International Journal of Biological Macromolecules

IDENTIFICATION OF A NOVEL BIOACTIVE GALACTOMANNAN FROM

ASTRAGALUS CRUCIATUS LINK. SEEDS
--Manuscript Draft--

Manuscript Number: IJBIOMAC-D-23-17757

Article Type: VSI: Carbohydrate hydrocolloids

Section/Category: Carbohydrates, Natural Polyacids and Lignins

Keywords: galactomannan, Astragalus cruciatus Link., antihyperglycemic.

Corresponding Author: Aicha TEDJANI

Kasdi Merbah Ouargla University
ALGERIA

First Author: Aicha TEDJANI

Order of Authors: Aicha TEDJANI

Zakaria Boual

Chemsa Ahmed Elkhalifa

Nouhad Amina Righi

Meriem Benyahkm

Nassima Hadri

Abstract: This study aims to identify novel polysaccharides present in Astragalus cruciatus Link.
seeds harvested from the septentrional Algerian Sahara, with a focus on isolating the
mucilage fraction using maceration and ethanol precipitation. The primary structural
analysis was performed using UV-visible spectroscopy, fourier-transform infrared
spectroscopy, X-ray diffraction, thin layer chromatography, high performance liquid
chromatography and gas chromatography-mass spectrometry with electron ionization.
The biological activity was evaluated by measuring the antihyperglycemic effects. The
extraction efficiency was 12.87%. The UV-visible scan indicated a reduced presence of
protein/nucleic acid impurities. Fourier-transform infrared spectroscopy revealed
characteristic bands corresponding to galactomannan. The X-ray diffraction pattern
demonstrated its amorphous structural arrangement. Thin layer chromatography
analysis confirmed the presence of mannose, galactose, glucuronic and galacturonic
acid. While, High performance liquid chromatography and gas chromatography-mass
spectrometry with electron ionization analysis confirm that 71.18% of the extract is
composed of galactomanan with 3.04 Man/Gal ratio. The investigations exposed a
dose-dependent antihyperglycemic effect through the inhibition of α-amylase
IC50=2.58mg/mL. These results suggest the potential suitability of the extract for the
preparation of drugs intended for diabetic subjects.

Suggested Reviewers: Jihen ELLEUCH

jihen.elleuch@gmail.com

Slim SMAOUI
slim.smaoui@cbs.rnrt.tn

Soukaina Bouissil
soukaina.bouissil@etu.uca.fr

Opposed Reviewers:

