INTRODUCTION:

Oxidative stress indicates to an imbalance between the production of reactive oxygen species (ROS) and the body's capacity to effectively neutralize and detoxify them. This imbalance can lead to the oxidation of biological molecules such as oxidizable lipids, proteins, and DNA, which can contribute to the evolution of acute and chronic diseases¹. To combat oxidative stress, the body has various mechanisms, such as producing its own antioxidants or obtaining them from a healthy diet rich in fruits and vegetables. Adequate intake of antioxidants through diet and/or supplements can help protect the body against the harmful effects of oxidative stress and may also have therapeutic benefits for the prevention and treatment of various maladies².

The exposure of the skin to ultraviolet (UV) radiation induces the generation of reactive oxygen species and reactive nitrogen species that can damage cells and increase the risk possibility of skin cancer^3,4. To protect against these harmful effects, topical sunscreens are used to block and absorb UV rays, which can help reduce the danger of skin cancer and retard photo-aging ⁵. In recent years, there has been a growing increase interest in replacing synthetic sunscreens with compounds derived from natural plants as alternatives to synthetic sunscreens due to their potential antioxidant, anti-inflammatory, and immuno-modulatory effects. These natural compounds may provide additional protection against the bad effects of UV exposure⁶, and the use of plants as a origin of UV-absorbing compounds could lead to the development of more effective and affordable sunscreen formulations.

Preventing diseases and finding new therapeutic agents is a priority for many people, increasing interest in natural antioxidants as alternatives to synthetic antioxidants. Synthetic antioxidants can sometimes have undesirable side effects and can cause permanent damage to visceral organs, making natural antioxidants a safer and more appealing option. Herbal treatments are coming back to the fore. Algeria has a diverse range of bioclimatic conditions, which contribute to its rich vegetal biodiversity and make it a potential source of natural antioxidants.

The oregano "Origanum vulgare" is a species of the family Lamiaceae that is commonly found in the Mediterranean diet and is used in traditional medicine in Algeria and Tunisia to treat cough, fever, respiratory diseases, and rheumatic pain. Previous research has shown that Origanum vulgare has several bioactive properties, including antiviral⁷, antimicrobial⁸, anti-inflammatory⁹, and antioxidant¹⁰ properties. In this context, this study aims to conduct a phytochemical analysis of Origanum vulgare and to investigate its potential antioxidant, and dermato-protective properties using in vitro methods.

MATERIALS AND METHODS:

Plant material:

Origanum vulgare was collected in April 2019 from the northeast of Algeria Skikda and identified at the botanical laboratory of Annaba University. The aerial part of the plant was dried in a dry place and protected from sunlight. Between 25 and 28C° for 3 weeks. Then crushed to give a powder from which the extracts were prepared.

Preparation of the extracts:

To extract the bioactive compounds from Origanum vulgare, 500g of the aerial part of the plant in powder underwent a maceration three times in a methanolic solution (80%) at room temperature protected from the light, to prevent oxidation. The residue was then filtered, concentrated, and successively extracted using four solvents of increasing polarity consecutively by petroleum ether, dichloromethane, ethyl acetate, and n-butanol.

Phytochemical screening:

a) Determination of total polyphenol content (TFC):

The total polyphenol content is determined by the Folin-Ciocalteu test¹¹, which involves mixing a volume of 20 μl of extract (1mg/ml) with 100μl of the solution of Folin-Ciolcalteu reagent (FCR) diluted with distilled water (1:10v ̸v) with the addition of 75μl of sodium carbonate (7.5% Na₂CO₃). The mixture was left in the dark for 120minutes at room temperature, and then the reading was obtained at 765nm. Gallic acid was the standard used to establish the calibration curve under the same conditions as the samples, from which the total phenol concentration of the extracts was calculated. The result is expressed as micrograms of gallic acid equivalents per milligram of extract (μgGAE/mg extract).

b) Determination of flavonol content:

The total flavonol content was determined by the aluminum trichloride (AlCl₃) colorimetric method reported by Kumaran A¹². Indeed, in a microplate reader, 50μl of the plant extract (1mg/mL) was thoroughly mixed with 50µl of aluminum trichloride (AlCl₃). Followed by the addition of 150µl of sodium acetate and incubated in the dark at room temperature for two and a half hours. The absorbance was then measured at 440nm and compared to a Quercetin calibration curve (0-200μg/mL). The results are expressed in microgram quercetin equivalents per milligram of extract (mg QE/mg dry extract).

