Antioxidative and ACE inhibitory activities in enzymatic hydrolysates of the cotton leafworm, Spodoptera littoralis

The larvae of the cotton leafworm, Spodoptera littoralis, were used as a source of food proteins exerting possible biological activities. A simulated gastrointestinal digestion (IC₅₀ = 320 μg/ml) and digestion by mucosal enzymes (IC₅₀ = 211 μg/ml) reveals a significantly higher in vitro ACE inhibitory activity compared to hydrolysis using thermolysin (IC₅₀ = 1392 μg/ml) and alcalase (IC₅₀ = 827 μg/ml) as pretreatment. This indicates that the choice of enzymes to generate ACE inhibitory peptides is important. All hydrolysates were also tested for antioxidant activity using two tests: a radical scavenging test using DPPH and the ferric reducing antioxidant power (FRAP) assay, and they showed a similar antioxidant activity which was relatively low compared to the standard antioxidants BHT and vitamin C. As a conclusion, the data obtained suggest that insect protein can be used to generate hydrolysates, exerting both ACE inhibitory and antioxidant activity, which might be incorporated as multifunctional ingredient into functional foods.     

Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS

Haruan myofibrillar protein was hydrolysed with proteinase K and thermolysin to isolate Angiotensin converting enzyme (ACE) inhibitory peptides. The thermolysin hydrolysate of myofibrillar protein with the highest ACE inhibition activity (IC₅₀ = 0.033 mg/ml) was fractionated by ultrafiltration and size exclusion chromatography to three fractions. Fraction F2 with higher ACE inhibitory activity was separated into five fractions (A–E) using reversed-phased high performance liquid chromatography (RP-HPLC). Fraction C showed 81% inhibition activity and was subjected to HPLC coupled to electrospray ionisation-time-of-flight mass spectrometry (ESI-TOF MS/MS). Two peptide sequences for the most abundant fragments were identified as VPAAPPK (IC₅₀ = 0.45 μM) at 791.155 m/z and NGTWFEPP (IC₅₀ = 0.63 μM) at 1085.841 m/z. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The results suggest that Haruan meat protein hydrolysate is a potent ACE inhibitor and may be used to decrease blood pressure.     

QSAR-aided in silico approach in evaluation of food proteins as precursors of ACE inhibitory peptides

An increasing prevalence of hypertension in the world implies the necessity of further study of antihypertensive peptides as an alternative means for hypertension management. The purpose of this study was to evaluate systematically the potential of major food proteins as precursors of ACE inhibitory peptides using QSAR-aided in silico approach, and thus to establish the rationale for choosing the appropriate substrate proteins in preparing ACE inhibitory peptides. In silico digestion of proteins from 15 common food commodities by thermolysin generated 5709 peptides ranging from 2 to 6 amino acid residues. Peptides were divided into three categories based on the potency of their predicted activities. Our results showed that meat proteins from pork, beef and chicken contain the largest number of potent peptides (IC₅₀ < 10 ??M), followed by proteins from milk, egg, soybean and canola, whereas proteins from fish (with the exception of salmon) and cereals (oat and barley) contain the least number of potent peptides. This study demonstrated that proteins from livestock meat, milk, egg, soybean and canola are good sources of ACE inhibitory peptides.     

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC₅₀ = 287 μg/mL) and (ii) from β-lactoglobulin (IC₅₀ = 128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC₅₀).     

The impact of fermentation and in vitro digestion on formation angiotensin converting enzyme (ACE) inhibitory peptides from pea proteins

Pea seeds were fermented by Lactobacillus plantarum 299v in monoculture under different time and temperature conditions and the fermented products were digested in vitro under gastrointestinal conditions. After fermentation and digestion ACE inhibitory activity was determined. In all samples after fermentation no ACE inhibitory activity was noted. Potentially antihypertensive peptides were released during in vitro digestion. The highest DH (68.62%) were noted for control sample, although the lowest IC₅₀ value (0.19 mg/ml) was determined for product after 7 days fermentation at 22 °C. The hydrolysate characterised by the highest ACE inhibitory activity was separated on Sephadex G10 and two peptides fractions were obtained. The highest ACE inhibitory activity (IC₅₀ = 64.04 μg/ml) for the first fraction was noted. This fraction was separated by HPLC and identified by LC–MS/MS and the sequence of peptide derived from pea proteins was determined as KEDDEEEEQGEEE.     

