Introduction

The Algerian Sahara has exceptional floral biodiversity, consisting of more than 1,200 species (Ozenda, 1991), of which approximately 162 species are endemic to the north of the Sahara alone to which is added a secular tradition of the traditional use of plants. Among the desert plants, there is the legume family (Leguminosae) which represents the third largest subfamily compared to flowering plants, and consists of 650 genera and about 18,000 species (Kass & Wink, 1997), and contributes effectively to food, agriculture, industrial and pharmaceutical due to the abundance of these genera and species (Al-Rejaboo & Jalaluldeen, 2019; Alshehri, 2020).

Glycyrrhiza glabra belongs to the Fabaceae family, is a fairly common species in the Mediterranean region throughout Algeria especially in arid and semi-arid regions (Baba Aissa, 2011). It is a plant used for both culinary and medicinal purposes (Hayashi & Sudo, 2009). The roots and rhizomes of G. glabra are commonly used in Algerian society, as a remedy for the digestive system, stomach, and duodenal ulcers (Bardhan et al., 1978), As well as for the treatment of spasmodic pain of chronic stomach (Zadeh et al., 2013). According to Bahmani et al. (2014), Licorice may reduce the symptoms of diabetes, such as frequent urination and polydipsia but cannot reduce blood glucose. The infusion prepared by the roots is considered to treat inflammatory diseases (Yang et al., 2017). According to the literature, Low bone mass, fractures, osteoporosis, bone defects, osteomalacia, osteogenesis imperfecta, bone disease, and periodontal illnesses have all been treated using G. glabra extract (Kumar et al., 2015).

The objective of our study was to estimate the number of polyphenols and flavonoids in Glycyrrhiza glabra L as well as the antioxidant and antimicrobial properties of these active compounds in the rhizomes parts.

Materials and Methods

Plant Material Collection and Identification

The rhizomes of the G.glabra plant were obtained from the Ghardaïa region in southern Algeria (Latitude: 32° 29' 24.81'' N, Longitude: 3° 40' 25.8276''E) in 2021. The plant was identified by Dr. Y. Halis Researcher in Scientific and Technical Research Center for Arid Areas (Touggourt) Algeria. The plant materials were cleaned and dried at room temperature for 15 days then ground into a powder, weighed, and stored in clean glass jars.

Phytochemical Screening

The phytochemical screening is carried out in the extracts prepared from the plant tested to demonstrate the presence of certain secondary metabolites. The analytical tests carried out are based on precipitation or coloring responses employing reagents that are particular to each chemical family (Table 1). The different chemical groups have been characterized according to the methods described by Trease and Evans  (2002).

Table 1. Primary qualitative tests in Phytochemical screening

Preparation of Phenolic Extracts from Rhizomes of the G. glabra

According to the Markham (1982) protocol, the different types of extracts were prepared from the pulverized rhizomes (100 g) using 1L of increasing polarity solvents (dichloromethane, ethyl acetate, butanol, methanol). At the end of the extraction, the four organic extracts were concentrated under vacuum at Rotavapor at temperatures of 35°C, 40°C and 50°C respectively. Each extract's dried sample was weighed, and the yield of soluble components was estimated using the equation: Yield (%) = [Final weight of dried extract / initial weight of licorice powder] x100 to determine the total phenol and flavonoid content as well as antioxidant activity, the extracts were dissolved in methanol (1 mg/ml). The experiment was carried out three times.

