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α-Glucosidase inhibition by Tunisian Scabiosa arenaria Forssk. extracts
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BIOMAC 4969 1–7
International Journal of Biological Macromolecules xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Review
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␣-Glucosidase inhibition by Tunisian Scabiosa arenaria Forssk. extracts
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Malek Besbes Hlila a,1 , Habib Mosbah b,∗,1 , Kawther Majouli c , Kamel Mssada d , Hichem Ben Jannet e , Mahjoub Aouni a , Boulbaba Selmi a a Laboratory of Transmissible Diseases and Biological Active Substances, Faculty of Pharmacy, University of Monastir, Avenue Avicenne, 5000 Monastir, Tunisia b Higher Institute of Biotechnology of Monastir, Avenue Taher Hadded, BP 74, 5000 Monastir, Tunisia c Research Laboratory in Biochemistry, Faculty of Medicine, University of Monastir, Avenue Avicenne, 5019, Tunisia d Laboratory of Bioactive Substances, Biotechnology Center in the Technopole of Borj-Cédria (CBBS), 2050 Hammam-lif, Tunisia e Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Team: Medicinal Chemistry and Natural Products, Faculty of Sciences of Monastir, University of Monastir, Avenue de l’ Environnement, 5019 Monastir, Tunisia
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Article history: Received 16 August 2014 Received in revised form 19 March 2015 Accepted 20 March 2015 Available online xxx
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Keywords: Scabiosa arenaria Forssk. ␣-Glucosidase Non-competitive inhibition RP-HPLC
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Contents
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1. 2.
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3.
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4.
Recent decades have witnessed a sharp increase in the incidence and prevalence of type 2 diabetes mellitus. One antidiabetic therapeutic approach is to reduce gastrointestinal glucose production and absorption through the inhibition of carbohydrate-digesting enzymes such as ␣-amylase and ␣glucosidase. In this study, crude extracts and their corresponding fractions of flowers, fruits, (stems and leaves) and roots of the endemic North African plant Scabiosa arenaria Forssk. were screened for their ability of ␣-glucosidase inhibition. It was found that the fruits ethyl acetate (EtOAc), the fruits butanolic (n-BuOH) and the flowers ethyl acetate (EtOAc) fractions inhibited ␣-glucosidase in a non competitive manner with IC50 values of 0.11 ± 0.09, 0.28 ± 0.04 and 0.221 ± 0.01 mg/ml, respectively. RP-HPLC analysis indicated that the major components of these active fractions are flavonoid aglycone, cinnamic acid and its derivatives. This result supports the conclusion that the three studied fractions could be a useful natural source for the development of a novel ␣-glucosidase inhibitory agent against diabetic complications. © 2015 Elsevier B.V. All rights reserved.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Reagents and standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. ␣-Glucosidase inhibition assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Kinetics study of ␣-glucosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Analysis of individual phenolic compounds by analytical RP-HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ␣-Glucosidase inhibition assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Kinetics of ␣-glucosidase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Phenolics identification by RP-HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Declaration of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗ Corresponding author. Tel.: +216 73463374; fax: +216 73465404. E-mail address: mosbah [email protected] (H. Mosbah). 1 Authors participated equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.03.035 0141-8130/© 2015 Elsevier B.V. All rights reserved.
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1. Introduction
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Thousands of scientific studies have shown the important therapeutic property of plant secondary metabolites in the prevention of many chronic diseases, such as neurodegenerative diseases and diabetes mellitus. These ailments affect people in a growing and alarming rate [1–5]. The dominance of diabetes in recent decades has increased dramatically worldwide [6]. Unless treated quickly, it can lead to serious complications, including hyperglycemia, diabetic ketoacidosis, cardiovascular disease and chronic kidney failure [7]. The adjustment of the blood sugar level is a very important and efficient approach to control diabetes. One of the ways to fight against these diseases is the inhibition of enzymes with critical roles in their evolution [8,9]. ␣-Glucosidase, frequently located on the surface of the brush border of the intestinal cells, is crucial for the digestion of oligosaccharides to monosaccharides which are absorbed easily by the intestine [10]. Inhibitors of ␣-glucosidase could delay the digestion of ingested carbohydrates, thus leading to the suppression of postprandial glucose [11]. Hence, these inhibitors are promising as therapeutic agents for non-insulin dependent diabetes mellitus [12]. However, the inhibition of the current ␣-glucosidase such as Acarbose and miglitol is not satisfactory and prolonged use often causes harmful side effects, including diarrhea [13], pain and intestinal flatulence [14]. Therefore, the search for new natural extracts and compounds inhibitors of ␣-glucosidase proves to be necessary. The Scabiosa genus belonging to the Dipsacaceae family, contains in Tunisia eleven recently identified species which are: Scabiosa rutifolia Vahl., S. succisa L., S. farinosa Coss., S. atropurpurea L., S. arenaria Forssk., S. stellata L., S. crenata Cyr., S. daucoides Desf., S. robertii Bonn and S. simplex Desf. [15]. An infusion of the flowers of S. atropurpurea L. is used orally by the peoples of the Iberian Peninsula in Spain as a hypoglycemic [16]. Several species of this genus have been investigated chemically or pharmacologically for their medicinal use and potential new drugs [17–21]. Luteolin 7-O-glucoside, luteolin, chlorogenic acid methyl ester, luteolin-7-rutinoside were isolated from S. atropurpurea [22]. Irridoid glucosides were identified from S. atropurpurea subsp. maritima [23], from S. columbaria [24] and from S. variifolia [25]. S. rotata [26] and S. tschiliensis [27] contained saponin glycosides, whereas flavonoids and oleanolic acid were identified in S. caucasica [28]. Coumarins, flavonoids and irridoid glycosides were identified in S. hymettia [29]. In our previous studies, the chemical composition of essential oils of flowers, fruits and (stems and leaves) of S. arenaria and their antimicrobial activities were analyzed [30]. The antioxidant and anti-acetylcholinesterase activities of the aerial parts of this species were also studied [31]. Following our research on Tunisian S. arenaria active secondary metabolites, we report the results relative to the ␣-glucosidase inhibition by some extracts of this plant. The inhibition mode of the more active extracts was also studied. Finally, the phenolic compounds in these extracts were characterized using RP-HPLC.
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2. Materials and methods
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2.1. Reagents and standards
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All solvents used in the experiments (ethyl acetate, butanol and methanol) were purchased from Merck (Darmstadt, Germany). ␣-Glucosidase (isolated from Aspergillus niger), p-nitrophenyl-␣-dglucopyranoside (pNPG) and Acarbose were purchased from Sigma Aldrich. Authentic standards of phenolic compounds were purchased from Sigma and Fluka. Stock solutions of these compounds
were prepared in HPLC-grade methanol. These solutions were wrapped in aluminum foil and stored at 4 ◦ C.
