In vitro investigation of antioxidant and antihemolytic activities of three Lamiaceae species from Morocco

Lkhzama (Lavandula officinalis), Mard-doch (Origanum majorana), and Lahbak (Ocimum basilicum) are aromatic and medicinal plants widely used in Moroccan folk medicine as a treatment for numerous diseases including liver diseases, rheumatism, and diabetes. This study was undertaken to examine the antioxidant and antihemolytic activities of the aqueous extracts of these plants. The antioxidant activity was evaluated using three in vitro tests: DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging activity, FRAP (ferric reducing antioxidant power assay), and ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay. The antihemolytic activity of plant extracts was evaluated against AAPH (2,2′-azobis(2-amidino-propane) dihydrochloride)-induced erythrocyte hemolysis. Our findings showed that all plant extracts displayed significant antioxidant and antihemolytic effects. In fact, among the studied plant extracts, the highest antioxidant power was recorded in Origanum majorana, based on DPPH (IC50 = 12.29 μg/mL), ABTS (226.13 μmol TE/g DW), and FRAP (477.82 μmol TE/g DW) assays. Moreover, the same plant also showed the best membrane protective effect (269.55%). Whereas, Ocimum basilicum exhibited the lowest antioxidant activity using DPPH (IC50 = 42.85 μg/mL), ABTS (IC50 = 226.13 μmol TE/g DW), and FRAP (IC50 = 172.84 μmol TE/g DW) and, thus, the lowest membrane protective effect (182.70%). This result supports the use of these plants in folk medicine for preventing and treating many diseases, especially those related to oxidative stress.


Background
Plants have been employed in many different forms since the earliest times. They have been used as food additives to improve their organoleptic properties apart from their use in traditional medicine because of their preservative and curative effects [1]. Numerous natural molecules extracted from plants, especially phenolic metabolites, are the main responsible for these biological activities due to their antioxidant potential [2]. Antioxidants play a key role against oxidative cell damage by inhibiting the formation of free radical species or enhancing their degradation and breakdown [3]. Recently, more and more epidemiological and experimental evidence strongly suggest that excessive free radicals and associated oxidative damage are mediators in several neurodegenerative disorders, cardiovascular problem such as hypertension, liver illnesses, aging, chronic kidney diseases, and cancer [4]. Moreover, in food industry, oxidation is the main cause for the development of unpleasant flavors and other changes that may affect the nutritional quality, the texture, and the appearance of the food, making them unfit for consumption [5].
Therefore, antioxidants are beneficial in human body as well as in the food system. Recently, antioxidants from natural sources have attracted an increasing attention because the use of synthetic antioxidants has been suspected to threaten human health [6].
According to Oyebode et al. report [7], 80% of Asian and African populations employ folk medicine and consult traditional practitioners to meet their primary health care needs because it is more affordable and accessible especially in low-and middle-income countries.
Lavandula officinalis, Origanum majorana, and Ocimum basilicum, known locally as Lkhzama, Mard-doch, and Lahbak, respectively, are among well-known aromatic and medicinal plants in Morocco, because they are widely used to improve the flavor and the taste of tea. Moreover, these plants are utilized to manage many ailments such as liver disease, rheumatism, diabetes, hypercholesterolemia, asthma, pain, kidney disease, cardiac disorders, and digestive problems [8,9].
In the light of the foregoing, the aim of this study is to determine total phenolic, flavonoid, and condensed tannin content, as well as to evaluate the antioxidant and antihemolytic properties of the aqueous extract of three Lamiaceae species.

Plant material
Three species of Lamiaceae family locally known as Lkhzama (Lavandula officinalis), Mard-doch (Origanum majorana), and Lahbak (Ocimum basilicum), respectively, were collected in Tinghir Region (southeastern Morocco), during the flowering period April-May 2019. The plants were taxonomically identified and authenticated by Pr Mohcine Elghazi (Ethnoobothanist at FSTE), and voucher specimen was deposited at the herbarium of the Faculty of Sciences and Techniques Errachidia: Origanum majora (HMOm185); Lavandula officinalis (HMLo 199); Ocimum basilicum (HMOb 205). The plant materials were dried and stored at room temperature (25°C) in the dark for extraction.