Determination of Total Polyphenols

The Folin-Ciocalteau method was used to determine the concentration of total phenols content in dry extracts of G.glabra rhizomes (Singleton & Rossi, 1965). 1 ml of Folin-Ciocalteu reagent (10%) was added to 200 µl of each extract and incubated for 4 minutes at room temperature. Then, 800 µl of sodium bicarbonate (7.5 %) was added to each mixture, after the second incubation of 2 hours, the total polyphenols content was measured at λ max = 765 nm with a spectrophotometer of CECIL2041 UV-VS. the gallic acid was used as a stander and the results were expressed in milligrams of equivalents of gallic acid per 100 g of dry matter (mg GAE / g Ext) 

Determination of Total Flavonoids 

The total flavonoids content was measured by the colorimetric method of aluminum chloride (AlCl ₃) (Bahorun et al., 1996). 1 ml of each extract tested as well as the reference compound (standard) were added to 1 ml AlCl ₃ solution (2% dissolved in methanol). The mixture was left to react for 10 min and the absorption was read at λ max = 430 nm. The flavonoid concentrations were deduced from the calibration curve established with quercetin. The results were measured in milligrams of quercetin equivalents per 100 grams of dried material (mg EQ / 100 g Ext) (Saptarini & Herawati, 2019).

DPPH Radical Scavenging Assay

The varied extracts were tested for the antioxidant potential by using the technique outlined by Mansouri et al. (2005) with some modification. 300 μl of the extract were added to 1300 μl of DPPH solution while the negative control is formed by blending 300 μl of methanol with 1300 μl of the DPPH solution.

After 30 minutes of incubation in the dark, the absorbance measurement was taken against a blank at 517 nm. The same method was followed for all the dilutions. The ascorbic acid was used as a reference compound and the results expressed as anti-free radical activity or inhibition of free radicals in percentages (I%) using the formula below:

The IC 50 value was computed as the concentration of extract that inhibited the 50% of DPPH radical

Evaluation of the Antibacterial Activity

Sources and maintenance of microorganisms Bacterial strains of Gram+: Bacillus cereus ATCC 11778 and Staphylococcus aureus ATCC25923; Bacterial strains of Gram^-: Escherichia coli ATCC25922 and ATCC 27853 Pseudomonas aeruginosa (American Type Culture Collection), obtained from the Pasteur Institute of Algiers, maintained by subculture on nutrient agar medium favorable to their growth and incubated at 37 ° C for 24^h. So we obtain a bacterial suspension colony-forming units per milliliter (cfu/ml) density of 10⁶ cfu/ml

Preparing Disks 

The phenolic extracts of rhizomes were recovered by dissolved in DMSO 2% (dimethyl-sulfoxide) A series of dilutions are carried out from C0 to reach an initial solution concentration of C 0 = 100mg/ml. 100 µl of each extract was impregnated into the sterile filter paper discs (6 mm diameter).

Concentrations of Each Extract

Diffusion Method on Agar Medium

The antibacterial activity of different plant extracts was evaluated using the method of agar diffusion (Benbott et al., 2012). For each strain, a bacterial suspension was produced in sterile distilled water from colonies that had grown for 18 to 24 hours. This suspension's turbidity is corrected to 0.5 McFarland before being diluted to 1/100. This produces a 10 6 cfu/ml inoculum estimate. This inoculum is inoculated by flooding Mueller-Hinton agar-coated Petri plates. Various concentrations of impregnated disks (12.5 %, 25 %, 50 %, and 100 %) were then placed on the agar's surface. Before being incubated at 37 ° C in an oven for 24 hours, the Petri plates were placed at room temperature for 1 hour to allow for pre-release chemicals. The diameter of the inhibition zone surrounding each disc is used to measure antibacterial activity.

Results and Discussion

Phytochemical Screening

The results of the phytochemical tests are shown in Table 2.

Table 2. Results of detection of secondary metabolism products of G. glabra root extracts

+: Presence, -: Absence

The results of phytochemical analysis of G. glabra rhizomes showed the presence of numerous chemical compounds, such as phenols, Volatile oils, tannins, flavonoids, Reducing compounds, saponins, coumarins, triterpenes, and sterols. On the other hand, we note the absence of the imodols. The presence of these active compounds in the roots of G. glabra indicates the importance of this plant in traditional and modern medicine, some of these results are close to those obtained by Rizzato et al. (2017) and Wang et al. (2015a).