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2.2. Sample preparation
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S. arenaria was collected in May 2010 from Monastir area at the flowering stage. The plant material was identified by Dr. Fethia Harzallah Skhiri (High Institute of Biotechnology of Monastir, Tunisia). A voucher specimen (S. arenaria) (Sa 110) was deposited in the Laboratory of Medicinal Chemistry and Natural Products at the faculty of Sciences of Monastir, Tunisia. S. arenaria flowers, fruits, (stems and leaves) and roots were airdried at room temperature for two weeks and reduced to coarse powder. The powdered samples (250 g) were extracted by maceration with MeOH–H2 O 80:20 (v/v) for 72 h at room temperature. The hydro-methanolic extraction was carried out in triplicate. After filtration, a portion of these extracts was dried completely for further biological activities and the residual aqueous layer was further subjected to a successive extraction with ethyl acetate (EtOAc) and butanol (n-BuOH) to yield dried fractions. The hydro-methanolic extracts and their fractions were maintained at 4 ◦ C before analysis.
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2.3. ˛-Glucosidase inhibition assay
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␣-Glucosidase inhibitory activity was determined as previously described by Tao et al. [32] with some modifications as detailed by Rengasamy et al. [33]. The ␣-glucosidase reaction mixture contained 2.5 mM p-nitrophenyl-␣-d-glucopyranoside (pNPG), 250 l of extract (varying concentrations) in DMSO and 0.3 U/ml of ␣glucosidase in phosphate buffer, pH 6.9. Control tubes contained only DMSO, enzyme and substrate, while in positive controls Acarbose replaced the plant extracts. Absorbance of the resulting pnitrophenol (pNP) was determined at 405 nm and was considered directly proportional to the activity of the enzyme. The S. arenaria extracts were tested for ␣-glucosidase inhibitory activity at different concentrations (10–0.15 mg/ml). Each sample was performed in triplicate. Inhibition Table 1 ␣-glucosidase inhibition by Scabiosa arenaria extracts. Extracts Flowers Crude extract EtOAc fraction n-BuOH fraction Water fraction Fruits Crude extract EtOAc fraction n-BuOH fraction Water fraction (Stems and leaves) Crude extract EtOAc fraction n-BuOH fraction Water fraction Roots Crude extract EtOAc fraction n-BuOH fraction Water fraction Positive control Acarbose
IC50 (mg/ml) ␣-Glucosidase inhibition n.d. 0.221 ± 0.014 0.547 ± 0.03 n.d. n.d. 0.11 ± 0.09 0.28 ± 0.04 n.d. n.d. 0.72 ± 0.2 0.454 ± 0.02 n.d. n.d. 0.285 ± 0.21 0.492± 0.03 n.d. 0.28 ± 0.01
Values were expressed as mean ± SE (n = 3). IC50 (mg/ml): the concentration at which 50% of enzyme is inhibited. n.d.: not determined because the inhibition at highest screened concentration (1 mg/ml) was less than 50%.
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The IC50 , which is the concentration of the sample required to inhibit 50% of the enzyme was determined for each sample. All extracts of S. arenaria were compared on the basis of their IC50 values estimated from the dose response curves.
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1/Vi(∆DO/min)
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The most active fractions showing the lowest IC50 value of S. arenaria were tested to find the type of inhibition exerted on ␣-glucosidase. The reaction mixture was as described above, except that the substrate concentration varied from 0.3 to 5 mM, and that of the extract was kept constant at 1.25 mg/ml. The reaction was started by the addition of enzyme, and monitored at 405 nm, at 5 min intervals during 30 min. The initial reaction rates were determined using calibration curves constructed using varying p-nitrophenol concentrations The results were used to construct Lineweaver–Burk plots to determine the type of inhibition,
DO sample Inhibition Percentage (%) = 1 − × 100. DO control
A Fruits EtOAc fracon
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1/Vi (∆DO/min)
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2.4. Kinetics study of ˛-glucosidase
Percentage by extracts and Acarbose were calculated using the following equation:
B
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Fruits n-BuOH fracon
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Fig. 1. Double-reciprocal plot of the initial velocity (Vmax ) of the hydrolysis reactions catalyzed by ␣-glucosidase at various substrate concentrations [S] in the absence and presence of Fruits EtOAc fraction (A), fruits n-BuOH fraction (B) and flowers EtOAc fraction. The results represent the means of three independent triplicate experiments.
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Michaelis–Menten constant (KM ) and maximum velocity (Vmax ) values. 2.5. Analysis of individual phenolic compounds by analytical RP-HPLC
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The dried active extracts were hydrolyzed according to the slightly modified method of Proestos et al. [34]. Forty milliliters of methanol containing BHT (1 mg/ml) were added to 0.5 g of dried fractions. Then, 10 ml of 6 M HCl were added. The mixture was stirred carefully and then sonicated for 15 min and refluxed in a water bath at 90 ◦ C for 2 h. The obtained mixture was filtered through a 0.45 m membrane filter and injected to HPLC/UV. The separation of phenolic compounds was performed with an Agilent 1100 series HPLC system equipped with on-line degasser (G 1322A), a quaternary pump (G 1311A), a thermostatic autosampler (G 1313A), a column heater (G 1316A) and a diode array detector (G 1315A). Instrument control and data analysis were carried out using Agilent HPLC Chemstation 10.1 edition through Windows 2000. The separation was carried out on a reverse phase ODS C18 (4 m, 250 mm × 4.6 mm, Hypersil) column used as a stationary phase at ambient temperature. The mobile phase consisted of acetonitrile (solvent A) and water with 0.2% sulfuric acid (solvent B). The flow rate was kept at 0.5 ml/min. The gradient program was as follows: 15% A/85% B (0–12 min), 40% A/60% B (12–14 min), 60% A/40% B (14–18 min), 80% A/20% B (18–20 min), 90% A/10% B (20–24 min), 100% A (24–28 min). The injection volume was 20 l and peaks were monitored at 280 nm. Phenolic compounds of S. arenaria extracts were identified by comparing their retention times with those of pure standards. The results were expressed as % of each compound from the total phenolic compounds.
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2.6. Statistical analysis
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The results were given as the average ± SE for at least three replicates for each sample. The IC50 (␣-glucosidase inhibition) values were calculated by linear regression analysis. The data were subjected to ANOVA, and Duncan’s multiple range test was used to compare means. Statistical analyses were per-formed with the SPSS statistical software program (SPSS v.16). p values < 0.05 were regarded as significant.
