Phytochemical profile, antioxidant and anti-inflammatory activities of Ephedra alata Decne growing in south Algeria

Ephedra alata Decne. (Ephedraceae) is a medicinal species commonly used to treat cancers, respiratory diseases, fever, and hypertension. The present study aimed to establish a phytochemical profile, evaluate the antioxidant potential and estimate the anti-inflammatory activities of E alata. Aqueous and methanolic extracts of E. alata aerial parts were phytochemically investigated using standard methods. DPPH, phosphomolybdenum total antioxidant capacity and reducing power assays were used to evaluate the antioxidant activity. Antioxidant activity was determined using total antioxidant capacity, the scavenging activity of the stable DPPH free radical and ferric reducing antioxidant power assays. The anti-inflammatory activity was determined using egg albumin membrane denaturation and human red blood cells membrane stabilizing assays. The anti-inflammatory potential of E. alata extracts was evaluated using human red blood cells membrane stabilization, egg albumin and BSA albumin denaturation assays. Quinones, anthraquinones, steroids, phytosteroids, phenols, terpenoids, flavonoids, saponins, glycosides, Cardiac glycosides, reducing sugars and anthocyanins were present in the E. alata’s aqueous extract, in addition to coumarins and proteins in the methanolic extract. The highest total phenolic and flavonoid content was recorded in the aqueous extract with 8.66 ±0.09 mg GA/g and 248.04 ±1.47 mg Q/g, respectively. On the other hand, E. alata methanolic extract had the highest tannin content of 62.12 ±0.10 mg C/g. The best radical scavenging activity (IC50 = 4.63±0.00 mg/ml) and total antioxidant capacity were exhibited by the E. alata aqueous (7.35±0.12 mg/ml AAE), whereas the methanolic extract possessed the highest reducing power activity (1.81±0.00 mg AAE/ml). Regarding the anti-inflammatory activities, E. alata methanolic extract exerted the highest HRBC stabilization of 34.72 ±0.08% whereas the aqueous extract exhibited the highest bovine serum and Egg albumin denaturation inhibition of 99.22 ±0.02% and 73.31 ±0.90, respectively. Taken together, our results suggest that E. alata aerial parts aqueous and methanolic extracts can be utilized as future antioxidants and anti-inflammatory ethnomedicines owing to their rich bioactive molecules content.


Introduction
Algeria is one of the richest Arabic and African countries regarding medicinal plants with 3164 species 1 , they are widely used by Algerians who still relay on traditional healers for therapy 2  Ephedra is one of the largest genera of the Ephedraceae family, it consists of 69 species mainly distributed in semi-arid environments, although some species are distributed through few Neotropical countries 6,7 . Ephedras are traditionally used to treat allergies, bronchial asthma, chills, colds, coughs, edema, fever, flu, headaches and nasal congestion 8 , they have been used in the US since the 20 th for weight loss and to treat syphilis and gonorrhea and in China for more than 5000 years and Ayurvedic medicine as a stimulant and an anti-asthmatic while Pharmacological trials on crude extracts, fractions and few isolated compounds of Ephedra species in vitro and in vivo demonstrated anti-inflammatory, anticancer, antibacterial, antioxidant, hepatoprotective, anti-obesity, antiviral, and diuretic activity 9 . However due to its side effects caused by misuse and the high alkaloid content (ephedrine and pseudoephedrine) the ephedra use has been controlled 8 , interestingly, among all the Ephedra species, only Ephedra alata is known for its low alkaloid content.
Ephedra alata, commonly known in Algeria as Alenda 10 , is a medicinal plant of the family Ephedraceae, it is a perennial and xerophytic (adapt to any environment to survive) gymnosperm and dioecious (has distinct male and female organisms which excludes self-fertilization) shrub with erect non climbing stems and short leaves that are united towards base and yellowish-green bracts that flowers in july 11,12 and have a strong pine like smell 13 The present study aimed to establish a phytochemical profile, evaluate the antioxidant potential and estimate the anti-inflammatory activities of E. alata growing in Algeria.

Extraction yield
According to our results, the highest extraction yield (38%) was obtained with the E. alata aqueous extract while the methanolic extract had the lowest extraction yield of Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 August 2021 doi:10.20944/preprints202108.0296.v1 15% (Table 1). This difference could be due to differences in solvent polarities which extract different types and amounts of metabolites.   According to our DPPH radical scavenging activity results (figure 2), there was a significant difference between the E. alata aqueous and methanolic extract (p< 0.05) and between the methanolic extract and the ascorbic acid control (p< 0.01). Table 4 and As shown in figure 1, the aqueous extract had the best free radical scavenging activity with an IC50 of 4.638±0.002 mg/ml, while the methanolic extract presented a low scavenging activity with a high IC50 of 20.943±2.903 mg/ml. The IC50 of the ascorbic acid used as a standard was found to be 2.786±0.019 mg/ml. alata extracts was almost six-fold higher than that of ascorbic acid (figure 2).