Hypotensive effect of a sweetpotato protein digest in spontaneously hypertensive rats and purification of angiotensin I-converting enzyme inhibitory peptides

We have investigated angiotensin I-converting enzyme (ACE) inhibitory activity in an enzyme digest of sweetpotato protein, the antihypertensive effect of the digest in spontaneously hypertensive rats (SHR), and the identification of an ACE inhibitory peptide. Protein was prepared from squeezed juice of sweetpotato by isoelectric focusing precipitation. Three kinds of proteases were selected for effective protein digestion. The digest, sweetpotato peptide (SPP), exhibited strong ACE inhibitory activity (IC₅₀: 18.2 μg/ml). SPP was orally administered by gavage to SHR at a dose of 100 mg/kg or 500 mg/kg. The systolic blood pressure and the diastolic blood pressure were measured at 0 (before administration), 2, 4, 8, and 24 h after administration. A dose-dependent decrease in systolic blood pressure in SHR was observed after oral administration of SPP. Significant differences between SPP-administered rats and control rats were observed 4 and 8 h after administration in the 500 mg/kg-administered group and 8 h after administration in the 100 mg/kg-administered group. Diastolic blood pressure also decreased in the SPP-administered groups, although the difference between SPP-administered rats and control rats was not significant. These results suggest that SPP may be useful in the prevention or treatment of hypertension. Peptides with ACE inhibitory activity were purified from SPP by absorption chromatography and preparative HPLC using an ODS column. The amino acid sequences of isolated peptides were I-T-P, I-I-P, G-Q-Y and S-T-Y-Q-T; their ACE inhibitory activities (IC₅₀) were 9.5 μM, 80.8 μM, 52.3 μM and 300.4 μM, respectively. In conclusion, I-T-P is a novel, strong ACE inhibitory peptide.     

Identification of angiotensin I-converting enzyme inhibitory peptides from koumiss, a traditional fermented mare's milk

Angiotensin I-converting enzyme (ACE) inhibitory activities in untreated koumiss and koumiss digested with ACE, pepsin, trypsinase, and chymotrypsin were compared and analyzed. Four novel ACE inhibitory peptides (P_I, P_K, P_M, and P_P) were purified using ultrafiltration and high performance liquid chromatography (HPLC). The classification study showed that these 4 peptides were of the true inhibitor type. The amino acid sequences of these peptides are YQDPRLGPTGELDPATQPIVAVHNPVIV, PKDLREN, LLLAHLL, and NHRNRMMDHVH, respectively. Their individual IC₅₀ (50% inhibitory concentration) values were as follows: 14.53 ± 0.21 μM, 9.82 ± 0.37 μM, 5.19 ± 0.18 μM, and 13.42 ± 0.17 μM. From sequence analysis, we determined that P_I was part of β-casein in mare's milk. The 3 peptides P_K, P_M, and P_P did not correspond with any known milk protein. The results suggest that koumiss is rich in ACE inhibitory peptides, and the ACE inhibitors in koumiss are of the pro-drug type or a mixture of the pro-drug type and the true inhibitor type. These results may provide evidence about the beneficial effects of koumiss, especially on cardiovascular health.     

LC–MS/MS coupled with QSAR modeling in characterising of angiotensin I-converting enzyme inhibitory peptides from soybean proteins

The importance of soy products in reducing the risk of cardiovascular disease is well documented. Our previous computation study has indicated the presence of several potent ACE inhibitory peptides within soybean proteins which needs to be identified. The aim of the study was to identify ACE inhibitory peptides from soy proteins using LC–MS/MS coupled with quantitative structure–activity relationship (QSAR) model. Soybean protein hydrolysate digested by thermolysin showed an IC₅₀ value of 53.6 μg/mL, decreased slightly to 51.8 μg/mL after adding pepsin, and increased to 115.6 μg/mL after adding trypsin. A total of 34 peptides were characterised from LC–MS/MS. Five novel tripeptides, IVF, LLF, LNF, LSW and LEF, with predicted IC₅₀ values lower than 10 μM were synthesized and validated. The results showed that soybean is an excellent source of ACE inhibitory peptides.     