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3. Results and discussion
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3.1. ˛-Glucosidase inhibition assay
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The ␣-glucosidase inhibitor effectiveness of the different S. arenaria extracts was compared on the basis of their resulting IC50 values. We could notice essentially that the EtOAc and the n-BuOH fractions of all the organs of our plant were more active than the hydro-methanolic extracts and the water fractions (Table 1). To evaluate the inhibitory power of different S. arenaria extracts, the Acarbose was used as positive control. Our results show that the IC50 value of Acarbose was 0.28 mg/ml which was 60-fold and 880-fold lower than those obtained by Shai et al. [35] and Rubilar et al. [36], respectively. As depicted in Table 1, the EtOAC of fruits fraction shows interesting antidiabetic activity with IC50 value (0.11 ± 0.09 mg/ml) two times lower than that of Acarbose. While, the EtOAC flowers and n-BuOH fruits fractions show similar antidiabetic activity to Acarbose with IC50 values of 0.221 ± 0.014 mg/ml and 0.28 ± 0.04 mg/ml, respectively. No reference was found for the anti ␣-glucosidase in vitro for the genus Scabiosa, or for the Dipsacaceae family. Only Elhawary et al. [22] showed that ethanolic, hexane, chloroform, ethyl acetate and
butanol extracts of S. atropurpurea L. exhibited an interesting antihyperglycemic power in vivo. These results support the traditional uses of the species to reduce blood glucose levels [16]. Recently, the aqueous and methanolic extracts of 500 Chinese medicinal plants were investigated with the aim of finding new sources of ␣-glucosidase inhibitors. As a result, the fruits extracts of Terminalia chebula, and Rosa rugosa flowers showed a high inhibition of ␣-glucosidase with an IC50 value of 9 g/ml [37]. Another recent study showed that the leaves ethyl acetate extract of Croton menyharthii was more potent than the reference product (Acarbose) with an IC50 value of 90.3 g/ml [38]. Comparing these two studies with our report, some differences in the value of IC50 between the studied extracts were noticed due to the difference in species and the solvent used. 3.2. Kinetics of ˛-glucosidase activity
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Kinetics was performed according to the procedure detailed in Section 2. For this purpose, the EtOAc fractions of fruits, flowers and the n-BuOH fraction of fruits which presented the lower values of IC50 were used to determine the type of inhibition (competitive, non-competitive or uncompetitive) of ␣-glucosidase. The Lineweaver–Burk plots indicated that these fractions inhibited ␣-glucosidase in a non-competitive manner (Fig. 1A–C). Thus, the addition of each fraction in the reaction medium led to a change in the maximum velocity (Vmax ) of ␣-glucosidase while keeping the same value of Michaelis–Menten constant (KM ). In the presence of the EtOAc, n-BuOH fraction of fruits and the EtOAc
Table 2 Main phenolic compounds identified by RP-HPLC in fruits EtOAc, fruits n-BuOH and flowers EtOAc fractions of S. arenaria. No
RT (min)
Compounds
% in Fr Et
% in Fl Et
% in Fr B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
3.89 5.84 8.6 9.05 9.10 9.85 10.09 10.26 10.5 10.8 11.09 11.53 11.99 12.24 12.43 12.47 12.65 13.06 13.56 14.08 14.33 14.68 15.18 15.20 15.37 15.59 16.19 16.8 17.59 18.10 18.13 19.34 19.5 19.79
Catechin Chlorogenic acid Unknown Unknown 2-Hydroxyphenylacetic acid Unknown Unknown Cinnamic acid p-Coumaric acid Taxifolin Ferulic acid Unknown Rutin Unknown Unknown Unknown Methyl 4-hydroxybenzoate Morin Daidzein Unknown Quercetin Hesperetin Genistein Unknown Unknown Unknown Unknown Methyl trans-cinnamate Unknown Unknown Chrysin Unknown Unknown Unknown
ND 2.02 ND 2.48 ND ND ND ND ND 1.19 2.06 ND ND ND 1.8 2.78 19.12 3.5 ND ND 9.33 ND ND ND ND 41.26 1.04 1.34 0.77 3.4 1.45 1.21 1.59 3.58
5.73 ND ND ND ND ND ND ND 5.79 ND 7.10 7.46 6.68 5.53 ND ND ND ND 2.54 3.10 6.53 23.6 ND 17.18 8.75 ND ND ND ND ND ND ND ND ND
ND ND 61.57 ND 6.54 5.08 3.81 6.73 ND ND 12.53 ND ND ND ND ND ND ND ND ND ND ND 3.71 ND ND ND ND ND ND ND ND ND ND ND
RT: retention times (min). ND: not detected. Fr Et: fruits EtOAc fraction; Fl Et: flowers EtOAc fraction; Fr B: fruits n-BuOH fraction.
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Fig. 2. RP-HPLC chromatograms of flowers EtOAc fraction (A), fruits EtOAc fraction (B) and fruits n-BuOH fraction (C) after acid hydrolysis. Signal was recorded at 280 nm. Numbering of peaks refers to their identification as shown in Table 2.
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fraction of flowers, the Michaelis–Menton constant (KM ) remained almost constant (2.27 mM, 2.56 mM and 2.32 mM, respectively). At the same extract concentration (1.25 mg/ml), the maximum velocity (Vmax ) decreased from 0.333 DO/min in the absence of any inhibitor to 0.15 DO/min, 0.13 DO/min and to 0.128 DO/min in the presence of fruits EtOAc, fruits n-BuOH and flowers EtOAc fraction, respectively. According to this type of inhibition, we can suggest that the active components in these extracts do not compete with the substrate to bind to the active site, rather the inhibitors bind to a separate site on the enzyme to retard the conversion of substrate to product. When comparing the inhibition manner of ␣-glucosidase by different fractions of S. arenaria with other previous studies, we found that the ethyl acetate extracts of Agatophora alopecuro, Anabasis articulata, Hammada elegans, Helianthemum kahiricum and Salsola baryosma harvested from Algeria presented interesting antidiabetic activities and exhibited varied types of inhibition [39].
