Microsoft Word - EJBR2023v13i1art18 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2023; 13(1): 18-30 DOI: http://dx.doi.org/10.5281/zenodo.7618530 Antidiabetic potential of mucilage fraction extracted from Astragalus gyzensis seeds Aicha Tedjani 1*, Zakaria Boual 1, Mohamed Didi Ould El Hadj 1, Touhami Lanez 2, Hakim Belkhalfa 3, Zainab El Alaoui-Talibi 4, Cherkaoui El Modafar 4, Slim Abdelkafi 5, Imen Fendri 6, Didier Le Cerf 7, Pascal Dubessay 7, Cédric Delattre 8, Pierre Guillaume 8, Philippe Michaud 8 1 Laboratory for the Protection of Ecosystems in Arid and Semi-Arid Zones, Kasdi Merbah-University, Ouargla 30000, Algeria 2 Laboratory of Valorisation and Technology of Sahara Resources (VTRS) University Echahid hamma lakhdar, El-Oued 39000, Algeria 3 Scientific and Technical Research Center in Physicochemical Analysis, Tipaza 42000, Algeria 4 Faculty of Sciences and Techniques, University of Cadi Ayyad, Marrakech 40000, Morocco 5 Laboratory of Enzymatic Engineering and Microbiology, Algae Biotechnology Team, National Engineering School of Sfax, Sfax University, Sfax 3038, Tunisia 6 Laboratory of Plant Biotechnology Applied to the Improvement of Plants, Faculty of Sciences, Sfax University, Sfax 3038, Tunisia 7 Department of Chemistry, University of Rouen Normandie, INSA Rouen, CNRS, PBS, 76000 Rouen, France 8 Institute of Pascal, University of Clermont Auvergne, CNRS, Clermont Auvergne INP, 63000 Clermont-Ferrand, France * Corresponding author e-mail: aichated94@gmail.com Received: 10 October 2022; Revised submission: 02 January 2023; Accepted: 03 February 2023 https://jbrodka.com/index.php/ejbr Copyright: © The Author(s) 2023. Licensee Joanna Bródka, Poland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: The objective of the current work is to extract a new mucilage fraction from Astragalus gyzensis Bunge. seeds, which are collected from the El-Oued province (septentrional Algerian Sahara) and evaluated for their antidiabetic potential. The mucilage fraction is obtained using hot water extraction followed by alcoholic precipitation of polysaccharides by cold ethanol (96%). The primary investigation was performed by describing the main structural features of the extract through colorimetric assays, Fourier-transform infrared spectroscopy and thin-layer chromatography analysis using two systems. Biological activity was also monitored by antidiabetic activity by testing the inhibition of α-amylase and α-glucosidase enzymes in vitro. The extraction yield was 20.69%. The chemical composition mainly consisted of 78.60±0.29% carbohydrates, among them 63.92±0.67% neutral sugar, 15.78±0.76% uronic acid, 8.08±0.04% proteins and 2.57±0.05% phenolic compounds. The results obtained by thin-layer chromatography analysis showed the dominance of mannose and galactose. Fourier-transform infrared spectrum showed characteristic bands expected galactomannans. The investigations highlighted the antihyperglycemic effect in a dose-dependent manner by the inhibition of the α- amylase enzyme (IC50=0.8±0.005 mg/mL). These factors make it suitable for the industrial application of dietary supplement fiber made for diabetic individuals. Keywords: Astragalus gyzensis Bunge; Mucilage; Antidiabetic; Galactomannans; Dietary supplement fiber. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 19 European Journal of Biological Research 2023; 13(1): 18-30 1. INTRODUCTION Ethnopharmacology serves as one of the best sources to find a plant molecule which can be used as a template or a lead compound for creating a drug. The traditional medical system continues to play a key role in health care [1]. Floral species earned a prominent place in pharmaceutics because of their therapeutic efficacy and the availability of their potentially active natural chemical constituents [2]. Polysaccharides isolated from plant sources have attracted a great deal of attention in the biomedical area, specifically for their broad spectrum of therapeutic properties and relatively low toxicity. Dietary fibers play a vital role in the prevention of various diseases [3]. Most plants from arid regions belong to the Fabaceae family, which is one of the main botanical families [4]. It is also a commonly used plant family in traditional African medicine [5]. Astragalus is likely the largest and most abundant genus of vascular plants on Earth, comprising nearly 2500–3000 annual and perennial species distributed in all continents, mainly around the Northern Hemisphere, Western North America, South America, Central Asia and tropical East Africa. However, it is not found in Australia. There are numerous species of Astragalus growing in North Africa and the Mediterranean, with 15 species found in the Sahara of Algeria [6]. The plants have been extensively analyzed for three main groups of biologically active compounds: polysaccharides, flavonoids and saponins [7]. Astragalus gyzensis Bunge. is traditionally used in North African medicine to treat snakebites. In Algeria, A. gyzensis is quite common in the deserts and is locally known as «Dlilia» [8]. Reactive oxygen species (ROS) are highly reactive molecules derived from the metabolism of oxygen. They are often byproducts of biological reactions. In vivo, some ROS play positive roles in cell physiology, but they may also damage cell membranes and DNA, inducing oxidation that causes membrane lipid peroxidation and decreased