Reducing power
The reducing power activity results showed that there was a dose-dependent increase in reducing power for both extracts ( Figure 3). The E. alata methanolic extract possessed the highest reducibility (1.81±0.00 mg of AAE /ml of extract) at 1000µg/ml, while the aqueous extract had 1.36±0.02 mg AAE/ml at the same concentration. However, this difference was not statistically significant.     contain alkaloids, which can be associated with a safe toxicological profile since Ephedra alkaloids (ephedrine and pseudoephedrine) are responsible for toxicity 18,21 . However, the Reducing power assay showed that the methanolic extract exhibited higher reducing power in all different concentrations. In correlation with our results, Al-Rimawi et al.
(2017) 52 reported that the reducing power of Palestinian E. alata significantly changed according to the solvent polarity.
Since human erythrocytes' membrane has similar structure to the lysosomal membrane, HRBC membrane stabilization assay is considered as a measure of anti-inflammatory activity of the plants extracts. According to our results, the E. alata methanolic extract had better anti-inflammatory activity than the standard widely used Diclofenac sodium, whereas the aqueous extract exhibited a membrane stabilizing activity similar to this standard NSAID. To further confirm our results, protein denaturation inhibition activity assay was conducted considering that protein denaturation is an indirect cause of inflammation through cell destruction or mutilation.
Therefore, substances able to inhibit protein denaturation can assumably inhibit inflammation. Both bovine serum albumin denaturation and egg albumin denaturation assays indicated that the aqueous extract had the highest protein denaturation inhibition percentage that was comparable to the anti-inflammatory potentials of diclofenac sodium. and IL-13 53 .
In conclusion, the present study showed that both aqueous and methanolic extracts of E. alata aerial parts contain various phytochemicals. Furthermore, E. alata

Plant material and extracts preparation
Ephedra alata whole plant was collected from Adrar (South Algeria) in January 2020.
Botanical identification was performed at the department of biology (University of Mascara, Algeria). Aerial parts were collected, cleaned, dried in semi shaded area for few weeks, and then finely grounded and stored in containers in dark. Preparation of E. alata aqueous extract The aqueous extract of E. alata aerial parts was prepared as follows: 20g of dried plant powder were decocted in 200 ml of water at 100°C for 10 mins, cooled to room temperature, and then filtered. This procedure was repeated twice [25][26][27] . After concentration, the crude aqueous extract was stored at +4°C until use.

Preparation of E. alata methanolic extract
The methanolic extract of E. alata aerial parts was prepared as follows: 20g of dried plant powder were macerated in 200 ml of methanol for one week at room temperature.
The extraction yield (%) was calculated using the following formula 28 : Phytochemical screening -The phytochemical profile of E. alata was performed using standard procedures described 29,30 with slight modifications.

Phytochemical quantitative analysis
Total phenolic content-Total phenolic content was determined using the Folin-Ciocalteu assay 31 . In brief, 100 µl of each extract and 500µl of 1% Folin-Ciocalteu reagent were added to 400 µl of 7.5% Sodium carbonate solution (w/v), mixed using a vortex, and then incubated for 10 min in the dark at room temperature. The absorbance was then measured at 760nm 32 . The phenolic content was calculated as gallic acid equivalents GAE/g of dry plant material. Values were determined in triplicate.
Total flavonoids content-The determination of the total flavonoid content of the extracts was carried out using the aluminum chloride colorimetric method 33  Values were expressed as mg AAE/ml of extract.
Reducing power (RP) assay-The reducing power was measured as described by  Egg albumin denaturation assay -The membrane denaturation assay was carried out as described by Shelke et al. (2020) 47 . 2ml of the extract (125, 250, 500, 1000 µg/ml) was mixed with 0.2mL of egg albumin (0.5% w/v aqueous solution) and 2.8 PBS (pH = 6.4), and incubated at 37°C for 15 min. The mixture was then heated at 70°C for five minutes in a water bath. Aabsorbance was measured at 660 nm against the solvent as a blank. for the control, distilled water was used instead of the extracts. A calibration curve was performed in parallel under the same operating conditions using sodium diclofenac as a positive control 43 : Funding: This study was supported by the General Directorate of Scientific Research and Technological Development, Algeria.