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New flavonoid glycosides from two Astragalus species (Fabaceae) and validation of their antihyperglycaemic activity using molecular modelling and in vitro studies
Industrial Crops & Products 118 (2018) 142–148
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
New flavonoid glycosides from two Astragalus species (Fabaceae) and validation of their antihyperglycaemic activity using molecular modelling and in vitro studies ⁎
Abdulaziz A. Janibekova,1, Fadia S. Youssefb,1, Mohamed L. Ashourb, , Nilufar Z. Mamadalievaa, a b
T
⁎
Institute of the Chemistry of Plant Substances, Academy of Sciences, Mirzo Ulugbek str. 77, 100170 Tashkent, Uzbekistan Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, 11566, Egypt
A R T I C LE I N FO
A B S T R A C T
Keywords: Astragalus Cytotoxicity α-Glucosidase Molecular modelling
Phytochemical investigation of the methanol extracts of the aerial parts of Astragalus turkestanus and A. xапthomeloides using various chromatographic techniques resulted in the isolation and structural elucidation of two new flavonoid glycosides, namely, 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) from the former and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D- galactopyranoside (6) from the latter. In addition five other known compounds were isolated for the first time from these species. In silico molecular modeling study was carried out on α-glucosidase (PDB ID 3TOP; 2.88 A°) to assess the α-glucosidase inhibitory activity of the isolated compounds. Both compounds 3 and 6 displayed the highest fitting score with ΔG equals to −82.10 and −67.66 Kcal/mol in the pH-based ionization method and −68.55 and −67.83 Kcal/mol in the rule-based ionization method, respectively. α-Glucosidase inhibitory activity was further asserted in vitro using an ELISA assay that is measured colorimetrically at 405 nm. Compound 3 showed a significant α-glucosidase inhibitory activity displaying IC50 of 50.31 μg/mL approaching that of acarbose (IC50 = 30.57 μg/mL). All the tested samples showed no significant cytotoxic activity against Caco-2 (human epithelial colorectal adenocarcinoma) cells using the MTT assay (IC50 > 400 μg/mL). Thus; both new compounds could offer promising natural antihyperglycaemic entities without any marked toxicity, which could be useful in the pharmaceutical industry.
1. Introduction
2017). Pharmaceutical industry worldwide is based mainly upon formulating the synthetic raw materials produced by companies that are of prominent side effects. Herein, an excellent opportunity is provided to use the treasure supplied by the natural environment as raw material to be formulated in a suitable dosage form by the pharmaceutical companies. These pharmaceutical products will be used by diabetic patients with minimum cost and accepted by several categories in the society than synthetic drugs. Astragalus represents one of the popular genera of flowering plants belonging to the family Fabaceae. It comprises nearly about 2000–3000 species of annual or perennial herbs, subshrubs, or shrubs that are prevalent in temperate and arid areas (Li et al., 2014). Traditionally, many of these species were widely adopted for the relief of depression and as diuretics and tonics (Avunduk et al., 2007; Choudhary et al.,
Diabetes mellitus has recently been recognized as one of the most popular spreading metabolic dysfunction with an evident occurrence of morbidity and mortality all over the globe. It is characterized by the consequent emergence of various complications on the micro- and macrovascular levels as neuropathy, retinopathy, and nephropathy as well as cardiovascular complications and ulceration, (Singab et al., 2014; Youssef et al., 2017). Although, insulin and synthetic drugs that control hyperglycaemia are considered the primary ways for proper adjustment of blood glucose levels, they showed vigorous undesirable side effects with concomitant failure to prohibit the complications associated with diabetes. Thus, the need for novel antidiabetic entities of herbal origin for effective combating of hyperglycaemia is felt mandatory worldwide (Youssef et al.,
Abbreviations: AG, α-glucosidase enzyme; AT, methanol extracts of the aerial parts of A. turkestanus; AX, methanol extracts of the aerial parts of A. xапthomeloides; PPARα, peroxisome proliferator-activated receptor alpha; PPARγ, peroxisome proliferator-activated receptor gamma ⁎ Corresponding authors. E-mail addresses: [email protected] (M.L. Ashour), [email protected] (N.Z. Mamadalieva). 1 These authors have contributed equally to this work. https://doi.org/10.1016/j.indcrop.2018.03.034 Received 28 December 2017; Received in revised form 16 February 2018; Accepted 17 March 2018 Available online 05 April 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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combined n-butanol fractions were then concentrated at 42 °C under reduced pressure to yield 152 g and 64 g, respectively. 150 g of the dried n-butanol fraction of A. turkestanus were chromatographed over a silica gel column using the dry loading method. The column was then eluted with CHCl3/CH3OH (9:1, v/v) accompanied by monitoring using TLC on silica gel 254 plates to give subfractions A1- A26 which contain a mixture of less polar compounds. Consequently elution with CHCl3/CH3OH/H2O in the ratio of 4:1:0.1, v/v followed by 70: 23:3, v/v accompanied by monitoring using TLC on silica gel 254 plates with subsequent pooling of similar subfractions was done in which the polar subfractions were collected in order to be B1B20 and C1-C25, respectively. Subfractions B12-B19 were pooled together and then subjected to column chromatography using silica gel with subsequent elution with CHCl3/CH3OH (6:1, v/v) to give a mixture (16 mg) of compounds 1 and 2. Additionally, the subfractions C7C10 were collected together, subjected to silica gel column chromatography and eluted using CHCl3/CH3OH/H2O (70:23:3 v/v) to give compound 3 (7 mg) and compound 4 (13.5 mg). In addition, 60 g of the dried n-butanol fraction of A. xапthomeloides were subjected to silica gel column and subsequently eluted using the following solvent systems successively CHCl3/CH3OH (9:1, v/v), followed by a mixtures of CHCl3/CH3OH/H2O (70:12:1, v/v), (4:1:0.1, v/ v), (70:23:3, v/v), (70:28:8, v/v) and (60:35:8, v/v) and then monitored by TLC on silica gel 254 plates with subsequent pooling of similar subfractions and gave X1-97 subfractions. Subfractions X71-77 were collected together and separated by silica gel column chromatography, then eluted with CHCl3/CH3OH/H2O (70:28:8, v/v) and followed by further purification to yield compound 5 (15 mg). Separation of sub fractions X88-90 by silica gel column chromatography using solvent system (60:35:8, v/v) resulted in the isolation of compound 6 (15.5 mg) and compound 7 (12 mg). TLC plates were visualized under UV light (λ = 254 and 365 nm) and by spraying with phosphorus wolfram acid solution followed by heating at 105° C for 10 min. A scheme summarizing the isolation of secondary metabolites from both Astragalus species was added in the in the supplementary data (Fig. S1).
2008). Recently, many of the Astragalus species have shown many biological activities, in particular, antiviral and hepatoprotective properties in addition to boosting the immune response (Linnek et al., 2011). Undoubtedly, this could be attributed to the existence of many bioactive secondary metabolites, including alkaloids, anthraquinones, flavonoids and saponins (Ibrahim et al., 2013). Meanwhile, many of the species are used as nutrients, substituting agents for many beverages as tea or coffee as well as medicinal agents and cosmetics relying on their richness by amino acids, polysaccharides and metallic elements (Li et al., 2014). In addition some Astragalus species have been reported to exert a potent antihyperglycaemic activity particularly the radix of many varieties belonging to Astragalus membranaceus that proved a high efficacy with a satisfying safety margins. This is relied upon their richness with polysaccharides and isoflavones. The polysaccharides resulted in a notable decline in serum glucose levels, triglycerides, and low density lipoproteins as well as in insulin resistance with concomitant elevation in high density lipoproteins in streptozotocin- induced diabetes in rats (Fu et al., 2014). However, the isolated isoflavones from Astragalus membranaceus showed a good antihyperglycaemic amelioration via in vitro activation of PPARα and PPARγ in addition to promoting adipocyte differentiation (Shen et al., 2006). Astragalus turkestanus Bge. and A. xапthomeloides Eug. Korr. et M. Pop. are endemic to the Uzbek flora. Tracing the current literature, nothing was found regarding either the chemical composition or the biological activities of A. turkestanus. However, only two compounds namely, D-pinitol (3-O-methyl-D-chiro-inositol) and kaempferol-3-O-αL-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside were reported earlier from A. xапthomeloides by one of the authors (Janibekov et al., 2015; Janibekov et al., 2016). Herein we reported the isolation and structural elucidation of two new antihyperglycaemic flavonoid glycosides from the aerial parts of Astragalus turkestanus and A. xапthomeloides, which are 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6). In addition, five other compounds namely; daucasterol (1) and stigmasterol glucoside (2), D-pinitol (4), azukisaponin V (5) and 7-methoxy kaempferol-3-O-β-D-glucopyranoside (7) were also isolated. Molecular modeling of the isolated compounds within the active sites of α-glucosidase was done followed by in vitro evaluation of their α-glucosidase inhibitory activity to validate their bioactivity. In addition, their cytotoxic effect on Caco-2 cell line was reported.
