Phenolic and Flavonoid Contents of Some Plant Extracts from Tunisia Southern Landscape by Using Different Extraction Techniques: The Case of Retama reatam

Nowadays traditional medicine, a source of several bioactive molecules for therapeutic purposes, has become a cure for various diseases. In fact, the evaluation of plant exploitation has become progressively significant and this for their therapeutic effects of many traditional medicines may be due to the immense presence of natural antioxidants. In this respect, Retama reatam was chosen among six herbal Tunisian plants traditionally known for their therapeutic virtues, as the best source of total polyphenol content (1122,927g GEA/g extract) with an important extraction yield as ethanol is the solvent. The Soxhlet extraction always has the lowest value in total polyphenol content (164,857 GEA/g extract) for such solvent. The total flavonoid content of the different extracts is not significantly different from each other. However, the yield extraction remains higher for ethanolic extraction exceeding a value of 26.7%. Concerning antioxidant activity of Retama reatam, results suggest that supercritical CO 2 extraction can be used as an efficient alternative for pre-treatment to eliminate fatty compounds and therefore evaluating oxygen radical absorbance capacity values.


INTRODUCTION
Traditionally, herbal materials are known as a prominent medicinal source of polyphenols particularly flavonoids and represent a class of bioactive molecules that are closely related to multiple health benefits. Saharian and southern Tunisian ancestors prepared folk medicaments to cure various diseases using several plants growing spontaneously, although there is little information about composition, contents and related effects.
The interest in plant and herbal materials has become gradually significant may be due to the presence of natural antioxidants such as phenolic plant compounds and flavonoids. In this respect, the development of a standard method for efficient and rapid extraction of these secondary metabolites remains a challenge.
Indeed, there is a diversity of species with therapeutic interests, but many of them are not studied. For example, very few books have been devoted to Retama reatam which is spontaneous shrub belonging to the Fabaceae family, locally known as R'tem. This virtue plays an important ecological role and is widely used in dune stabilization and soil fixation [1]. In addition, it is usefully used for the treatment of several diseases such as diabetes, Conforti et al. [2], eczema and hypertension, Alfalluos et al. [3].
The aim of this study is to investigate a qualitative and quantitative phytochemical analysis and antioxidant activity for medicinal plants as Ratema raetem, Artemisia alba, Elymus repens, Diplotaxis harra, Kochia scoparia, Lawsonia inermis, especially. Firstly, this study will focus on determining the richest TPC source among tested plants using maceration as a conventional extraction technique. Secondly, to give data: extraction yield efficiency, TPC, TFC and antioxidant activity of the chosen herbal materiel (Retama reatam) following several extraction techniques.

Plant sampling
All plants are traditional medicinal plants present in humid bioclimatic regions in arid zones of Tunisia and used for their therapeutic effect. Different leaves and seeds plants were handily gathered in Tunisia southern landscape on spring season between March and May in 2018. The identity of plants is confirmed at Laboratory of Bio-resources: Integrative Biology and Valorization (LR14-ES06), University of Monastir (Tunisia). The harvested products are well cleaned and rinsed with distilled water then dried in shade at room temperature (~20°C) for 3 weeks. Such dying process may avoid undesirable thermal degradation of the finished products until reaching a new stable form and a constant weight. Materials are chopped and ground separately to fine powders in a mechanical blender. The obtained powders are well-kept in hygienic plastic containers in a dark room to avoid effect of light, heat and moisture until use.
Our interest is focused on metabolic properties of 6 types of plants belonging to the Tunisia southern landscape. They are considered as those occurring spontaneously in the Tunisian Sahara and steppes. Hence, we propose a comparative study of various extraction methods applied to leaves of Retama raetem, Artemisia alba, Elymus repens, Diplotaxis harra, Kochia scoparia, Lawsonia inermis ( Figure 1). These spontaneous plants are usefully proved in local folk medicine and studied by several previous works [4][5][6].

Chemicals
All chemicals are of analytical reagent grade chemicals. Carburos Metálicos S.A. (Spain) supplied carbon dioxide (mass fraction purity 0.999 in the liquid phase). Folin-Ciocalteau reagent, gallic acid, catechin, are gotten from Sigma Aldrich (Spain). Other reagents are of analytical grade or higher available purity.

