Saponins from Glycine Merrill (soybean)

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Mudasir A. Tantry a,⁎, Ikhlas A. Khan a,b a National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS 38677, USA b Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA a r t i c l e i n f o a b s t r a c t Article history: Received 6 February 2013 Accepted in revised form 19 March 2013 Available online 1 April 2013 Saponins are a diverse group of plant secondary metabolites with a wide array of activities, as well as a significant role in nutrition and health. Saponins occur as multi-component mixtures of compounds with very similar polarities. Soysaponins are a special group of saponins. These represent the main source of saponins in Glycine max (soybeans, Fabaceae). In a study of the chemical profiling of plants, to investigate the possible misidentification and authentication of dietary supplements, the hydro-alcoholic extract of G. max was investigated. Three new saponins, designated as soysaponins M1 (1), M2 (2) and M3 (3) along with seven known soysaponins (4–10) were isolated by normal and reverse phase liquid chromatography. All compounds were characterized by spectroscopic techniques including 2D NMR spectroscopy. Published by Elsevier B.V. Keywords: Glycine max Fabaceae Soybeans Soysaponins 1. Introduction The identification and characterization of specific dietderived chemicals, and an increased understanding of their biological activity have given rise to increased interest in the role for natural products in disease prevention and treatment [1]. Legumes play a pivotal role in the traditional diets of many regions throughout the world [2]. Soybeans are unique among legumes because they are a primary source of soysaponins, flavonoids and other secondary metabolites [3]. Plant-derived saponins are considered to play a significant role in plant defense systems against pathogens and herbivores. Numerous reports emphasize the fungicidal [4–6], antimicrobial [7], allelopathic [8], insecticidal [9–12] and molluscicidal [13–16] activity of various saponins. The presence of saponins in soybeans has also attracted considerable interest because of both their health benefits and adverse sensory characteristics. Soysaponins are the primary dietary sources of saponins from foods. Soysaponins have been demonstrated to posses multiple health-promoting properties, such as lowering of cholesterol by inhibiting its absorption, being anticarcinogenic, antihepatotoxic, promoting antiinfectivity of HIV, antimutagenic, immunostimulatory and antimurogenic activities [17–23]. The primary objective of the current study was the elucidation of chemical markers from plants and the chemical profiling of dietary supplements. In the study, as a part of our continued work on Glycine max three previously undiscovered saponins (1–3) along with seven known saponins (4–10) were isolated from G. max. All saponins were isolated from hydroalcoholic extract of G. max. The compounds were characterized by NMR including 2D spectroscopic techniques. Electrospray ionization coupled with a multi-stage tandem mass spectrometry (ESI-MSn) with an ion-trap mass analyzer was used to obtain typical fragmentation. In positive mode, molecule adducted hydrogen ions [M + H]+, sodium ions [M + Na]+ and [M + 2Na − H]+ are easily identified. However, since the existence of β-D-glucopyranosiduronic acid, negative mode showed clear fragment [M − H]−, [M − H-sugar(s)]− and [aglycone]−. 