2.3. General experimental procedures 1 H and 13C (APT) NMR analyses were carried out on a Bruker Ascend 400/R spectrometer (Burker Avance III, Fallanden Switzerland) at the operating frequencies of 400 and 100 MHz, respectively. Chemical shifts were expressed in δ ppm and were related to that of the solvents. The measured samples were dissolved in various deuterated solvents (Sigma Aldrich, Germany) depending upon their solubility and transferred to 3 mm NMR tubes (Bruker). Spectra were recorded at 25 °C; δ ppm rel. to Me4Si as internal standard. Two-dimensional (2D) NMR experiments (H, H −COSY; H, C HSQC; H, C HMBC) were performed utilizing the pulse sequences from the Bruker user library. ESI–MS analysis was done on a Waters Xevo TQD mass spectrometer with UPLC Acquity mode (Milford, USA). HR-ESI–MS analyses were performed using Bruker micro-TOF-Q Daltonics (API) Time-of-Flight mass spectrometer (Bremen, Germany) applying the method previously described (Nováková et al., 2010). Normal phase column chromatography was performed using silica gel (63–100 μm; Tianjin, Sinomed Pharmaceutical Co. Ltd., China). TLC analysis was done utilizing normal phase silica gel precoated plates F254 (Merck, Germany).
2. Materials and Methods 2.1. Plant material The aerial parts of Astragalus turkestanus Bge. and Astragalus xапthomeloides Eug. Korr. et M. Pop. were collected from the Qashqadaryo and Tashkent regions of Uzbekistan in 2015. The authenticated voucher specimens of the species (Accession no. 20081101 and 20081106, respectively) are kept in the Department of Herbal Plants (Institute of the Chemistry of Plant Substances, Uzbekistan). 2.2. Extraction and isolation The air-dried powdered aerial parts of A. turkestanus and A. xапthomeloides (2.5 kg each) were macerated in neat methanol (5 × 12 L) at room temperature till exhaustion (no considerable yield was further obtained) i.e. the extraction was carried out five times, using 12 L of neat methanol each. The solvent was evaporated at 40 °C using a rotary vacuum evaporator (BÜCHI Labortechnik AG, Switzerland) and concentrated to give 233 and 190 g dried methanol extracts, respectively. Consequently, the dried methanol extracts were successively fractionated using chloroform then n-butanol. The
2.4. Compound characterization 2.4.1. 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyrano side (3) Yellowish amorphous powder; UV (MeOH) λmax: 267 and 349 nm; 1 H (400 MHz) and 13C NNR (100 MHz) in DMSO see Table 1; HRESI–MS [M-H]− m/z 593.1530 (calculated. for m/z C27H30O15, 593.1584). Compound different spectra are available in the 143
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Table 1 1 H (400 MHz) and ppm, J in Hz).
C (100 MHz) data of compounds 3 and 6 in DMSO (δ in
3
#
δH (multi, J values) 1 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 7 (OCH3)
glucosidase (PDB ID 3TOP; 2.88 A°) downloaded from protein data bank (www.pdb.org) that is co-crystalized with acarbose, lead compound, using Discovery Studio 2.5 (Accelrys Inc., San Diego, USA). Docking was carried out using C-docker protocol applying both pHbased ionization and rule-based ionization methods. Docking was performed in a similar manner as previously described (Youssef et al., 2016, 2017; Ashour et al., 2017). However, the free binding energies were calculated in Kcal/mol using the following equation:
13
6.71 (d, 2.0, 1H) 6.36 (d, 2.0, 1H)
8.06 (d, 8.5, 1H) 6.90 (d, 8.5, 1H) 6.90 (d, 8.5, 1H) 8.06 (d, 8.5, 1H) 3.86 (s, 3H)
6 δC
160.51 (C) 133.91 (C) 178.00 (C]O) 161.31 (C) 98.40 (CH) 165.55 (C) 92.81 (CH) 157.27 (C) 105.54 (C) 121.24 (C) 131.45 (CH) 115.59 (CH) 156.86 (C) 115.59 (CH) 131.45 (CH) 56.56
δH (multi, J values)
δC
6.88 (d, 8.4, 1H) 8.10 (d, 8.4, 1H)
160.63 (C) 134.00 (C) 178.09 (C]O) 161.31 (C) 95.10 (CH) 162.07 (C) 99.79 (CH) 157.52 (C) 106.03 (C) 121.13 (C) 131.5 (CH) 115.58 (CH) 156.47 (C) 115.58 (CH) 131.5 (CH)
6.82 (d, 2.0, 1H) 6.46 (d, 2.0, 1H)
8.10 (d, 8.4, 1H) 6.88 (d, 8.4, 1H)
ΔGbinding = Ecomplex − (EAG + E
ligand)
Where; ΔGbinding: The ligand–enzyme interaction binding energy, Ecomplex: The potential energy for the complex of AG bound with the ligand, Eprotein: The potential energy of AG alone and Eligand: The potential energy for the ligand alone. 2.7. In vitro evaluation of the α-glucosidase inhibitory activity The inhibitory effect of the samples on α-glucosidase was determined in vitro based upon the previously reported method with certain modifications (You et al., 2011). Basically, 1 mg of each of the examined samples or acarbose (positive control) at several concentrations in the range of 1.95–500 μg/mL was incubated together with 500 μL of 1.0 U/mL α-glucosidase solution in 100 mM phosphate buffer, pH = 6.8, kept at 37 °C for 20 min. The absorbance of the obtained pnitrophenol was determined at a λmax = 405 nm utilizing a microplate reader (Bio Tek Instruments, Inc., Winooski, VT). Noteworthy to mention that the control is the control reaction that contains all the reagents except the tested sample. Moreover, the IC50 is the concentration of the tested sample resulting in 50% inhibition of α-glucosidase activity under the experiment conditions. The inhibition percentage was computed using the following equation
Sugar 1” 2” 3” 4” 5” 6”
Galactose 3.97 (d, 7.1, 1H) 3.12 (m, 1H) 2.79 (m, 1H) 3.12 (m, 1H) 3.20 (m, 1H) 3.76 (m, 1Ha), 2.70 (m, 1Hb)
Galactose 104.07 (CH) 70.24 (CH) 73.56 (CH) 69.84 (CH) 74.52 (CH) 68.24 (CH2)
Rhamnose 5.56 (d, 1.5, 1H) 3.40 (m, 1H) 3.64 (m, 1H) 3.38 (m, 1H) 3.09 (m, 1H) 1.13 (d, 6.1, 3H)
Rhamnose 98.85 (CH) 70.87 (CH) 70.54 (CH) 68.72 (CH) 72.04 (CH) 18.38 (CH3)
Sugar 1”' 2”' 3”' 4”' 5”'
Arabinose 5.38 (d, 1.5, 1H) 3.12 (m, 1H) 2.79 (m, 1H) 3.28 (m, 1H) 3.50 (m, 1Ha), 3.42 (m, 1Hb)
Arabinose 101.34 (CH) 76.66 (CH) 76.67 (CH) 76.86 (CH) 65.75 (CH2)
Galactose 5.35 (d, 7.5, 1H) 3.57 (m, 1H) 3.35 (m, 1H) 3.41 (m, 1H) 3.85 (d, 6.4, 1H)
Galactose 102.23 (CH) 74.06 (CH) 71.55 (CH) 73.40 (CH) 70.29 (CH)
6”'
3.62 (m, 1Ha), 3.36 (m, 1Hb)
65.65 CH2)
2.8. Cytotoxicity assay
Sugar 1”” 2”” 3”' 4”” 5”” 6””
Rhamnose 4.41(d, 1.5, 1H) 3.31 (m, 1H) 3.64 (m, 1H) 3.62 (m, 1H) 3.09 (m, 1H) 1.06 (d, 6.1, 3H)
Rhamnose 100.46 (CH) 71.05(CH) 70.71 (CH) 68.43 (CH) 72.30 (CH) 18.38 (CH3)
MTT assay was performed as previously mentioned by (Saliba et al., 2002) with certain modifications. Briefly, 100 μL of the examined sample after dissolution in 5% DMSO and RPMI tissue culture medium were added to each well in order to attain a final concentration of (10–4000 μg/mL). Control wells contained two aliquots of 100 μL of tissue culture medium (MEM + fetal bovine serum at 9:1 ratio) and 100 μL of 5% sterile DMSO and RPMI tissue culture medium. After 24 h incubation period at 37 °C, the wells were washed with PBS with subsequent incubation with MTT solution (2 mg/mL) at 100 μL per each well for 1 hat 37 °C. Supernatants were then decanted and the cells were treated with 100 μL of DMSO per each well to dissolve the formazan crystals formed in the viable metabolically active cells. Elutes of the 8 wells of each sample were collected and the absorbance was measured at 540 nm. Control wells were similarly treated (Elissawy et al., 2017). The percentage cytotoxicity was determined applying the following equation (Murakami et al., 2000).
