In Vitro Assessment of Antioxidant Activity, Total Phenolic and Flavonoid Contents of Sweet Marjoram (Origanum majorana L.) Extract

Sweet marjoram (Origanum majorana L.) is cultivated as a condiment for its aromatic leaves for culinary purposes and utilized as a medicinal plant for many diseases. The aim of this study was to evaluate in vitro antioxidant activity of marjoram extract by the 2, 2–diphenyl–1–picrylhydrazyl–hydrate (DPPH) free radical scavenging method while total phenolic and flavonoid contents were quantified by spectrophotometry using Folin−Ciocalteu and aluminum chloride colorimetric methods, respectively. The extraction yield of sweet marjoram obtained by maceration in absolute ethanol at a ratio of 1/5 (w/v) for 24h at room temperature was 8.41 ± 0.76 % (w/w). The obtained results showed that the investigated extract contained a higher amount of phenolics: 164.96 ± 4.61 mg GAE/g of dry plant, lower flavonoid contents: 44.61 ± 2.08 mg QE/g of dry plant, and exhibited a strong antioxidant activity (IC50 value: 40.09 μg/ml) almost like those of the used standard products, namely ascorbic acid and butylated hydroxytoluene (BHT). Based on the obtained results, marjoram (Origanum majorana L.) features a potential application as natural antioxidants that could be exploited by the pharmaceutical and food industries. Keywords— Antioxidant activity, DPPH, ethanolic extract, flavonoid contents, marjoram, phenolic


I. INTRODUCTION
In recent decades, the employment of natural antioxidants from plant sources as green chemicals has attracted major interest and has provided a potential alternative to the commonly used synthetic antioxidant molecules that present potential human health risks especially butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (Rodrigues et al., 2019;Suhaj, 2006).
Polyphenols and flavonoids are considered as natural compounds widely presents in plants ( San-Feliciano et al., 2012). They are products of the secondary plant metabolism that are involved in hormone regulation of plant growth, in protection against UV rays and microbial infections, in the attraction of pollinators and contribute to the plant pigmentation (Naczk and Shahidi, 2004). Polyphenol and flavonoid compounds arouse important interest due to their potential beneficial effects on public's health as they have demonstrated a multiple biological activities, such as antioxidant, antibacterial, antifungal, anti-spasmodic, analgesic and anti-hyperglycemic activities (Chishti et al., 2013). Their natural antioxidant activities are attracting increasing interest widely exploited in food and pharmaceutical industries (Chen et al., 2004). In food industry, phenolic compounds are used as additives and contribute particularly to slowing the oxidative degradation of lipids, thus improving the microbial and nutritional quality of food (Jukic et al., 2015). In addition, their presence in human diet is associated with beneficial Open Access pharmacological effects that reduce the risk of various chronicle diseases related to the oxidative stress (Zhou et al., 2006).
Origanum majorana L. also called sweet marjoram is a species of perennial plant in the Lamiaceae family, indigenous from Mediterranean and cultivated as a condiment for its aromatic leaves for culinary purposes (Sud and Kumar, 2004). Marjoram is also used as a medicinal plant for various diseases because it contains high amount of phenolic compounds such as carvacrol and thymol (Burt, 2004). Various authors have previously studied the effects of sweet marjoram extracts from different origin and demonstrated their antioxidant The present study is part of the context of exploiting and enhancing the biodiversity of aromatic plants for their natural properties. The aim consisted in assessing the total phenolic contents and the concentration of flavonoids, as well as to evaluate the antioxidant activity of the ethanolic sweet marjoram extract (Origanum majorana L.).

Plant material
Fresh marjoram was purchased from local market in April 2019. The plants were botanically identified as Origanum majorana L. Aerial parts of Marjoram were dried at room temperature (20 °C) in the dark to preserve their molecular integrity. The plant materials were individually ground into a fine powder (500 µm, ISO, 1999).

Preparation of plant extract
Extraction was made in line with the method reported by Bensid et al. (2014) with slight modifications. Sample was macerated at room temperature in absolute ethanol in a ratio of 1:5 for 24h. Extract was filtered over Whatman No. 1 filter paper. Activated carbon was appended to filtrate (20 g of activated carbon / 100 g of plant material) and was immediately removed by filtration. After that, all ethanol was evaporated under reduced pressure using a Büchi Rotavapor R-200 at 50 °C. Then, extract was stored in amber flasks at refrigerated conditions until use.

Determination of total phenolic compounds
Total phenolic contents (TPC) were evaluated using the spectrophotometric analysis with Folin−Ciocalteu's phenol reagent (Waterhouse, 2002). Briefly, 100 µL of appropriately diluted sample was added to 400 µL of a 1:10 diluted Folin−Ciocalteu's reagent. After 5 min, 500 µL of a saturated sodium carbonate (Na2CO3) solution and 1.5 mL of ultrapure water were added. The mixture was incubated in dark at room temperature for 2 h and the absorbance was determined at 765 nm against water blank on spectrophotometer. Calibration curve was made using gallic acid standard solution (100, 80, 60, 40 and 20 µg/mL) under the same procedure as above and results are expressed as milligram of gallic acid equivalents (GAE) per g of dry Plant.