membrane fluidity [9]. On the other hand, many diseases, such as cardiovascular diseases and diabetes, are associated with oxidative stress [10]. Type 2 diabetes is a serious metabolic disorder characterized by defects in the control of blood glucose [11]. Like other nutrients, carbohydrates are mostly digested in the small intestine. However, it is the salivary amylase in the mouth that begins 5% of the initial breakdown of carbohydrates. The glucosidase enzymes (maltase, lactase and sucrase) secreted by intestinal mucosa complete the breakdown of oligosaccharides into monosaccharide units, which are then absorbed by the body and transported to the liver through the portal vein. The body uses these monosaccharides as a direct source of energy [12]. Therapeutic treatments include commercial insulin and oral hypoglycemic medication that help control a patient's glycaemia. Both physical activity and proper diet are key factors in managing diabetes. Dietary fibers, especially soluble fibers, stand out as an important part of a healthy diet directed at treating diabetes due to their ability to increase peripheral insulin sensitivity and reduce blood lipid levels [13]. Reduced postprandial hyperglycemia is one treatment method for early-stage diabetes. This is accomplished by suppressing the carbohydrate-hydrolyzing enzymes, α-glucosidase and α-amylase in the digestive system, to prevent glucose absorption. As a result, inhibitors of these enzymes slow the absorption of glucose, dampening the postprandial plasma glucose spike [14]. The hypoglycemic and antidiabetic effects of several plants used as traditional antidiabetic remedies have been proven, and the mechanisms of hypoglycemic activity in these plants has been studied effectively [15]. Moreover, increasing evidence has confirmed that polysaccharides with different structures from various plants possess different inhibitory activities of α-amylase and α-glucosidase [16]. Antidiabetic drugs combat diabetes by reducing hyperglycemia and its complications through different modes of action, one being improved peripheral utilization of glucose and another that increases antioxidant activity which scavenges free radicals formed due to hyperglycemia [17]. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 20 European Journal of Biological Research 2023; 13(1): 18-30 To our knowledge, the chemical composition and biological benefits of the water-soluble polysaccharides extracted from Astragalus gyzensis Bunge. seeds (PSAG) have not been reported. This work was done to evaluate if the water-soluble polysaccharides could resist the carbohydrate hydrolyzing enzymes and reduce hyperglycemia. The goals of our work were the isolation and partial characterization of the mucilage fraction using colorimetric assays, UV-vis Scanning, Fourier Transform Infrared spectroscopy (FT-IR) and Thin Layer Chromatography analysis (TLC), and the in vitro evaluation of the biological activities by antioxidant and antihyperglycemic activities, which were tested against α-amylase and α-glucosidase enzymes. 2. MATERIALS AND METHODS 2.1. Raw material and chemicals The pods of A. gyzensis were collected in March and April 2019 at their full maturity from Hassi Khalifa in the El-Oued province (septentrional Algerian Sahara). The identification of the plant specimen was confirmed by Dr. Slimani Noureddine (Faculty of Biology, University Echahid Hamma Lakhdar, El-Oued, Algeria). The pods were dried in the shade away from sunlight and moisture for three weeks. The seeds were then manually isolated from the dry pods and stored in kraft paper bags at room temperature. Standard monosaccharides (arabinose, rhamnose, galactose, glucose, mannose, glucuronic acid and galacturonic acid), metahydroxydiphenyl, trifluoroacetic acid (TFA), α-amylase, α-glucosidase, acarbose, p-nitrophenyl α-D- glucopyranoside (p-NPG), 2-chloro-p-nitrophenylα-D-maltotrioside (CNPG3), and 2,2'-diphenyl-1- picrylhydrazyle (DPPH) were purchased from Sigma-Aldrich in Germany. All other chemicals used were of an analytical grade. 2.2. Polysaccharides extraction procedure The polysaccharides extraction procedure followed the method of Addoun et al. [18] with a slight modification. A. gyzensis seeds were ground to a fine powder using an agate mortar and pestle. Ten grams of unground seeds were extracted by hot maceration using distilled water (20% w/v) at 70°C for two hours with moderate stirring (450 rpm). The highly viscous dispersion was put through a fine filter to remove residual debris. The same process was repeated three times before it was centrifugated at 4000 rpm for 15 minutes. The macerate was concentrated (1/3V) by a rotary evaporator at 65°C. The polysaccharides were precipitated by adding three volumes of cold ethanol (96%) and refrigerating for 24 hours in a 4°C environment. The pellet was recovered after centrifugation (4000 g, 4°C, 15 minutes), then washed with acetone multiple times. Finally, the polysaccharides fractions obtained (PSAG) were dried at 50°C for 48 hours, then crushed into a fine powder (<3 mm) by a mechanical blender. Figure 1. Astragalus gyzensis Bunge. (a) Astragalus gyzensis Bunge. plant (b) Astragalus gyzensis Bunge. seeds (April 2019). Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 21 European Journal of Biological Research 2023; 13(1): 18-30 2.3. Chemical Determination 2.3.1. Determination of total sugars content The total sugar content was evaluated by phenol-sulfuric acid method using Glc as the standard [19] with a small modification. Two hundred microliters of each dilution of PSAG was mixed with 200 µL of 5% aqueous solution of phenol in a test tube. Subsequently, 1 mL of concentrated sulfuric acid was rapidly added to the mixture. After allowing the test tubes to stand for ten minutes, they were vortexed for 30 seconds then placed for 30 minutes in a water bath at 90°C for color development. Lastly, light absorption was used at 490 nm. 2.3.2. Determination of neutral sugars content The neutral sugar levels were measured by 1,3-dihydroxybenzen method using Glc as the standard [20] with a minor alteration. Two hundred microliters of each dilution of PSAG was mixed with 200 µL of 0.6% aqueous solution of resorcinol in a test tube. One milliliter of concentrated sulfuric acid was added rapidly to the mixture. The test tubes were left to rest for ten minutes, then vortexed for 30 seconds and placed in a water bath for 30 minutes at 80°C for color development. Then, light absorption was used at 450 nm. 2.3.3. Determination of the uronic acids content The uronic acid content was quantified by m-hydroxydiphenyl assay using Gal. A as the standard [21] with a slight modification. Two hundred microliters of each dilution of PSAG was mixed with 1.2 mL of a 0,12M solution of tetraborate of sodium in a test tube. After ten minutes, the test tubes were vortexed for 30 seconds and placed in a water bath for five minutes at 100°C. Next, the test tubes were removed from the water bath and 20 μL of 0,15% solution of m-hydroxydiphenyl was added, Then, light absorption was used at 520 nm. 2.3.4. Determination of the proteins content Protein content was estimated by Bradford assay using bovine serum albumin as reference [22] with a minor change. First, 500 µL of each dilution of PSAG is mixed with 500 µL of Bradford reagent. Then, they are vortexed for 30 seconds and placed in a dark, in room temperature environment for 30 minutes. Then, light absorption was used at 595nm. 2.3.5. Determination of the phenol content The phenolic content was evaluated with the Folin-ciocalteu reagent using gallic acid as the standard [23] with a slight alteration. One milliliter of each dilution of PSAG was mixed with 0.5 mL of Folin-ciocalteu reagent (diluted ten times with distilled water), then the mixture was left in the dark for five minutes. Next, 2 mL of sodium carbonate (7.5%) was added to the tube, stirred, and stored in the dark at laboratory temperature for 30 minutes. The final absorbance of the solution obtained was read at λ=765 nm. All test reference solutions were prepared in an identical manner, except that the volume of the extract was replaced with distilled water. The results were calculated according to the standard curve and expressed in triplicates (means ±SD, n=3). 2.4. Spectroscopic analyses Ultraviolet-visible spectra of 2.5 mg/mL of PSAG solution (dissolved in distilled water) was scanned with a UV-visible (Shimadzu-1800) scanner using a wavelength of 200–900 nm at 25°C. Fourier transform infrared spectra of PSAG powder (2 mg) was measured according to the spectrum of FT-IR (Nicolet iS5, Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 22 European Journal of Biological Research 2023; 13(1): 18-30 Thermo Fisher Scientific) in the spectral range 400–4000 cm-1. The spectrum performed a smoothing and a correction of the baseline using Origin Pro8 software. 2.5. Monosaccharide composition by Thin Layer Chromatography (TLC) PSAG monosaccharides content was determined through its hydrolysis by TFA 2M at 100°C for four hours [24] followed by thin layer chromatography (TLC) analysis. Standard solutions and hydrolyzed polysaccharides were applied in a Silica gel 60 F254 chromatoplate. After elution with System 1; ethyl acetate, pyridine, water, n-butanol, acetic acid in the proportions 5/4/4/10/2 [25]. System 2; Chloroform, n-butanol, methanol, acetic acid, water in the proportions 4.5/12.5/5/1.5/1.5 [26]. The plates were dried at 100°C for two minutes and detected with nigrum. The Rf values for the separated spots were calculated and compared with Rf values of the pure standards. 2.6. Antihyperglycemic activity The antihyperglycemic activity of PSAG was investigated by evaluating the inhibition of both α-amylase and α-glucosidase activities. 2.6.1. Inhibition of α-amylase activity The inhibition of α-amylase activity was estimated using the methods of Kumar et al. [27] and Kajaria et al. [28] with a slight modification. One hundred eighty microliters of each dilution from 0.1–5 mg/mL of PSAG was added to dry test tubes, with acarbose as the positive control and PBS as the negative control. Then, 90 µL of α-amylase solution (5 IU/L) was added to each tube. The reaction mixtures were pre-incubated for 15 minutes at 37◦C. Next, 500 µL of the substrate CNPG3 solution (0.5 mg/mL) was added using gentle stirring, followed by incubation for ten minutes at 37◦C. The absorbances were measured at λ=405 nm using a (Shimadzu-1800 spectrophotometer). 