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3.3. Phenolics identification by RP-HPLC
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of the compounds contained therein. However, some researchers have reported a relationship between the chemical composition of the major compounds in the extract and their biological activities [41]. Moreover, today, it is known that the synergistic or antagonistic effect of a compound present in minor proportion in a mixture must be considered as well [42]. As previously demonstrated, the EtOAc fractions of fruits, flowers and the n-BuOH fraction of fruits are potent inhibitors of ␣-glucosidase which may be due to the presence of flavonoids. Indeed, the major compounds mentioned in Table 2 (catechin, cinnamic acid, ferulic acid, quercetin and hesperetin) have been found by several researchers as good ␣-glucosidase inhibitors [43–50]. The anti-␣-glucosidase activity of these three fractions may be due to the presence and the possible synergistic effect of these phenolic compounds. In addition, up to 18 compounds were unknown and are the subject of future purification and characterization efforts. 4. Conclusion
261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305
As mentioned above, the fruits EtOAc, n-BuOH and the flowers EtOAc fractions showed an interesting ␣-glucosidase inhibition. This result motivated us to identify the constituents of each fraction by RP-HPLC and localize the eventual researched activities. It is known that the free forms of phenolic compounds are rarely present in plants. More often, they occur as esters, glycosides or are bound to the cell wall. For this reason, before HPLC analysis, the hydrolysis of glycosides or esters was necessary, so that phenolic compounds can be identified, since a considerable fraction is in bounded form. Moreover, BHT, a powerful antioxidant, was added to prevent phenolics degradation during hydrolysis [40]. RP-HPLC coupled with UV–vis multiwavelength detector was employed to separate phenolic compounds. Compounds have been identified according to their retention times and the spectral characteristics of their peaks against those of standards, as well as by spiking the sample with standards. The compounds identified in the three fractions are reported in Table 2 and Fig. 2A–C. It was found that the EtOAc fraction of flowers contained twelve compounds numbered according to their retention time (Fig. 2A; Table 2). It is rich in flavonoids such as hesperetin (22) (23.6%), rutin (13) (6.68%), quercetin (21) (6.53%) and catechin (1) (5.73%). This fraction also contained phenolic acids in high proportion such as ferulic acid (11) (7.10%) and p-coumaric acid (9) (5.79%). Five unknown compounds were found in this fraction. On the other hand, the EtOAc fruits fraction contained 18 compounds numbered according to their retention time (Fig. 2B; Table 2). Among the major compounds, we identified an unknown molecule (26) in high percentage (41.26%), methyl 4hydroxybenzoate (19.12%), quercetin (21), (9.33%) and morin (18), (3.5%). This fraction also contained several minor compounds such as phenolic acid: Chlorogenic acid (2) (2.02%) and ferulic acid (11) (2.06%). We also noted that 9 molecules of this fraction were still unknown. However, among the compounds of the butanolic fruits fraction, we detected product (5) 2-hydroxyphenylacetic acid with a percentage of 6.54%. We also obtained ferulic acid (11) (12.53%), cinnamic acid (8) (6.73%) and genistein (23) (3.71%). The major product in this fraction numbered (3) (61. 57%) was also still unknown (Fig. 2C; Table 2). The chemical analysis of the three S. arenaria fractions showed their richness in secondary metabolites which could be considered responsible for a more or less important part of enzymatic inhibition activities. These biological activities are difficult to correlate with a specific compound, because of the complexity and variability
This is the first study on enzymatic inhibition by S. arenaria extracts. Our results showed that some of these extracts exhibited a potent ability to inhibit ␣-glucosidase. The type of inhibition by the most active extracts was confirmed as non-competitive. These results may be due to the presence of the above mentioned compounds. The data pointed out that S. arenaria containing these flavonoids could be a useful natural source for the development of ˛-glucosidase inhibitory agent. Future studies are needed to isolate and identify the bioactive metabolites which are effective in the ␣-glucosidase inhibitory activity. Declaration of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors are grateful to Dr. Fethia Harzallah-Skhiri (High Institute of Biotechnology of Monastir, Monastir, Tunisia) for the botanical identification. We are grateful to Pr. Bejaoui Hafedh (FSS, Sfax-Tunisia) for his help with English. References [1] G. Rea, A. Antonacci, M. Lambreva, A. Margonelli, C. Ambrosi, M.T. Giardi, U.S. Q3 Springer, vol. 698, pp. 1–16 (Chapter 1). [2] S.I. Cameron, R.F. Smith, K.E. Kierstead, Pharm. Biol. 43 (2005) 425–433. [3] Z.M. Evangelista, A.E. Moreno, Biotecnologia 11 (2007) 37–50. [4] K. Colvine, A.J. Kerr, A. McLachlan, P. Gow, S. Kumar, J. Ly, C. Wiltshire, E. Robinson, N. Dalbeth, N. N. Z. Med. J. 121 (2008) 73–81. [5] A. Corrado, F. D’Onofrio, N. Santoro, N. Melillo, F.P. Cantatore, Minerva Med. 97 (2006) 495–509. [6] J. Gorelick, A. Kitron, S. Pen, T. Rosenzweig, Z. Madar, J. Ethnopharmacol. 137 (2011) 1245–1249. [7] Z.P. Yu, Y.G. Yin, W.Z. Zhao, Y.D. Yu, B.Q. Liu, F. Chen, Food Chem. 129 (2013) 1376–1382. [8] N. Amessis-Ouchemoukh, K. Madani, M.P. Falé, L. Serralheiro, M.E. Araújo, Ind. Crops Prod. 53 (2014) 6–15. [9] J. Yan, G. Zhang, J. Pan, Y. Wang, Int. J. Biol. Macromol. 64 (2014) 213–223. [10] S.D. Kim, Food Chem. 136 (2013) 297–300. [11] P.M. Heacock, S.R. Hertzler, J.A. Williams, B.W. Wolf, J. Am. Diet. Assoc. 105 (2005) 65–71. [12] A.D. Baron, Diabetes Res. Clin. Pract. 40 (1998) 51–55. [13] H.E. Lebovitz, Endocrinol. Metab. Clin. North Am. 26 (1997) 539–551. [14] T. Fujisawa, H. Ikegami, K. Inoue, Y. Kawabata, T. Ogihara, Metabolism 54 (2005) 387–390. [15] E. Le floc’h, L. Boulos, E. Vela, Catalogue synonymique commenté de la Flore de Tunisie, République Tunisienne, Ministère de L’environnement et du Développement Durable, banque nationale de gènes, 2010, pp. 195–196. [16] M.A. Bonet, M. Parada, A. Selga, J. Vallès, J. Ethnopharmacol. 68 (1999) 145–168. [17] R.W. Bussmann, G. Malca-García, A. Glenn, D. Sharon, G. Chaitc, D. Díaz, K. Pourmand, B. Jonat, S. Somogy, G. Guardado, C. Aguirrf, R. Chan, K. Meyer,
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Contents lists available at ScienceDirect
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Review
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␣-Glucosidase inhibition by Tunisian Scabiosa arenaria Forssk. extracts
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Malek Besbes Hlila a,1 , Habib Mosbah b,∗,1 , Kawther Majouli c , Kamel Mssada d , Hichem Ben Jannet e , Mahjoub Aouni a , Boulbaba Selmi a a Laboratory of Transmissible Diseases and Biological Active Substances, Faculty of Pharmacy, University of Monastir, Avenue Avicenne, 5000 Monastir, Tunisia b Higher Institute of Biotechnology of Monastir, Avenue Taher Hadded, BP 74, 5000 Monastir, Tunisia c Research Laboratory in Biochemistry, Faculty of Medicine, University of Monastir, Avenue Avicenne, 5019, Tunisia d Laboratory of Bioactive Substances, Biotechnology Center in the Technopole of Borj-Cédria (CBBS), 2050 Hammam-lif, Tunisia e Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Team: Medicinal Chemistry and Natural Products, Faculty of Sciences of Monastir, University of Monastir, Avenue de l’ Environnement, 5019 Monastir, Tunisia
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Article history: Received 16 August 2014 Received in revised form 19 March 2015 Accepted 20 March 2015 Available online xxx
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Keywords: Scabiosa arenaria Forssk. ␣-Glucosidase Non-competitive inhibition RP-HPLC
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Contents
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4.