% Inhibition = (A 405 of the control − A 405 of the tested sample ) × 100
supplementary data (Fig. S2). 2.4.2. Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-β-D-glucopyranosyl(1 → 6)-α-L-rhamnopyranoside(6) Light yellowish amorphous powder; UV (MeOH) λmax: 266 and 352 nm; 1H (400 MHz) and 13C NNR (100 MHz) in DMSO see Table 1; HR-ESI–MS [M-H]− m/z 739.2090 (calculated. for m/z C33H40O19, 739.2091). Compound different spectra are available in the supplementary data (Fig. S3).
A540 of the test culture ⎫ Cytotoxcity % = 1 − ⎧ × 100 ⎨ ⎩ A540 of the control culture ⎬ ⎭
2.5. Acid hydrolysis of the isolated flavonoids A solution of each of the tested compounds was treated with HCl using the same method and conditions as previously reported (Kim et al., 1999). Identification of D- glucose, D-galactose, L-rhamnose; and L-arabinose resulted from the tested compounds hydrolysis was done by comparing their retention time and retention factors with those of authentic samples using co-chromatography.
3. Results and Discussion 3.1. Phytochemical characterization Comprehensive phytochemical characterization of the methanol extracts of the aerial parts of A. turkestanus (AT) and A. xапthomeloides (AX) resulted in the isolation and structural elucidation of two new flavonoid glycosides which are 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) from the former and
2.6. Molecular modelling study on α-glucosidase Virtual screening of the isolated compounds was done on α144
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Fig. 1. Secondary metabolites isolated from the aerial parts of Astraglus species.
kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D- galactopyranoside (6) from the latter in addition to other five known compounds that were isolated for the first time from these species. Their structures were fully elucidated by comparing their 1D and 2D NMR data with those reported in literature for previously isolated compounds (Fig. S4). They were identified as daucasterol (1) and stigmasterol glucoside (2) (Ashour et al., 2012) in addition to D-pinitol (4) (Sharma et al., 2016) from A. turkestanus whereas an oleanane type triterpenoidal saponin, azukisaponin V (5) (Gromova et al., 2001), and a flavonoid glycoside, 7-methoxy kaempferol-3-O-β-D-glucopyranoside (7) (Khalfallah et al., 2014) were isolated from A. xапthomeloides (Fig. 1) The structures of the two new compounds were comprehensively elucidated using UV, NMR and HR-ESI–MS techniques (Table 1). Compound 3 was obtained as a yellow amorphous powder, its molecular formula C27H30O15, was clarified using HR-ESI–MS showing m/z: 593.1530 [M-H]−, (calculated. 593.1584). The UV spectra of the methanol solution of compound 3 showed two characteristic λ maxima existing at 267 and 349 nm for bands I and II, respectively confirming the presence of a flavanol skeleton. Upon addition of sodium acetate no bathochromic shift was shown in band I that consequently declared the absence of a free hydroxyl group at position 7. However upon addition of AlCl3 reagent, a bathochromic shift (38 nm) was observed only in band I (229 nm) indicating the presence of a free hydroxyl group at position 5 with no bathochromic shift observed in band II that revealed the absence of a free hydroxyl group at position 3. Its 1H NMR revealed the existence of four doublet signals corresponding to six aromatic protons which are: ortho-coupled at δH 8.06 (d, J = 8.5, 2H) and 6.90 (d, J = 8.5, 2H) and meta- coupled at 6.71 (d, J = 2.0, 1H) and 6.36 (d, J = 2.0, 1H) that confirmed compound 3 to be a kaempferol derivative. Moreover, two distinct signals in both 1H NMR at δH 3.97 (d, J = 7.1, 1H) and 5.38 (d, J = 1.5, 1H) and 13C NMR spectra at δc 104.07 and
101.34 ppm, respectively were found and were further confirmed from HSQC spectrum. These presented data further ascertained the presence of two anomeric protons and carbons and proved compound 3 to be a flavanol diglycoside. However, a sharp singlet signal in the 1HNMR spectrum integrated for three protons at δH 3.86 ppm that is correlated with a carbon signal at δc 56.56 ppm as revealed from 13C NMR and HSQC correlation declared the presence of a OCH3 group. From HMBC spectrum of compound 3 a long range HMBC correlation exist between the protons of OCH3 group at δH 3.86 ppm and the C7 carbon at δc 165.55 ppm suggesting the presence of OCH3 group at position 7 and thus the sugars moieties will be at position 3. The mass spectral data together with the above displayed analytical ones, suggested a kaempferol derivative containing one hexoside and one pentoside moieties. 1H NMR,13C NMR and HSQC correlations declared the assignment of one galactose (Sikorska and Matlawska, 2000; Bubb, 2003) and one arabinose molecule (Xiao et al., 2006). The large JH1″-H2″ coupling constant 7.1 Hz established the β-configuration of the galactose moiety meanwhile the small JH1‴-H2‴ coupling constant 1.5 Hz established the α-configuration of the arabinose molecule (de Sousa et al., 2017). The glycone chain (galactose- arabinose) was established to be between C1”” and C6”' as revealed from the downfield shift of C6‴ (68.24 ppm) of the galactose moiety (Dudek-Makuch and Matławska, 2011).In addition, upon acid hydrolysis D-galactose and L-arabinose were identified by using co-chromatography and after being compared with authentic sugars. Thus compound 3 was finally identified as 7methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (Fig. 2). Compound 6 was obtained as a light yellowish amorphous powder, its molecular formula was determined to be C33H40O19, as revealed from HR-ESI–MS that showed a molecular ion peak in the negative mode equals to 739.2090 [M-H]−, calculated. 739.2091. Comprehensive interpretation of its UV spectral data clarified the 145
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glucosidase inhibitory activity of the isolated compounds that consequently predicted their antihyperglycaemic activity using both pHbased ionization and rule-based ionization methods. The former was done in a pH range of 6.5 and 8.5 with an average of 7.5 to mimic the biological circumstances; however the latter was done to examine the effect of various ionisable groups on the attitude of the molecules at the active site. Both 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-αL-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6) displayed the highest fitting score in both ionization methods with ΔG equals to −82.10 and −67.66 Kcal/mol in the pH-based ionization method and −68.55 and −67.83 Kcal/mol in the rule-based ionization methods for both compounds, respectively approaching that of acarbose. On the contrary, azukisaponin V (5) did not show any binding affinity and failed to dock in the active site using either ionization methods. Results were displayed in Table 2.
