation process over time55.

In conclusion, although O. dubium is not characterised by the presence of rosmarinic acid and its derivatives, it is a rich source of carvacrol and polar phenolic compounds, giving evidence to its anti-oxidative capacity, anti-lipid peroxidation and lipoxygenase inhibitory activity.

Acknowledgements

The authors wish to thank Dr. T. Vrahimi-Hadjilouca, and Dr. D. Droushiotis (Agricultural Research Institute, Ministry of Agriculture, Natural Resources & Environment, Nicosia, Cyprus) for the collection and identification of plant material.

Declaration of interest

The authors report no declarations of interest.