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1
IDENTIFICATION OF A NOVEL BIOACTIVE
2 GALACTOMANNAN FROM ASTRAGALUS CRUCIATUS LINK.
3
4 SEEDS
5
Aicha Tedjani1*, Zakaria Boual1, Chemsa2, Nouhad Amina Righi1, Meriem Benyahkm1 and Nassima
6
7 Hadri1
1
8 Laboratory for the Protection of Ecosystems in Arid and Semi -Arid Zones, Kasdi Merbah-University,
9 Ouargla 30000, Algeria
2
10 Department of Biology, Faculty of Life and Natural Sciences, University of El Oued 39000, Algeria
11 *Corresponding author: aichated94@gmail.com
12
13 Abstract
14
15 This study aims to identify novel polysaccharides present in Astragalus cruciatus Link. seeds harvested
16 from the septentrional Algerian Sahara, with a focus on isolating the mucilage fraction using
17 maceration and ethanol precipitation. The primary structural analysis was performed using UV-visible
18 spectroscopy, fourier-transform infrared spectroscopy, X-ray diffraction, thin layer chromatography,
19 high performance liquid chromatography and gas chromatography-mass spectrometry with electron
20 ionization. The biological activity was evaluated by measuring the antihyperglycemic effects. The
21
22 extraction efficiency was 12.87%. The UV-visible scan indicated a reduced presence of protein/nucleic
23 acid impurities. Fourier-transform infrared spectroscopy revealed characteristic bands corresponding to
24 galactomannan. The X-ray diffraction pattern demonstrated its amorphous structural arrangement. Thin
25 layer chromatography analysis confirmed the presence of mannose, galactose, glucuronic and
26 galacturonic acid. While, High performance liquid chromatography and gas chromatography-mass
27 spectrometry with electron ionization analysis confirm that 71.18% of the extract is composed of
28
29 galactomanan with 3.04 Man/Gal ratio. The investigations exposed a dose-dependent
30 antihyperglycemic effect through the inhibition of α-amylase IC50=2.58mg/mL. These results suggest
31 the potential suitability of the extract for the preparation of drugs intended for diabetic subjects.
32 Keywords: galactomannan, Astragalus cruciatus Link., antihyperglycemic.
33
34 INTRODUCTION
35
36 The role of natural products in drug discovery is paramount, given their distinctive features that set
37 them apart from conventional synthetic molecules, offering both advantages and challenges to the drug
38 discovery process [1]. Natural products, sourced from diverse environments such as plants, animals,
39 microorganisms, and marine organisms, encompass a broad spectrum of metabolites, serving as
40 intermediate products in biological systems' metabolic processes [2,3]. Mucilage, a high molecular
41 weight gelatinous substance, widely produced by various plant species, has garnered attention.
42 Composed primarily of polysaccharides with trace amounts of proteins, minerals, and lipids, mucilage
43 has been investigated for its monosaccharide composition and polysaccharide structure. Recently, there
44 has been a shift towards understanding the functional properties of mucilage in connection with its
45 underlying chemistry [4]. Plant-derived mucilage, recognized for its diverse health and food-related
46 benefits, including anticancer, enzyme inhibition, immune-stimulation, and anti-diabetic activity, is a
47 valuable active ingredient in pharmaceutical, functional, and nutraceutical product formulations.
48 Structurally, mucilage presents as a complex of polymeric polysaccharides, featuring highly branched
49 structures with monomer units such as L-arabinose, D-xylose, D-galactose, L-rhamnose, and
50 galacturonic acid. Glycoproteins, along with a variety of bioactive compounds like tannins, alkaloids,
51 and steroids, contribute to its composition. Commonly extracted from the seed coat of various plant
52 species, mucilage's structural variations have been linked to biotic/abiotic parameters and changes in
53
plant physiology, as noted in the literature [5-7]. Understanding these structural characteristics is
54
crucial, as multiple studies suggest that the applications and effects of polysaccharides are intricately
55
tied to their specific structural features [7].
56
Exploring the flora of arid environments is crucial for unraveling the biological traits and
57
biogeographical distribution of plant species. While floristic studies offer insights, numerous aspects of
58
plant biology, taxonomy, and ecology remain poorly understood [8]. The specialized plant community
59
thriving in severe climatic conditions of arid steppic rangelands, covering 15 million hectares in the
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 Algerian steppe, holds significant ecological importance [9]. Despite the Algerian Sahara constituting
1 approximately 80% of the total surface area, limited research has been dedicated to understanding the
2 biological resources unique to this region, which are representative of arid environments [10]. The
3 septentrional Sahara boasts a notably scarce flora, a consequence of harsh conditions and limited
4 resources that challenge the spontaneous survival of living organisms [11]. The Saharan flora exhibits
5 distinct modes of adaptation to drought, categorized into ephemeral plants that emerge after rainfall,
6 completing their vegetative cycle before the soil dries, and permanent plants [12]. Within the extensive
7 Fabaceae family of angiosperms [13], Astragalus, the largest genus, stands out for its diverse bioactive
8 metabolites, commonly utilized in pharmaceutical and food industries. Found predominantly in
9 Mediterranean climatic regions of Europe and North Africa, the Sahara Desert in Algeria is home to
10 fifteen documented Astragalus species [14]. Notably, A.cruciatus holds particular renown intraditional
11 North African medicine [15].
12 Disorders related to hyperglycemia, prominently diabetes, pose a significant global public health
13 challenge [16]. While various therapeutic strategies, including insulin replacement, enhancing insulin
14 sensitivity, and oral antidiabetic drugs, have been employed to manage diabetes, inhibiting crucial
15 enzymes such as α-amylase and α-glucosidase remains a popular approach to delay glucose release
16 from dietary starch [17]. Inhibiting α-amylase disrupts starch hydrolysis into oligosaccharides, while
17 inhibiting α-glucosidase suppresses disaccharide hydrolysis [18]. The clinical inhibitor of α-
18 glucosidase, acarbose, is associated with gastrointestinal side effects, including flatulence and diarrhea
19 [19]. These adverse effects are attributed to increased abnormal fermentation of partially digested
20 carbohydrates by gut microbiota, often leading to treatment discontinuation. This emphasizes the
21 necessity for searching new inhibitors with improved safety profiles [20]. Plant polysaccharides have
22 demonstrated an inhibitory effect on carbohydrate digestion enzymes, offering a promising alternative
23 to existing inhibitors with fewer associated side effects [21].
24 To our knowledge, no prior studies have explored the chemical composition and potential health
25 benefits of mucilage extracted from Astragalus cruciatus Link seeds. This study aims to isolate novel
26 polysaccharides from A.cruciatus seeds, utilizing a comprehensive analytical approach, including
27 colorimetric assays, UV-Vis Scanning, Fourier Transform Infrared spectroscopy (FT-IR), X-ray
28 diffraction (XRD), Thin Layer Chromatography (TLC), High Performance Liquid Chromatography
29 (HPLC), and Gas Chromatography-Mass Spectrometry with Electron Ionization (GC-MS-EI) for
30 structural characterization. Additionally, we aim to assess the inhibitory effects of the extracted
31 mucilage on α-amylase and α-glucosidase and the antioxidant activities as part of this investigation.
32
33
MATERIALS AND METHODS
34
35 2.1. General experimental procedures
36 Spectrophotometer (the device is Shimadzu UV-1800, UV–Vis Spectrophotometer) controlled by
37 Agilent Scan software. Fourier transform infrared spectrometer (FT-IR, the Agilent Cary 630 type
38 (Agilent Technologies)). X-Ray Powder Diffraction (PROTO AXRD Benchtop). The HPLC system