Extraction procedures
There are several methods for extracting polyphenols and for selecting one plant among others, maceration with ethanol and hexane is used. Two types of sampling are prepared for each plant (two solvents of different polarity): hexane (apolar) and ethanol (polar). We also use Solid liquid, Soxhlet and Supercritical fluid extraction techniques to extract Retama reatam and evaluate TPC, TFC and antioxidant activity. Solvent choice: Two solvent systems are selected: ethanol as polar solvent (96%) and hexane as apolar solvent (99%) where solubility of such phenolic compound is strongly related to the solvent polarity. Indeed, the nature of extraction solvent can have a significant impact on the extraction yield of polyphenols from plant material [7].
Maceration: It is the simplest mode of extraction where the powdered plant material is taken inside an airtight container and soaked with the solvent for a specified period of time until the soluble portions are dissolved in the solvent (60, 90, and 180 minutes). A series of experiments aims at varying the extraction parameters (temperature, extraction time and solvent volume) are evaluated in order to optimize the TPC as well as the TFC. The operation is repeated several times to identify the best parameters. The resulting mixture is filtered with filter paper and put in a box knowing that the moisture of Retama is 13.60%. 10 g of each plant are weighed and placed in the filter cartridge, then in a box in which 200 mL of solvent (96% ethanol then hexane) is added. This extraction step is stopped after 6 days when the liquid surrounding the cartridge takes the plant color. This color indicates that the solvent no longer extracts anything from the solid. The various extracts obtained are filtered on Whatman paper No. 4 (Ashless filter paper) and stored at 4°C. for subsequent analyzes.

Solid liquid (SL) extraction:
The conventional SL extraction is carried out by batch ethanolic extraction from Retama reatam in a stirred vessel. Several experiments for the investigated system are carried out in order to obtain the optimum conditions for extraction. The formed solution is stirred for eliminating external mass transfer resistance. Experiments are performed at both 40°C and 60°C. Each extraction experiment lasts 90 minutes in order to reach equilibrium state of the process.

Soxhlet extraction:
Crushed Retama reatam leaf powder (10 g) is packed into a Soxhlet apparatus and extracted with 100 mL hexane at 40°C for 6 hours. The extract is filtered through Whatman filter paper No. 1, and the filtrate is concentrated under evaporation to dryness at 100°C using an oven. The extract is dried, weighed (8.98

SFE extraction: Supercritical Fluid Extraction (SFE) is considered
an environmentally friendly and a novel extraction technique that allows for selective extraction using a supercritical solvent. Supercritical uid extraction technology has advanced tremendously since its inception and currently represents the method of choice in many food processing industries [8]. The extraction tank is loaded with 90 g dried Retama reteam leaves. The system is used at a constant temperature of 45°C and a pressure of 280 bars to eliminate the waxy cuticles and other fatty compounds from the ethanolic extraction. For the experiment, conditions in the recovery section are defined at 46 bars/15°C in the separator. The CO 2 flow rate is 2 kg/h and the extraction is complete after 75 min. The obtained mass from the extractor weighs 87.5 g. Then this extract is subjected to a series of solid liquid extractions with ethanol to get a final mass from separator close to 7.26 g. ). An aliquot of 100 μL of the extract is poured into 1 mL of distilled water and mixed with 300 μL of NaNO 2 (5% w/v). After 5 min, 500 μL of AlCl 3 (2% w/v) is added. The solution has reacted for 6 min followed by neutralization with 500 μL of 1 M NaOH. The solution is stirred vigorously and further diluted by adding 10 mL of milliQ water. After 10 min, the absorbance is measured against a blank at 510-nm UV 2550 Shimadzu spectrophotometer. Measurements are quantified using a calibration curve made with catechin (60-500 ppm), and results are expressed as milligrams of catechin equivalents per gram of dry product (mgCAT/gDB). All samples are analyzed in triplicate.

Antioxidant activity:
The oxygen radical absorbance capacity (ORAC) assay is used as a standard tool to measure the antioxidant activity of polyphenols in extracted Retama reatam seeds via different methods. The antioxidant activity of polyphenols is due to their ability to scavenge free radicals, donate hydrogen atoms or electron, or chelate metal cations. The fundamental of these assays relies on the fluorescence quenching of disodium fluorescein (FS) salt after exposure to AAPH (2,2-azobis(2-amidino-propane) dihydrochloride), which generates oxygen radicals (ROO•) at a constant rate [9]. ORAC assay is carried out following the method developed by Huang et al. [10].