Antioxidant activity:

In this study, the antioxidant activity of the Origanum vulgare extracts was determined in vitro using several different methods. These methods included the trapping of free radical α,α-diphenyl-1-picrylhydrazyl (DPPH), Reducing power assay, Cupric reducing antioxidant capacity (CUPRAC) and β-carotene bleaching assay. These methods are used to evaluate the ability of the extracts to neutralize free radicals and protect against oxidative damage.

a) DPPH free radical scavenging activity:

The DPPH assay is a widely used method for evaluating the antioxidant activity of different compounds. It was initially described by Blois MS¹³, and then has been extensively modified by many researchers. The stock solutions of the extract were prepared in methanol at a concentration of 800μg/ml. A series of dilutions in geometric progression was prepared at a ratio of 2. A volume of 40μl of these diluted extract solutions, at different concentrations, was added to 160μl of the 0.4 mM DPPH solution. The reaction mixture was shaken vigorously and, then incubated for 30 min in the dark at room temperature. The absorbance values were measured at 517nm. In parallel, standard antioxidant solutions, including BHT, BHA, α-tocopherol, and ascorbic acid, were used as positive controls and their absorbance was measured under the same conditions as the test samples. Each concentration tested was performed in triplicate¹⁴. The DPPH radical scavenging activity was estimated as the percentage of DPPH solution decolorization, which is the amount of an antioxidant required to decrease the initial amount of DPPH by 50% (IC50). The percentage reduction of the DPPH free radical is expressed by the following formula:

% Inhibition = [(Abs c -Abs t)/Abs c] x 100

Abs c: Absorbance of the control,

Abs t: Absorbance of the tested sample.

b) Reducing power assay:

The chelation activity of metal ions in extracts by the ferrous ion Fe⁺² was measured according to the method described by Oyaizu¹⁵. A volume of 10μl of the extracts at different concentrations, 40μl of phosphate buffer (0.2 M, pH 6.6), and 50μl of 1% potassium ferricyanide K3Fe (CN)6 solution were mixed and incubated at 50°C for 20 minutes. After that, 50μl of trichloroacetic acid (TCA) (10%) was added to the mixture for stopping the reaction. Then, 40μl H₂O and 10μl of ferric chloride FeCl3 (0.1%) were added to the mix. The absorbance of the reaction mixture was measured at 700nm and compared to standard antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), Ascorbic acid, and α-Tocopherol were used as standard antioxidants. The antioxidant activity of the extracts was expressed as an effective concentration (μg/ml) required to reduce power by 0.5 absorbance units, which is obtained from linear regression analysis.

c) Cupric reducing antioxidant capacity (CUPRAC):

The cupric ion-reducing antioxidant capacity (CUPRAC) assay is used to measure the ability of extracts of Origanum vulgare to reduce copper ions. This assay is based on tracking the diminution of the increased absorbance of the neocupronin (NC), copper (Cu⁺²) Nc2-Cu⁺² complex¹⁶. In a 96-well microplate, 60 μl of ammonium acetate NH₄Ac (1 M, pH = 7.0) and 50 μl of neocupronin (7.5mM), and 50μl of (Cu Cl₂, 2H₂O) (10mM) were added to 40µl of the extract at different concentrations. After 1h in the dark, the absorbance was measured at 450nm. The results were given as absorbance calculated as A0.5(μg/ml) corresponding to the concentration indicating 0.50 absorbance, and compared with those of BHA and BHT used as antioxidant standards.

d) Bleaching activity of β-carotene:

The β-carotene bleaching activity of the plant extracts was estimated by measuring the inhibition of the oxidative degradation of β-carotene by linoleic acid oxidation products according to the method described by Marco GJ¹⁷. The β-carotene/linoleic acid emulsion was prepared by solubilizing 0.5mg of β-carotene dissolved in 1ml of chloroform then adding to a 25μl volume of linoleic acid and 200mg of Tween 40, obtaining an emulsifying mixture. After the evaporation of chloroform under vacuum, 100ml of oxygen-saturated distilled water (H₂O₂) was added with vigorous agitation. The absorbance of the β-carotene solution should be between 0.8 and 0.9nm. (If it is found higher than 1.2, oxygenated water H₂O₂ is added). A 160μl volume of a prepared solution was mixed with a 40μl volume of extract at various concentrations, with methanol serving as a negative control. The same procedure was repeated with BHT and BHA as positive control. The kinetics of the activity was measured at 470 nm using a microplate reader at intervals of regular 30 min during 2h of incubation at room temperature in the dark. The antioxidant activity was expressed as a percentage and calculated by the following equation:

AA (%) = [1- (AH0 -AHt)/(AC0 - ACt)] × 100

AA (%): Antioxidant activity

AH0: absorbance value of β-carotene in the presence of the extract measured at t=0.