Preparation of plant extracts
The aqueous extracts were obtained according to the method of Bouhlali et al. [10] with slight modifications. In brief, 30 g of plant material was extracted in 500 mL of distilled water for 12 h at 35°C with an orbital shaking incubator. Then, the mixture was filtered and the filtrate was concentrated under reduced pressure at 40°C until total evaporation of the water, by a rotary evaporator. The crude extracts were stored at − 20°C in dark glass bottles until use. The extracts were dissolved in a known dilution in distilled water in order to measure the phenol, flavonoid, condensed tannin content, and their antioxidant properties.

Measurement of total phenolic compounds
The total phenolic content of plant extracts was determined following the method of Hmidani et al. [11]. In brief, 100 μL of the extract was added to 500 μL of a 1/ 10 dilution of the Folin-Ciocalteau reagent in distilled water, and then 400 μL of sodium carbonate solution (7.5% w/v) was added. The mixture was left for 60 min at room temperature, where the absorbance was measured at 765 nm. Gallic acid was used to prepare the calibration curve. Total phenolic compounds were reported as milligram gallic acid equivalent (GAE) per gram of plant dry weight (DW).

Measurement of flavonoid content
The total flavonoid content of plant was determined by the method of Kim et al. [12]. One milliliter of plant extract was mixed with 4 mL of distilled water. Then, 0.3 mL of sodium nitrite solution (5%) was added, followed by 0.3 mL aluminum chloride solution (10%). Test tubes were incubated for 5 min at ambient temperature, and then 2 mL of sodium hydroxide (1 M) was added to the mixture and then the final volume was made up to 10 mL with distilled water. The mixture was thoroughly vortexed and the absorbance was determined at 510 nm. Measurements were calibrated to a standard curve of prepared rutin solution, and the results were expressed as milligram rutin equivalent (RE) per gram dry weight (DW).

Measurement of total condensed tannins
The total condensed tannins were measured according to the method modified by Heimler et al. [13]. In brief, 400 μL of the plant extract was added in 3 mL of methanolic solution of vanillin (4%) and 1.5 mL of concentrated hydrochloric acid. The mixture was allowed to incubate at room temperature for 15 min and the absorbance was recorded at 500 nm. A calibration curve of catechin was prepared, and the results were expressed as mg CE (Catechin Equivalent)/g DW (plant dry weight).

ABTS radical scavenging assay
The ABTS radical scavenging was evaluated in accordance with the method of Re et al. [14]. The ABTS radical cations (ABTS+) were generated by reacting aqueous solution of ABTS (7 mM) with an aqueous solution of potassium persulphate (2.45 mM). Before the use of the mixture, it was allowed to stand in the dark at room temperature for 12-16 h, and then diluted with distilled water to get an absorbance of 0.700 ± 0.005 at 734 nm. Thirty microliters of the sample added to 3 mL of the ABTS radical solution was incubated at room temperature for 6 min and the absorbance at 734 nm was calculated immediately. A standard curve was obtained using an aqueous solution of Trolox. The total antioxidants were expressed as grams of Trolox Equivalent (TE) per gram of plant dry weight (DW).

Ferric reducing antioxidant power assay
The ferric reducing ability of plant extract was assessed following the procedure of Benzie and Strain method [15]. In brief, the FRAP reagent was obtained using a mixture of 50 mL acetate buffer (0.3 M) at pH 3.6, 5 ml tripydyltriazine (TPTZ) solution dissolved in HCl (40 mM) and 5 ml ferric chloride (FeCl3) solution (20 mM). Two milliliters of the freshly prepared FRAP reagent has been added to 10 μL of plant extract. The absorbance was calculated at 593 nm against the blank after 10 min at room temperature. The standard curve was plotted using Trolox. The result was reported as Trolox equivalent (TE) in milligram per gram of plant dry weight (DW).