Yield
Extraction of the phenolic compounds using maceration is carried out with two types of solvents of different polarity: 96% ethanol as a polar solvent and hexane as a polar solvent. The extraction yield (mass of extract/mass of dry matter) is used as an indicator of the effects of the maceration conditions. In reflux, ethanol extract yield (15.36%) is maximum for Retama reatam. For Lawsonia inermis also, the trend is similar as observed in the case of refluxing. Whereas the extract yield is relatively lower than that with hexane (6.95%). Figure 2 shows the comparison between yields of two solvents per plant. Generally, the yield is higher in ethanolic extracts than those treated with hexane. More precisely, the best yield is obtained for the ethanolic extract of Retama reatam with 15.36%; therefore, ethanol is about six times more effective than hexane in extracting phenolic compounds from Retama reatam. Similarly, for the other plants, Artemisia is twice, kochia almost three times, while for the Elymus repens, the difference between the two solvents is practically negligible.   Experimentally, ethanol as a polar solvent is recommended and frequently used for the extraction of the phenolic compounds therefore several studies indicated that the phenols are moderate polar compounds [11]. In the case of hexane, the best yield is obtained with Elymus repens (6.296%), followed by Artemisia alba (4.143%). The other plants have almost the same yield, with a slight difference of 2.4% on average.

Total phenolic content
Results show that Retama reatam extract has the highest yield and the most important TPC compared to the other extracts. Comparing the total polyphenol content value by maceration technique lasted after 6 days, 1123-mgGEA/g dry extract, TPC for other extracts following different extractions is lower, this may be due to the extraction time at a given temperature and influenced by ethanol concentration [12]. In fact, a longer extraction time can lead to increase exposure to temperature, light and therefore to increase TPC values relatively to different bioactive compound with diverse structures.
A similar trend is observed for TPC when maceration technique results are compared to SFE extraction ones while Soxhlet extraction still has the lowest TPC value. Indeed, Retama reatam chromatograms from different saharian regions were published in several papers such as Hammouche-Mokrane et al. and Awen et al. [13,14]. These data allowed us to valid the richness of Retama reatam with phenolic compounds.
Similarly, the TFC of the other extracts do not show significant difference from each other, the value varies between 5.366 g EC/g. extract for the SFE extraction and 6.0474 for the ethanolic extraction. On the other hand, the yield is however higher for stirred maceration with a value of 26.7%.
Based on the comparison of the different extraction methods used for polyphenols, the best results are shown for the extracts obtained through the conventional extraction method (ethanolic) indicating a higher selectivity for the extraction of antioxidants compounds.
The advantage of this technique is that it is a high efficiency extraction and can be done easily. In addition, easy maintenance of extraction vessels and improved recovery and repeatability are also better with this conventional technique than with new extraction techniques.

Total flavonoid content
As reported in histogram plots (Figure 3), we notice that Retama reatam TPC values exceeds 20 mg GAE/g for all used extraction techniques except soxhlet method. The obtained data are also validated through comparison with experimental results (ref 1 in histograms, (Figure 4)

Determination of antioxidant activity
The ORAC assay is able to differentiate the suitable Retama reatam extraction method which had a superior antioxidant activity. According to Figure 5 SFE (supercritical fluid extraction) has the highest ORAC value when is followed by SL (solid liquid extraction) and maceration, and soxhlet had mid-range ORAC values.
Furthermore, the finding showed that, there is no correlation between ORAC and TPC Retama extracts at a given extraction method. The lowest Retama TPC is seen when Retama reatam is extracted with SFE technique cannot reflect the high value of ORAC of Retama extracted with the same technique.

CONCLUSION
In this study Retama reatam extracts obtained through ethanolic extraction and supercritical extraction, in three samples, indicates a higher TPC and ORAC for the extraction of herbal materiel from Tunisia southern landscape. The effect of using various extraction methods on polyphenols, flavonoids, and antioxidant activity of Retama reatam plant is shown. The results indicate that TFC is slightly higher in ethanol maceration, whereas between SFE and soxhlet there is no statistically significant difference. Depending on the polyphenol content, the highest grade is for the first maceration (6 days). This study allowed the optimization of a simple, fast and precise method for the determination of the TPC TFC and ORAC in leaves of Retama reatam, which can be used to support the quality assessment of this Tunisia southern herbal material.