Fig. 2. 1H–1H COSY and HMBC key correlations of compounds 3 and 6.
presence of two distinctive λ maxima appearing at 266 and 352 nm corresponding to bands I and II, respectively that consequently proved the existence of a flavanol skeleton. Using sodium acetate as a UV shifting reagent did not cause any obvious bathochromic shift in band I and thus declared the absence of a free hydroxyl group at position 7. On the contrary the addition of AlCl3 reagent resulted in a bathochromic shift (26 nm) in band I (240 nm) ascertaining the presence of a free hydroxyl group at position 5, however no bathochromic shift was observed in band II declaring the absence a free hydroxyl group at position 3. Similarly as compound 3, a kaempferol derivative was suggested owing to the presence of four doublet signals in the 1H NMR spectrum corresponding to six aromatic protons which are; ortho-coupled at δH 8.10 (d, J = 8.4, 2H) and 6.88 (d, J = 8.4, 2H) and meta- coupled at 6.82 (d, J = 2.0, 1H) and 6.46 (d, J = 2.0, 1H). 1H NMR and 13C NMR showed signals for three anomeric protons and carbons. HSQC confirmed the correlation between the anomeric protons and their respective carbons to be as follows δH 4.41(d, J = 1.5, 1H) and δc 100.46 ppm; δH 5.35 (d, J = 7.5, 1H) and δc 102.23 ppm; 5.56 (d, J = 1.5, 1H) and δc 98.85 ppm. From mass spectral information as well as from the above displayed analytical ones, we concluded that compound 6 is a flavanol triglycoside containing three hexoses, the large JH1‴-H2‴ coupling constant 7.5 Hz established the β-configuration of one hexose moiety meanwhile the small JH1″-H2″ and JH1″″-H2″″ coupling constant 1.5 Hz established the α-configuration for the other two hexoses (de Sousa et al., 2017). 1H NMR, 13C NMR and HSQC correlations declared the assignment of one galactose and two rhamnoses (Jung et al., 1999; Sikorska and Matlawska, 2000; Bubb, 2003). From HMBC spectral data of compound 6, it was obvious that a long range HMBC correlation occurs between the anomeric proton of the galactose moiety at δH 5.35 ppm and C8 at δc 99.79 ppm and this in turn suggested the existence of a galactose- rhamnose chain at position 7. Additionally, the linkage between galactose- rhamnose was established to be between C1″″ and C6‴ as revealed from the long range HMBC correlation that exists between the anomeric proton of one rhamnose moiety at δH 4.41 ppm and C6‴ of the galactose moiety at δc 65.65 ppm in addition to the downfield shift of C6‴ (65.65 ppm) of the galactose moiety (Dudek-Makuch and Matławska, 2011). Additionally, a long range HMBC correlation exists between the anomeric proton of rhamnose H1” at δH 5.56 ppm and C2 at δc 160.63 indicating the existence of a single rhamnose moiety at position 3. Besides, upon acid hydrolysis Dgalactose and L-rhamnose were identified by using co-chromatography and upon comparison with authentics. Thus compound 6 was finally identified as kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (Fig. 3).
3.3. In vitro α-glucosidase inhibitory activity Accordingly, in vitro α-glucosidase inhibitory activity was evaluated for the previously mentioned flavonoid glycosides to validate their antihyperglycaemic activity and to confirm the obtained molecular modelling results. Moreover, α-glucosidase inhibitory activity was also assessed for azukisaponin V (5) to ascertain the validity of the docking study. 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) showed a significant α-glucosidase inhibitory activity displaying IC50 of 50.31 μg/mL approaching that of acarbose that is 30.57 μg/mL (Table 3). This undoubtedly could be explained in virtue of its 2D and 3D binding modes as displayed in Fig. 3 in which the compound is tightly bonded to the active site via the formation of nine firm hydrogen bondings with Asp 1157, Asp 1279, Asp 1420, Asp 1526, Arg 1510, Thr 1586 and Lys 1466 amino acid residues at the active site. However, kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6) exhibited a substantial activity with IC50 equals to 79.60 μg/mL. Moreover, azukisaponin V (5) showed a mild inhibitory activity showing 117.96 μg/mL as an IC50 and thus it comes in accordance with the results of the molecular docking studies. Previous studies have been done to evaluate the α-glucosidase inhibitory activity of other Astragalus species particularly Astragalus membranaceus root in which its ethanol extract showed α-glucosidase inhibitory activity of 49.71 μg/mL. The α-glucosidase inhibitory activity of the ethanol extract was mainly attributed to the presence of flavonoids and phenolic compounds. The formers were estimated by 44.93 μg/mL quercetin and 70.32 μg/mL rutin whereas the latter were estimated by 26.13 μg/mL catechins (Yin et al., 2009). In addition, previous studies showed that the number of hydroxyl groups in ring B considerably affect the inhibitory activity. In conclusion, it was proved that the minimum structures requirements involves the presence of an unsaturated C ring in addition to the presence of a hydroxyl group at the 3 position, 4-CO, the linkage of the B ring at the 3 position, and the hydroxyl substitution on the B ring significantly increase the α-glucosidase inhibitory activity (Tadera et al., 2006).
3.4. Cytotoxic activity The total methanol extracts, (AT and AX) in addition to the isolated compounds, were tested for their cytotoxicity against Caco-2 (human epithelial colorectal adenocarcinoma) cells using the MTT assay (Table 4) but none of the tested samples showed significant cytotoxic activity indicating their safety. This came in accordance to some previously carried studies that showed the safety of some flavonoids glycosides (Chung et al., 2007).
3.2. In silico molecular modelling In silico molecular modelling study was done to examine the α146
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Fig. 3. 2D and 3D binding modes of compound 3 within α-glycosidase active site. Table 2 Free binding energies (ΔG) of the isolated compounds from Astragalus species in the active site of α-glucosidase using molecular modeling experiments calculated in Kcal/mol applying both pH-based and rule-based ionization methods. Compound
Acarbose Daucasterol (1) Stigmasterol glucoside (2) 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) D-Pinitol (4) Azukisaponin V (5) Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-Lrhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6) 7-Methoxy kaempferol-3-O-β-D-glucopyranoside (7)
Table 4 In vitro cytotoxicity evaluation using MTT assay on Caco-2 cell line.
Binding energy (Kcal/mol) pH-based
Rule based
−84.89 −63.64 −61.56 −82.10
−98.71 −63.64 −61.56 −68.55
−30.78 FD −67.66
−35.46 FD −67.83
−52.08
−51.81
Table 3 In vitro α-glucosidase inhibitory activity of 7-methoxy kaempferol-3-O-α-Larabinosyl-(1 → 6)-β-D-galactopyranoside (3), azukisaponin V (5) and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6). IC50 (μg/mL)
Acarbose 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) Azukisaponin V (5) Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-Lrhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6)
30.57 ± 3.27 50.31 ± 4.98
IC50 (μg/mL)
Astagalus xапthomeloides methanol extract Astagalus turkestanus methanol extract β-Sitosterol glucoside (1) 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) D-Pinitol (4) Azukisaponin V (5) Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl(1 → 6)-β-D- galactopyranoside (6)
1180.00 918.05 622.00 455.00 440.00 914.40 615.50
(1 → 6)-β-D-galactopyranoside (6) could offer a promising natural antihyperglycaemic entities without any marked toxicity that are of great usefulness in the pharmaceutical industries for the manufacturing of new safe antihyperglycaemic drugs that are recognized to be more welcomed by a large category of patients comparable to the synthetic drugs owing to its natural ancestry. However, in vivo studies followed by clinical trials should be conducted to ascertain their activity.
FD: fail to dock.
Compound
Sample
Declaration of interest The authors declare no conflict of interest. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
117.96 ± 10.96 79.60 ± 7.74
Appendix A. Supplementary data
All determinations were carried out in triplicate manner and values are expressed as the mean ± S.D.
Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.03.034.