Angiotensin I-converting enzyme inhibitory activity of low-molecular-weight peptides from Atlantic salmon (Salmo salar L.) skin

Collagen extracted from Atlantic salmon (Salmo salar L.) skin (which is normally discarded in the process of manufacture) was hydrolyzed with Alcalase and papain, and treated by multistage separation. The salmon skin collagen peptides (SSCP) obtained had high protein content (91.20 ± 1.03%) and low molecular weights, 90.79% of which were less than 1000 Da. SSCP was then separated by reversed-phase high performance liquid chromatography. Eleven major fractions were collected and their angiotensin I-converting enzyme (ACE) inhibitory activity was assayed. Fractions 5 and 7 displaying higher ACE inhibitory activity were subjected to mass spectrometer to identify the ACE inhibitory peptides. A total of eleven peptide sequences were identified, and two dipeptides, Ala-Pro and Val-Arg, were selected for further ACE inhibitory activity analysis. The ACE inhibitory activities of Ala-Pro (IC₅₀ = 0.060 ± 0.001 mg/ml) and Val-Arg (IC₅₀ = 0.332 ± 0.005 mg/ml) were found to be approximately 20- and 4-fold higher than that of SSCP (1.165 ± 0.087 mg/ml), respectively.     

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC₅₀ = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC₅₀ = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.     

Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates

A tryptic gliadin hydrolysate was fractionated into peptide fractions, which were assigned to either the central domain (CD) or terminal domains (TD) of gliadins. The domains were expected to contain amino acid (AA) sequences which, when released from the parent protein, inhibit the angiotensin I-converting enzyme (ACE), which plays a key role in regulating blood pressure. A proline (Pro) poor TD related fraction, containing the smallest peptides, showed the highest ACE inhibitory activity (IC₅₀ = 0.33 mg/ml). Additional peptidases were selected based on their in silico predicted ability to release ACE inhibitory peptides. Further hydrolysis of the tryptic hydrolysate fractions with thermolysin, Clarex, Alcalase and Esperase increased ACE inhibitory activities. Immobilised Ni²⁺-ion affinity chromatography (IMAC) purification of a TD related peptide fraction obtained by sequential hydrolysis with trypsin and thermolysin yielded a fraction with an IC₅₀ value of 0.02 mg/ml. This IMAC fraction was enriched in histidine and hydrophobic AA (Pro, Val, Ile, Leu and Phe).     

Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds

This study investigated the antioxidant and antihypertensive activities of peptides obtained from protein fractions of Adzuki bean seeds. Peptides were obtained by the use of hydrolytic enzymes in vitro under gastrointestinal conditions. A determination was made of the activity of the peptide inhibitors of the angiotensin I converting enzyme (ACE), and the antiradical and ion chelating activity of peptides from different protein fractions. The highest peptide levels after the absorption process (<7 kDa) were noted in the albumin fraction (50.69 μg/ml). Furthermore, it was found that peptides from the prolamin fraction were characterised by the highest antiradical activity and ACE inhibitory activity (IC₅₀ = 0.17 mg/ml). Peptides obtained from the globulin fraction showed the highest ability to chelate iron ions, and peptides from the glutelin fraction were characterised as being the most effective in the chelation of copper ions.     

Anti-inflammatory effects of compounds from Kaempferia parviflora and Boesenbergia pandurata

Kaempferia parviflora and Boesenbergia pandurata are perennial herbs in the Zingiberaceae family. The rhizomes of these two plants have been used as food ingredients and in Thai traditional medicine for treatment of several inflammatory-related diseases, such as gout, allergy, apthous ulcer and peptic ulcer. The compounds isolated from the rhizomes of K. parviflora and B. pandurata were, therefore, examined for their inhibitory activities against nitric oxide (NO) production. For K. parviflora, compound 5 (5-hydroxy-3,7,3′,4′-tetramethoxyflavone) exhibited the highest activity against the NO inhibitory effect, with an IC₅₀ value of 16.1 μM, followed by 4 (IC₅₀ = 24.5 μM) and 3 (IC₅₀ = 30.6 μM). Regarding the NO inhibitory activity of B. pandurata, compound 2 (panduratin A) displayed the most potent effect with an IC₅₀ value of 5.3 μM, followed by 3 (hydroxypanduratin A, IC₅₀ = 13.3 μM) and 7 (cardamonin, IC₅₀ = 24.7 μM), respectively. The 5-hydroxy-3,7,3′,4′-tetramethoxyflavone (5), panduratin A (2) and hydroxypanduratin A (3), were also tested on prostaglandin E₂ (PGE₂) and tumour necrosis factor-alpha (TNF-α) production. 5-Hydroxy-3,7,3′,4′-tetramethoxyflavone (5) exhibited a potent inhibitory effect on PGE₂ production (IC₅₀ = 16.3 μM), but a mild effect on TNF-α (IC₅₀ > 100 μM). Panduratin A and hydroxypanduratin A showed strong activity against PGE₂ with IC₅₀ values of 10.5 and 12.3 μM, respectively, and a moderate effect on TNF-α (IC₅₀ = 60.3 and 57.3 μM, respectively). This study indicated that compound 5 (5-hydroxy-3,7,3′,4′-tetramethoxyflavone) is responsible for anti-inflammatory activity of K. parviflora, while 2 (panduratin A) and 3 (hydroxypanduratin A), the prenylated chalcones, are responsible for that of B. pandurata.     

Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design

The importance of water-to-substrate ratio, protease type, percent enzyme and incubation time on hydrolysates produced from shrimp processing byproducts was investigated using Taguchi’s L₁₆ (4⁵) experimental design. Protease type significantly (p < 0.05) influenced soluble yield, degree of hydrolysis (DH), angiotensin-I-converting enzyme (ACE) inhibitory activity and bitterness of hydrolysates, while percent enzyme only affected the DH. Hydrolysates produced by Alcalase and Protamex possessed strong ACE inhibitory activity (IC₅₀ = 100–200 μg/ml and 70 μg/ml, respectively), accompanied by high yield, high DH and strong bitterness. Furthermore, ACE inhibition was positively correlated (r² = 0.87) with bitterness of the hydrolysates. Fractionation by size-exclusion chromatography revealed that the bitter substances, which also showed strong ACE inhibition, were <3 kDa in size and contained many hydrophobic residues, including Tyr, Phe, Leu, Ile, Val and Lys. Despite the bitterness, these hydrolysates may have potential health benefits, arising from their potent ACE inhibitory activity.     

Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk

Milk was fermented for up to 5 h at 43 °C with two lactic acid bacteria (Streptococcus thermophilus, Lactobacillus bulgaricus). A protease, flavourzyme, was added at the beginning of fermentation. The whey fraction was separated from the fermented milk and freeze-dried. During the 5 h of fermentation, the soluble protein content increased from 4.9 to 57.4 mg/g and peptide content increased from 2.1 to 32.8 mg/g, while inhibition of angiotensin I-converting enzyme (ACE) increased by a decrease of IC₅₀ from 0.708 to 0.266 mg/ml, respectively. The whey was fractionated into four fractions by size exclusion chromatography on a Sephadex G-15 column. The fourth fraction of the whey showed the highest inhibitory efficiency ratio (IER) being 1329%/mg/ml. The amino acid sequence of the inhibitory peptide was Tyr-Pro-Tyr-Tyr, of which the IC₅₀ was 90.9 μM. The whey showed mixed-type inhibition kinetics, while Captopril, the positive control showed competitive inhibition on ACE. Their K_i values were 0.188 mg/ml and 0.0067 μg/ml, respectively. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) was reduced to 15.9 and 15.6 mm Hg, respectively, in spontaneously hypertensive rat (SHR), after 8 weeks of oral administration of diluted whey (peptide concentration 4.9 mg/ml).     

Angiotensin converting enzyme inhibitory activity of proteolytic digests of peanut (Arachis hypogaea L.) flour

Defatted raw and roasted peanut flour were hydrolyzed with alcalase or sequentially with pepsin and pancreatin, and then the hydrolyzates were fractionated by RP-HPLC and tested for hypotensive potential. This research revealed that proteolytic peanut digests have an inhibitory effect on the activity of angiotensin converting enzyme (ACE). Three fractions from the hydrophobic end of the chromatogram of each hydrolyzate were the most potent for inhibiting ACE activity in comparison to seven other fractions. These potentially potent fractions were then assayed for IC₅₀. Fractions from the alcalase digestion of raw peanut exhibited IC₅₀ values of 8.7-122 μg/ml, and those from roasted flour exhibited values of 12-235 μg/ml. IC₅₀ values of 7.9-65.9 μg/ml, and 11-36 μg/ml for raw and roasted peanut, respectively, from the pepsin-pancreatin system were observed. These values compare to the IC₅₀ value of 0.36 μg/ml of a known commercial ACE inhibitor (pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro).     

Identification and characterization of novel angiotensin-converting enzyme inhibitors obtained from goat milk