DPPH radical scavenging power
As described by Blois method with slight modifications [16], the plants were evaluated for their abilities to scavenge the DPPH radical. Brief, 100 μL of plant extract, at different concentrations, was added in 1.9 mL of methanolic DPPH (0.3 mM). The absorbance was measured at 517 with a spectrophotometer after incubation of mixture at room temperature for 20 min in the dark. The IC 50 values were calculated as the concentration providing 50% inhibition of the DPPH radical based on the plotted graph of scavenging capacity against sample concentrations.

The protective effect of plant extracts against AAPH induced erythrocyte oxidative hemolysis
The antihemolytic activity caused by a peroxyl radical initiator, AAPH, was determined by the method reported by Blache and Prost [17] with slight modifications. Two hundred microliters of rabbit blood taken from heparin bulbs has been mixed with 10 μL of plant extract. Then 600 μL of AAPH (10%) was added. Incubation of the mixture was carried out at 37°C. Absorbance of the mixture was recorded at 450 nm every 5 min. Plant extract was substituted with Trolox and saline water (0.9% NaCl) in the positive and negative controls, respectively. The protective effects of the plant extract on free radical-induced hemolysis of erythrocytes were estimated on the basis of the time needed for 50% of hemolysis, the percentage of hemolysis induced by 600 μL AAPH (10%) was taken here as 100% hemolysis.

Statistical analysis
Statistical analysis was carried out with StatView 5.0 software. Results were expressed as the average of the repetitions of all experiments ± SE (standard error). Analysis of variance (ANOVA) and post hoc Bonferroni tests were utilized to compare the experimental groups.
The Pearson correlation coefficient (r) has been used for measuring the correlation between two variables. Differences at p < 0.05 were reported as significant.

Phenolic content 3.1.1 Total phenolic content
Standard calibration curve gallic acid was used to evaluate the total phenolic content in the aqueous extracts of studied plants. Results are shown in Table 1, and it has been noted that all studied plant extracts exhibited a large amount of phenolic content, where the amount of polyphenols is varied in different tested plants and ranged from 52.35 ± 2.15 mg GAE/g DW in Origanum majorana to 27.82 ± 1.53 mg GAE/g DW in Ocimum basilicum. Table 1 reports total flavonoid content data in milligram rutin equivalent per gram of dry weight (mg RE/g DW).

Total flavonoid content
The flavonoid content in the selected species ranged from 24.41 (Origanum majorana) to 11.94 mg RE/g DW (Ocimum basilicum).

Total condensed tannins
The total condensed tannin content of selected plants of Lamiaceae is shown in Table 1. Where the tannin content was found highest in Origanum majorana (8.73 ± 0.91 mg CE/g DW) and lowest in Lavandula officinalis (3.04 ± 0.82 mg CE/g DW).

In vitro antioxidant activity
In vitro antioxidant power of plants was assessed using three different tests: DPPH, ABTS, and FRAP assays. The findings from these assays are detailed below.

Ferric reducing antioxidant power assay
As summarized in Table 1, the best reducing power based on FRAP assay was shown in Origanum majorana (477.82 μmol TE/g DW), followed by Lavandula