AC0: absorbance value of β-carotene in the presence of negative control measured at t=0.

AHt: absorbance value of β-carotene in the presence of the extract measured at t=120 min.

ACt: absorbance value of β-carotene in the presence of negative control measured at t=120 min.

Determination of the sun protection factor (SPF) in vitro:

The sun protection factor (SPF) is determined in vitro by the method of Mansur¹⁸. The plant extracts were dissolved in methanol. The absorbance is measured in the range of 290 to 320nm every 5nm (UV-B), and the SPF value is calculated by applying the mathematical equation of Mansur (1986).

SPF _{spectrophotometric}=

CF х

EE: erythemal effect spectrum

I: solar intensity spectrum

Abs: absorbance of sunscreen product

CF: correction factor (= 10)

Statistical analysis:

In the analysis of the results of the study, statistical analyses were realized using SPSS 23 software. For this, we performed an analysis of variance ANOVA, with one factor, followed by the Tukey test for multiple comparisons and determination of significance levels. The means were considered significantly different for a P value of less than 0.05.

RESULTS AND DISCUSSION:

Bioactive compounds (total phenolic content and flavonol)

Various solvents were used to realize the extraction of the active substances with diversity in their polarity. The results of the total phenolic and flavonol contents of the extracts presented in Table 1 were determined in petroleum ether, dichloromethane, ethyl acetate, and butanol extracts of Origanum vulgare.

Table 1: Total phenolic content and flavonol content of Origanum vulgare extracts.

extract	Total phenolic content (µg GAE/mg extract)	Flavonol content (µg QE/mg extract)
Petroleum ether extract	103,65±3,67	58,61±0,58
Dichloromethane extract	157,03±3,56	83,73±2,89
Ethyl acetate extract	195,12±3,84	57,96±1,99
n-Butanol extract	226,88±0,29	34,34±0,43

The results are expressed as means±SD of three measurements.

Micrograms of gallic acid equivalents per milligram of extract for total phenolic content.

Micrograms of quercetin equivalents per milligram of extract for flavonol.

The total phenol content (TPC) of Origanum vulgare extracts was estimated using the Folin-Ciocalteu method. The results (Table 1) revealed that all four extracts were rich in polyphenols (>100μg GAE/mg extract). The n-butanol extract composed the most content (226.88±0.29μg GAE/mg) as followed by the ethyl acetate extract with 195.12±3.84μg GAE/mg. A considerable content was determined in the dichloromethane extract (157.03±3.56μg GAE/mg) followed by the petroleum ether extract (103.65±3.67μg GAE/mg). Contrary to the total phenol content, the flavonol content of the dichloromethane extract was important with a value of 83.73±2.89μg EEQ/mg, compared with the petroleum ether and ethyl acetate extracts (57.96±1.99 and 58.61±0.58μg EEQ/mg), respectively. The minimum content was recorded by the n-butanol fraction (34.34±0.43μg QE/mg). The obtained results are coherent with those of Kaurinovic B¹⁹ who found that the highest content of phenolic compounds was recorded in the ethyl acetate extract, followed by the n-butanol extract. This is in accordance with the previously reported by Ličina who measured a very high content of total phenols (166mg GAE/g extract) in the ethyl acetate extract²⁰. A study by Babbar and his collaborators revealed that ethyl acetate was superior to dichloromethane in the extraction of phenolic compounds and that the polarity of the extraction solvent could influence the TPC of the extracts²¹.

Phenolic content, as previously reported by Benchikha N and Tusevski O, was found to be high in methanolic extracts, with values of TPC = 194.78±1.49 and 123.41 ±8.77μg GAE/mg extract. These results suggest that these extracts can be used as a potentially rich source of phenolic contents and other phytochemicals in medicine, pharmacy, cosmetics, and the food industry^22,23.