Recent decades have witnessed a sharp increase in the incidence and prevalence of type 2 diabetes mellitus. One antidiabetic therapeutic approach is to reduce gastrointestinal glucose production and absorption through the inhibition of carbohydrate-digesting enzymes such as ␣-amylase and ␣glucosidase. In this study, crude extracts and their corresponding fractions of flowers, fruits, (stems and leaves) and roots of the endemic North African plant Scabiosa arenaria Forssk. were screened for their ability of ␣-glucosidase inhibition. It was found that the fruits ethyl acetate (EtOAc), the fruits butanolic (n-BuOH) and the flowers ethyl acetate (EtOAc) fractions inhibited ␣-glucosidase in a non competitive manner with IC50 values of 0.11 ± 0.09, 0.28 ± 0.04 and 0.221 ± 0.01 mg/ml, respectively. RP-HPLC analysis indicated that the major components of these active fractions are flavonoid aglycone, cinnamic acid and its derivatives. This result supports the conclusion that the three studied fractions could be a useful natural source for the development of a novel ␣-glucosidase inhibitory agent against diabetic complications. © 2015 Elsevier B.V. All rights reserved.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Reagents and standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. ␣-Glucosidase inhibition assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Kinetics study of ␣-glucosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Analysis of individual phenolic compounds by analytical RP-HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ␣-Glucosidase inhibition assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Kinetics of ␣-glucosidase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Phenolics identification by RP-HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Declaration of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗ Corresponding author. Tel.: +216 73463374; fax: +216 73465404. E-mail address: mosbah [email protected] (H. Mosbah). 1 Authors participated equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.03.035 0141-8130/© 2015 Elsevier B.V. All rights reserved.
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Thousands of scientific studies have shown the important therapeutic property of plant secondary metabolites in the prevention of many chronic diseases, such as neurodegenerative diseases and diabetes mellitus. These ailments affect people in a growing and alarming rate [1–5]. The dominance of diabetes in recent decades has increased dramatically worldwide [6]. Unless treated quickly, it can lead to serious complications, including hyperglycemia, diabetic ketoacidosis, cardiovascular disease and chronic kidney failure [7]. The adjustment of the blood sugar level is a very important and efficient approach to control diabetes. One of the ways to fight against these diseases is the inhibition of enzymes with critical roles in their evolution [8,9]. ␣-Glucosidase, frequently located on the surface of the brush border of the intestinal cells, is crucial for the digestion of oligosaccharides to monosaccharides which are absorbed easily by the intestine [10]. Inhibitors of ␣-glucosidase could delay the digestion of ingested carbohydrates, thus leading to the suppression of postprandial glucose [11]. Hence, these inhibitors are promising as therapeutic agents for non-insulin dependent diabetes mellitus [12]. However, the inhibition of the current ␣-glucosidase such as Acarbose and miglitol is not satisfactory and prolonged use often causes harmful side effects, including diarrhea [13], pain and intestinal flatulence [14]. Therefore, the search for new natural extracts and compounds inhibitors of ␣-glucosidase proves to be necessary. The Scabiosa genus belonging to the Dipsacaceae family, contains in Tunisia eleven recently identified species which are: Scabiosa rutifolia Vahl., S. succisa L., S. farinosa Coss., S. atropurpurea L., S. arenaria Forssk., S. stellata L., S. crenata Cyr., S. daucoides Desf., S. robertii Bonn and S. simplex Desf. [15]. An infusion of the flowers of S. atropurpurea L. is used orally by the peoples of the Iberian Peninsula in Spain as a hypoglycemic [16]. Several species of this genus have been investigated chemically or pharmacologically for their medicinal use and potential new drugs [17–21]. Luteolin 7-O-glucoside, luteolin, chlorogenic acid methyl ester, luteolin-7-rutinoside were isolated from S. atropurpurea [22]. Irridoid glucosides were identified from S. atropurpurea subsp. maritima [23], from S. columbaria [24] and from S. variifolia [25]. S. rotata [26] and S. tschiliensis [27] contained saponin glycosides, whereas flavonoids and oleanolic acid were identified in S. caucasica [28]. Coumarins, flavonoids and irridoid glycosides were identified in S. hymettia [29]. In our previous studies, the chemical composition of essential oils of flowers, fruits and (stems and leaves) of S. arenaria and their antimicrobial activities were analyzed [30]. The antioxidant and anti-acetylcholinesterase activities of the aerial parts of this species were also studied [31]. Following our research on Tunisian S. arenaria active secondary metabolites, we report the results relative to the ␣-glucosidase inhibition by some extracts of this plant. The inhibition mode of the more active extracts was also studied. Finally, the phenolic compounds in these extracts were characterized using RP-HPLC.
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2. Materials and methods
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2.1. Reagents and standards
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All solvents used in the experiments (ethyl acetate, butanol and methanol) were purchased from Merck (Darmstadt, Germany). ␣-Glucosidase (isolated from Aspergillus niger), p-nitrophenyl-␣-dglucopyranoside (pNPG) and Acarbose were purchased from Sigma Aldrich. Authentic standards of phenolic compounds were purchased from Sigma and Fluka. Stock solutions of these compounds
were prepared in HPLC-grade methanol. These solutions were wrapped in aluminum foil and stored at 4 ◦ C.