2. Experimental 2.1. General NMR spectra were recorded on a Varian AS 400 & 600 NMR spectrometer instrument using TMS as internal standard. Chemical shifts were reported in δ units and coupling constants (J) in Hz. ESIMS and HRESIMS were obtained on Agilent Series 1100 SL mass spectrometer. IR spectra were recorded using Fitoterapia 87 (2013) 49–56 ⁎ Corresponding author. Tel.: +1 910119906500051; fax: +1 662 915 7821. E-mail address: mudaek@gmail.com (M.A. Tantry). 0367-326X/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.03.021 Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote KBr pellet on a Bruker Tensor 27 FT-IR spectrometer. Optical rotations were measured on a Rudolph Research AutoPol IV polarimeter. Melting points were determined on Meltemp melting point apparatus and are uncorrected. Column chromatography was performed by using silica gel (40 μm mesh, JT Baker) and reversed-phase RP-C18 silica (Polarbond; JT Baker). Medium Pressure Liquid Chromatography was performed on Biotage Inc. Horizon MPLC System. TLC analysis was carried out on silica gel 60F254 plates (Merck) and spots on TLC plates were observed under UV light (254/365 nm). Spraying reagents p-anisaldehyde-H2SO4 (Sigma-Aldrich), 10% H2SO4 in ethanol and water, followed by heating were used for the detection of spots. 2.2. Plant material The concentrated (40%) hydro-alcoholic soybean extract was purchased from Organic Technologies. The authenticity of the commercial extract was checked by co-TLC (silica gel; CHCl3–MeOH–H2O, 13:7:2, lower layer) with the freshly prepared methanolic extract of soybeans (G. max Merrill). The voucher specimen with reference numbers (3681-GlMAF: 02-11-2008 & 9352-GlMAF: 01-05-2011) has been deposited in Botanical Division at the National Center for Natural Products Research, University of Mississippi. 2.3. Extraction and isolation The finely powdered G. max extract (495 g) was mixed with an equal amount of silica gel and subjected to column chromatography (CC) over a silica gel (2.8 kg) column (135 × 6.0 cm) and eluted with CHCl3–MeOH (9:1) to obtain fractions SS-A (33.2 g) and SS-B (15.1 g). The polarity of eluant was changed to CHCl3–MeOH–H2O (13:7:2; lower layer, labeled as solvent system A) to afford eight fractions labeled as SS-C to SS-J. Fraction SS-C (5.1 g) after recolumn-chromatography gave soysaponin IV (4; 42.2 mg) and soysaponin M1 (1; 8.2 mg) (silica gel, 1.5 kg; column 100 × 2.5 cm; CHCl3–MeOH–H2O, 13:7:2, lower layer). Fraction D (12 g) was further fractionated and resolved (silica gel, 1.2 kg; column 100 × 2.5 cm; CHCl3– MeOH–H2O, 13:7:2, lower layer) to give soysaponin III (5; 88.2 mg), soysaponin II (6; 702.5 mg) and soysaponin I (7; 1002.4 mg). Fraction E (18 g) and Fraction F (22 g) after resolving was subjected to MPLC normal