39 consisted of a Shimadzu LC-9A isocratic pump (Kyoto) equipped with a refractive index detector, the
40 HPLC system was fitted with a ShodexAsahipak NH2P-50 4E column. TLC analyses were carried out
41 on the precoated silica gel GF254 plates (Qingdao Marine Chemical Co., Ltd., P.R.). GC/MS-EI
42 system was an Agilent 6890 GC / 5973 Network Mass Selective Detector, equipped with an OPTIMA-
43 1MS Accent column (Macherey-Nagel; 30 m, 0.32 mm, 0.25 μm). α-amylase (E.C.3.2.1.1) and α-
44 glucosidase (E.C.3.2.1.20) and all the solvents used for isolation were of analytical grade and the
45 solvents used for HPLC were of HPLC grade.
46 2.2. Plant material
47 The botanical specimen (A. cruciatus Figure 1) was carefully collected from Hassani Abdelkarim,
48 El-Oued (septentrional Algerian Sahara) (GPS: 6.53205N 33.27159E) during the months of March and
49 April in 2019 and was identified through a combination of morphological and taxonomic analyses,
50 including examination of key features such as leaf shape, flower morphology and seed characteristics
51 by Pr. SLIMANI Noureddine (Faculty of Biology, University Echahid Hamma Lakhdar, El-Oued,
52 Algeria).
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 1
2
3
4
5
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7
8
9
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17 Figure1:(a) Astragalus cruciatus Link. plant (b) Astragalus cruciatus Link. Seeds (April 2019).
18 2.3. Extraction and isolation of the mucilage
19 The mucilage component of A. cruciatus was isolated using our previously established method [22],
20 involving hot maceration of seeds (10g) in distilled water (20%w/v) at 70°C for 2 hours with moderate
21 stirring (450rpm). The resulting highly viscous dispersion was subjected to filtration through a fine
22 filter to remove residual debris and the process was repeated three more times, followed by
23 centrifugation (4000rpm/15min). The macerate was then concentrated by rotary evaporation at 65°C to
24 a volume of one-third of its original amount and precipitated by the addition of three volumes of cold
25 ethanol (96%), followed by refrigeration for 24 hours at 4°C. The resulting pellet was recovered via
26 centrifugation (4000g, 4°C, 15min) and washed multiple times with acetone. The resulting mucilage
27 fractions called (PSAC) were then dried at 50°C for 48 hours and pulverized into a fine powder with a
28 mechanical blender with a particle size less than 3mm.
29 2.4. Chemical Determination
30 The mucilage (PSAC) composition was analyzed using colorimetric assays. Total sugars were
31 quantified via the phenol-sulfuric acid method with glucose as the standard [23], while neutral sugars
32 were measured using the 1,3-dihydroxybenzene method with glucose as the standard [24].The content
33 of uronic acids was determined using the m-hydroxydiphenyl assay, with galacturonic acid as the
34 standard [25].The protein content was estimated using the Bradford assay with bovine serum albumin
35 as the reference [26]. The total phenolic compounds were quantified by means of the Folin–Ciocalteu
36 method with gallic acid as the reference compound [27].
37 2.5. UV-visible Spectrum Scanning
38 The extract (PSAC) was dissolved in distilled water at a concentration of 2.5 mg/mL and analyzed
39 using a UV-visible spectrophotometer in the 200–500 nm wavelength range at a temperature of 25°C.
40 2.6. FT-IR spectroscopy
41 The identification of organic functional groups and primary structure of PSAC was performed by
42 analyzing the FT-IR spectrum in the spectral range of 400–4000 cm-1.
43 2.7. XRD characterization
44 The crystal structure of the extract was determined using single crystal X-ray diffraction analysis.
45 Diffraction data was collected at room temperature with Cu Kα radiation (λ = 0.154281 nm) in 2θ
46 ranging from 10◦ to 90◦ with a determinate scanning speed of 0.5◦/min.
47 2.8. Chemical hydrolysis
48 To initiate preliminary investigations, PSAC was subjected to acid hydrolysis using 2M
49 trifluoroacetic acid (TFA) at 100°C for 4 hours. The hydrolyzed product was then analyzed using thin
50 layer chromatography (TLC) and high performance liquid chromatography (HPLC) [28].
51 2.9. Monosaccharide compositions by Thin Layer Chromatography (TLC)
52 TLC analysis of the hydrolysate was performed using two different mobile phases. System 1
53 consisted of a mixture of ethyl acetate, pyridine, water, n-butanol and acetic acid in the proportions
54 5/4/4/10/2[29]. While System 2 consisted of a mixture of Chloroform, n-butanol, methanol, acetic acid
55 and water in the proportions 4.5/12.5/5/1.5/1.5 [30]. To visualize the separated compounds, the plates
56 were developed using the nigrum as revelator and the Rf values for the separated spots were calculated
57
and compared with the Rf values of pure standards.
58
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 2.10. Monosaccharide compositions by High Performance Liquid Chromatography (HPLC)
1 In this study, the sample was analyzed using high-performance liquid chromatography (HPLC)
2 according to a modified method of [31]. The mobile phase consisted of acetonitrile:water (80:20, v/v),
3 and was retained for 15 minutes at a flow rate of 1.0 mL/min. The sample injection volume was 10 μL
4 and the column temperature was maintained at 35°C. The HPLC analysis allowed for the separation
5 and quantification of the monosaccharide compositions present in the sample, based on their retention
6 times and refractive index signals.
7 2.11. Monosaccharide compositions bygas chromatography-mass spectrometry with electron
8 ionization
9 The monosaccharide composition analysis using GC/MS-EI was conducted following the method
10 described by Pierre et al.[32,33].Polysaccharides weighing ten milligrams were hydrolyzed using 2M
11 trifluoroacetic acid (TFA) at a temperature of 120 °C with stirring for 90 minutes. The resulting
12 hydrolysates were then derivatized by trimethylsilylation using BSTFA:TMCS (N,O
13 bis[trimethylsilyltrifluoroacetamide] with 1% trimethylchlorosilane) in a ratio of 99:1. Afterward, the
14 samples were evaporated under dry nitrogen and dissolved in dichloromethane. The same procedure
15 was applied to the standards containing the monosaccharidesAra (Arabinose), Glc (Glucose), Gal
16 (Galactose), GalA (Galacturonic acid), Rha (Rhamnose), and Xyl (Xylose). During the GC/MS-EI
17 analysis, the following parameters were employed: target ion range of 40-800 m/z, injector line
18 temperature set to 250 °C, trap temperature at 150 °C, split ratio of 50:1, helium pressure of 8.8 psi,
19 and helium flow rate of 2.3 mL/min. Ionization was performed at 70 eV. The temperature program
20 involved initially starting at 100 °C for 3 minutes, followed by an increase of 8 °C/min until reaching
21 200 °C, which was maintained for 1 minute. Finally, the temperature was further increased at a rate of
22 95°C/minupto215°C.
23 2.12. Inhibition of α-amylase activity
24 To estimate the inhibition of α-amylase activity, a modified method of Kumar et al.[34] and Kajaria
25 et al.[35]was employed. Briefly, dry tubes were prepared with 180 µL of each dilution of the PSAC
26 sample, ranging from 0.1 to 5mg/mL, along with acarbose as a positive control or phosphate buffer
27 solution (PBS) as a negative control. To each tube, 90 µL of α-amylase solution (5IU/L) was added,
28 and the reaction mixtures were pre-incubated for 15 minutes at 37°C. Following this, 500 µL of 2-
29 chloro-p-nitrophenylα-D-maltotrioside (CNPG3) solution (0.5mg/mL) was added with gentle stirring
30 and the reaction mixtures were incubated for an additional 10 minutes at 37°C. The absorbances were
31 then measured at a wavelength of λ=405 nm to determine the degree of α-amylase inhibition.
32 2.13. Inhibition of α-glucosidase activity
33 To estimate the inhibition of α-glucosidase activity, a modified method of Bisht et al.[36] and Qian et
34 al.[37] was employed. Briefly, dry tubes were prepared with 500µL of α-glucosidase solution (2IU/L)
35 and 100 µL of each dilution of the PSAC sample, ranging from 0.1 to 5mg/mL, along with acarbose as