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2.2. Sample preparation
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S. arenaria was collected in May 2010 from Monastir area at the flowering stage. The plant material was identified by Dr. Fethia Harzallah Skhiri (High Institute of Biotechnology of Monastir, Tunisia). A voucher specimen (S. arenaria) (Sa 110) was deposited in the Laboratory of Medicinal Chemistry and Natural Products at the faculty of Sciences of Monastir, Tunisia. S. arenaria flowers, fruits, (stems and leaves) and roots were airdried at room temperature for two weeks and reduced to coarse powder. The powdered samples (250 g) were extracted by maceration with MeOH–H2 O 80:20 (v/v) for 72 h at room temperature. The hydro-methanolic extraction was carried out in triplicate. After filtration, a portion of these extracts was dried completely for further biological activities and the residual aqueous layer was further subjected to a successive extraction with ethyl acetate (EtOAc) and butanol (n-BuOH) to yield dried fractions. The hydro-methanolic extracts and their fractions were maintained at 4 ◦ C before analysis.
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␣-Glucosidase inhibitory activity was determined as previously described by Tao et al. [32] with some modifications as detailed by Rengasamy et al. [33]. The ␣-glucosidase reaction mixture contained 2.5 mM p-nitrophenyl-␣-d-glucopyranoside (pNPG), 250 l of extract (varying concentrations) in DMSO and 0.3 U/ml of ␣glucosidase in phosphate buffer, pH 6.9. Control tubes contained only DMSO, enzyme and substrate, while in positive controls Acarbose replaced the plant extracts. Absorbance of the resulting pnitrophenol (pNP) was determined at 405 nm and was considered directly proportional to the activity of the enzyme. The S. arenaria extracts were tested for ␣-glucosidase inhibitory activity at different concentrations (10–0.15 mg/ml). Each sample was performed in triplicate. Inhibition Table 1 ␣-glucosidase inhibition by Scabiosa arenaria extracts. Extracts Flowers Crude extract EtOAc fraction n-BuOH fraction Water fraction Fruits Crude extract EtOAc fraction n-BuOH fraction Water fraction (Stems and leaves) Crude extract EtOAc fraction n-BuOH fraction Water fraction Roots Crude extract EtOAc fraction n-BuOH fraction Water fraction Positive control Acarbose
IC50 (mg/ml) ␣-Glucosidase inhibition n.d. 0.221 ± 0.014 0.547 ± 0.03 n.d. n.d. 0.11 ± 0.09 0.28 ± 0.04 n.d. n.d. 0.72 ± 0.2 0.454 ± 0.02 n.d. n.d. 0.285 ± 0.21 0.492± 0.03 n.d. 0.28 ± 0.01
Values were expressed as mean ± SE (n = 3). IC50 (mg/ml): the concentration at which 50% of enzyme is inhibited. n.d.: not determined because the inhibition at highest screened concentration (1 mg/ml) was less than 50%.
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The IC50 , which is the concentration of the sample required to inhibit 50% of the enzyme was determined for each sample. All extracts of S. arenaria were compared on the basis of their IC50 values estimated from the dose response curves.
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1/Vi(∆DO/min)
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-1
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The most active fractions showing the lowest IC50 value of S. arenaria were tested to find the type of inhibition exerted on ␣-glucosidase. The reaction mixture was as described above, except that the substrate concentration varied from 0.3 to 5 mM, and that of the extract was kept constant at 1.25 mg/ml. The reaction was started by the addition of enzyme, and monitored at 405 nm, at 5 min intervals during 30 min. The initial reaction rates were determined using calibration curves constructed using varying p-nitrophenol concentrations The results were used to construct Lineweaver–Burk plots to determine the type of inhibition,
DO sample Inhibition Percentage (%) = 1 − × 100. DO control
A Fruits EtOAc fracon
wihou nhibitor 0.5
1
1.5
2
2.5
3
3.5
1/ [S ] (mM) 100
1/Vi (∆DO/min)
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2.4. Kinetics study of ˛-glucosidase
Percentage by extracts and Acarbose were calculated using the following equation:
B
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Fruits n-BuOH fracon
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Withou nhibitor
10 -1
0 -10 0
-0.5
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1
1.5
2
2.5
3
3.5
1/[S] (mM) 100
1/Vi( ∆DO/min)
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C
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Flowers EtOAc fracon
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Withou nhibitor
10 -1
-0.5
0
-10 0
0.5
1
1.5
2
2.5
3
3.5
1/[S] (mM)
Fig. 1. Double-reciprocal plot of the initial velocity (Vmax ) of the hydrolysis reactions catalyzed by ␣-glucosidase at various substrate concentrations [S] in the absence and presence of Fruits EtOAc fraction (A), fruits n-BuOH fraction (B) and flowers EtOAc fraction. The results represent the means of three independent triplicate experiments.
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Michaelis–Menten constant (KM ) and maximum velocity (Vmax ) values. 2.5. Analysis of individual phenolic compounds by analytical RP-HPLC
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The dried active extracts were hydrolyzed according to the slightly modified method of Proestos et al. [34]. Forty milliliters of methanol containing BHT (1 mg/ml) were added to 0.5 g of dried fractions. Then, 10 ml of 6 M HCl were added. The mixture was stirred carefully and then sonicated for 15 min and refluxed in a water bath at 90 ◦ C for 2 h. The obtained mixture was filtered through a 0.45 m membrane filter and injected to HPLC/UV. The separation of phenolic compounds was performed with an Agilent 1100 series HPLC system equipped with on-line degasser (G 1322A), a quaternary pump (G 1311A), a thermostatic autosampler (G 1313A), a column heater (G 1316A) and a diode array detector (G 1315A). Instrument control and data analysis were carried out using Agilent HPLC Chemstation 10.1 edition through Windows 2000. The separation was carried out on a reverse phase ODS C18 (4 m, 250 mm × 4.6 mm, Hypersil) column used as a stationary phase at ambient temperature. The mobile phase consisted of acetonitrile (solvent A) and water with 0.2% sulfuric acid (solvent B). The flow rate was kept at 0.5 ml/min. The gradient program was as follows: 15% A/85% B (0–12 min), 40% A/60% B (12–14 min), 60% A/40% B (14–18 min), 80% A/20% B (18–20 min), 90% A/10% B (20–24 min), 100% A (24–28 min). The injection volume was 20 l and peaks were monitored at 280 nm. Phenolic compounds of S. arenaria extracts were identified by comparing their retention times with those of pure standards. The results were expressed as % of each compound from the total phenolic compounds.