phase column (Biotage, 40 + M) to afford robinioside E (8; 18.2 mg) and on reverse phase column (Biotage, 25 + M) gave soysaponin M2 (2; 10.3 mg) respectively. Fraction G (52 g) was subjected to CC over reversed phase silica (RP-C18 silica 60 g, column 90 × 2.5 cm) to give soysaponin A2 (9; 32.0 mg) and soysaponin M3 (3; 11.3 mg). Soysaponin A1 (10; 15.5 mg) was obtained after subjecting fraction SS-J over reversed phase silica (RP-C18 silica 30 g, column 30 × 1.0 cm). 2.3.1. Compound 1 (soysaponin M1) White amorphous powder; m.p.: 202.2 °C; [α]D 25: −32.1 (0.20, CH3OH); IR (KBr): νmax = 3445 (\OH), 1732 (\COO) cm−1; HRESIMS (positive mode): m/z 729.4534 [M + Na]+ (calcd. for C40H66O10); 1H NMR (C5D5N–CD3OD, 7:3; 600 MHz) and 13C NMR (C5D5N–CD3OD, 7:3; 150 MHz) date, see Tables 1 & 2. 2.3.2. Compound 2 (soysaponin M2) Amorphous powder; m.p.: 253.4 °C; [α]D 25: −48.1 (0.20, CH3OH); IR (KBr): νmax = 3450 (\OH), 1692 (\CO) cm−1; HRESIMS (positive mode): m/z 981.4084 [M + Na]+ (calcd. for C48H78O19); 1H NMR (C5D5N–CD3OD, 7:3; 400 MHz) and 13C NMR (C5D5N–CD3OD, 7:3; 100 MHz) date, see Tables 1 & 2. 2.3.3. Compound 3 (soysaponin M3) Amorphous powder; m.p.: 281.2 °C; [α]D 25: −33.4 (0.20, CH3OH); IR (KBr): νmax = 3452 (\OH), 1689 (\CO) cm−1; HRESIMS (positive mode): m/z 1159.5485 [M + Na]+ (calcd. for C54H88O25); 1H NMR (C5D5N–CD3OD, 7:3; 400 MHz) and 13C NMR (C5D5N–CD3OD, 7:3; 100 MHz) date, see Tables 1 & 2. 2.4. Sugar analysis Compounds (1–3) (3 mg) were hydrolyzed with 1 N HCl (2 ml) for 3 h at 90 °C. The reaction mixture was cooled, neutralized and partitioned between EtOAc (3 ml) and H2O (3 ml). The aqueous layer was treated with NaBH4 (3 mg) at room temperature for 3 h and excess of NaBH4 was removed by glacial acetic acid. The residue was dissolved in pyridine (0.5 ml) and 0.1 M L-cysteine methyl ester hydrochloride in pyridine (1 ml) was added. The mixture was heated at 70 °C for 1 h. An equal volume of Ac2O was added with heating continued for another 1 h. Acetylated thiazolidine derivatives were subjected to GC analysis (conditions: a ThermoQuest Trace 2000 GC; Column, Phenomenex DB-5 column(30 m × 0.25 mm × 0.25 μ m);carrier gasHe; injection Table 1 13C NMR (100 & 150 MHz, C5D5N–CD3OD (7:3), δC) data of compounds 1–3. Position δC Position δC 1 2 3 1 2 3 1 38.1 t 38.2 t 38.1 t 1′ 104.6 d 102.6 d 103.4 d 2 26.2 t 24.2 t 24.9 t 2′ 73.8 d 81.4 d 82.1 d 3 90.2 d 90.3 d 90.3 d 3′ 76.7 d 77.5 d 76.9 d 4 45.5 s 42.1 s 44.4 s 4′ 72.2 d 72.6 d 72.5 d 5 55.2 d 45.6 d 46.5 d 5′ 78.3 d 78.8 d 77.8 d 6 28.8 t 17.2 t 18.1 t 6′ 175.5 s 174.0 s 173.3 s 7 33.5 t 29.9 t 30.4 t 7′ 77.5 d 8 40.1 s 40.9 s 41.4 s 8′ 68.2 t 9 46.4 d 46.6 d 45.4 d 9′ 22.2 t 10 35.5 s 37.8 s 38.8 s 10′ 15.3 q 11 25.5 t 23.9 t 24.1 t 1″ 102.1 d 102.3 d 12 123.5 d 124.0 d 124.2 d 2″ 77.4 d 77.5 d 13 144.2 s 144.5 s 143.9 s 3″ 75.8 d 75.6 d 14 42.4 s 41.5 s 44.3 s 4″ 72.2 d 73.5 d 15 28.8 t 25.5 t 25.8 t 5″ 79.0 d 78.8 d 16 27.8 t 28.8 t 27.6 t 6″ 64.0 t 64.6 t 17 37.2 s 36.6 s 39.1 s 1‴ 102.4 d 101.3 d 18 48.0 d 43.3 d 43.4 d 2‴ 74.2 d 76.6 d 19 42.3 t 46.6 t 45.5 t 3‴ 75.3 d 76.0 d 20 45.4 s 32.0 s 33.7 s 4‴ 72.4 d 72.5 d 21 40.1 