36 a positive control or phosphate buffer solution (PBS) as a negative control. Then the reaction mixtures
37 were pre-incubated for 15 minutes at 37°C. Following this, 100 µL of p-nitrophenyl α-D-
38 glucopyranoside (p-NPG) solution (4mM) was added with gentle stirring and the reaction mixtures
39 were incubated for an additional 20 minutes at 37°C. The absorbances were then measured at a
40 wavelength of λ=405 nm to determine the degree of α- glucosidase inhibition.
41 The inhibition of both α-amylase and α-glucosidase activities was determined and the results were
42
expressed using the following equation [38]:
43
Inhibition (%) = ((A(control) − A(sample))/A(control)) × 100
44
All the experiments were done in triplicate.
45
2.14. DPPH radical scavenging assay
46
The assessment of the antioxidant potential of PSAC and ascorbic acid was conducted through the
47
utilization of the DPPH radical-scavenging method, as elucidated by Delattre et al. in their prior work
48
[32]. In this analytical procedure, 1 milliliter of various dilutions, ranging from 0.1 to 5 milligrams per
49
milliliter (mg/mL), of PSAC and ascorbic acid, respectively, were introduced into 1 milliliter of a 0.1
50
51 mM DPPH solution in an ethanol medium. Subsequently, this concoction was subjected to vigorous
52 agitation, followed by a 30 minute incubation period at a consistent ambient temperature of 25°C
53 within a light-obscured environment. The optical absorbance of the resultant mixture was quantified at
54 a wavelength of 517 nanometers, utilizing a Shimadzu-1800 spectrophotometer, renowned for its
55 precise light absorption measurements.
56 The DPPH inhibition percentage, indicative of the degree of radical scavenging and thus the
57 antioxidant efficacy, was computed employing the ensuing formula:
58 Inhibition (%) = (1 - (Asample/Acontrol)) × 100
59
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 Herein, Asample signifies the absorbance attributed to the sample, encompassing either PSAC or ascorbic
1 acid in conjunction with DPPH, while Acontrol pertains to the absorbance value corresponding to
2 ultrapure water, providing a baseline reference for the absorbance of the DPPH solution.
3 2.15. Statistical analysis
4 The data obtained from the experiments were analyzed using Microsoft Excel 2007 and origin Pro8.
5 3. RESULTS AND DISCUSSIONS
6 3.1. Chemical composition of PSAC
7 The mucilage fraction extracted from A cruciatus seeds appeared as a white powder and its
8 extraction yield is presented in the Table1, i.e., 128.7g/kg (12.87% w/w). The high proportion (85.47 ±
9 0.2%) of total sugar in the mucilage confirms that it is predominantly composed of
10 carbohydrates.Compared to other Astragalus species seeds, such as A. cicer (5.9%) [39], A.
11 lehmannianus (4.8%) [40], A. sericeocanus (3.6%) [28], A. danicus (3.4%) [41], A. alpinus (0.6%), and
12 A. tibetanus[42], the carbohydrate yield from the seeds of the studied plant was notably higher. It also
13 surpassed the carbohydrate content of seeds from endemic plant species in the septentrional Algerian
14 Sahara, such as A. armatus (4.21%) [43] and A. gombo (6.8%) [44], but was lower than A. gyzensis
15 seeds (20.69%) [22]. This suggests the potential of A. cruciatus seeds as a rich source of dietary
16 carbohydrates.
17 Allocation of resources to carbohydrate storage in plants is a key trait influencing ecological and
18 economic strategies. Species storing water-soluble poly- and oligosaccharides as primary carbon
19 storage compounds often exhibit a slow economic strategy, characterized by conservative resource use
20 and long-term persistence under stressful conditions, especially drought [45]. Modifications in
21 structural sugars, including those forming part of cell wall components, play a crucial role in conferring
22 drought tolerance to plants. The major constituents of the plant cell wall, such as cellulose,
23 hemicelluloses (mannan, xyloglucan, xylan, and mixed-linked glucan), and lignin, provide mechanical
24 strength and support to the plant cell. Changes in the biosynthesis and deposition of these cell wall
25 components can enhance a plant's resistance to drought stress by strengthening the cell wall and
26 improving water retention [46].
27 According to Tang et al.[47], Plant polysaccharides can be classified into intracellular polysaccharides,
28 extracellular polysaccharides and cell wall polysaccharides. Different methods are used to extract these
29 polysaccharides from different plant tissues and the extraction efficiency can be improved by
30 optimizing the extraction conditions. Studies have shown that the extraction rate of polysaccharides is
31 particularly sensitive to temperature, with optimal extraction rates observed at temperatures below
32 92°C. However, the use of certain acidic extraction and purification methods, such as trichloroacetic
33 acid (TCA), can lead to the degradation of certain polysaccharides. Therefore, careful selection of
34 extraction and purification methods is critical for obtaining high-quality plant polysaccharides [48].
35 Biochemical analysis of the extracted mucilage revealed that it is primarily composed of neutral sugars
36 (64.13± 1.6%) and uronic acid (19.97± 1.48%). The neutral sugar content is consistent with the typical
37 biochemical composition of other polysaccharides extracted from the seeds of Astragalus species
38 [40,28,41,39,42,43,22]. The uronic acid content of the extracted polysaccharides was found to be
39 consistent with that of polysaccharides from A. gyzensis seeds [22], and notably higher than the values
40 reported for polysaccharides extracted from other types of seeds. A. armatus and A. gombo[43,44].The
41 observed differences in biochemical composition can likely be attributed to variations in both the plant
42
species and the harvest area. The soil type, climatic conditions and geographical location can all impact
43
the biochemical composition of plant tissues, including polysaccharides. These findings suggest that
44
the mucilage extracted from this species could have similar physicochemical properties and potential
45
applications as other Astragalus-derived polysaccharides, such as in the food, pharmaceutical and
46
cosmetic industries. The extract was found to contain detectable levels of both protein (4.84±0.16%)
47
and polyphenolic compounds (2.72 ± 0.37%).Efforts to fully remove the protein component were
48
unsuccessful, possibly due to the presence of polysaccharide conjugates challenging to separate [47].
49
Caution is warranted in using protein separation techniques, as they may lead to polysaccharide
50
51 degradation and loss of native structure due to the covalent linkage between protein and polysaccharide
52 moieties within glycoproteins [49].
53
54 Table 1 Biochemical characterization of polysaccharides from Astragalus cruciatus Link. seeds.
55 Extraction Total sugar Neutral Uronic acid Proteins Phenolic
56 yield (% w/w) sugar (% w/w) (% w/w) compounds
57 (% w/w) (% w/w) (% w/w)
58 PSAC 12.87 85.47 ± 0.2 64.13± 1.6 19.97± 1.48 4.84±0.16 2.72±0 0.37
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 3.2. UV-visible spectrum scanning
1 To ensure an accurate structural analysis of the mucilage, it is important to first determine its purity by
2 estimating the proportion of its polysaccharide components and detecting the presence of impurities.
3 The term "total sugars" encompasses monosaccharides, oligosaccharides and polysaccharides and
4 distinguishes them from other non-sugar components (such as proteins, lipids, aromatic compounds,
5 minerals, etc.) that may be present [50].
6 The Figure 2 displays the UV-visible absorption spectra of the (PSAC) extract, which showed
7 distinct peaks at 210 nm and between 250 and 300 nm. The peak at around 190 nm is characteristic of
8 polysaccharides, while the peak appear around 280 nm is indicative of lower protein and/or nucleic
9 acid impurities [50,51].Additionally, there was no absorption observed at 620 nm, indicating complete
10 removal of the pigment [47].These findings are consistent with previous results obtained through
11 colorimetric assays.
12
13
14 PSAC
4,0
15
16 3,5