The aim of the present study was to identify and characterize new inhibitory peptides of angiotensin I-converting enzyme (ACE) from goat milk and to analyze the effect of long-term intake of a goat milk hydrolysate-supplemented (GP-hyd) diet on the development of hypertension in spontaneously hypertensive rats (SHR). Three new inhibitory peptides for ACE (TGPIPN, SLPQ, and SQPK) were isolated. The inhibitory concentration 50% (IC₅₀) values of individual peptides were 316, 330, and 354 μmol/L, respectively. Only TGPIPN was found to pass intact a monolayer of Caco-2 cells in small amounts. The SHR fed for 12 wk a diet (GP-hyd) enriched in a hydrolysate containing these peptides (estimated intake of TGPIPN was 230 mg/kg per d) showed lower (approximately 15 mmHg) systolic blood pressure than animals fed a control diet. The ACE activities in the aorta, left ventricle, and kidney were significantly decreased in the GP-hyd group compared with those of the control group and were similar to those found in SHR fed captopril (130 mg/kg per d). Impaired endothelium-dependent relaxation to acetylcholine by aortic rings from SHR was improved in those fed the GP-hyd diet. The left ventricle weight and kidney weight index were significantly reduced in the GP-hyd group and captopril groups. Moreover, long-term treatment of SHR with a diet enriched in goat milk hydrolysate, or captopril, attenuated the development of hypertension, cardiac and renal hypertrophy, and endothelial dysfunction. These effects might be related to the in vivo inhibitory effects of the hydrolysate on tissue ACE activity.     

Comparison of analytical methods to assay inhibitors of angiotensin I-converting enzyme

The linearity, precision and repeatability of visible spectrophotometric (VSP) and high-performance liquid chromatography (HPLC) methods for analysis of inhibitory activity of angiotensin I-converting enzyme (ACE) were compared by using several inhibitors and Hip-His-Leu (HHL) as substrates. IC₅₀ values (concentration at which ACE activity is inhibited by 50%) of 0.00206 ± 0.00005 μg/mL for captopril, 192 ± 4.53 μg/mL for soybean peptides, and 153 ± 4.29 μg/mL for grass carp peptides determined by the VSP method, and these values were 1.07, 1.07, 1.18 and 1.44-fold, respectively, higher than those from the HPLC method. In addition, the inhibitory constant (K_i value) of captopril was determined to be 7.09 nM and 4.94 nM using VSP and HPLC method, respectively. These results showed that the HPLC method revealed a higher level of sensitivity and precision, suitable for assaying ACE inhibition activity of antihyper-sensitive peptides. In contrast, the VSP method can simultaneously measure several samples with simple operations, suitable for analysis of ACE inhibition activity of food protein enzymatic hydrolysates.     

Phenolic compounds in cowpea and horse gram flours in comparison to chickpea flour: Evaluation of their antioxidant and enzyme inhibitory properties associated with hyperglycemia and hypertension

Evaluation of phenolic contents and their in vitro bioactive functional properties of cowpea and horse gram flours in comparison to chickpea flour revealed that horse gram flour, which contain highest concentrations of total phenolics and total flavonoids was found to be the most active 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenger. Hydrogen peroxide scavenging capacity, reducing power and metal chelating abilities were significantly higher in cowpea and horse gram than chickpea flour. Phenolic extracts from flours showed distinct variations in the inhibition of enzymes associated with hyperglycemia and hypertension. Although, α-glucosidase inhibitory activity was superior in cowpea (IC₅₀ 52.8 μg/ml), α-amylase (IC₅₀ 96.4 μg/ml) and angiotensin-I converting enzyme (IC₅₀ 32.8 μg/ml) inhibitory activities were predominant in horse gram. These results provided useful information for effective utilisation of legume flours as ingredients in composite flours and for the development of food products with health promoting functions.     

In vitro activity of the essential oils of Origanum vulgare, Satureja montana and their main constituents in peroxynitrite-induced oxidative processes

The essential oil obtained from aerial parts of Satureja montana L. and Origanum vulgare L. (Labiatae) along with four of its main components, p-cymene, carvacrol, thymol and γ-terpinene were tested in models of in vitro peroxynitrite-induced formation of both 3-nitrotyrosine and malondialdehyde, two biomarkers of the oxidative stress of recognised pathological and toxicological significance. The essential oils showed a significant activity, thus decreasing 3-nitrotyrosine formation (IC₅₀ values of 43.9 μg/ml for S. montana and 19.2 μg/ml for O. vulgare), and also inhibited the peroxynitrite induced malondialdehyde formation (IC₅₀ values of 27.2 μg/ml and 17.0 μg/ml respectively). Thymol and carvacrol inhibited 3-nitrotyrosine formation (IC₅₀ values of 81.3 μM and 106.3 μM; ascorbic acid IC₅₀ = 400 μM) and reduced malondialdehyde formation (IC₅₀ values of 43.9 μM and 70.1 μM respectively; trolox IC₅₀ = 240 μM). On the contrary, p-cymene and γ-terpinene were completely inactive in both assays under the concentration of 300 μg/ml. These results support, in particular for origanum, the nutraceutical value of these spices and the potential of thymol and carvacrol in preventing the formation of toxic products by the action of reactive nitrogen species.