t 43.5 t 71.1 d 5‴ 78.4 d 77.9 d 22 77.7 d 74.0 d 92.1 d 6‴ 65.2 t 65.0 t 23 22.5 q 17.1 q 17.2 q 1″″ 101.6 d 24 69.4 t 68.9 t 67.4 t 2″″ 77.1 d 25 19.4 q 15.8 q 17.2 q 3″″ 75.1 d 26 17.7 q 17.8 q 17.8 q 4″″ 72.9 d 27 25.4 q 25.8 q 25.8 q 5″″ 76.9 d 28 26.5 q 28.8 q 19.2 q 6″″ 64.9 t 29 25.4 q 27.5 q 26.6 q 30 25.2 q 26.5 q 27.6 q 50 M.A. Tantry, I.A. Khan / Fitoterapia 87 (2013) 49–56 temperature 250 °C, detection temperature 270 °C; column temperature, 100 °C (1 min), 20 °C/min to 300 °C (30 min)). The configurations were determined by comparing their retention times (tR D-glucose 13.38 min, tR D-galactose 13.43 min and tR D-glucuronic acid 14.2 min) with acetylated thiazolidine derivatives prepared in a similar way from standard sugars. Compounds 1–3 (1 mg) were hydrolyzed with 1 N HCl (3 ml) for 4 h at 95 °C. The reaction mixture was cooled, neutralized and partitioned between EtOAc (3 ml) and H2O (3 ml). The aqueous layer was analyzed using TLC (CHCl3– MeOH–H2O, 13:7:2) with comparison to authentic standard samples of D-glucose, D-galactose and D-glucuronic acid. The spots were visualized by spraying with p-anisaldehyde-H2SO4 followed by heating. The sugars obtained on hydrolysis showed comparable Rf values to those of D-glucose (Rf 0.38), D-galactose (Rf 0.33) and D-glucuronic acid (Rf 0.29). 3. Results and discussion Compounds (1–3) along with seven known compounds (4–10) were isolated from hydro-alcoholic extract of G. max by using normal and reverse phase column chromatography. Their structural assignments were determined by analysis of spectroscopic data including 2D NMR experiments (Fig. 1). Compound 1 was isolated as white amorphous powder from the saponin concentrate by normal phase column chromatography. The HRESIMS showed the molecular ion at m/z 729.4534 [M + Na]+ in agreement with the molecular formula C40H66O10. This C-40 carbon skeleton was again supported by 13C NMRand DEPT experiments. The IR spectrum revealed characteristic acetoxy absorptions at 1732 cm−1 and 3445 cm−1 ascribable to hydroxyl function. The 13C NMR spectrum showed 40 resonance signals from both the glycone and aglycone parts of the molecule. The DEPT experiment revealed eight methyls, twelve methylenes, twelve methines and eightquaternarycarbons. The 1Hand 13CNMR of 1showed typical glycone part as β-glucuronopyranose [δH = 5.02 (d, J = 8.2 Hz, H-1′), 3.78 (m, H-2′), 3.62 (m, H-3′), 3.73 (m, H-4′), 3.82 (d, J = 7.4 Hz, H-5) and δC = 104.6 (C-1′) 73.8 (C-2′), 76.7 (C-3′), 72.2 (C-4′), 78.3 (H-5′) and 175.5 (H-6′)]. The downfield resonances at δC 123.5 and 144.2 were assigned to tetrasubstituted double bond between C-12 and C-13. The connectivity of monoglycosidic moiety as glucuronopyranoside was supported by long range HMBC and HMQC correlations. The connectivity observed was between the signals H-1′ and C-3 and H-3 with C-1′. As allthe 1Hand 13CNMRspectraresemblewithtypicalsubstituted monoglycosidic as glucuronic acid saponin, this helped to reveal the side chain of butyl alcohol asan ester linkage withcarboxylic acidofglucuronicacid,whichwasagainsupportedbykeyHMBC correlations of H-7′ with C-6′, which was