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2.6. Statistical analysis
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The results were given as the average ± SE for at least three replicates for each sample. The IC50 (␣-glucosidase inhibition) values were calculated by linear regression analysis. The data were subjected to ANOVA, and Duncan’s multiple range test was used to compare means. Statistical analyses were per-formed with the SPSS statistical software program (SPSS v.16). p values < 0.05 were regarded as significant.
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3. Results and discussion
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The ␣-glucosidase inhibitor effectiveness of the different S. arenaria extracts was compared on the basis of their resulting IC50 values. We could notice essentially that the EtOAc and the n-BuOH fractions of all the organs of our plant were more active than the hydro-methanolic extracts and the water fractions (Table 1). To evaluate the inhibitory power of different S. arenaria extracts, the Acarbose was used as positive control. Our results show that the IC50 value of Acarbose was 0.28 mg/ml which was 60-fold and 880-fold lower than those obtained by Shai et al. [35] and Rubilar et al. [36], respectively. As depicted in Table 1, the EtOAC of fruits fraction shows interesting antidiabetic activity with IC50 value (0.11 ± 0.09 mg/ml) two times lower than that of Acarbose. While, the EtOAC flowers and n-BuOH fruits fractions show similar antidiabetic activity to Acarbose with IC50 values of 0.221 ± 0.014 mg/ml and 0.28 ± 0.04 mg/ml, respectively. No reference was found for the anti ␣-glucosidase in vitro for the genus Scabiosa, or for the Dipsacaceae family. Only Elhawary et al. [22] showed that ethanolic, hexane, chloroform, ethyl acetate and
butanol extracts of S. atropurpurea L. exhibited an interesting antihyperglycemic power in vivo. These results support the traditional uses of the species to reduce blood glucose levels [16]. Recently, the aqueous and methanolic extracts of 500 Chinese medicinal plants were investigated with the aim of finding new sources of ␣-glucosidase inhibitors. As a result, the fruits extracts of Terminalia chebula, and Rosa rugosa flowers showed a high inhibition of ␣-glucosidase with an IC50 value of 9 g/ml [37]. Another recent study showed that the leaves ethyl acetate extract of Croton menyharthii was more potent than the reference product (Acarbose) with an IC50 value of 90.3 g/ml [38]. Comparing these two studies with our report, some differences in the value of IC50 between the studied extracts were noticed due to the difference in species and the solvent used. 3.2. Kinetics of ˛-glucosidase activity
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Kinetics was performed according to the procedure detailed in Section 2. For this purpose, the EtOAc fractions of fruits, flowers and the n-BuOH fraction of fruits which presented the lower values of IC50 were used to determine the type of inhibition (competitive, non-competitive or uncompetitive) of ␣-glucosidase. The Lineweaver–Burk plots indicated that these fractions inhibited ␣-glucosidase in a non-competitive manner (Fig. 1A–C). Thus, the addition of each fraction in the reaction medium led to a change in the maximum velocity (Vmax ) of ␣-glucosidase while keeping the same value of Michaelis–Menten constant (KM ). In the presence of the EtOAc, n-BuOH fraction of fruits and the EtOAc
Table 2 Main phenolic compounds identified by RP-HPLC in fruits EtOAc, fruits n-BuOH and flowers EtOAc fractions of S. arenaria. No
RT (min)
Compounds
% in Fr Et
% in Fl Et
% in Fr B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
3.89 5.84 8.6 9.05 9.10 9.85 10.09 10.26 10.5 10.8 11.09 11.53 11.99 12.24 12.43 12.47 12.65 13.06 13.56 14.08 14.33 14.68 15.18 15.20 15.37 15.59 16.19 16.8 17.59 18.10 18.13 19.34 19.5 19.79
Catechin Chlorogenic acid Unknown Unknown 2-Hydroxyphenylacetic acid Unknown Unknown Cinnamic acid p-Coumaric acid Taxifolin Ferulic acid Unknown Rutin Unknown Unknown Unknown Methyl 4-hydroxybenzoate Morin Daidzein Unknown Quercetin Hesperetin Genistein Unknown Unknown Unknown Unknown Methyl trans-cinnamate Unknown Unknown Chrysin Unknown Unknown Unknown
ND 2.02 ND 2.48 ND ND ND ND ND 1.19 2.06 ND ND ND 1.8 2.78 19.12 3.5 ND ND 9.33 ND ND ND ND 41.26 1.04 1.34 0.77 3.4 1.45 1.21 1.59 3.58
5.73 ND ND ND ND ND ND ND 5.79 ND 7.10 7.46 6.68 5.53 ND ND ND ND 2.54 3.10 6.53 23.6 ND 17.18 8.75 ND ND ND ND ND ND ND ND ND
ND ND 61.57 ND 6.54 5.08 3.81 6.73 ND ND 12.53 ND ND ND ND ND ND ND ND ND ND ND 3.71 ND ND ND ND ND ND ND ND ND ND ND
RT: retention times (min). ND: not detected. Fr Et: fruits EtOAc fraction; Fl Et: flowers EtOAc fraction; Fr B: fruits n-BuOH fraction.
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Fig. 2. RP-HPLC chromatograms of flowers EtOAc fraction (A), fruits EtOAc fraction (B) and fruits n-BuOH fraction (C) after acid hydrolysis. Signal was recorded at 280 nm. Numbering of peaks refers to their identification as shown in Table 2.
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fraction of flowers, the Michaelis–Menton constant (KM ) remained almost constant (2.27 mM, 2.56 mM and 2.32 mM, respectively). At the same extract concentration (1.25 mg/ml), the maximum velocity (Vmax ) decreased from 0.333 DO/min in the absence of any inhibitor to 0.15 DO/min, 0.13 DO/min and to 0.128 DO/min in the presence of fruits EtOAc, fruits n-BuOH and flowers EtOAc fraction, respectively. According to this type of inhibition, we can suggest that the active components in these extracts do not compete with the substrate to bind to the active site, rather the inhibitors bind to a separate site on the enzyme to retard the conversion of substrate to product. When comparing the inhibition manner of ␣-glucosidase by different fractions of S. arenaria with other previous studies, we found that the ethyl acetate extracts of Agatophora alopecuro, Anabasis articulata, Hammada elegans, Helianthemum kahiricum and Salsola baryosma harvested from Algeria presented interesting antidiabetic activities and exhibited varied types of inhibition [39].