17 3,0

18 2,5

19
Abs

2,0
20
1,5
21
1,0
22
0,5
23
24 0,0
200 300 400 500 600 700 800 900
25 Wavelength(nm)
26
27 Fig. 2: UV-vis absorption spectra of PSAC..
28
29 3.3. FT-IR spectroscopy analysis
30 In the field of polysaccharide structural analysis, IR spectroscopy has emerged as a potent
31 technique. This approach is highly sensitive to the position and anomeric configuration of glycosidic
32 bonds, enabling the identification and characterization of various polysaccharides [53].The Figure 3
33 displayed the characteristic bands that have been frequently reported in the literature for
34 galactomannans [43]. Analysis of PSAC structure was conducted using FT-IR in the frequency range
35 of 400 cm-1 to 4000 cm-1, which allowed for the identification of key absorption peaks. Results
36 revealed the presence of broad absorption peak at approximately 3421.72 cm-1 that suggest the
37 presence of hydroxyl group, as reported by Chien et al.[54]. The peak observed in the 2800 to 3000 cm-
38
1
region may be attributed to the vibrational motion of the methyl group –CH. This finding is consistent
39 with previous studies, which have reported similar spectral features [55].The absorption peak observed
40 at 1647.21 cm-1 is likely attributed to the presence of bound water in the sample [43]. The peak
41 observed at 1373.32 cm-1 is indicative of the deformation of the C–H group [56]. The spectral features
42 observed in the 900-1200 cm-1 region correspond to the C–C–O, C–OH and C–O–C groups [57].The
43 absorption peaks observed at 1029.99 cm-1 and 1149.57 cm-1 are indicative of the constituent sugar
44 cycles in the PSAC, specifically belonging to the pyranose cycle. These peaks are generated by the
45 vibrational motion of the COC ether bond. The absorption peaks observed at 815–820 cm-1 and 873–
46 878 cm-1 are indicative of the presence of α and β anomeric configurations and glycosidic bonds in the
47 sample, attributed to α-D-galactopyranose and β-D-mannopyranose, respectively [58].
48
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 PSAC
1 100

2 90
3
80
4

Transmittance (%)
5 70

6 60
7
50
8
9 40

10
30
11
12 20
4000 3500 3000 2500 2000 1500 1000 500
13 Wavenumber (cm )
-1

14
15
16 Fig.3FT-IR spectra of PSAC.
17
18 3.4. XRD characterization
19 The X-ray diffraction (XRD) patterns obtainedthe (Figure 4)confirm the amorphous nature of the
20 PSAC, as indicated by the presence of a broad peak around 2θ = 20 ◦, which is characteristic of
21 amorphous structures. This finding is consistent with previous studies that have reported similar XRD
22 patterns related to amorphous galactomannan extracted from Delonixregia seeds[59]. Polysaccharides
23 are known to interact with water molecules, leading to structural transitions that can affect their
24 molecular mobility and functional properties, particularly in relation to amorphous-crystalline
25 transitions [60]
26
27 500 PSAC