2.6.2. Inhibition of α-glucosidase activity The inhibition of α-glucosidase activity was estimated using the methods of Bisht et al. [29] and Qian et al. [30] with a slight difference. First, 500 µL of α-glucosidase solution (2IU/L) was introduced to dry test tubes with 100 µL of each dilution from 0.1–5 mg/mL of PSAG, using acarbose as the positive control and PBS as the negative control. The mixture was pre-incubated for 15 minutes at 37◦C. Then, 100 µL of the substrate p- NPG solution (4 mM) was added. The tubes were shaken and incubated for 20 minutes at 37◦C. One milliliter of Na2CO3 (0.2 M) was added to stop the reaction and the absorbances were measured at λ=405 nm using a (Shimadzu-1800 spectrophotometer). The results of the inhibition of both α-amylase and α-glucosidase activities was expressed using the equation of Telagari et al. [31]: Inhibition (%) = ((Acontrol-Asample)/Acontrol)×100 All the experiments were done in triplicate. 2.7. Antioxidant activity by DPPH radical scavenging assay The antioxidant activity of PSAG and ascorbic acid were evaluated by using the DPPH procedure described by Delattre et al. [32]. One milliliter of each dilution from 0.1–5 mg/mL of PSAG or ascorbic acid was added into 1 mL of a DPPH solution at 0.1 mM in ethanol. The solution was aggressively stirred then incubated for 30 minutes at room temperature (25°C) in obscurity. The absorbance was measured at 517 nm using a (Shimadzu-1800 spectrophotometer). The DPPH inhibition (%) was calculated using the equation below: Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 23 European Journal of Biological Research 2023; 13(1): 18-30 Inhibition (%) = (1 - ( Asample/Acontrol)) × 100 where Asample and Acontrol are the absorbances of the sample and ultra-pure water, respectively. 2.8. Statistical analysis The data were analyzed via Origin Pro8 software and Microsoft Excel 2007. 3. RESULTS AND DISCUSSION 3.1. Extraction yield of PSAG Our method was quite effective in the extraction of polysaccharides. The mucilage fraction isolated from Astragalus gyzensis Bunge. seeds were amorphous white powders. The extraction yield was 20.69% w/w. Regarding the extractions procedure, this number of polysaccharides is a higher yield compared to the seeds of other plants in the Fabaceae family, such as fenugreek (10%) [33], Alhagi maurorum Medik. (12.58%) [34], and Caesalpinia ferrea Mart. seeds (9%) [35]. However, it is lower than the yield from Senna tora seeds (35%) [36]. Seed gums are mostly obtained from leguminous plants, the endosperms of the seeds mainly responsible for water solubility [37]. Moreover, this value was higher than amounts of polysaccharides found in other seeds species of the Astragalus genus such as A. lehmannianus (4,8%), A. sericeocanus (3,6%), A. danicus (3,4%), A. cicer (5,9%), A. alpinus (0,6%) and A. tibetanus [38, 24, 39, 40, 41]. Some seeds species collected in the same Saharan zone reported higher values than these, such as A. armatus (4.21%) [42] and A. gombo (6.8%) [43]. 3.2. Chemical composition of PSAG The mucilage fraction is mainly composed of (78.6%) total sugar. This high sugar content confirmed the efficiency of the extraction process [44], among them (63.92%) neutral sugar and (15.78%) uronic acid. Neutral sugar contents are consistent with the biochemical composition of other polysaccharides extracted from Astragalus sp. [24, 38, 39, 40-42]. While the uronic acid content was higher than the values described for other polysaccharides seeds of A. armatus and A. Gombo [42, 43], these changes in biochemical compositions could be attributed to differences in species and the harvest area. Both PSAG extracts contained traces of proteins (8.08%) and polyphenols compounds (2.57%). The elimination of proteins might be difficult, especially when conjugate proteins are present [44]. The use of protein separation techniques can lead to the degradation of polysaccharides and cause them to lose their native structure, because the glycoprotein protein is linked covalently to the polysaccharides moiety [45]. Table 1. Chemical composition of polysaccharides from Astragalus gyzensis Bunge. Seeds. Total sugar (% w/w) Neutral sugar (% w/w) Uronic acid (% w/w) Proteins (% w/w) Phenolic compounds (% w/w) 78.60 ± 0.29 63.92 ± 0.67 15.78 ± 0.76 8.08± 0.04 2.57±0.05 3.3. Spectroscopic analyses 3.3.1. UV-visible Spectrum Scanning The absorption spectra (UV-visible) of the PSAG extract are presented in figure 2. As illustrated, PSAG reaches UV absorption peak at 210 nm, which corresponds with the absorption of the polysaccharides and a larger absorption peak between 250–300 nm, indicated by the low amount of proteins and/or nucleic acids impurities in the extract, which is consistent with previous results [46, 47]. There was no absorption at 620 nm, suggesting the pigment was completely removed as the result described by Tang et al. [44]. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 24 European Journal of Biological Research 2023; 13(1): 18-30 Figure 2. Uv-vis absorption spectra of PSAG (polysaccharides extracted from Astragalus gyzensis Bunge. seeds). 