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of the compounds contained therein. However, some researchers have reported a relationship between the chemical composition of the major compounds in the extract and their biological activities [41]. Moreover, today, it is known that the synergistic or antagonistic effect of a compound present in minor proportion in a mixture must be considered as well [42]. As previously demonstrated, the EtOAc fractions of fruits, flowers and the n-BuOH fraction of fruits are potent inhibitors of ␣-glucosidase which may be due to the presence of flavonoids. Indeed, the major compounds mentioned in Table 2 (catechin, cinnamic acid, ferulic acid, quercetin and hesperetin) have been found by several researchers as good ␣-glucosidase inhibitors [43–50]. The anti-␣-glucosidase activity of these three fractions may be due to the presence and the possible synergistic effect of these phenolic compounds. In addition, up to 18 compounds were unknown and are the subject of future purification and characterization efforts. 4. Conclusion
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As mentioned above, the fruits EtOAc, n-BuOH and the flowers EtOAc fractions showed an interesting ␣-glucosidase inhibition. This result motivated us to identify the constituents of each fraction by RP-HPLC and localize the eventual researched activities. It is known that the free forms of phenolic compounds are rarely present in plants. More often, they occur as esters, glycosides or are bound to the cell wall. For this reason, before HPLC analysis, the hydrolysis of glycosides or esters was necessary, so that phenolic compounds can be identified, since a considerable fraction is in bounded form. Moreover, BHT, a powerful antioxidant, was added to prevent phenolics degradation during hydrolysis [40]. RP-HPLC coupled with UV–vis multiwavelength detector was employed to separate phenolic compounds. Compounds have been identified according to their retention times and the spectral characteristics of their peaks against those of standards, as well as by spiking the sample with standards. The compounds identified in the three fractions are reported in Table 2 and Fig. 2A–C. It was found that the EtOAc fraction of flowers contained twelve compounds numbered according to their retention time (Fig. 2A; Table 2). It is rich in flavonoids such as hesperetin (22) (23.6%), rutin (13) (6.68%), quercetin (21) (6.53%) and catechin (1) (5.73%). This fraction also contained phenolic acids in high proportion such as ferulic acid (11) (7.10%) and p-coumaric acid (9) (5.79%). Five unknown compounds were found in this fraction. On the other hand, the EtOAc fruits fraction contained 18 compounds numbered according to their retention time (Fig. 2B; Table 2). Among the major compounds, we identified an unknown molecule (26) in high percentage (41.26%), methyl 4hydroxybenzoate (19.12%), quercetin (21), (9.33%) and morin (18), (3.5%). This fraction also contained several minor compounds such as phenolic acid: Chlorogenic acid (2) (2.02%) and ferulic acid (11) (2.06%). We also noted that 9 molecules of this fraction were still unknown. However, among the compounds of the butanolic fruits fraction, we detected product (5) 2-hydroxyphenylacetic acid with a percentage of 6.54%. We also obtained ferulic acid (11) (12.53%), cinnamic acid (8) (6.73%) and genistein (23) (3.71%). The major product in this fraction numbered (3) (61. 57%) was also still unknown (Fig. 2C; Table 2). The chemical analysis of the three S. arenaria fractions showed their richness in secondary metabolites which could be considered responsible for a more or less important part of enzymatic inhibition activities. These biological activities are difficult to correlate with a specific compound, because of the complexity and variability
This is the first study on enzymatic inhibition by S. arenaria extracts. Our results showed that some of these extracts exhibited a potent ability to inhibit ␣-glucosidase. The type of inhibition by the most active extracts was confirmed as non-competitive. These results may be due to the presence of the above mentioned compounds. The data pointed out that S. arenaria containing these flavonoids could be a useful natural source for the development of ˛-glucosidase inhibitory agent. Future studies are needed to isolate and identify the bioactive metabolites which are effective in the ␣-glucosidase inhibitory activity. Declaration of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors are grateful to Dr. Fethia Harzallah-Skhiri (High Institute of Biotechnology of Monastir, Monastir, Tunisia) for the botanical identification. We are grateful to Pr. Bejaoui Hafedh (FSS, Sfax-Tunisia) for his help with English. References [1] G. Rea, A. Antonacci, M. Lambreva, A. Margonelli, C. Ambrosi, M.T. Giardi, U.S. Q3 Springer, vol. 698, pp. 1–16 (Chapter 1). [2] S.I. Cameron, R.F. Smith, K.E. Kierstead, Pharm. Biol. 43 (2005) 425–433. [3] Z.M. Evangelista, A.E. Moreno, Biotecnologia 11 (2007) 37–50. [4] K. Colvine, A.J. Kerr, A. McLachlan, P. Gow, S. Kumar, J. Ly, C. Wiltshire, E. Robinson, N. Dalbeth, N. N. Z. Med. J. 121 (2008) 73–81. [5] A. Corrado, F. D’Onofrio, N. Santoro, N. Melillo, F.P. Cantatore, Minerva Med. 97 (2006) 495–509. [6] J. Gorelick, A. Kitron, S. Pen, T. Rosenzweig, Z. Madar, J. Ethnopharmacol. 137 (2011) 1245–1249. [7] Z.P. Yu, Y.G. Yin, W.Z. Zhao, Y.D. Yu, B.Q. Liu, F. Chen, Food Chem. 129 (2013) 1376–1382. [8] N. Amessis-Ouchemoukh, K. Madani, M.P. Falé, L. Serralheiro, M.E. Araújo, Ind. Crops Prod. 53 (2014) 6–15. [9] J. Yan, G. Zhang, J. Pan, Y. Wang, Int. J. Biol. Macromol. 64 (2014) 213–223. [10] S.D. Kim, Food Chem. 136 (2013) 297–300. [11] P.M. Heacock, S.R. Hertzler, J.A. Williams, B.W. Wolf, J. Am. Diet. Assoc. 105 (2005) 65–71. [12] A.D. Baron, Diabetes Res. Clin. Pract. 40 (1998) 51–55. [13] H.E. Lebovitz, Endocrinol. Metab. Clin. North Am. 26 (1997) 539–551. [14] T. Fujisawa, H. Ikegami, K. Inoue, Y. Kawabata, T. Ogihara, Metabolism 54 (2005) 387–390. [15] E. Le floc’h, L. Boulos, E. Vela, Catalogue synonymique commenté de la Flore de Tunisie, République Tunisienne, Ministère de L’environnement et du Développement Durable, banque nationale de gènes, 2010, pp. 195–196. [16] M.A. Bonet, M. Parada, A. Selga, J. Vallès, J. Ethnopharmacol. 68 (1999) 145–168. [17] R.W. Bussmann, G. Malca-García, A. Glenn, D. Sharon, G. Chaitc, D. Díaz, K. Pourmand, B. Jonat, S. Somogy, G. Guardado, C. Aguirrf, R. Chan, K. Meyer,
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