again confirmed by electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn), with m/z 633 [M-BuOH + H] and m/z 457 [M-glucuronic acid–BuOH + H]. The point of attachment of butyl side chain was supported by carboxylic group as ester and key correlations in HMBC spectrum between H-7′ and C-6′. The further assignments of 1H and 13C NMR data were correlated with soysaponin IV [24] and confirmed by HMBC Table 2 1H NMR (400 & 600 MHz, C5D5N–CD3OD (7:3), δH, J/Hz) data of compounds 1–3. Position δH Position δH 1 2 3 1 2 3 1 1.32, 1.09 1.52, 1.21 1.50, 1.18 1′ 5.02 d 4.52 d 4.58 d 2 1.42, 1.12 1.45, 1.23 1.38, 1.19 2′ 3.78 3.36 3.38 3 3.56 (br, d) 3.32 3.31 3′ 3.62 3.73 3.73 4 4′ 3.73 3.63 3.45 5 1.34 d 1.30 1.31 5′ 3.82 3.80 3.76 6 1.51, 1.18 1.48, 1.27 1.47, 1.24 6′ 7 1.50, 1.21 1.51, 1.29 1.49, 1.29 7′ 3.92 8 8′ 3.89, 3.53 9 1.48 d 1.38 1.40 9′ 1.62, 1.03 10 10′ 0.91 11 1.72, 1.42 1.80, 1.52 1.79, 1.61 1″ 4.58 d 4.59 d 12 5.22 (br, s) 5.25 (br, s) 5.27 (br, s) 2″ 3.36 3.72 13 3″ 3.72 3.50 14 4″ 3.45 3.44 15 0.92, 1.13 1.36, 1.10 1.34, 1.18 5″ 3.76 3.72 16 0.94, 1.12 1.32, 1.12 1.30, 1.16 6″ 3.78, 3.53 3.77, 3.58 17 1‴ 4.60 d 4.62 d 18 1.54 1.84 1.82 2‴ 3.71 3.70 19 1.25, 1.45 1.48, 1.18 1.47, 1.17 3‴ 3.50 3.53 20 4‴ 3.42 3.45 21 1.81, 1.31 1.58, 1.25 3.27 d 5‴ 3.73 3.70 22 3.72 3.52 3.29 d 6‴ 3.78, 3.50 3.79, 3.49 23 1.01 1.02 1.04 1″″ 4.68 d 24 3.65, 3.40 3.78, 3.21 3.62, 3.29 2″″ 3.69 25 1.02 0.99 0.98 3″″ 3.51 26 1.18 0.98 0.99 4″″ 3.43 27 0.98 0.96 0.97 5″″ 3.69 28 0.99 0.99 0.99 6″″ 3.75, 3.48 29 0.94 1.00 0.98 30 0.96 0.99 0.99 Multiplicity is not clear for some signals due to overlapping. M.A. Tantry, I.A. Khan / Fitoterapia 87 (2013) 49–56 51 Fig. 1. Structure of compounds 1–3 and 4–10. 52 M.A. Tantry, I.A. Khan / Fitoterapia 87 (2013) 49–56 Fig. 2. Selected HMBC correlations of compounds 1, 2 and 3. M.A. Tantry, I.A. Khan / Fitoterapia 87 (2013) 49–56 53 Fig. 3. Possible biogenesis of soysaponins 1–3 by successive sugar chain transfers from UDP-sugars to soysapogenol B. 54 M.A. Tantry, I.A. Khan / Fitoterapia 87 (2013) 49–56 and HMQC experiments. The configuration at C-3 was determined as S and by NOESY experiments. Configuration of glucuronic acid was found to be D by preparing its thiazolidine derivatives [25]. On the basis of abovementioned data and conclusion drawn out of it, compound 1 was characterized as 3β-O-[β-D-glucuronopyranosyl]-6′-O-[butan-2-ol]-12-eneolean-22,24-diol. Compound 2 was isolated as gray amorphous powder. It showed the molecular ion at m/z 981.4084 [M + Na]+ in HRESIMS. The HRESIMS and 13C NMR helped in determining the molecular formula to be C48H78O19. The IR absorptions indicated the presence of hydroxyl (3450 cm−1), carbonyl (1692 cm−1) and olefin (1630 and 1060 cm−1). The resonances in the 1H and 13C NMR spectrum revealed seven tert-methyls [δH/δC 1.02 (s)/17.1 (Me-23), 0.99 (s)/15.8 (Me-25), 0.98 (s)/17.8 (Me-26), 0.96 (s)/25.8 (Me-27), 0.99 (s)/28.8 (Me-28), 1.00 (s)/27.5 (Me-29), 0.99 (s)/26.5 (Me-30)] and one sec-methyl [δH/δC 3.78, 3.21/68.9 (Me-24)], indicating aglycone similar to robinioside E [26] with one sec-methyl replaced by tert-methyl. The