Discussion
Plants are an interesting source of bioactive natural compounds such as flavonoids, ascorbic acid, and phenolic acids [18]. Thanks to their beneficial effects on human health and their several biological activities including antioxidant and antihemolytic capacities, many scientific researchers have been focusing on the study of phenolic acids [19,20]. In this regard, the purpose of this study was to assess the phenolic content, antioxidant, and antihemolytic properties of the aqueous extracts of three plants from the Lamiaceae family, commonly used in Moroccan folk medicine. Interestingly, our data demonstrate that all selected plant extracts contain an appreciable level of total phenolic content, flavonoid, and total condensed tannins, and the best amount was recorded in Origanum majorana, followed by Lavandula officinalis and, then, Ocimum basilicum. Our results are higher than those reported by Méabed et al. [21] who have found that total phenolic content in Origanum majorana from Egypt was 9.2 mg GAE/g DW. However, Surveswaran and co-workers [22] reported that the amount of total phenolic content in Ocimum basilicum (26.3 mg GAE/g DW) was slightly higher compared to this found in the current study. In the same way, Spiridon et al. [23] mentioned a greater level of polyphenols in Lavandula angustifolia (50.6 mg GAE/g DW). In addition, this investigation confirms the previous results found in several plants, mainly those from Lamiaceae family. In this respect, khomdram and Singh (Khomdram and Singh, 2011) indicate that total phenolic content in eight plants (Lamiaceae family) is ranged between 46.28 and 23.80 mg CE/g DW. Concerning other classes of polyphenols, a remarkable amount of flavonoid content also recorded in selected plant species. In this respect, Méabed et al. [21] showed that Egyptian Origanum majorana contains appreciable total flavonoid content (57.3 mg quercetin equivalent (QE)/g DW). Other scientific researchers confirm the notable amount of flavonoids in Ocimum basilicum and Lavandula species [24,25]. Moreover, the tested plants also possess a significant amount of condensed tannins. This result is in agreement with other researches which confirmed the presence of condensed tannins in plant extracts [26]. Indeed, Kpètèhoto et al. [27] demonstrated that the level of condensed tannins in Ocimum gratissimum linn harvested in southern Benin (2017) was in the   [28], it seems that the majority of these plants show low condensed tannins, except for Rhus tripartitum and Periploca laevigat. The difference found in phenolic compounds could be attributed in part to the genetic diversity, plant part, geographical distribution, phytochemical content [29].
Since the methods of antioxidant activities differ in their mechanisms of action and offer more insights about the sample's antioxidant potential, tested plants, in this study, have been evaluated using three antioxidant methods : DPPH, ABTS, and FRAP assays.
The DPPH method consists of measuring the capacity of an antioxidant substance to reduce the DPPH• radicals to the corresponding hydrazine, and simultaneously changing its color from violet to yellow, with concomitant decrease in absorbance at 517 nm [30]. Data are expressed as IC 50 , defined as the substrate concentration causing a loss of 50% of DPPH activity (Table 1). A lower IC 50 reflects a higher antioxidant effect. In the present investigation, all plant extracts exhibit considerable antioxidant activities based on DPPH method in a concentration-dependent manner. Thus, Origanum majorana extract (12.29 μg/mL) displayed the highest antioxidant capacity. The ABTS method is largely accepted to evaluate the antioxidant capacity of food extracts, natural products, and pure compounds [31]. This assay measures the potential of the antioxidant to scavenge the radical (ABTS+•) produced in the aqueous phase at 734 nm [32]. In addition, the decolorization of ABTS radical cation reflects the capacity of the plant extracts to donate electrons or hydrogen atoms and to deactivate these radical species [32]. The finding recorded during this investigation demonstrated that overall extracts exhibit potent abilities to quench the free radical ABTS. The antioxidant potential of plant extracts was also estimated for their ability to reduce the ferric tripyridyltriazine complex to the colored ferrous tripyridyltriazine [33]. Data from the present work display a significant reducing power in all plant extracts using FRAP assay. The findings of this paper conform to the previous reports which have demonstrated the interesting potential antioxidant capacity of the species studied here. In fact, Petrovic and his collaborators [34] indicated that the antioxidant capacities of Serbian Lavandula officinalis through DPPH, ABTS, and FRAP were 4.35, 10.35, and 15.53 mmol Trolox