4. Conclusion
References
Thus it can be concluded that the two new flavonoid glycosides 7methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-glactopyranoside (3) and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-
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New flavonoid glycosides from two Astragalus species (Fabaceae) and validation of their antihyperglycaemic activity using molecular modelling and in vitro studies ⁎
Abdulaziz A. Janibekova,1, Fadia S. Youssefb,1, Mohamed L. Ashourb, , Nilufar Z. Mamadalievaa, a b
T
⁎
Institute of the Chemistry of Plant Substances, Academy of Sciences, Mirzo Ulugbek str. 77, 100170 Tashkent, Uzbekistan Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, 11566, Egypt
A R T I C LE I N FO
A B S T R A C T
Keywords: Astragalus Cytotoxicity α-Glucosidase Molecular modelling
Phytochemical investigation of the methanol extracts of the aerial parts of Astragalus turkestanus and A. xапthomeloides using various chromatographic techniques resulted in the isolation and structural elucidation of two new flavonoid glycosides, namely, 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) from the former and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D- galactopyranoside (6) from the latter. In addition five other known compounds were isolated for the first time from these species. In silico molecular modeling study was carried out on α-glucosidase (PDB ID 3TOP; 2.88 A°) to assess the α-glucosidase inhibitory activity of the isolated compounds. Both compounds 3 and 6 displayed the highest fitting score with ΔG equals to −82.10 and −67.66 Kcal/mol in the pH-based ionization method and −68.55 and −67.83 Kcal/mol in the rule-based ionization method, respectively. α-Glucosidase inhibitory activity was further asserted in vitro using an ELISA assay that is measured colorimetrically at 405 nm. Compound 3 showed a significant α-glucosidase inhibitory activity displaying IC50 of 50.31 μg/mL approaching that of acarbose (IC50 = 30.57 μg/mL). All the tested samples showed no significant cytotoxic activity against Caco-2 (human epithelial colorectal adenocarcinoma) cells using the MTT assay (IC50 > 400 μg/mL). Thus; both new compounds could offer promising natural antihyperglycaemic entities without any marked toxicity, which could be useful in the pharmaceutical industry.
1. Introduction
2017). Pharmaceutical industry worldwide is based mainly upon formulating the synthetic raw materials produced by companies that are of prominent side effects. Herein, an excellent opportunity is provided to use the treasure supplied by the natural environment as raw material to be formulated in a suitable dosage form by the pharmaceutical companies. These pharmaceutical products will be used by diabetic patients with minimum cost and accepted by several categories in the society than synthetic drugs. Astragalus represents one of the popular genera of flowering plants belonging to the family Fabaceae. It comprises nearly about 2000–3000 species of annual or perennial herbs, subshrubs, or shrubs that are prevalent in temperate and arid areas (Li et al., 2014). Traditionally, many of these species were widely adopted for the relief of depression and as diuretics and tonics (Avunduk et al., 2007; Choudhary et al.,
Diabetes mellitus has recently been recognized as one of the most popular spreading metabolic dysfunction with an evident occurrence of morbidity and mortality all over the globe. It is characterized by the consequent emergence of various complications on the micro- and macrovascular levels as neuropathy, retinopathy, and nephropathy as well as cardiovascular complications and ulceration, (Singab et al., 2014; Youssef et al., 2017). Although, insulin and synthetic drugs that control hyperglycaemia are considered the primary ways for proper adjustment of blood glucose levels, they showed vigorous undesirable side effects with concomitant failure to prohibit the complications associated with diabetes. Thus, the need for novel antidiabetic entities of herbal origin for effective combating of hyperglycaemia is felt mandatory worldwide (Youssef et al.,
Abbreviations: AG, α-glucosidase enzyme; AT, methanol extracts of the aerial parts of A. turkestanus; AX, methanol extracts of the aerial parts of A. xапthomeloides; PPARα, peroxisome proliferator-activated receptor alpha; PPARγ, peroxisome proliferator-activated receptor gamma ⁎ Corresponding authors. E-mail addresses: [email protected] (M.L. Ashour), [email protected] (N.Z. Mamadalieva). 1 These authors have contributed equally to this work. https://doi.org/10.1016/j.indcrop.2018.03.034 Received 28 December 2017; Received in revised form 16 February 2018; Accepted 17 March 2018 Available online 05 April 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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combined n-butanol fractions were then concentrated at 42 °C under reduced pressure to yield 152 g and 64 g, respectively. 150 g of the dried n-butanol fraction of A. turkestanus were chromatographed over a silica gel column using the dry loading method. The column was then eluted with CHCl3/CH3OH (9:1, v/v) accompanied by monitoring using TLC on silica gel 254 plates to give subfractions A1- A26 which contain a mixture of less polar compounds. Consequently elution with CHCl3/CH3OH/H2O in the ratio of 4:1:0.1, v/v followed by 70: 23:3, v/v accompanied by monitoring using TLC on silica gel 254 plates with subsequent pooling of similar subfractions was done in which the polar subfractions were collected in order to be B1B20 and C1-C25, respectively. Subfractions B12-B19 were pooled together and then subjected to column chromatography using silica gel with subsequent elution with CHCl3/CH3OH (6:1, v/v) to give a mixture (16 mg) of compounds 1 and 2. Additionally, the subfractions C7C10 were collected together, subjected to silica gel column chromatography and eluted using CHCl3/CH3OH/H2O (70:23:3 v/v) to give compound 3 (7 mg) and compound 4 (13.5 mg). In addition, 60 g of the dried n-butanol fraction of A. xапthomeloides were subjected to silica gel column and subsequently eluted using the following solvent systems successively CHCl3/CH3OH (9:1, v/v), followed by a mixtures of CHCl3/CH3OH/H2O (70:12:1, v/v), (4:1:0.1, v/ v), (70:23:3, v/v), (70:28:8, v/v) and (60:35:8, v/v) and then monitored by TLC on silica gel 254 plates with subsequent pooling of similar subfractions and gave X1-97 subfractions. Subfractions X71-77 were collected together and separated by silica gel column chromatography, then eluted with CHCl3/CH3OH/H2O (70:28:8, v/v) and followed by further purification to yield compound 5 (15 mg). Separation of sub fractions X88-90 by silica gel column chromatography using solvent system (60:35:8, v/v) resulted in the isolation of compound 6 (15.5 mg) and compound 7 (12 mg). TLC plates were visualized under UV light (λ = 254 and 365 nm) and by spraying with phosphorus wolfram acid solution followed by heating at 105° C for 10 min. A scheme summarizing the isolation of secondary metabolites from both Astragalus species was added in the in the supplementary data (Fig. S1).
2008). Recently, many of the Astragalus species have shown many biological activities, in particular, antiviral and hepatoprotective properties in addition to boosting the immune response (Linnek et al., 2011). Undoubtedly, this could be attributed to the existence of many bioactive secondary metabolites, including alkaloids, anthraquinones, flavonoids and saponins (Ibrahim et al., 2013). Meanwhile, many of the species are used as nutrients, substituting agents for many beverages as tea or coffee as well as medicinal agents and cosmetics relying on their richness by amino acids, polysaccharides and metallic elements (Li et al., 2014). In addition some Astragalus species have been reported to exert a potent antihyperglycaemic activity particularly the radix of many varieties belonging to Astragalus membranaceus that proved a high efficacy with a satisfying safety margins. This is relied upon their richness with polysaccharides and isoflavones. The polysaccharides resulted in a notable decline in serum glucose levels, triglycerides, and low density lipoproteins as well as in insulin resistance with concomitant elevation in high density lipoproteins in streptozotocin- induced diabetes in rats (Fu et al., 2014). However, the isolated isoflavones from Astragalus membranaceus showed a good antihyperglycaemic amelioration via in vitro activation of PPARα and PPARγ in addition to promoting adipocyte differentiation (Shen et al., 2006). Astragalus turkestanus Bge. and A. xапthomeloides Eug. Korr. et M. Pop. are endemic to the Uzbek flora. Tracing the current literature, nothing was found regarding either the chemical composition or the biological activities of A. turkestanus. However, only two compounds namely, D-pinitol (3-O-methyl-D-chiro-inositol) and kaempferol-3-O-αL-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside were reported earlier from A. xапthomeloides by one of the authors (Janibekov et al., 2015; Janibekov et al., 2016). Herein we reported the isolation and structural elucidation of two new antihyperglycaemic flavonoid glycosides from the aerial parts of Astragalus turkestanus and A. xапthomeloides, which are 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6). In addition, five other compounds namely; daucasterol (1) and stigmasterol glucoside (2), D-pinitol (4), azukisaponin V (5) and 7-methoxy kaempferol-3-O-β-D-glucopyranoside (7) were also isolated. Molecular modeling of the isolated compounds within the active sites of α-glucosidase was done followed by in vitro evaluation of their α-glucosidase inhibitory activity to validate their bioactivity. In addition, their cytotoxic effect on Caco-2 cell line was reported.