28
29 400
30
31 300
-1

32
Counts S

33
200
34
35
100
36
37
38 0
10 20 30 40 50 60 70 80
39 2 Theta (deg)
40
41 Fig.4XRD pattern of PSAC.
42
43 3.5. Thin Layer Chromatography (TLC)
44 The hydrolysis of the PSAC extract in an acidic environment resulted in the liberation of individual
45 monosaccharide units, which were then subjected to characterization through the use of thin layer
46 chromatography (TLC) utilizing two different systems. The first system (Figure 5.a)revealed two
47 distinct spots in the sample's migration pattern, Rf1= 0.43 and Rf2= 0.54 which corresponded to the
48 migration patterns of galactose and mannose, respectively. In the second system(Figure 5.b) four spots
49 were observed in the sample's migration pattern, Rf1= 0.18, Rf2= 0.22, Rf3 = 0.45 and Rf4 = 0.531 which
50 corresponded to the migration patterns of galacturonic acid, glucuronic acid, galactose and mannose,
51 respectively. These findings are in agreement with the results obtained previously from the colorimetric
52 analysis of neutral sugars and uronic acids. The outcome of the thin layer chromatography (TLC)
53 separation of the extract using the first system indicated the presence of a galactomannan-type
54 polysaccharide in the sample. On the other hand, the use of the second system suggested the presence
55 of galactomannan and pectin-type polysaccharides in the extract. Pectin, which is mainly composed of
56 D-Galacturonic, is a predominant component in many plant tissues. D-Glucuronic, a key component of
57 various natural polysaccharides, is a vital element in metabolic processes that occur within living
58 organisms [61].The findings of our study exhibit a resemblance to the thin layer chromatography
59
(TLC) analysis of a hydrolyzate of guar galactomannansusing two monophasic solvent systems: first,
60
61
62
63
64
65
 n-butanol/ethanol/water (4/1/1) and subsequently, n-butanol/acetic acid/water (4/1/1). The obtained
1 results from the TLC revelation demonstrated the presence of two distinct spots that corresponded to
2 D-mannose and D-galactose [62].The hydrolysis of the polysaccharide isolated from unripe pods of
3 Caesalpiniaspinosa in an acidic medium liberated free monosaccharide units, which were subsequently
4 characterized using thin layer chromatography (TLC) techniques. The results of the TLC analysis
5 revealed that the hydrolyzed galactomannan sample migrated with the same R f value as a 1:1 mixture
6 of galactose and mannose monosaccharide units[63].
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Fig.5Chemical characterization of PSAC (a) TLC chromatograms of PSAC by the First system. (b)
23
24 TLC chromatograms of PSAC by the Second system (A. Gal : galacturonic acid, A. Glc: glucuronic
25 acid, Ara: arabinose, Gal: galactose, Glc: glucose, Man: mannose and Rha: rhamnose).
26
27 3.6. High Performance Liquid Chromatography (HPLC)
28 The chromatogram in Figure 6 and 7 displays the monosaccharide composition of the pure standards
29 (galactose and mannose) and the extract, respectively.Which reveals the separation of various sugars
30 present in the PSAC hydrolysat. The detailed information for each sugar is summarized in Table 2. It is
31 important to note that the total percentage for neutral sugar did not add up to 100% The total amount of
32 neutral sugar obtained by HPLC was approximately 49.556%, which is lower than the value obtained
33 from the colorimetric assay for neutral sugar analysis, where the result showed 64.13%. The
34 monosaccharide analysis of PSAC revealed that the extract contained approximately 71.82% mannose
35 and 28.16% galactose, which suggests the presence of galactomannan. The ratio Man/Gal of the
36 galactomannan in the present study was (2.54) and this value can significantly impact its properties.
37 Moreover, the solubility of galactomannan from the present work was found to be lower in water. This
38 finding suggests that the solubility of galactomannan in water decreases as the number of galactose side
39 chains decreases [64]. Varying ratios have been reported in numerous plant species, such as locust bean
40 gum (approximately 4:1), tara gum (about 3:1), guar gum (about 2:1) and fenugreek gum (about 1:1),
41 according to several studies [65].
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57 Fig. 6 HPLC chromatogram of the pure standards (galactose and mannose).
58
59
60
61
62
63
64
65
 1
2
3
4
5
6
7
8
9
10
11
12
13
14 Fig. 7 HPLC chromatogram of monosaccharide compositions of PSAC from Astragaluscruciatus Link.
15
16 Table 2 Monosaccharide composition of PSAC from Astragaluscruciatus Link. seeds.
17
18 PSAC
19
20 Man (%) 71.82
21
22 Gal (%) 28.16
23
24
Neutral sugar (%) 49.556
25 (Man/Gal) ratio 2.54
26
27
28 3.7. Monosaccharide compositions by gas chromatography-mass spectrometry with electron
29 ionization (GC-MS-EI)
30 The determination of the monosaccharide composition of the plant extract is confirmed by GC-
31 MS-EI analysis after trimethylsilylation (Figure 8and Table 3). It is important to note that the total
32 percentage of neutral sugars did not reach 100% due to the presence of uronic acids, proteins, phenolic
33 compounds and other constituents still present in the mucilage fraction even after the extraction
34 process. The total amount of neutral sugar present in the extract was approximately 71.18% for PSAC,
35 suggesting the presence of galactomannans. The monosaccharide composition for PSAC clearly
36 demonstrates the predominant presence of galactomannan with 74.22% D-mannose and 24.37% D-
37 galactose, with a Man/Gal ratio of 3.04, Small traces of pectic derivatives (visible between 10-12 min)
38 and a small trace of D-glucose were also observed.
39 This result is consistent with the presence of galactomannan found in polysaccharide extracts
40 from Fabaceae seeds. The monosaccharide composition of the seed polysaccharides and the ratio
41 between galactose and mannose are characteristic of Fabaceae galactomannans. Molecular, chemical
42 and structural variations leading to differences in functional behavior and distribution contribute to the
43 wide variety of plant polysaccharides that exist [66]. The Man/Gal ratio of seed galactomannan is close
44 to that of tara gum. Compared to other galactomannans extracted from seeds of different leguminous
45 plants, the Man/Gal ratio was higher than those estimated for Astragalus gombo Bunge (1.7) [44],
46 Astragalus armatus (1.6) [43] and Alhagi maurorum Medik. (2.2) [67].
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23 Fig. 8 GC-MS-EI profile after trimethylsilyl-O-glycosides derived from polysaccharides of
24
25 Astragaluscruciatus Link. seeds.
26
27 Table 3 GC-MS-EI profile after trimethylsilyl-O-glycosides derived from polysaccharides of
28 Astragaluscruciatus Link. seeds.
29 PSAC
30
31 Man (%) 74.22±2.82
32
33 Gal (%) 24.37±2.76
34
35 Neutral sugar (%) 71.18
36 3.04
(Man/Gal) ratio
37
38
39 3.8. Evaluation of antihyperglycemic activity
40
The mucilage fraction of A. cruciatus seeds was evaluated for its in vitro antihyperglycemic
41
properties by quantifying its inhibitory effect on the activities of α-amylase and α-glucosidase and
42
comparing it with acarbose, a positive control (as illustrated in Figure 9 and 10).
43
The data presented in Figure 8 illustrated that the percentage of inhibition of α-amylase increased in
44
a dose-dependent manner from 0 to 1 mg/mL of PSAC and acarbose, followed by a slight increase
45
from 1 to 2.5 mg/mL. Subsequently, there was a plateau in the inhibitory effect of PSAC and acarbose
46
from 2.5 to 5 mg/mL. The results of this study demonstrated that PSAC exerts a potent inhibitory effect
47
48 on α-amylase with an IC50 value of 2.58±0.56 mg/mL, which is superior to that of acarbose
49 (0.295±0.006 mg/mL). Additionally, Figure 9 depicted a similar trend where the percentage of
50 inhibition of α-glucosidase increased in a dose-dependent manner from 0 to 0.75 mg/mL of PSAC and
51 acarbose, followed by a marginal increase from 0.75 to 2.5 mg/mL. Subsequently, there was a plateau
52 in the inhibitory effect of the extract and positive control from 2.5 to 5 mg/mL. The extract exhibited a
53 weak inhibitory effect on α-glucosidase, with an inhibition value that exceeded 12.38% at 5 mg/mL,
54 while acarbose exhibited complete inhibition at the same concentration. As per the findings reported in
55 other study, it was observed that Guar galactomannan has a direct noncompetitive inhibitory effect on
56 α-amylase, which suggests the possibility of a direct binding between the galactomannan and the
57 enzyme. The resultant formation of a galactomannan–α-amylase complex may render the enzyme
58 inactive, leading to the observed inhibitory effect [68].The inhibitory effect of the fenugreek seed
59 endosperm galactomannan on both α-amylase and α-glucosidase was observed to be dose-dependent.
60 The IC50 values are 21.08±0.085 and 67.17±5.15 μg/mL for α-amylase and α-glucosidase, respectively
61
62
63
64
65
 [69].Either, the polysaccharides extracted from Astragalus gyzensis Bunge. was observed to exhibit a
1 low inhibitory effect on α-glucosidase, while highlighting a prominent antihyperglycemic effect due to
2 the inhibition of α-amylase enzyme (IC50=0.8 mg/mL) [22].Our findings are inconsistent with a
3 previous study which reported a significant α-glucosidase inhibitory effect of an APS extract obtained
4 from dried Radix Astragalus. This inconsistency may be due to differences in the chemical
5 composition of the polysaccharides [70]. In addition, an in vitro evaluation of the effect of a
6 galactomannan from Fenugreek (Trigonella foenum-graecum) seeds on glucose uptake by
7 hemidiaphragm was conducted. The results of this study demonstrate that the galactomannan exhibited
8 enhanced glucose uptake [71].Furthermore, the administration of Adenanther apavonina L.
9 galactomannan at concentrations of 1% and 2% for a duration of 21 days resulted in a significant
10 reduction in glycemia when compared to diabetic control groups [72].
11
12 100 Acarbose
13
14 PSAC
15 80
16
17
Inhibition (%)