Antioxidant capacity:

Oxidative stress occurs under conditions of imbalance in the ratio of oxidants to antioxidants and is related to many serious health disorders and diseases. The organism needs to obtain its antioxidants from food and other complements. Several research works have reported that dietary antioxidants, which come from a wide range of sources and have diverse biological and pharmacological effects, may be useful as adjunctive therapy in certain cases. In addition, natural substances with antioxidant properties may be preferable to synthetic alternatives due to their lack of side effects²⁴. In this context, and in order to study the phytochemicals and biological activity of plant extracts, a range of tests are used to analyze their composition, which act through a variety of systems such as avoidance of chain initiation, conversion metal ion catalyst binding, peroxide decomposition, prevention of continuous hydrogen abstraction, reduction capacity, and radical scavenging. The antioxidant activity of different Origanum vulgare extracts was measured using four different methods: DPPH radical scavenging, reducing power assay, CUPRAC assay, and β-carotene bleaching. These results were correlated to those of standards in order to better understand the extracts' antioxidant potential and are summarized in Table 2.

In this study, the methods chosen for evaluating the antioxidant potential of oregano extracts were selected for their efficiency in quickly identifying and measuring this activity. The outcome of the tests demonstrated that the extracts generally had significant antioxidant activity at various levels, especially the ethyl acetate and n-butanol extracts. The values of IC50 and A0,5 are different depending on the extract.

The results of the assays showed that all extracts had antioxidant activity, with the potency increasing with the increase of the concentration of the extracts from 1.5625 and 800μg/mL. The low IC50 and A0.5 values indicate a high antioxidant activity.

The DPPH assay is a common method for identifying molecules with antioxidant activities present in plant extracts. The mechanisms involved in the reduction of these radicals are based on the capacity of certain compounds to give hydrogen, and the data collected and analyzed (presented in Table 2) show that the extracts are effective at scavenging 2,2-diphenyl-1- picrylhydrazyl (DPPH) free radicals compared to the standard. The extracts of ethyl acetate (EA) and n-butanol were found to have a high level of inhibition with IC50 equal to 11.35±0.34 and 13.15±0.44μg/mL, respectively. These values are similar to that of α-Tocopherol and BHT at the same concentration (13, 02 ±5.17 and 12.99±0.41μg/mL). There was no significant difference between the EA and n-butanol extracts and the standards (P<0.05). The dichloromethane extract also showed a good antioxidant activity, with an IC50 of 21.32±0.45μg/mL, but this was significantly different from the standards. Although the petroleum ether fraction had considerable activity, no extract showed better antioxidant properties than BHA. These results are consistent with those of Pezzani R and Kaurinovic B ^9,19 who found that the n-butanol extract had the best antioxidant capacity, followed by the ethyl acetate extract (12.91 and 14.11μg/mL). However, our results differ from those of Ličina BZ²⁰ who found that the ethyl acetate extract had an IC50=56μg/ml, but are similar to those of Krimat S²⁵, who found that hydro-methanolic extract induced high inhibition with an IC50 = 12.8±0.2μg/ml. According to Tusevski "The DPPH values of Origanum vulgare extracts were significantly lower than those of reference compounds, These results suggest that Origanum vulgare extracts contain different classes of phenolic antioxidant compounds with high hydrogen donating capacity to scavenge DPPH radicals"²³.

To better explain the mode of antioxidant action, the reducing power and CUPRAC tests were employed to appraise the capacity of 'extracts to reduce iron and copper ions, respectively. The reducing power of antioxidants is the capacity of an extract to reduce ferric iron (Fe⁺³) to ferrous iron (Fe⁺²), which is a low value of A0.5 representing the highest iron-reducing power. The best reducing power was obtained by ethyl acetate (EA) extract A0.5 equal to 6.69±0.48μg/ml. This value is lower than that of reference antioxidants; ascorbic acid and BHA A0.5 is equal to 6.77±1.15 and 8.41±0.67 with a non-significant difference as well as α-Tocopherol and BHT (A0.5 equal to 34.93±2.38 and >50μg/ml), respectively with a significant difference. Thus, n-butanol and Dichloromethane extracts showed a very good reduction with A0.5 equal to 13.39±0.98 and 15.03±1.02μg/mL compared to Tocopherol and BHT. while petroleum ether extract recorded the lowest power. It has been found that the extracts of Origanum vulgare can effectively respond with free radicals to transform them into more stable radicals. Several research works on Origanum vulgare extracts confirm our result. This has been confirmed by previous studies, including Tusevski O and Martins N, who found that hydromethanolic extracts also had good iron reducing power; Tusevski O (1108.82±18.72μmol AAE/g DW) and Martins N (EC50= 237.45±8.51). Even in the study of Oniga I for ethanolic extract (794.40±25.80μ M TE/g) ^10,23,26. These findings suggest that Origanum vulgare extracts may be effective at neutralizing free radicals and potentially providing antioxidant effects.