3.3.2. FT-IR analysis FT-IR analysis is a significant tool for obtaining first-hand knowledge and preliminary identification of biopolymers from diversified sources. Galactomannan as a biopolymer has been characterized using FT-IR previously in literature [33]. Figure 3 represents FT-IR spectra of PSAG in the frequency range between 400 cm-1–4000 cm-1. The IR spectra of polysaccharides showed the peaks at 814 cm−1 and 871 cm−1 are related to the presence of anomeric configurations (α and β conformers) and glycosidic linkages attributed to α-D- galactopyranose and β units -D-mannopyranose units, respectively [35]. The absorption peaked in 1029.99 cm- 1, and in 1149.57 cm-1 showed that the constituent sugar cycles in PSAG belong to the pyranose cycle, which are the absorption peaks generated by the vibration of the COC ether bond [48]. The broad band between 1198 cm−1 and 983 cm−1 results from the stretching vibration of C O in C O H bonds (e.g., glycosidic bonds). The peak at 1149 cm−1 corresponds to bending vibrational modes of C O, present in the pyranose ring, while the broad band between 1134 cm−1 and 983 cm−1 is a characteristic contribution of C OH bending [36]. While the broad band at around 2924.09 cm-1 (between 3000 cm−1–2800 cm-1) is attributed to the vibration of the methyl group –CH [49], the peaks between 3200 cm−1–3600 cm-1 are attributed to OH stretching vibration [50]. The absorption of 1647.21 cm-1 was due to bound water [42]. Figure 3. FT-IR spectra of PSAG (polysaccharides extracted from Astragalus gyzensis Bunge. seeds). Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 25 European Journal of Biological Research 2023; 13(1): 18-30 3.3.3. Thin Layer Chromatography (TLC) The polysaccharides extract obtained from the seeds of Astragalus gyzensis Bunge. was hydrolyzed in an acid medium to obtain free monosaccharide units, which were characterized by the TLC technique. The first system showed the sample migrate with two spots: Rf1=0.43 and Rf2=0.54, like those obtained from the migration of the galactose and mannose patterns, respectively. The second system showed that it migrates with four similar spots: Rf1=0.18, Rf2=0.22, Rf3=0.45 and Rf4=0.531, as those obtained from the migration of galacturonic acid, glucuronic acid, galactose and mannose profiles, respectively. This is consistent with the results obtained previously for neutral sugar and uronic acid. Our results are similar to the result of TLC when the hydrolysate galactomannan from the Senna tora (Fabaceae) seeds showed two spots. The spots were identified as galactose and mannose by comparing their Rf values with standards of pure galactose and mannose [36]. Like the result of TLC of galactomannan from Adenanthera avonine L. (Fabaceae), it showed that it was comprised of mannose and galactose unities [13]. This result suggests that the constituent polysaccharides of PSAG are of a galactomannan type. It is well known that seeds of the genus Astragalus are a valuable source of galactomannans, which have been found in 16 species of this genus [24]. Table 2. The Rf values of the separated spots of PSAG (polysaccharides extracted from Astragalus gyzensis Bunge. seeds) and the pure standards (A. Gal : galacturonic acid, A. Glc: glucuronic acid, Ara: arabinose, Gal: galactose, Glc: glucose, Man: mannose and Rha: rhamnose) using system 1 and 2. A. Gal A. Glc Ara Gal Glc Man Rha Standards (system1) 0.15 0.21 0.54 0.43 0.48 0.54 0.69 PSAG (system1) / / / 0.43 / 0.54 / Standards (system2) 0.18 0.22 0.537 0.45 0.51 0.531 0.64 PSAG (system2) 0.18 0.22 / 0.45 / 0.531 / 3.4. Evaluation of antihyperglycemic activity In vitro antihyperglycemic effects of polysaccharides extracted from A. gyzensis Bunge. seeds was quantified and compared to acarbose using a positive control measuring the inhibition of α-amylase and α- glucosidase activity. 3.4.1. Inhibition of α-amylase activity The inhibitory activity of PSAG extract on α-amylase was investigated in this study and the results shown in figure 4. From 0–1 mg/mL of PSAG and acarbose, there was a dose dependent increase in the percentage of inhibition. Then, from 1–2.5 mg/mL of PSAG and acarbose, there was a slight increase. From 2.5–5 mg/mL, PSAG and acarbose possessed certain stability. PSAG extract had a strong inhibitory effect on α-amylase activity with an IC50 value of 0.8±0.005 mg/mL, compared with acarbose as a positive control with an IC50 value of 0.295±0.006 mg/mL. The inhibitory effects of polysaccharides extracted from the plant seeds were considerably better against α-amylase. Two galactomannans fractions from Alhagi maurorum Medik. seeds showed IC50 values of 5.43 mg/mL and 6.81 mg/mL [34]. However, the inhibition of α-amylase activity by a polysaccharides fraction extracted from the seeds of Plantago ciliata Desf. showed an IC50 value of 3.60 mg/mL [18]. Another galactomannan from soybeans, Glycine max (L.) Merrill, is under investigation, as it possesses inhibitory activity against some pancreatic amylase associated with digestion of starches [51]. Two polysaccharides fractions isolated from wheat bran exhibited a competitive inhibition of α-amylase [16]. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 26 European Journal of Biological Research 2023; 13(1): 18-30 Figure 4. Inhibition of α-amylase activity using PSAG (polysaccharides extracted from Astragalus gyzensis Bunge) and acarbose. 