2.3. General experimental procedures 1 H and 13C (APT) NMR analyses were carried out on a Bruker Ascend 400/R spectrometer (Burker Avance III, Fallanden Switzerland) at the operating frequencies of 400 and 100 MHz, respectively. Chemical shifts were expressed in δ ppm and were related to that of the solvents. The measured samples were dissolved in various deuterated solvents (Sigma Aldrich, Germany) depending upon their solubility and transferred to 3 mm NMR tubes (Bruker). Spectra were recorded at 25 °C; δ ppm rel. to Me4Si as internal standard. Two-dimensional (2D) NMR experiments (H, H −COSY; H, C HSQC; H, C HMBC) were performed utilizing the pulse sequences from the Bruker user library. ESI–MS analysis was done on a Waters Xevo TQD mass spectrometer with UPLC Acquity mode (Milford, USA). HR-ESI–MS analyses were performed using Bruker micro-TOF-Q Daltonics (API) Time-of-Flight mass spectrometer (Bremen, Germany) applying the method previously described (Nováková et al., 2010). Normal phase column chromatography was performed using silica gel (63–100 μm; Tianjin, Sinomed Pharmaceutical Co. Ltd., China). TLC analysis was done utilizing normal phase silica gel precoated plates F254 (Merck, Germany).
2. Materials and Methods 2.1. Plant material The aerial parts of Astragalus turkestanus Bge. and Astragalus xапthomeloides Eug. Korr. et M. Pop. were collected from the Qashqadaryo and Tashkent regions of Uzbekistan in 2015. The authenticated voucher specimens of the species (Accession no. 20081101 and 20081106, respectively) are kept in the Department of Herbal Plants (Institute of the Chemistry of Plant Substances, Uzbekistan). 2.2. Extraction and isolation The air-dried powdered aerial parts of A. turkestanus and A. xапthomeloides (2.5 kg each) were macerated in neat methanol (5 × 12 L) at room temperature till exhaustion (no considerable yield was further obtained) i.e. the extraction was carried out five times, using 12 L of neat methanol each. The solvent was evaporated at 40 °C using a rotary vacuum evaporator (BÜCHI Labortechnik AG, Switzerland) and concentrated to give 233 and 190 g dried methanol extracts, respectively. Consequently, the dried methanol extracts were successively fractionated using chloroform then n-butanol. The
2.4. Compound characterization 2.4.1. 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyrano side (3) Yellowish amorphous powder; UV (MeOH) λmax: 267 and 349 nm; 1 H (400 MHz) and 13C NNR (100 MHz) in DMSO see Table 1; HRESI–MS [M-H]− m/z 593.1530 (calculated. for m/z C27H30O15, 593.1584). Compound different spectra are available in the 143
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Table 1 1 H (400 MHz) and ppm, J in Hz).
C (100 MHz) data of compounds 3 and 6 in DMSO (δ in
3
#
δH (multi, J values) 1 2 3 4 5 6 7 8 9 10 1' 2' 3' 4' 5' 6' 7 (OCH3)
glucosidase (PDB ID 3TOP; 2.88 A°) downloaded from protein data bank (www.pdb.org) that is co-crystalized with acarbose, lead compound, using Discovery Studio 2.5 (Accelrys Inc., San Diego, USA). Docking was carried out using C-docker protocol applying both pHbased ionization and rule-based ionization methods. Docking was performed in a similar manner as previously described (Youssef et al., 2016, 2017; Ashour et al., 2017). However, the free binding energies were calculated in Kcal/mol using the following equation:
13
6.71 (d, 2.0, 1H) 6.36 (d, 2.0, 1H)
8.06 (d, 8.5, 1H) 6.90 (d, 8.5, 1H) 6.90 (d, 8.5, 1H) 8.06 (d, 8.5, 1H) 3.86 (s, 3H)
6 δC
160.51 (C) 133.91 (C) 178.00 (C]O) 161.31 (C) 98.40 (CH) 165.55 (C) 92.81 (CH) 157.27 (C) 105.54 (C) 121.24 (C) 131.45 (CH) 115.59 (CH) 156.86 (C) 115.59 (CH) 131.45 (CH) 56.56
δH (multi, J values)
δC
6.88 (d, 8.4, 1H) 8.10 (d, 8.4, 1H)
160.63 (C) 134.00 (C) 178.09 (C]O) 161.31 (C) 95.10 (CH) 162.07 (C) 99.79 (CH) 157.52 (C) 106.03 (C) 121.13 (C) 131.5 (CH) 115.58 (CH) 156.47 (C) 115.58 (CH) 131.5 (CH)
6.82 (d, 2.0, 1H) 6.46 (d, 2.0, 1H)
8.10 (d, 8.4, 1H) 6.88 (d, 8.4, 1H)
ΔGbinding = Ecomplex − (EAG + E
ligand)
Where; ΔGbinding: The ligand–enzyme interaction binding energy, Ecomplex: The potential energy for the complex of AG bound with the ligand, Eprotein: The potential energy of AG alone and Eligand: The potential energy for the ligand alone. 2.7. In vitro evaluation of the α-glucosidase inhibitory activity The inhibitory effect of the samples on α-glucosidase was determined in vitro based upon the previously reported method with certain modifications (You et al., 2011). Basically, 1 mg of each of the examined samples or acarbose (positive control) at several concentrations in the range of 1.95–500 μg/mL was incubated together with 500 μL of 1.0 U/mL α-glucosidase solution in 100 mM phosphate buffer, pH = 6.8, kept at 37 °C for 20 min. The absorbance of the obtained pnitrophenol was determined at a λmax = 405 nm utilizing a microplate reader (Bio Tek Instruments, Inc., Winooski, VT). Noteworthy to mention that the control is the control reaction that contains all the reagents except the tested sample. Moreover, the IC50 is the concentration of the tested sample resulting in 50% inhibition of α-glucosidase activity under the experiment conditions. The inhibition percentage was computed using the following equation
Sugar 1” 2” 3” 4” 5” 6”
Galactose 3.97 (d, 7.1, 1H) 3.12 (m, 1H) 2.79 (m, 1H) 3.12 (m, 1H) 3.20 (m, 1H) 3.76 (m, 1Ha), 2.70 (m, 1Hb)
Galactose 104.07 (CH) 70.24 (CH) 73.56 (CH) 69.84 (CH) 74.52 (CH) 68.24 (CH2)
Rhamnose 5.56 (d, 1.5, 1H) 3.40 (m, 1H) 3.64 (m, 1H) 3.38 (m, 1H) 3.09 (m, 1H) 1.13 (d, 6.1, 3H)
Rhamnose 98.85 (CH) 70.87 (CH) 70.54 (CH) 68.72 (CH) 72.04 (CH) 18.38 (CH3)
Sugar 1”' 2”' 3”' 4”' 5”'
Arabinose 5.38 (d, 1.5, 1H) 3.12 (m, 1H) 2.79 (m, 1H) 3.28 (m, 1H) 3.50 (m, 1Ha), 3.42 (m, 1Hb)
Arabinose 101.34 (CH) 76.66 (CH) 76.67 (CH) 76.86 (CH) 65.75 (CH2)
Galactose 5.35 (d, 7.5, 1H) 3.57 (m, 1H) 3.35 (m, 1H) 3.41 (m, 1H) 3.85 (d, 6.4, 1H)
Galactose 102.23 (CH) 74.06 (CH) 71.55 (CH) 73.40 (CH) 70.29 (CH)
6”'
3.62 (m, 1Ha), 3.36 (m, 1Hb)
65.65 CH2)
2.8. Cytotoxicity assay
Sugar 1”” 2”” 3”' 4”” 5”” 6””
Rhamnose 4.41(d, 1.5, 1H) 3.31 (m, 1H) 3.64 (m, 1H) 3.62 (m, 1H) 3.09 (m, 1H) 1.06 (d, 6.1, 3H)
Rhamnose 100.46 (CH) 71.05(CH) 70.71 (CH) 68.43 (CH) 72.30 (CH) 18.38 (CH3)
MTT assay was performed as previously mentioned by (Saliba et al., 2002) with certain modifications. Briefly, 100 μL of the examined sample after dissolution in 5% DMSO and RPMI tissue culture medium were added to each well in order to attain a final concentration of (10–4000 μg/mL). Control wells contained two aliquots of 100 μL of tissue culture medium (MEM + fetal bovine serum at 9:1 ratio) and 100 μL of 5% sterile DMSO and RPMI tissue culture medium. After 24 h incubation period at 37 °C, the wells were washed with PBS with subsequent incubation with MTT solution (2 mg/mL) at 100 μL per each well for 1 hat 37 °C. Supernatants were then decanted and the cells were treated with 100 μL of DMSO per each well to dissolve the formazan crystals formed in the viable metabolically active cells. Elutes of the 8 wells of each sample were collected and the absorbance was measured at 540 nm. Control wells were similarly treated (Elissawy et al., 2017). The percentage cytotoxicity was determined applying the following equation (Murakami et al., 2000).