18 60
19
20
21 40
22
23
20
24
25
26 0
27
0 1 2 3 4 5
28 Concentration (mg/mL)
29
30
Fig.9 Inhibition of α-amylase activity using PSAC and Acarbose.
31
32
33 100 Acarbose
34 PSAG
35
36 80
37
Inhibition (%)

38
39 60
40
41
40
42
43
44 20
45
46
47 0
48 0 1 2 3 4 5
49
50 Concentration (mg/mL)
51
52
Fig.10Inhibition of α-glucosidase activity using PSAC and Acarbose.
53
54 3.9. Evaluation of DPPH radical scavenging activity
55 Oxidative stress stands as a pivotal factor in both the onset and advancement of diabetes and its related
56 complications. The equilibrium between the generation of reactive oxygen species (ROS) and the
57 body's inherent antioxidant defense mechanisms is a key determinant of cellular vitality. Diabetes, in
58 particular, showcases a proclivity for chronic hyperglycemia and mitochondrial aberrations, which
59 converge to augment ROS production, thereby intensifying the state of oxidative stress. In case of
60
61
62
63
64
65
 diabetes, It is crucial to implement approaches focused on diminishing oxidative stress, among which
1 antioxidant therapy emerges as a promising intervention[73].DPPH, a widely used free radical, serves
2 as a rapid, cost-effective method to assess antioxidant capabilities. It is a popular choice for antioxidant
3 assessment. In various in vitro studies, plant extracts have demonstrated the ability to effectively
4 scavenge DPPH radicals, highlighting their antioxidant potential[74].
5 In the context of this study, our objective was to evaluate the scavenging capacity of PSAC concerning
6 the DPPH radical, with a comparative analysis against ascorbic acid employed as a positive control.
7 The Figure 11 illustrates the electronic absorption spectra depicting the interaction between DPPH• and
8 PSAC. Notably, the polysaccharide extract PSAC demonstrated a diminishing effect on absorption as
9 its concentration increased. However, both PSAC and ascorbic acid exhibited relatively modest
10 antioxidant activity when challenged by the DPPH radical, as evidenced by a relatively high IC50 value
11 of 1.34 mg/mL.This IC50 value markedly contrasts with the findings of Boual et al.[42], who reported a
12 substantially lower IC50 value of 33 μg/mL for a galactomannan extracted from Astragalusarmatus.
13 Also, it is noteworthy that fenugreek galactomannan, demonstrated limited antioxidant activity, which
14 was associated with a reduction in lipid peroxidation and an elevation in the levels of antioxidant
15 enzymes [57].
16 The biological activities of polysaccharides are intricately linked to a range of structural factors.
17 These include molecular weight, the adoption of the triple-helix conformation, the composition of
18 monosaccharides, the mode of glycosidic bond linkage, and the presence of polysaccharide conjugates.
19 These structural characteristics collectively influence the bioactive properties and functional
20 capabilities of polysaccharides[75].
21 00 mg/ml
22 0,025 mg/ml
0,75
0,05 mg/ml
23 0,70 0,1 mg/ml
0,7mg/ml
24 0,65
25
0,60
26
0,55
27
Abs