The Yield of Phenolic Compounds in Various Extracts

The findings of this investigation revealed that the optimum yield of phenolic compounds in G. glabra rhizomes was with the methanol extract (9.2 ± 0.21 %) knowing that the extraction was done with 80% methanol The dichloromethane had the lowest yield. extract (5.8 ± 0.13%) (Table 3). There is a correlative relationship between the polarity of the solvent and its solubility due to the solubility nature of these compounds in the solvents (Benbott et al., 2018). Note that the yield of the methanol extract is high compared to the rest of the extracts. Indeed, alcoholic solvents can increase the permeability of cell walls by facilitating the extraction of more polar, middle, and low polar molecules.

Table 3. The yield of phenols in each extract of G.glabra

Content of Total Phenolic and Flavonoid Compounds

Table 4 reports the contents of phenolic compounds in the extracts of the rhizomes of G. glabra. A considerable phenolic content was observed by the extracts tested, while the methanol extract has the highest concentration 50.37 ± 2.14 mg EAG / g E. On the other hand, we find that the content of flavonoids in the butanoic extract of the rhizomes of the plant G. glabra is higher compared to the other extracts.

Table 4. Total phenols and flavonoids content of G. glabra rhizomes

Our results differ from those of Asan-Ozusaglam and Karacoka (2014), which obtained higher levels of phenolic compounds in dichloromethane than those obtained from aqueous and ethanolic extracts of Turkish licorice root.

Several factors can have an impact on the distribution of phenolic compounds in the fractions, such as climatic and environmental parameters.        

DPPH Free Radical-Scavenging Activity

The results of the change in the inhibition percent of DPPH as a function of the concentration of the extracts and the standard are shown in Figures 1 and 2.

We find that the extracts exert an inhibitory power of the free radical DPPH. This power gradually increases with increasing concentration of extracts as well as standard.

The greatest percentage of inhibition was recorded by the methanol extract at a concentration of 0.1 mg / mL (94.04% ± 0. 14). According to Tohma and Gulçin (2010), the DPPH radical inhibition percent of G. glabra aqueous and ethanol root extracts at a concentration of 0.03 g/ml were 52.2 % and 54.4%, respectively. These percentages of inhibitions allowed us to calculate the IC ₅₀ and compare the effectiveness of the extracts. We recall that the lower the value of the IC ₅₀, the more the extract is powerful against free radicals.

According to the values obtained, the methanol extract has the lowest IC ₅₀ compared to that of the other extracts, and therefore the best activity with an IC ₅₀ of approximately 0.035 ± 0.24 mg / mL followed by the butanol extract and the ethyl acetate extract. Whereas, the highest

IC ₅₀ was recorded by dichloromethane extract which means the lowest antioxidant activity.

However, when compared with the standard antioxidant, the extracts tested were found to be moderately active.

Our results are in agreement with the study conducted by Sharma et al. (2013) which indicated that the methanol extract of the roots of G. glabra was more effective as a free radical scavenger and had good reducing power.

According to Biondi et al. (2003). The dihydrostilbene derivates found in G. glabra leaves have been shown to have high antioxidant activity. On the other hand, G. glabra also contains licochalcones B and D, which have a significant DPPH radical scavenging action as well as the capacity to suppress microsomal lipid peroxidation (Biondi et al., 2003; Sharma et al., 2018).

Determination of the Antibacterial Activity

The sensitivity of the strains to extracts of the plant of G. glabra was achieved by the technique of diffusion in an agar medium. The results of the diameters of the zones of inhibition of extracts from the rhizomes of the G. glabra plant against Gram-positive and Gram-negative bacteria are shown in Table 5.

The results reveal variable responses depending on the strains tested and the concentration of the extract studied. The highest antibacterial activity against B. subtilus was recorded with the methanol extract at the highest concentration with an inhibition diameter of 19±0.8 mm, compared to butanol extract with an 11±0.3 mm inhibition diameter. Concerning the E. coli and P. aeruginosa bacterial strains, the diameter of the inhibition zones is between 6. ±0.1 and 9±0.37mm.

Table 5. Determination of the activity of methanolic extract and butanol against four reference bacterial strains