most downfield signals displayed in 1H and 13C NMR spectrum at [δH/δC 1.80, 1.52/124.0 (C-11) and 5.25 (br, s)/144.5 (C-12)] were ascribable to tetra-substituted olefinic bond characteristic in soysaponins at C11–C12 which was supported by HMBC correlation of H-11 (δH 1.80, 1.52) with C-9 (δC 46.6) and H-18 (δH 1.84) with C-12 (δC 124.0). The presence of three anomeric resonances in NMR spectrum [δH/δC 4.52 (d)/102.6 (C-1′), 4.58 (d)/102.1 (C-1″) and 4.60 (d)/102.4 (C-1‴)], indicated the presence of three sugars in the molecule. The 1H and13C NMR of 2, robinioside E and soysaponin I [27] revealed resonance signals assignable to β-glucuronopyranose [δH/δC 4.52 (d)/1.02 (C-1′), 3.36 (m)/81.4 (C-2′), 3.73 (m)/77.5 (C-3′), 3.63 (m)/72.6 (C-4′), 3.80 (m)/78.8 (C-5′) and 174.0 (C-6′)], β-galactopyranose [δH/δC 4.58 (d)/102.1 (C-1″), 3.36 (m)/77.4 (C-2″), 3.72 (m)/75.8 (C-3″), 3.45 (m)/72.2 (C-4″), 3.76 (m)/79.0 (C-5″), 3.78, 3.53/64.0 (C-6″)] and β-glucopyranose [δH/δC 4.60 (d)/102.4 (C-1‴), 3.71 (m)/74.2 (C-2‴), 3.50 (m)/75.3 (C-3‴), 3.42 (m)/72.4 (C-4‴), 3.73 (m)/78.4(C-5‴), 3.78, 3.50/65.2 (C-6‴)]. The fragment ions observed in ESIMS gave the sequence of sugars at m/z 795 [M-glucose], m/z 633 [M-glucose-galactose], m/z 457 [M-glucose-galactose-glucuronic acid] which was supported by HMBC correlation of H-1″ (δH 4.58) with C-2′ (δC 81.4) and H-1‴ (δH 4.60) with C-2″ (δC 77.4). The connectivity of oligosaccharide moiety and the aglycone of 2 was confirmed by long range HMBC correlation observed between H-1′ (δH 4.52) with C-3 (δC 90.3), which was further supported by connectivity of H-23 (δH 1.02) with C-3 (δC 90.3) and H-24 (δH 3.78, 3.53) with C-3 (δC 90.3). The configuration at C-3 of 2 was found to be S by comparing the chemical shift of C-3 and C-5 with soysaponin I and robinioside E. The configuration of sugars in oligosaccharide was found to be D-glucuronic acid, D-galactose and D-glucose by preparing thiazolidine derivatives of 2 and analyzing them through GC/MS. On the basis of above evidence compound 2 was elucidated to be 3β-O- [β-D-glucopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 2)- β-D-glucronopyranosyl]-12-ene-olean-22,24-diol. Compound 3 was obtained as white amorphous powder. The HRESIMS displayed the molecular ion peak at m/z 1159.5485 [M + Na]+, in which together with 13C NMR spectroscopic data revealed a molecular formula C54H88O25. The 13C NMR displayed 54 resonance signals, of which 30 coincided with oleanane triterpene skeleton and 24 to four sugar moieties as two oligomers, representing a bidesmosidic saponin. DEPT experiments revealed 7 methyls, 12 methylenes, 27 methines and 8 quaternary carbons in the molecule. The seven methyls at [δH/δC 1.04/17.2 (C-23), 0.98/17.2 (C-25), 0.99/17.8 (C-26), 0.97/25.8 (C-27), 0.99/19.2 (C-28), 0.98/26.6 (C-29) and 0.99/27.6 (C-30)] describe the agylone similar to 2 and four anomeric resonances [δH/δC 4.58/103.4, 4.59/102.3, 4.62/101.3, and 4.68/101.6] revealed that the molecule contained four sugars. The attachment of sugars to be bidesmosidic rather than monodesmosidic tetrasaccharide, was obtained by observing the HMBC correlation