3.4.2. Inhibition of α-glucosidase activity α-Glucosidase is a key digestive enzyme that participates in the body's carbohydrate metabolism and cuts glucose from the non-reducing end of the polysaccharides by hydrolyzing the α-1,4-glycosidic bond. α- glucosidase inhibitors are an effective strategy in reducing post-prandial hyperglycemia [52]. The inhibitory activity of PSAG extract on α-glucosidase was investigated in this study and the results shown in figure 5. From 0–0.75 mg/mL of PSAG and acarbose, there was a dose dependent increase in the percentage of inhibition. Then, from 0.75–2.5 mg/mL of PSAG and acarbose, there was a small increase. From 2.5–5 mg/mL, PSAG and acarbose possessed certain stability. The results of the inhibition of α-glucosidase activity showed that PSAG had a weak inhibitory effect on α-glucosidase with an inhibition value of 12.93% at 5 mg/ml respectively, compared with acarbose as a positive control with an inhibition value of 100% at 5 mg/ml. However, the inhibition of α-glucosidase activity by a polysaccharides fraction extracted from the seeds of Plantago ciliata Desf. showed a better IC50 value of 10 mg/mL [18]. Zhu et al. [53] reported a good inhibitory effect of α-glucosidase by a polysaccharides (APS) extract from dried Radix Astragalus. Two polysaccharides fractions that were isolated from wheat bran exhibited a mixed-type non-competitive inhibition of α-glucosidase [16]. The α-glucosidase inhibitory activities of polysaccharides were closely related to their monosaccharide compositions, molecular weights, and type of glycosidic linkages [54]. Further, in vivo experiments confirmed that galactomannan extracted from Retama reatam can reduce the glycemic index of starchy foods and inhibit the surge of postprandial blood glucose level [48]. Figure 5. Inhibition of α-glucosidase activity using PSAG (polysaccharides extracted from Astragalus gyzensis Bunge seeds) and acarbose. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 27 European Journal of Biological Research 2023; 13(1): 18-30 3.5. Evaluation of antioxidant activity by DPPH radical scavenging assay Diabetes is closely associated with oxidative stress because hyperglycemia causes oxidative stress and produces free radicals, which can induce diabetic complications such as endothelial dysfunction and atherosclerosis [55]. The DPPH utilizes a scavenging mechanism causing hydrogen transfer from the antioxidant to hydrazyl DPP (radical) to convert it to hydrazine DPP. This is to avoid the presence of active free radicals, which can degenerate proteins, lipids and DNA in the human body or food, leading to degenerative diseases. Hydrogen transfer occurs through a possible reaction between the radical and the amine or amide groups present in the antioxidant [56]. In this context, the scavenging ability of PSAG on DPPH radical was investigated by comparison with ascorbic acid. PSAG possessed a weak antioxidant activity on DPPH radical with a high value of IC50=1.339 mg/mL. This value is higher than that obtained by Boual et al. [42], a value of IC50=33 μg/mL from a galactomannan extracted from Astragalus armatus. Furthermore, fenugreek galactomannan exhibits little antioxidant activity through lowered lipid peroxidation and elevated levels of antioxidant enzymes [57]. As inferred from FT-IR analysis, amide or amine groups are absent in the spectra of PSAG, therefore, it has low antioxidant activity. The biological activities of polysaccharides are correlated to their structure. The bond type as well as the number and position of branches present in the polymer chain strongly influence the three-dimensional arrangement, and in addition to the molecular size, these factors determine the behavior of the polysaccharides. Physical properties, such as solubility, viscosity, and gelation, can also influence biological activity as they can affect bioavailability. Some studies have proposed that polysaccharides have significantly different average molecular weights, however, fractions with similar monosaccharides compositions may display the same biological activity. Therefore, elucidating the molecular structures of polysaccharides present in medicinal plants is very important to predict their biological behaviour [44]. 4. CONCLUSION In this study, we reported for the first time the isolation, partial characterization and in vitro biological investigations of the water-soluble polysaccharides extracted from Astragalus gyzensis Bunge. seeds. The extraction method used gave a high yield. The chemical composition mainly consisted of neutral sugar. The partial characterization of the polysaccharides proposed they are of a galactomannans type by using TLC and FT-IR. The result showed an anti-diabetic effect through the inhibition of α-amylase, but a low inhibition of α- glucosidase and a low antioxidant effect, thus making it suitable for use in the supplemental dietary fiber of diabetic individuals. Further investigation on purification, total characterization, in silico, in vitro and in vivo studies will be carried out to understand the specific inhibitory mechanisms of antihyperglycemic activity of the polysaccharides extracted from the seeds of Astragalus gyzensis Bunge. Authors’ contributions: AT: conception and design, methodology development, data acquisition, analysis and data interpretation. ZB: revision of the manuscript, study supervision. MDOEH: project administration, methodology development, conception revision. LT and HB: laboratory administration. ZEAT, CEM, SA, IF, DLC, PD, CD, GP and PM: conception revision. All authors discussed the results and contributed to the final manuscript. Conflict of interest: The authors declare no potential conflict of interest. Acknowledgments: The authors would like to thank Mr Ali Tliba from the Laboratory of Valorisation and Technology of Saharan Resources (VTRS), for his assistance. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 28 European Journal of Biological Research 2023; 13(1): 18-30 REFERENCES 1. Noronha M, Pawar V, Prajapati A, Subramanian RB. A literature review on traditional herbal medicines for malaria. South Afr J Bot. 2020; (128): 292-303. 2. Chelladurai GRM, Chinnachamy C. Alpha amylase and alpha glucosidase inhibitory effects of aqueous stem extract of Salacia oblonga and its GC-MS analysis. Braz J Pharmaceut Sci. 2018; 54(1): 1-10. 3. Ullah S, Khalil AA, Shaukat F, Song Y. Extraction and Biomedical Properties of Polysaccharides. Foods. 2019; 8(304): 1-23. 4. Chopra C, Abrol BK, Handa KL. Medicinal Plants of Arid Regions. [in French]. Paris-7e. 1960. 5. Wojciechowski MF, Lavin M, Sanderson MJ. A phylogeny of Legumes (Leguminosae) based on analysis of the plastid MATK gene resolves many well-supported subclades within the family. Am J Bot. 2004; 11: 1846. 6. Abd El-Ghani MM, El-Sayed ASA, Moubarak A, Rashad R, Nosier H, Khattab A. Biosystematic Study on Some Egyptian Species of Astragalus L. (Fabaceae). Agriculture. 2021; 11(125): 1-16. 7. Bratkov VM, Shkondrov AM, Zdraveva PK, Krasteva IN. Flavonoids from the Genus Astragalus: Phytochemistry and Biological Activity. Pharmacogn Rev. 2016; 10(19): 11-32. 8. Chehma A. Catalog of spontaneous plants of the northern Algerian Sahara. [in French]. 2006; 141. 9. Juan CA, de la Lastra JMP, Plou FJ, Pérez-Lebeña E. The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies. Int J Mol Sci. 2021; 22(4642): 1-21. 10. Daiber A, Hahad O, Andreadou I, Steven S, Daub S, Münzel T. Redox-related biomarkers in human cardiovascular disease - classical footprints and beyond. Redox Biol. 2021; 42(101875): 1-21. 11. Dandekar P, Ameeta S, Kumar R. Structure-activity relationships of pancreatic α-amylase and α-glucosidase as antidiabetic targets. Stud Nat Prod Chem. 2021; 70: 381-410. 12. Shah SB, Sartaj L, Ali F, Shah SIA, Khan MT. Plant extracts are the potential inhibitors of α-amylase: a review. MOJ Bioequiv Availab. 2018; 5(5): 270-273. 13. Pinto Vieira, I. G, Mendes FN, da Silva SC, Paim RT, da Silva BB, Benjamin SR, Florean EP, Florindo Guedes MI. Antidiabetic effects of galactomannans from Adenanthera pavonina L. in streptozotocin-induced diabetic mice. Asian Pac J Trop Med. 2018; 11: 116-122. 14. Kashtoh H, Baek K. Recent Updates on Phytoconstituent Alpha-Glucosidase Inhibitors: An Approach towards the Treatment of Type Two Diabetes. Plants. 2022; 11(2722): 1-25. 15. Tran N, Pham B, Le L. Review Bioactive Compounds in Anti-Diabetic Plants: From Herbal Medicine to Modern Drug Discovery. Biology. 2020; 9(252): 1-31. 16. Lv Q, Cao J, Liu R, Chen H. Structural characterization, α-amylase and α-glucosidase inhibitory 2 activities of polysaccharides from wheat bran. Food Chem. 2021; 341(Pt 1): 128218. 17. Khan RU, Rashid AM, Khan S, Ozturk E. Impact of humic acid and chemical fertilizer application on growth and grain yield of rainfed wheat Triticum aestivum L. Pakistan J Agric Res. 2010; 23(3-4): 113-121. 18. Addoun N, Boual Z, Delattre C, Chouana T, Gardarin C, Dubessay P, et al. Beneficial Health Potential of Algerian Polysaccharides Extracted from Plantago ciliata Desf.(Septentrional Sahara) Leaves and Seeds. Appl Sci. 2021; 11(4299): 1-15. 19. Dubois M, Gilles KA, Hamilton JK, Pebers PA, Smith F. Colorimetric method for determination of sugar and relayed substances. Anal Chem. 1956; (28): 350-356. 20. Monsigny M, Petit C, Roche AC. Colorimetric determination of neutral sugars by a resorcinol sulfuric acids micromethod. Anal Biochem. 1988; 175: 525-530. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 29 European Journal of Biological Research 2023; 13(1): 18-30 21. Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal Biochem. 1973; 54: 484-489. 22. Bradford MMA. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.1976; 72: 248-254. 23. Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Vitic. 1965; 16: 144-158. 24. Olennikov DN, Rokhin AV. Polysaccharides of Fabaceae. I. Galactomannan of Astragalus sericeocanus seeds. Chem Nat Comp. 2008; 44(6): 685-687. 25. Hoton-Dorge M. Separation of aldoses and polysaccharides by thin layer chromatography on cellulose and new spraying reagent allowing their sensitive detection. [in French]. Chromatography. 1976; 116: 417-423. 26. Cheng Y, Jia G, Jiang-Sheng Z, Shao-Ping L. Use of HPTLC to Differentiate Among the Crude Polysaccharides in Six Traditional Chinese Medicines. J Planar Chromatogr. 2010; 23: 46-49. 27. Kumar A, Lakshman K, Jayaveera K, Shekar S, Swamy N, Khan S, et al. In vitro α-amylase inhibition and antioxidant activities of methanolic extract of Amaranthus caudatus Linn. Oman Med J. 2011; 26: 166-170. 28. Kajaria D, Tripathi J, Tripathi YB, Tiwari S. In vitro α-amylase and glycosidase inhibitory effect of ethanolic extract of antiasthmatic drug-Shirishadi. J Adv Pharm Technol Res. 2013; 4: 206-209. 29. Bisht S, Kant R, Kumar V. α-glucosidase inhibitory activity of polysaccharide isolated from Acacia tortilis gum exudate. Int J Biol Macromol. 2013; 59):214-220. 30. Qian JY, Bai YY, Tang J, Chen W. Antioxidation and α-glucosidase inhibitory activities of barley polysaccharides modified with sulfation. LWT-Food Sci Technol. 2015; 64: 104-111. 31. Telagari M, Hulltti K. In vitro α-amylase and α-glucosidase inhibitory activity of Adiantum caudatum Linn. and Celosia argentea Linn. extracts and fractions. Ind J Pharmacol. 2015; 47: 425-429. 