equivalent/L, respectively. Furthermore, Duletić-Laušević and coauthors [35] examined the ethanolic and the aqueous extracts of Origanum majorana L. originating from Serbia, Greece, Egypt, and Libya. They showed that the highest antioxidant ability was manifested by Serbian aqueous extracts based on DPPH (IC 50 = 28.25 mg/mL), ABTS (2.06 mg ascorbic acid equivalent/mL), and FRAP (826.45 umol Fe (II)/g). Moreover, in a recent study, Rezzoug et al. [36] measured the antioxidant activity of Algerian Ocimum basilicum ethanolic extract using DPPH, ABTS, and FRAP assays. The extract displayed moderate activities against DPPH radical (IC 50 = 0.629 mg/mL), ABTS radical (IC 50 = 0.629 mg/mL), and a considerable reducing power of ferric complex (3657 mg ascorbic acid equivalent/mL).
To correlate these results with the different methods, a linear regression analysis was carried out (r 2 , Table 4). Accordingly, a significant correlation was found between phenolic compounds and the antioxidant capacities assessed using DPPH, ABTS, and FRAP methods. This high correlation suggests that the antioxidant power found in the studied extracts could possibly be due to their phenolic content (r 2 polyphenols/FRAP = 0.86; r 2 polyphenols/DPPH = 0.87). The present data were supported by several earlier reports that highlight on the most important relationship between antioxidant activities and phenolic content [37][38][39].
Erythrocytes are very sensitive cells to oxidative stress. This sensitivity is related to their rich membrane lipid composition of polyunsaturated fatty acids, the constant interactions with oxygen, and the production of ROS by activated inflammation cells [40,41]. Additionally, the presence of hemoglobin in large quantities also exposes red blood cells to high levels of auto-oxidation [42]. Therefore, hemolysis is widely employed as an important indicator of free radical damage affecting the membrane of erythrocytes [43]. On the other hand, the antioxidant compounds are known to have the ability to scavenge free radicals and protect the cells against hemolysis induced by oxidative stress. In this study, an assessment was performed on the ability of plant extracts to protect red blood cells (RBCs) from oxidative damage caused by AAPH-induced hemolysis. Our data, expressed as the half-hemolysis time (HT 50% , which is the time required for hemolysis of 50% of erythrocytes) and the percentage of protective effect on erythrocyte membrane, indicated that all studied plant extracts significantly protected the membrane of the red blood cells from hemolysis better than Trolox (1%), used as a standard antioxidant. These results clearly demonstrated that the selected plant extracts were able to scavenge AAPH-derived peroxyl radicals, thereby protecting erythrocyte membrane. Furthermore, tested extracts induced a significant increase of half-hemolysis time compared to the control containing only blood. The finding clearly demonstrated the important stabilizing effect of selected plants on the erythrocyte membrane. The analysis of linear correlation displayed a significant interaction between hemolytic inhibition activities (AAPH) and polyphenols content (r 2 = 0.60), flavonoids content (r 2 = 0.90), while a weak correlation was detected for condensed tannins (r 2 = 0.30). On the basis of these results, some of this activity seems to be explained by the presence of polyphenols in plant extracts, especially flavonoids. Our results are also in the same line with previous investigations that have proven the remarkable antihemolytic activity of several plant extracts, and the capacity of the phenolic content to protect erythrocytes from oxidative stress or to increase their resistance to oxidativeinduced damage. Indeed, in a recent scientific work, Deruich et al. [44] assessed the protective effect of erythrocyte membrane against AAPH-induced hemolysis of three Apiaceae plants from Morocco and, thus, they concluded that Petroselinum crispum manifested the highest protective effect (HT 50% = 273.64 min) and Apium graveolens recorded the lowest effect (HT 50% = 194.71 min). Moreover, Bhat and his collaborators [45] displayed that an alcoholic extract of Caralluma quadrangula from the Northwestern region of Saudi Arabia exhibited an inhibition of red blood cell lysis close to 90% at the highest concentration 2000 μg/mL.

Conclusion
The present scientific research indicates that all studied plant extracts possess considerable antihemolytic and antioxidant capacities and, thus, the Origanum majorana species has shown the best results. These results confirm the use of these plants in popular medicine for the prevention and treatment of many diseases, especially those related to oxidative stress. In addition, more research is needed to identify and characterize the bioactive compounds responsible for these observed biological activities.