0,50
28
29 0,45

30 0,40

31 0,35

32 0,30
33 400 450 500 550 600
34 Wavelength (nm)
35
36 Fig.11Electronic absorption spectra of 0.1 mM DPPH· interaction with PSAC.
37
38 4. CONCLUSION
39
40 Our research has successfully extracted and identified a novel galactomannan from the mucilage
41 fraction of A.cruciatus seeds, demonstrating a mannose to galactose (Man/Gal) ratio of 3.04. In vitro
42 biological assays were conducted to explore its potential as an antidiabetic agent. The results indicate
43 that this galactomannan exhibits significant antihyperglycemic effects, primarily by inhibiting α-
44 amylase activity. Although it shows limited inhibition of α-glucosidase and possesses minimal
45 antioxidant activity against DPPH• radicals, these findings highlight its potential for incorporation into
46 antidiabetic drug formulations. Future studies will focus on the purification and comprehensive
47 characterization of this galactomannan. This will involve in silico, in vitro, and in vivo approaches to
48 elucidate its specific inhibitory mechanisms and evaluate its efficacy as a therapeutic agent for
49 managing hyperglycemia.
50
51 Declarations
52
53 Ethical Approval This study is not applicable to both human and/oranimal studies that require ethical
54 approval.
55 Competing Interests The authors declare that they have no knowncompeting financial interests or
56 personal relationships that could haveappeared to influence the work reported in this paper.
57 Author contributions All authors contributed to the study AT: corresponding author, conception and
58 design, methodology development, data acquisition, analysis and data interpretation, english revision.
59 ZB: study supervision. AEC: conception revision. NAR, MB, NH: data acquisition. All authors
60
61
62
63
64
65
 commented on previous versions of the manuscript. All authors read and approved the final
1 manuscript.
2 Funding The authors declare that no funding was received for this study.
3 Data Availability The data that supports the findings of this study are available within the article.
4
5
6
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Abstract

IDENTIFICATION OF A NOVEL BIOACTIVE

GALACTOMANNAN FROM ASTRAGALUS CRUCIATUS LINK.
SEEDS
Aicha Tedjani1*, Zakaria Boual1, Chemsa2, Nouhad Amina Righi1, Meriem Benyahkm1 and Nassima
Hadri1
1
Laboratory for the Protection of Ecosystems in Arid and Semi -Arid Zones, Kasdi Merbah-University,
Ouargla 30000, Algeria
2
Department of Biology, Faculty of Life and Natural Sciences, University of El Oued 39000, Algeria
*Corresponding author: aichated94@gmail.com

Abstract
This study aims to identify novel polysaccharides present in Astragalus cruciatus Link. seeds harvested
from the septentrional Algerian Sahara, with a focus on isolating the mucilage fraction using
maceration and ethanol precipitation. The primary structural analysis was performed using UV-visible
spectroscopy, fourier-transform infrared spectroscopy, X-ray diffraction, thin layer chromatography,
high performance liquid chromatography and gas chromatography-mass spectrometry with electron
ionization. The biological activity was evaluated by measuring the antihyperglycemic effects. The
extraction efficiency was 12.87%. The UV-visible scan indicated a reduced presence of protein/nucleic
acid impurities. Fourier-transform infrared spectroscopy revealed characteristic bands corresponding to
galactomannan. The X-ray diffraction pattern demonstrated its amorphous structural arrangement. Thin
layer chromatography analysis confirmed the presence of mannose, galactose, glucuronic and
galacturonic acid. While, High performance liquid chromatography and gas chromatography-mass
spectrometry with electron ionization analysis confirm that 71.18% of the extract is composed of
galactomanan with 3.04 Man/Gal ratio. The investigations exposed a dose-dependent
antihyperglycemic effect through the inhibition of α-amylase IC50=2.58mg/mL. These results suggest
the potential suitability of the extract for the preparation of drugs intended for diabetic subjects.
Keywords: galactomannan, Astragalus cruciatus Link., antihyperglycemic.
Declaration of Interest Statement

DECLARATION OF INTEREST STATEMENT

Not applicable.
Cover Letter

Aicha Tedjani November 22th, 2023

Laboratory for the Protection of Ecosystems

in Arid and Semi-Arid Zones
Kasdi Merbah-University,
Ouargla 30000, Algeria
Email: aichated94@gmail.com

Cover letter

Dear Editor,

On behalf of my co-authors, I wish to submit a research article entitled “ I

IDENTIFICATION OF A NOVEL BIOACTIVE GALACTOMANNAN FROM
ASTRAGALUS CRUCIATUS LINK. SEEDS”.
For consideration by the Journal: International Journal of Biological
Macromolecules
I confirm that this work is original and has not been published elsewhere, nor it is
currently under consideration for publication in another journal.
In this paper, We have successfully identified a novel galactomannan from the
mucilage fraction of Astragalus cruciatus Link. seeds and investigated its potential as
an antidiabetic agent through in vitro biological assays.

We have no conflicts of interest to disclose.

Please address all correspondence concerning this manuscript to me at:
aichated94@gmail.com
Thank you for your consideration of this manuscript.
Sincerely,
Aicha Tedjani