Determination of flavonoid contents
Total flavonoids were determined using a colorimetric assay (Kim and lee, 2004). A 500 µL aliquot of appropriately diluted sample was put into a volumetric flask containing 2 mL of ultrapure water. At zero time, 150 µL of 5 % sodium nitrite (NaNO2) was added to the flask. At 5 min, 150 µL of 10 % aluminum chloride (AlCl3) was added. At 11 min, 1 mL of 1M sodium hydroxide (NaOH) was appended into the mixture. Forthwith, the contents of the reaction flask were diluted with 1.2 mL of ultrapure water and carefully blended. Mixture absorbance was read at 510 nm against water blank. Calibration curve for flavonoids was set using quercetin standard solution (100, 80, 60, 40 and 20 µg/mL) under the same procedure as above and results are expressed as milligram of quercetin equivalents (QE) per g of dry Plant.

Statistical analysis
Average values and standard deviations were obtained from triplicate data. One-way ANOVA followed by Duncan's post-hoc was performed to compare differences of the data at P value < 0.05.

III. RESULTS AND DISCUSSION
Ethanolic extracts were prepared to examine the total phenolic compounds, flavonoid contents and antioxidant activity. The extraction yield of phenolic compounds obtained from plant material was determined to be 8. The concentration of flavonoids present in the Marjoram extract was determined using spectrophotometric method with aluminum chloride. The flavonoid contents were calculated using the equation: y = 0.013x -0.053, r 2 = 0.998 extracted from standard curve of quercetin. The concentration of flavonoids was found to be 44.61 ± 2.08 mg QE/g of dry plant, which was very similar to the value reported by Gawlic-Dziki (2012).
The recorded differences in the extraction yield and in the contents of polyphenol compounds (total phenolics and flavonoids) contained in the studied plant extract might be due on one hand to extrinsic factors related to the plant such as; the origin, plant species and considered organ (Valnet, 1980). Indeed, Smallfield (2001) and Bruneton (1993) report that environmental and climatic conditions, the stage and harvest period, the vegetative cycle and/or the techniques and time of conservation can influence the extraction yields and the contents of compounds. On the other hand, due to factors related to the extraction procedures used such as; particle size, choice and concentration of solvent, exhaustion, temperature, pressure, time, ratio and / or extraction methods (maceration, decoction, infusion, hydro distillation, etc.) ( . Therefore, the assay carried out by this reagent represents a crude evaluation of all hydroxyl groups contained in the extract because it is not specific to phenolic, but many compounds can react with this reagent, resulting in high values (Tawaha et al., 2007). For this reason, High Performance Liquid Chromatography (HPLC) analysis is interesting for the determination of the individual phenolic constituents in the plant extract.
The antioxidant activity of ethanolic plant extract from marjoram was evaluated using a methanol solution of DPPH reagent and was compared to that of natural and synthetic antioxidants currently used in therapy and food industry, namely; ascorbic acid and BHT, respectively. The antioxidant activity of all samples: the studied plant extract, natural and synthetic antioxidants (ascorbic acid and BHT) was determined using a spectrophotometer by following the transition of color from purple to yellow once the reduction of DPPH free radical (Majhenic et al., 2007). All the samples showed concentration-dependent increases significantly (P < 0.05) in free radical scavenging capacity (Fig. 1). In all experiment, free radical scavenging Open Access activity of ascorbic acid was significantly different (P < 0.05) from that of BHT and ME which they showed no significant difference (P > 0.05). Ascorbic acid had relatively high free radical scavenging activity with 93.34 % at 0.1 mg/mL while BHT and marjoram extract showed values of 76.32 % and 72.53 % respectively at the same concentration. Our extract has an important antioxidant power which can replace synthetic antioxidant because it presents a potential free radical scavenging activity similar to those observed for the used reference standard antioxidant products. This potential free radical scavenging ability can be attributed to the active hydrogen donor ability of hydroxyl substitution due probably to the high level of phenolic compounds containing in extract (Siddhuraju, 2007). The results showed that ascorbic acid had the most powerful antioxidant activity (P < 0.05). However, natural extract obtained in our study (marjoram) and BHT exhibited similar anti-radical activities (P > 0.05), thus claiming that our extract had a significant antioxidant activity which can replace the synthetic antioxidant (BHT).

IV. CONCLUSION
Based upon the obtained results in the present study, we can conclude that ethanolic extract of marjoram (Origanum majorana L.) contains a considerable amount of phenols (phenolic and flavonoid contents), exhibits a strong antioxidant power and a potential free radical scavenging ability that can replace the synthetic antioxidant (BHT). These indicate that marjoram represents a significant source of natural antioxidants which might be helpful in the pharmaceutical and food industries. Nevertheless, further studies are required to determine and quantify the individual phenolic constituents containing in the extract.