of H-1′ (δH 4.58) with C-3 (δC 90.3) and H-1‴ (δH 4.62) with C-22 (δC 92.1). The NMR data of aglycone of 3 matches well with soysaponin A2 [28] but displayed additional signal (δH/δC 3.79, 3.49/65.0) in sugars which corresponds to glucose rather than arabinose, which was fully supported by HMBC correlation of H-5‴ (δH 3.70) with C-6‴ (δC 65.0) and H-6‴ (δH 3.79, 3.49) with C-5‴ (δC 77.9) and C-4‴ (δC 72.5). The identification of sugars was established after acid hydrolysis of 3, which was found to be D-glucuronic acid, D-galactose and D-glucose by comparing to those of standard sugar samples of D-glucuronic acid, D-galactose and D-glucose respectively, on Co-TLC. The configuration of sugars was again confirmed to be D-glucuronic acid, D-galactose and D-glucose by preparing their thiazolidine derivatives. Accordingly structure of 3 was determined to be 3β-O-[β-O-galactopyranosyl-(1 → 2)-β-Dglucuronopyranosyl], 22β-O-[β-D-glucopyranosyl-(1 → 2)-β-Dglucopyranosyl]-12-ene-olean-21,24-diol. The HMBC correlations for compounds 1–3 are shown in Fig. 2. The carbon and proton data are presented in Tables 1 and 2, respectively. 3.1. Possible biogenetic pathways for compounds 1–3 The enzymes belonging to multigene families of oxidosqualene cyclases (OSCs), cytochromes and UDPglycosyltransferases are key players in biogenesis of plant triterpenoid saponins [29]. The members of these three multigene families presumably perform comparable reactions throughout all saponin producing plants. Family Fabaceae (G. max) appears to employ these genes for the synthesis of structurally closely related saponins. Glycosyltransferases (GTs) contribute greatly for generating the structural diversity of triterpene saponins, and are thus important targets in investigating the origin of structural diversity within this class of natural products. The enzyme transferases act as UDPs (uridine-diphosphoglucuronate-decarboxylase) by successive sugar transfers from UDP-sugars to soysapogenol moiety (aglycone) [30]. Thus biogenesis of triterpenoid seems to have evolved independently several times during plant evolution. The possible biogenesis paths of isolated soysaponins 1–3 are illustrated in Fig. 3. Acknowledgments This research is supported in part by ‘Science Based Authentication of Dietary Supplements’ and ‘Botanical Dietary Supplement Research’ funded by the Food and Drug Administration grant numbers 5U01FD002071-10 and 1U01FD003871-02, the United States Department of Agriculture, Agriculture Research Service, Specific Cooperative M.A. Tantry, I.A. Khan / Fitoterapia 87 (2013) 49–56 55 Agreement No. 58-6408-2-0009 and the Global Consortium of Medicinal Plants, supported by King Saud University, Riyadh. We thank Dr B. Avula for conducting the high resolution mass spectra, Dr A. Ali for experimenting larvicidal activity, Dr V. Raman for proving the plant material and Dr J. Parcher for his helpful discussion. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.03.021. References [1] Willer WC. Diet and health: what should we eat? Science 1994;264: 532. [2] Messina MJ. 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