Phytochemicals screening, cytotoxicity and antioxidant activity of the Origanum majorana growing in Casablanca, Morocco

Origanum majorana L. is a medicinal plant of the Lamiaceae family, knowing as Zaatar in traditional Moroccan medicine [2]. This plant is distributed around the Mediterranean regions, Asia, and North Africa, in particular, Morocco, Algeria, Egypt, Spain, and Portugal [3]. Origanum majorana showed various biological activities such as allergies, fever, hypertension [4,5], respiratory infections [6], antidiabetic [7], painful menstruation, Kidney Yang defi ciency, stomach ache, cough [8], rheumatism, headache, insomnia [9], also in intestinal antispasmodic [10]. Moreover, Origanum majorana L. exhibits a wide effect spectrum with antioxidant, antibacterial, antifungal, nephroprotective, anti-proliferative, anti-cancer activities [11-16]. These effects are mediated by the presence of bioactive compounds such as thymol, carvacrol, tannins, hydroquinone, sitosterol, cis-sabinene hydrate, limonene, terpinene, camphene, and fl avonoids like diosmetin, quercetin, luteolin, and apigenin [17,18].

Citation: Ennaji  to the development of antibiotic resistance and without a doubt, the incidence of multidrug-resistant pathogens is considered a major disadvantage in medication strategy, which has led attention towards innovative antibiotic sources. On the other hand, the plant had a therapeutic potential against drug-resistant microbial infections [19]. The herbal made four major groups of antimicrobial compounds like phenolics and polyphenols, terpenoids and essential oils, lectins and polypeptides, and alkaloids, those bioactive plant extracts could yield an enhanced effect (Cowan,1999). A lot of mechanisms against the bacteria were used via these compounds, including inactivation of proteins, adhesins, and enzymes, among other targets.
Some recent research revealed that certain molecules in the plant could also block cell-to-cell signaling pathways, extinguish the production of virulence factors and disrupt or inhibit the formation of biofi lms, which bestow a protective advantage to pathogens during infection [20]. control [10] The purpose of the present study was to characterize the fl avonoid, polyphenols, and tannins content, antioxidant properties, and cytotoxic activity of O. majorana. From the moisture content we can determine the dry matter content which is calculated by the following formula:

Plant material and extract preparation
Dry matter content (%) = 100 -Humidity

Preliminary qualitative phytochemicals screening
The extract of the plant material was subjected to phytochemical screening to qualitatively determine some types of interested phyto-organic constituents which are responsible for biological activities, alkaloids, polyphenols, quinones, saponins, tannins, sterols, carbohydrates, glycoside cardiac, triterpenes, and terpenes which were the major cheeked groups using standard methods.

Determination of total phenolic content
Folin-Ciocalteu reagent method described by Zhishen, et al. 1999 [22] was used to detect total phenolic content (TPC) present in the hydro-ethanolic extract of O. majorana. A volume of 1.5 mL of 10% Folin-Ciocalteu reagent was added to 0.5 ml of extract and mixed for 5 min. Then, 3 mL of sodium carbonate solution 7.5% (Na 2 CO 3 ) was added and further incubated at 30 0 C for 2 h. Finally, the absorbance was calculated at 760 nm using a UV-visible spectrophotometer against a blank composed of the same previously reagents except for the extract. The quantity of total phenolic compounds was detected from the standard curve of gallic acid and expressed in mg gallic acid equivalent (GAE) per g of the dry weight of extract (mg GAE/g DW).

Determination of total fl avonoid content
Aluminum Chloride (AlCl 3 ) colorimetric method is described by Dewanto, et al. 2002 [23]. Was used to detect the Total Flavonoids Content (TFC). Briefl y, one mL of 2% AlCl 3 solution was added to 1 mL of the hydro-ethanolic extract of O. majorana and incubated for 30 min. The absorbance was measured at 420 nm and the fl avonoid content was detected from a quercetin standard curve and expressed in mg quercetin equivalents per g of the dry weight of extract (mg QuE/g DW).

Determination of condensed tannins content
Condensed Tannins Content (CTC) was determined according to the method of Sun, et al. 1998. An aliquot of the extract with different concentrations (0.5-2-4mg/mL) was mixed with 3mL of vanillin (4%) and 1.5 mL of sulfuric acid concentred. After homogenization, the tubes are incubated in darkness at ambient temperature for 15min.
Then, the absorbance was measured at 500nm. The

Antioxidant activity
The DPPH free radical scavenging activity to hydroethanolic extract of O. majorana was quantifi ed according to the method of Wu, et al. 2019 [24], with some modifi cations.
Absorbance was recorded at 517 nm. The scavenging activity was calculated using the formula: Where A 0 and A E are the absorbance of the control and extract after 30 min, respectively.