However, the reduction of Cu²⁺ ions was evaluated by Cupric reducing antioxidant capacity (CUPRAC). This test is colorimetric and the reagent changes from blue green to yellow in the presence of an antioxidant. This is evident in our results that exposed that the ethyl acetate extract had a great activity, with an A0.5 value of 6.06±0.31μg/mL, which was not significantly different from the known potent BHT (9.62±0.87μg/mL), and more below than BHA (3.64±0.19μg/mL). Moreover, the two extracts n-butanol and Dichloromethane revealed a good activity with A0.5 =13.35±0.10 and 13.01±0.99μg/mL respectively, which were close to the BHT. This is in good agreement with the results of Oniga and Tusevski showing that Origanum vulgare extracts exhibited a very high CUPRAC activity (>1000 μmol TR/g DW)^10,23. The results of the CUPRAC test may be particularly relevant for understanding the potential antioxidant effects of Origanum vulgare extracts in vivo, as CUPRAC reaction is realized at pH 7.0, comparative to the physiological pH¹⁶.

The β-carotene assay is largely used to test the antioxidant capability of 'extracts. The obtained results approved that Dichloromethane extract has a moderate antioxidant activity (IC50=14.77±1.13μM) followed by petroleum ether extract which showed some low activity (IC50=94.25±2.67μg/mL). In contrast, the ethyl acetate and n-butanol extracts had a very low activity;IC50 < 800. These findings were significantly distinctive from those of the standards and still far from those of the standard antioxidants, including BHT and BHA, which had an IC50 value of 0.91±0.01, 1.05±0.03μg/mL, which are consistent with the study by Natalia. The latter reported a low inhibitory bleaching activity of b-carotene (EC50=371.45±12.40μg/mL) with the hydroalcoholic extract²⁶.

Table 3: Sun Protection Factor (SPF) of the different extracts.

Extracts

FPS ± SD

Petroleum ether extract

Dichloromethane extract

Ethyl acetate extract

N-butanol extract

22.51±0.59

47.29±0.00

47.30±0.00

47.21±0.14

The photoprotective effect was evaluated by measuring the sun protection factor (SPF), the four extracts. Indeed, the dichloromethane, ethyl acetate, and butanol were found to have an excellent ability to absorb UV radiation, providing high protection with estimated sun protection factors of 47.29±0.00, 47.30±0.00 and 47.21±0.14, respectively. These values are considered to be excellent filters against UV radiation, according to the latest Australian regulatory guidelines for sunscreens, which classify products as below (SPF 4 - 10), intermediate (SPF 15 - 25), high (SPF 30 - 50), or very high (SPF 50+)²⁷. The chemical composition of the extracts, including flavonoids and phenolic compounds, was found to be effective sunscreens with significant photoprotective properties⁶.

CONCLUSION:

Oregano, a medicinal plant known as Origanum vulgare, has long been used to treat a range of maladies . Extracts of the plant's aerial parts, obtained through the use of various solvents, were found to consist of high levels of phenolic compounds and flavonols, particularly when extracted using butanol and ethyl acetate. These extracts also exhibited strongest antioxidant activity, surpassing certain standards in some cases. These results have confirmed the interesting potential of oregano as a natural surrogate source to fabricate antioxidants. In addition, oregano was found to have photoprotective properties, as demonstrated by its high sun protection factor (SPF). These findings suggest that oregano has significant potential for use in the medical, cosmetic, and health fields.

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Received on 19.05.2023 Modified on 27.07.2023

Research J. Pharm. and Tech 2023; 16(12):5631-5636.

DOI: 10.52711/0974-360X.2023.00910

Tests Extrait	DPPH IC50 (μg/mL)	Reducing power A0.5 (μg/mL)	CUPRAC A0.5 (μg/mL)	β-carotene bleaching IC50 (μg/mL)
Petroleum ether extract	117,16±1,34	150,08±1,92	39,07±2,73	94,25±2,67
Dichloromethane extract	21.32±0.45	15,03±1,02^b	13,01±0,99^b	14,77±1,13
Ethyl acetate extract	11,35±0,34ª	6,69±0,48ª	6,06±0,31ª	>800
n-butanol extract	13.15±0,44^b	13,39±0,98^b	13,35±0,10^b	>800
BHT*	12.99±0.41ª^b	>50	9.62±0.87ª	0.91±0.01ª
BHA*	6.14±0.41	8.41±0.67ª	3.64±0.19ª	1.05±0.03ª
α-Tocopherol*	13.02±5,17^b	34.93±2.38	NT	NT
Ascorbic acid*	NT	6.77±1.15ª	NT	NT