% Inhibition = (A 405 of the control − A 405 of the tested sample ) × 100
supplementary data (Fig. S2). 2.4.2. Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-β-D-glucopyranosyl(1 → 6)-α-L-rhamnopyranoside(6) Light yellowish amorphous powder; UV (MeOH) λmax: 266 and 352 nm; 1H (400 MHz) and 13C NNR (100 MHz) in DMSO see Table 1; HR-ESI–MS [M-H]− m/z 739.2090 (calculated. for m/z C33H40O19, 739.2091). Compound different spectra are available in the supplementary data (Fig. S3).
A540 of the test culture ⎫ Cytotoxcity % = 1 − ⎧ × 100 ⎨ ⎩ A540 of the control culture ⎬ ⎭
2.5. Acid hydrolysis of the isolated flavonoids A solution of each of the tested compounds was treated with HCl using the same method and conditions as previously reported (Kim et al., 1999). Identification of D- glucose, D-galactose, L-rhamnose; and L-arabinose resulted from the tested compounds hydrolysis was done by comparing their retention time and retention factors with those of authentic samples using co-chromatography.
3. Results and Discussion 3.1. Phytochemical characterization Comprehensive phytochemical characterization of the methanol extracts of the aerial parts of A. turkestanus (AT) and A. xапthomeloides (AX) resulted in the isolation and structural elucidation of two new flavonoid glycosides which are 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) from the former and
2.6. Molecular modelling study on α-glucosidase Virtual screening of the isolated compounds was done on α144
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Fig. 1. Secondary metabolites isolated from the aerial parts of Astraglus species.
kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D- galactopyranoside (6) from the latter in addition to other five known compounds that were isolated for the first time from these species. Their structures were fully elucidated by comparing their 1D and 2D NMR data with those reported in literature for previously isolated compounds (Fig. S4). They were identified as daucasterol (1) and stigmasterol glucoside (2) (Ashour et al., 2012) in addition to D-pinitol (4) (Sharma et al., 2016) from A. turkestanus whereas an oleanane type triterpenoidal saponin, azukisaponin V (5) (Gromova et al., 2001), and a flavonoid glycoside, 7-methoxy kaempferol-3-O-β-D-glucopyranoside (7) (Khalfallah et al., 2014) were isolated from A. xапthomeloides (Fig. 1) The structures of the two new compounds were comprehensively elucidated using UV, NMR and HR-ESI–MS techniques (Table 1). Compound 3 was obtained as a yellow amorphous powder, its molecular formula C27H30O15, was clarified using HR-ESI–MS showing m/z: 593.1530 [M-H]−, (calculated. 593.1584). The UV spectra of the methanol solution of compound 3 showed two characteristic λ maxima existing at 267 and 349 nm for bands I and II, respectively confirming the presence of a flavanol skeleton. Upon addition of sodium acetate no bathochromic shift was shown in band I that consequently declared the absence of a free hydroxyl group at position 7. However upon addition of AlCl3 reagent, a bathochromic shift (38 nm) was observed only in band I (229 nm) indicating the presence of a free hydroxyl group at position 5 with no bathochromic shift observed in band II that revealed the absence of a free hydroxyl group at position 3. Its 1H NMR revealed the existence of four doublet signals corresponding to six aromatic protons which are: ortho-coupled at δH 8.06 (d, J = 8.5, 2H) and 6.90 (d, J = 8.5, 2H) and meta- coupled at 6.71 (d, J = 2.0, 1H) and 6.36 (d, J = 2.0, 1H) that confirmed compound 3 to be a kaempferol derivative. Moreover, two distinct signals in both 1H NMR at δH 3.97 (d, J = 7.1, 1H) and 5.38 (d, J = 1.5, 1H) and 13C NMR spectra at δc 104.07 and
101.34 ppm, respectively were found and were further confirmed from HSQC spectrum. These presented data further ascertained the presence of two anomeric protons and carbons and proved compound 3 to be a flavanol diglycoside. However, a sharp singlet signal in the 1HNMR spectrum integrated for three protons at δH 3.86 ppm that is correlated with a carbon signal at δc 56.56 ppm as revealed from 13C NMR and HSQC correlation declared the presence of a OCH3 group. From HMBC spectrum of compound 3 a long range HMBC correlation exist between the protons of OCH3 group at δH 3.86 ppm and the C7 carbon at δc 165.55 ppm suggesting the presence of OCH3 group at position 7 and thus the sugars moieties will be at position 3. The mass spectral data together with the above displayed analytical ones, suggested a kaempferol derivative containing one hexoside and one pentoside moieties. 1H NMR,13C NMR and HSQC correlations declared the assignment of one galactose (Sikorska and Matlawska, 2000; Bubb, 2003) and one arabinose molecule (Xiao et al., 2006). The large JH1″-H2″ coupling constant 7.1 Hz established the β-configuration of the galactose moiety meanwhile the small JH1‴-H2‴ coupling constant 1.5 Hz established the α-configuration of the arabinose molecule (de Sousa et al., 2017). The glycone chain (galactose- arabinose) was established to be between C1”” and C6”' as revealed from the downfield shift of C6‴ (68.24 ppm) of the galactose moiety (Dudek-Makuch and Matławska, 2011).In addition, upon acid hydrolysis D-galactose and L-arabinose were identified by using co-chromatography and after being compared with authentic sugars. Thus compound 3 was finally identified as 7methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (Fig. 2). Compound 6 was obtained as a light yellowish amorphous powder, its molecular formula was determined to be C33H40O19, as revealed from HR-ESI–MS that showed a molecular ion peak in the negative mode equals to 739.2090 [M-H]−, calculated. 739.2091. Comprehensive interpretation of its UV spectral data clarified the 145
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glucosidase inhibitory activity of the isolated compounds that consequently predicted their antihyperglycaemic activity using both pHbased ionization and rule-based ionization methods. The former was done in a pH range of 6.5 and 8.5 with an average of 7.5 to mimic the biological circumstances; however the latter was done to examine the effect of various ionisable groups on the attitude of the molecules at the active site. Both 7-methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-αL-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6) displayed the highest fitting score in both ionization methods with ΔG equals to −82.10 and −67.66 Kcal/mol in the pH-based ionization method and −68.55 and −67.83 Kcal/mol in the rule-based ionization methods for both compounds, respectively approaching that of acarbose. On the contrary, azukisaponin V (5) did not show any binding affinity and failed to dock in the active site using either ionization methods. Results were displayed in Table 2.