32. Delattre C, Pierre G, Gardarin C, Traikia M, Elboutachfaiti R, Isogai A, Michaud P. Antioxidant activity of a polyglucuronic acid sodium salt obtained from TEMPOmediated oxidation of xanthan. Carbohydrate Polymers. 2015; 116: 34-41. 33. Rashid F, Hussain S, Ahmed Z. Extraction purification and characterization of galactomannan from fenugreek for industrial utilization. Carbohydrate Polymers. 2017; 180: 88-95. 34. Chakou FZ, Boual Z, Hadj MDOE, Belkhalfa H, Bachari K, El Alaoui-Talibi Z, et al. Pharmacological Investigations in Traditional Utilization of Alhagi maurorum Medik. in Saharan Algeria: In Vitro Study of Anti-Inflammatory and Antihyperglycemic Activities of Water-Soluble Polysaccharides Extracted from the Seeds. Plants. 2021; 10: 2658. 35. Gallão MI, Normando LO, Vieira ÍGP, Mendes FNP, Ricardo NMPS, Brito ES. Morphological, chemical and rheological properties of the main seed polysaccharide from Caesalpinia ferrea Mart. Indust Crops Prod. 2013; 47: 58-62. 36. Pawar HA, Lalitha KG. Isolation, purification and characterization of galactomannans as an excipient from Senna tora seeds. Int J Biol Macromol. 2014; 65: 167-175. 37. Srivastava M, Kapoor VP. Seed galactomannans: an overview. Chem Biodivers. 2005; 2: 295-317. 38. Mestechkina NM, Anulov OV, Smirnova NI, Shcherbukhin VD. Composition and structure of a galactomannan macromolecule from seeds of Astragalus lehmannianus bunge. Appl Biochem Microbiol. 2000; 36(5): 502-506. 39. Olennikov DN, Rokhin AV. Polysaccharides of Fabaceae. II. Galactomannan from Astragalus danicus seeds. Chem Nat Comp. 2009; 45(3): 297-299. 40. Olennikov DN, Rokhin AV. Fabaceae polysaccharides. III. Galactomannan from Astragalus cicer seeds. Chem Nat Comp. 2010; 46(2): 165-168. Tedjani et al. Antidiabetic potential of mucilage fraction from Astragalus gyzensis 30 European Journal of Biological Research 2023; 13(1): 18-30 41. Olennikov DN, Rokhin AV. Polysaccharides of Fabaceae. VI. Galactomannans from seeds of Astragalus alpinus and A. tibetanus. Chem Nat Comp. 2011; 47(3): 343-346. 42. Boual Z, Pierre G, Delattre C, Benaoun F, Petit E, Gardarin C, et al. Mediterranean semi-arid plant Astragalus armatus as a source of bioactive galactomannan. Bioactive Carbohydr Dietary Fibre. 2015; 5: 10-18. 43. Chouana T. Pierre G. Vial C. Gardarin C. Wadouachi A. Cailleu D. et al. Structural characterization and rheological properties of a galactomannan from Astragalus gombo Bunge. seeds harvested in Algerian Sahara. Carbohydrate Polymers. 2015; 1-32. 44. Tang Y, Xiao Y, Tang Z, Jin W, Wang Y, Chen H, et al. Extraction of polysaccharides from Amaranthus hybridus L. by hot water and analysis of their antioxidant activity. PeerJ. 2019; 7: 7149. 45. Horan NJ, Eccles CR. Purification and characterization of extracellular polysaccharide from activated sludges. Pergamon Journals Ltd. 1986; 20(11): 1427-1432. 46. Liu P, Xue J, Tong S, Dong W, Wu P. Structure Characterization and Hypoglycaemic Activities of Two Polysaccharides from Inonotus obliquus. Molecules. 2018; 23: 1-15. 47. Zhang ZP, Shen CC, Gao FL, Wei H, Ren DF, Lu J. Isolation, Purification and Structural Characterization of Two Novel Water-Soluble Polysaccharides from Anredera cordifolia. Molecules. 2017; 22(1276): 1-13. 48. Chouaibi M, Rezig L, Lakoud A, Boussaid A, Hassouna M, Ferrari G, et al. Exploring potential new galactomannan source of Retama reatam seeds for food, cosmetic and pharmaceuticals: Characterization and physical, emulsifying and antidiabetic properties. Int J Biol Macromol. 2019; 124: 1167-1176. 49. Feng L, Yin J, Nie S, Wan Y, Xie M. Fractionation, physicochemical property and immunological activity of polysaccharides from Cassia obtusifolia. Int J Biol Macromol. 2016; 1-35. 50. Hammi KM, Hammami M, Rihouey C, Cerf DL, Ksouri R, Majdoub H. Optimization extraction of polysaccharide from Tunisian Zizyphus lotus fruit by response surface methodology: Composition and antioxidant activity. Food Chem. 2016; 1(184): 9-80. 51. Kashef RKH, Hassan HMM, Afify AS, Ghabbour S, Saleh NT. Effect of Soybean Galactomannan on the Activities of á-Amylase, Trypsin, Lipase and Starch Digestion. J Appl Sci Res. 2008; 4(12): 1893-1897. 52. Gong L, Feng D, Wang T, Ren Y, Liu Y, Wang J. Inhibitors of α-amylase and α-glucosidase: Potential linkage for whole cereal foods on prevention of hyperglycemia. Food Sci Nutr. 2020; 8(12): 6320-6337. 53. Zhu ZY, Luo Y, Dong GL, Ren YY, Chen LJ, Guo MZ, et al. Effects of the ultra-high pressure on structure and α- glucosidase inhibition of polysaccharide from Astragalus. Int J Biol Macromol. 2016; 87: 570-576. 54. Le B, Anh P, Yang S. Polysaccharide Derived from Nelumbo nucifera Lotus. Plumule Shows Potential Prebiotic Activity and Ameliorates Insulin Resistance in HepG2 Cells. Polymers. 2021; 13(11): 1780. 55. Kim M, Kim E, Kwak HS, Jeong Y. The ingredients in Saengshik, a formulated health food, inhibited the activity of α-amylase and α-glucosidase as anti-diabetic function. Nutr Res Pract. 2014; 8(5): 602-606. 56. Abou El Azm N, Fleita D, Rifaat D, Mpingirika EZ, Amleh A, El‐Sayed MMH. Production of Bioactive Compounds from the Sulfated Polysaccharides Extracts of Ulva lactuca: Post‐extraction Enzymatic Hydrolysis Followed by Ion‐ exchange Chromatographic Fractionation. Molecules. 2019; 24(2132): 1-17. 57. Anwar S, Desai S, Mandlik R. Exploring Antidiabetic Mechanisms of Action of Galactomannan: A Carbohydrate Isolated from Fenugreek Seeds. J Complem Integr Med. 2009; 6(1): 1-10.