PBMCs isolation and cell culture
The whole blood was collected from healthy adults informed with written consent. The PBMCs were isolated using Ficoll-

MTT assay
Viability and proliferation were measured by the MTT (3- test [25] in each well and compared to that of the untreated group. After incubation, 20μL (5mg/mL) (Sigma, USA) was added to each well and the plates were incubated 4h at 37°C under 5% CO2 and 95% humidity.
Thereafter, the supernatant was removed from each well after centrifugation, and 100μL DMSO was added to dissolve the formazan crystals produced following the viable cells able to metabolize MTT salts. The extent of the formazan product was measured at 492nm using FLUO star Omega microplate reader.
Cell viability was calculated using the following formula: Cell viability (%) = (mean of optical density of treated cells /mean of optical density of negative control cells) x100 And cell proliferation was calculated using the following formula:

Statistical analysis
The statistical signifi cance between the mean values was performed by an independent t-test. using SPSS statistical software version (15.0.1) (Chicago, IL) to determine the difference between groups. All the [* p < 0.05; ** p < 0.01; *** p < 0.001] were considered signifi cant.

Ethics
The study was assessed and approved by the Faculty of Medicine and Pharmacy, Hassan II University, Ethics Committee, following the declaration of Helsinki. Consents were obtained for the collection of PBMCs samples.

Extracts yields and humidity
The yield extracted from O. majorana by four solvents namely, hydro-methanol, hydro-ethanol, diethyl ether, and dichloromethane is presented in Figure 1. The highest extract yield was obtained by hydro-methanol extraction followed by hydro-ethanol, dichloromethane, and fi nally by diethyl ether. The yields of both hydro-methanol and hydro-ethanol were 9.98 and 10 % respectively. Diethyl ether showed as a solvent was lower than the effi ciencies of all other solvents. terpenoids, sterols, and triterpenoids were found in moderate concentration while quinone and glycosides cardiac was not found. Although, the alkaloids were present by the Mayer test but not by Dragendorff ( Table 1).

Determination of total polyphenols, fl avonoids, and tannins content
Based on the absorbance values of the hydro-ethanolic extract solution, which reacts with the Folin-Ciocalteu reagent and compared to standard solutions of gallic acid equivalents (y=0.0064x -0.1213, R 2 =0.9288) a polyphenol content of (3.87 to 5.15 mg GAE/g DW) was found ( Table 2).
The result of fl avonoids content revealed their presence of a concentration between 3.02 to 5.62mg QuE/g DW this result was differentiated with a standard curve of quercetin. The fl avonoid contents were calculated using the equation y=0.0352x+0.0273, R 2 =0.9889.
While the number of condensed tannins varied by increasing concentration in the hydro-ethanolic extract of O. majorana and ranged from 1.9 to 4.4 mg CE/g of dry material ( Table 2). Catechin was used as a standard compound and the condensed tannins content were expressed as mg/ml catechin equivalent using the standard curve equation: y = 0.003x + 0.021, (R2= 0.995).

Antioxidant activity
The radical Scavenging activity of hydro-ethanolic extract of O. majorana was examined using the DPPH radical at different concentrations. Scavenging activity against the DPPH radical was concentration dependant. The IC 50 , the concentration at which 50% of DPPH molecules are inhibited, is obtained from the point of intersection of the scavenging activity and the antioxidant activity curves (Figure 2). IC 50 corresponds to 2.308 mg/mL of O. majorana. The maximum scavenging activity of the hydro-ethanolic extract was 53.068% at 4 mg/mL ( Table  2).

Discussion
Recently, medicinal plants are known for their various biological activities to cure humans against many diseases.
The extraction method adopted is successive exhaustion Our results are a little high with those of Shahidi, et al.
2018 [28] who stated that the moisture content of O.majorana is between 6.5 and 8%. Numerous studies have shown that the humidity level is only relative can be related to environmental factors such as climatic conditions, geographical origin as well as the conditions, methods, and applied drying time.  [29] and similar to those of Bhardwaj, et al. [30].
However, Vasudeva reported the absence of alkaloids and glycosides in this species, which is comparable to our results [31]. Another study carried out on the methanolic extract of O. majorana in Yemen confi rms the results of our work by confi rming the absence of alkaloids and the presence of other chemical compounds [32]. Contrariwise, the literature has shown the existence of various alkaloid molecules while they were not detected in our plant sample. This disagreement on the chemical composition of the same species can be justifi ed by differences in geographical location and extraction method [33].
The choice of using hydro-ethanol extract was because the ethanol was a better solvent than the others in extracting phenolic compounds from the extracts due to his polarity and good solubility for phenolic components from plant materials [34].
Under our study, we found a slight variation between total phenolic and fl avonoids content in the three concentrations while a low concentration of tannins (Table 2). That was enabled the correlation between the total fl avonoid, polyphenols content, and antioxidant activity. Our results are in an agreement with those [35] and disagreed with [36]. We suggest that could be concerning high levels of polar compounds in the plant materials which are soluble in solvents with a high polarity such as water, methanol, or ethanol.  Citation: Ennaji  therefore, the effect observed will be that of the extract used.
Our results are consistent with an Indian study evaluating the effect of ethanolic, methanolic, and aqueous extract of O.
majorana on normal lymphocytes at a concentration range of 40 to 120 μg / mL. The ethanolic and aqueous extracts were found to be non-cytotoxic on peripheral blood lymphocytes, while the methanolic extract was slightly toxic [14].

Conclusion
In conclusion, in vitro effi cacy of the hydro-ethanolic extract of Origanum majorana in the proliferation of the cells.
Theis herbs contain high phenolic and Flavonoid compounds that showed antioxidant and free radical scavenging activities.
More studies will be required to purify and isolate the bioactive fractions of this extract also the effect on the immune system.