Fig. 2. 1H–1H COSY and HMBC key correlations of compounds 3 and 6.
presence of two distinctive λ maxima appearing at 266 and 352 nm corresponding to bands I and II, respectively that consequently proved the existence of a flavanol skeleton. Using sodium acetate as a UV shifting reagent did not cause any obvious bathochromic shift in band I and thus declared the absence of a free hydroxyl group at position 7. On the contrary the addition of AlCl3 reagent resulted in a bathochromic shift (26 nm) in band I (240 nm) ascertaining the presence of a free hydroxyl group at position 5, however no bathochromic shift was observed in band II declaring the absence a free hydroxyl group at position 3. Similarly as compound 3, a kaempferol derivative was suggested owing to the presence of four doublet signals in the 1H NMR spectrum corresponding to six aromatic protons which are; ortho-coupled at δH 8.10 (d, J = 8.4, 2H) and 6.88 (d, J = 8.4, 2H) and meta- coupled at 6.82 (d, J = 2.0, 1H) and 6.46 (d, J = 2.0, 1H). 1H NMR and 13C NMR showed signals for three anomeric protons and carbons. HSQC confirmed the correlation between the anomeric protons and their respective carbons to be as follows δH 4.41(d, J = 1.5, 1H) and δc 100.46 ppm; δH 5.35 (d, J = 7.5, 1H) and δc 102.23 ppm; 5.56 (d, J = 1.5, 1H) and δc 98.85 ppm. From mass spectral information as well as from the above displayed analytical ones, we concluded that compound 6 is a flavanol triglycoside containing three hexoses, the large JH1‴-H2‴ coupling constant 7.5 Hz established the β-configuration of one hexose moiety meanwhile the small JH1″-H2″ and JH1″″-H2″″ coupling constant 1.5 Hz established the α-configuration for the other two hexoses (de Sousa et al., 2017). 1H NMR, 13C NMR and HSQC correlations declared the assignment of one galactose and two rhamnoses (Jung et al., 1999; Sikorska and Matlawska, 2000; Bubb, 2003). From HMBC spectral data of compound 6, it was obvious that a long range HMBC correlation occurs between the anomeric proton of the galactose moiety at δH 5.35 ppm and C8 at δc 99.79 ppm and this in turn suggested the existence of a galactose- rhamnose chain at position 7. Additionally, the linkage between galactose- rhamnose was established to be between C1″″ and C6‴ as revealed from the long range HMBC correlation that exists between the anomeric proton of one rhamnose moiety at δH 4.41 ppm and C6‴ of the galactose moiety at δc 65.65 ppm in addition to the downfield shift of C6‴ (65.65 ppm) of the galactose moiety (Dudek-Makuch and Matławska, 2011). Additionally, a long range HMBC correlation exists between the anomeric proton of rhamnose H1” at δH 5.56 ppm and C2 at δc 160.63 indicating the existence of a single rhamnose moiety at position 3. Besides, upon acid hydrolysis Dgalactose and L-rhamnose were identified by using co-chromatography and upon comparison with authentics. Thus compound 6 was finally identified as kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (Fig. 3).
3.3. In vitro α-glucosidase inhibitory activity Accordingly, in vitro α-glucosidase inhibitory activity was evaluated for the previously mentioned flavonoid glycosides to validate their antihyperglycaemic activity and to confirm the obtained molecular modelling results. Moreover, α-glucosidase inhibitory activity was also assessed for azukisaponin V (5) to ascertain the validity of the docking study. 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-D-galactopyranoside (3) showed a significant α-glucosidase inhibitory activity displaying IC50 of 50.31 μg/mL approaching that of acarbose that is 30.57 μg/mL (Table 3). This undoubtedly could be explained in virtue of its 2D and 3D binding modes as displayed in Fig. 3 in which the compound is tightly bonded to the active site via the formation of nine firm hydrogen bondings with Asp 1157, Asp 1279, Asp 1420, Asp 1526, Arg 1510, Thr 1586 and Lys 1466 amino acid residues at the active site. However, kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6) exhibited a substantial activity with IC50 equals to 79.60 μg/mL. Moreover, azukisaponin V (5) showed a mild inhibitory activity showing 117.96 μg/mL as an IC50 and thus it comes in accordance with the results of the molecular docking studies. Previous studies have been done to evaluate the α-glucosidase inhibitory activity of other Astragalus species particularly Astragalus membranaceus root in which its ethanol extract showed α-glucosidase inhibitory activity of 49.71 μg/mL. The α-glucosidase inhibitory activity of the ethanol extract was mainly attributed to the presence of flavonoids and phenolic compounds. The formers were estimated by 44.93 μg/mL quercetin and 70.32 μg/mL rutin whereas the latter were estimated by 26.13 μg/mL catechins (Yin et al., 2009). In addition, previous studies showed that the number of hydroxyl groups in ring B considerably affect the inhibitory activity. In conclusion, it was proved that the minimum structures requirements involves the presence of an unsaturated C ring in addition to the presence of a hydroxyl group at the 3 position, 4-CO, the linkage of the B ring at the 3 position, and the hydroxyl substitution on the B ring significantly increase the α-glucosidase inhibitory activity (Tadera et al., 2006).
3.4. Cytotoxic activity The total methanol extracts, (AT and AX) in addition to the isolated compounds, were tested for their cytotoxicity against Caco-2 (human epithelial colorectal adenocarcinoma) cells using the MTT assay (Table 4) but none of the tested samples showed significant cytotoxic activity indicating their safety. This came in accordance to some previously carried studies that showed the safety of some flavonoids glycosides (Chung et al., 2007).
3.2. In silico molecular modelling In silico molecular modelling study was done to examine the α146
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Fig. 3. 2D and 3D binding modes of compound 3 within α-glycosidase active site. Table 2 Free binding energies (ΔG) of the isolated compounds from Astragalus species in the active site of α-glucosidase using molecular modeling experiments calculated in Kcal/mol applying both pH-based and rule-based ionization methods. Compound
Acarbose Daucasterol (1) Stigmasterol glucoside (2) 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) D-Pinitol (4) Azukisaponin V (5) Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-Lrhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6) 7-Methoxy kaempferol-3-O-β-D-glucopyranoside (7)
Table 4 In vitro cytotoxicity evaluation using MTT assay on Caco-2 cell line.
Binding energy (Kcal/mol) pH-based
Rule based
−84.89 −63.64 −61.56 −82.10
−98.71 −63.64 −61.56 −68.55
−30.78 FD −67.66
−35.46 FD −67.83
−52.08
−51.81
Table 3 In vitro α-glucosidase inhibitory activity of 7-methoxy kaempferol-3-O-α-Larabinosyl-(1 → 6)-β-D-galactopyranoside (3), azukisaponin V (5) and kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6). IC50 (μg/mL)
Acarbose 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) Azukisaponin V (5) Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-Lrhamnopyranosyl-(1 → 6)-β-D-galactopyranoside (6)
30.57 ± 3.27 50.31 ± 4.98
IC50 (μg/mL)
Astagalus xапthomeloides methanol extract Astagalus turkestanus methanol extract β-Sitosterol glucoside (1) 7-Methoxy kaempferol-3-O-α-L-arabinosyl-(1 → 6)-β-Dgalactopyranoside (3) D-Pinitol (4) Azukisaponin V (5) Kaempferol-3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranosyl(1 → 6)-β-D- galactopyranoside (6)
1180.00 918.05 622.00 455.00 440.00 914.40 615.50
(1 → 6)-β-D-galactopyranoside (6) could offer a promising natural antihyperglycaemic entities without any marked toxicity that are of great usefulness in the pharmaceutical industries for the manufacturing of new safe antihyperglycaemic drugs that are recognized to be more welcomed by a large category of patients comparable to the synthetic drugs owing to its natural ancestry. However, in vivo studies followed by clinical trials should be conducted to ascertain their activity.
FD: fail to dock.
Compound
Sample
Declaration of interest The authors declare no conflict of interest. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
117.96 ± 10.96 79.60 ± 7.74
Appendix A. Supplementary data
All determinations were carried out in triplicate manner and values are expressed as the mean ± S.D.
Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.03.034.
4. Conclusion
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