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A further amendment to the classical core structure of gum arabic (Acacia senegal)
Food Hydrocolloids 31 (2013) 42e48
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A further amendment to the classical core structure of gum arabic (Acacia senegal) Shao-Ping Nie a, b, Cathy Wang b, Steve W. Cui a, b, *, Qi Wang b, Ming-Yong Xie a, Glyn O. Phillips c, d a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 c Gyln O. Hydrocolloid Research Centre, Glyndwr University, Wrexham LL11 2AW, Wales, UK d Phillips Hydrocolloids Research Ltd, 45 Old Bond Street, London W1S 4QT, UK b
ˇ
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2012 Accepted 24 September 2012
Using the more recently available techniques such as methylationeGCeMS, 1D (1H, 13C) and 2D (COSY, TOCSY, HMQC and HMBC) NMR spectral analysis, we have revisited the classical structure of gum arabic (Acacia senegal). Methylation and GCeMS analysis confirmed that gum arabic (A. senegal) is a highly branched polysaccharide with the backbone composed of 1,3-linked galactopyransyl (Galp) residues substituted at O-2, O-6 or O-4 positions. The terminal sugar residues are 59.5% of the total sugars. The residues of /2,3,6-b-D-Galp1/, /3,4-Galp1/, /3,4,6-Galp1/ and substitutions at O-2 and O-4 position were not identified in previous studies. Ó 2012 Published by Elsevier Ltd.
Keywords: Gum arabic (Acacia senegal) Structural characterization Methylation analysis 2D NMR
1. Introduction Gum arabic is a tree exudate, mainly from African Sahelian countries, and is an important additive and ingredient used in the food and pharmaceutical industries (Phillips & Williams, 2009, chap. 11). Some 93% of the structure is associated with the core carbohydrate. It was the classical work of the late Douglas Anderson in the UK and Alistair Stephens in South Africa that led to the structure that we are currently using for this core arabinogalactan carbohydrate component (Anderson & Stoddart, 1966; Churms, Merrifield, & Stephen, 1983). Subsequently, we have seen, of course, that gum arabic (Acacia senegal) is a polydisperse molecule which includes morphological structures in the form of a compact arabinogalactan protein (AGP) (Mahendran, Williams, Phillips, AlAssaf, & Baldwin, 2008) and a disk arabinogalactan (AG) (Mahendran et al., 2008). The AGP consists of a polypeptide chain possibly containing w250 amino acids with short arabinose side chains and much larger blocks of carbohydrate of molecular mass w4.0 104 Da attached. The carbohydrate is highly branched. The molecule adopts a very compact conformation with Rg of w36 nm (Fig. 1) (Mahendran et al., 2008). Sanchez (2010) has also proposed a structure for the AGP which is consistent with this model. A thin
oblate ellipsoid structure has been proposed for the AG component (Fig. 2). The branched structure of this disc-like structure is mainly composed of 1,3-linked b-D-galactopyranosyl units with 1,6-linked b-D-galactopyranosyl side chains to which there are linked many aarabinosyl, uronic acid and rhamnose residues (Anderson & Stoddart, 1966; Churms et al., 1983; Sanchez et al., 2008). The classical AndersoneStephen structure has stood the test of time (Fig. 3) (Street & Anderson, 1983; Wang, Burchard, Cui, Huang, & Phillips, 2008). Here we revisit the structure of these carbohydrate blocks of A. senegal using more recently available techniques. The methods used were methylation analysis and 2D NMR spectroscopy including homonuclear 1H/1H correlation spectroscopy (COSY, TOCSY), and heteronuclear 13C/1H multiple quantum coherence experiments (HMQC, HMBC). 2. Materials and methods 2.1. Materials The A. senegal var. senegal samples used in this investigation were from the Gum Arabic Company, Sudan and had been well authenticated (Al-Assaf, Phillips, & Williams, 2005a, 2005b). 2.2. Methylation and GCeMS for gum arabic
* Corresponding author. Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9. Tel.: þ1 519 780 8028; fax: þ1 519 829 2600. E-mail addresses: [email protected] (S.-P. Nie), [email protected] (C. Wang), [email protected] (S.W. Cui), [email protected] (Q. Wang), [email protected] (M.-Y. Xie), [email protected] (G.O. Phillips). 0268-005X/$ e see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodhyd.2012.09.014
Uronic acids were reduced to neutral polysaccharide before methylation following the previous procedure (Taylor & Conrad, 1972; York, Darvill, McNeil, Stevenson, & Albersheim, 1986), with minor modifications. Duplicated samples of gum arabic (5 mg)
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Fig. 2. Different views of the thin oblate ellipsoid AG structural component of gum arabic (Sanchez et al., 2008).
Fig. 1. Schematic illustration of the structure of the gum arabic arabinogalactan protein complex (Mahendran et al., 2008).
were dissolved in deuterium oxide D2O (2 mL). To the solution, 50 mg of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide methyl-p-toluenesulfonate was added while using 0.1 mol/L HCl in D2O to keep the pH at 4.75. The solution was left for 1 h while stirring, and followed by adding 5 mL of sodium borodeuteride (160 mg/mL) drop wise over a period of 0.5 h, and the reaction mixture pH was maintained at 7.0, using 2.0 mol/L HCl in D2O during the reduction reaction. The reaction continued with constant stirring for 0.5 h at pH 7.0 after the addition of sodium borodeuteride. The solution pH was then brought back to pH 4.0. The reduced polysaccharide was separated from salts by dialysis against distilled water overnight at 25 C (3500 Da molecular weight cut off), then lyophilized. The polysaccharide was redissolved in 0.5 mL distilled water and 0.5 mL 10% acetic acid in methanol was added. The mixture was dried under a stream of nitrogen to remove boric acid. Another 0.5 mL of 10% acetic acid in methanol was added to the residue and evaporated under nitrogen stream. This process was repeated 3e4 times to ensure that most of the boric acid was removed. Finally, a few drops of methanol were added and the solution evaporated (two times) to remove any boric acid remaining. Methylation analysis of gum arabic was conducted according to the method of Ciucanu and Kerek (1984) with slight modification. The dried samples (about 2e3 mg) were dissolved in anhydrous
DMSO (0.5 mL), sonicated at 50 C for 6.5 h, then heated at 85 C for 2 h with constant stirring. The sample in DMSO solution was stirred at room temperature overnight to obtain a clear solution. Dry sodium hydroxide (20 mg) was added, and the mixture was stirred for 3 h at room temperature before introducing methyl iodide. The mixture was stirred for another 2.5 h after adding 0.3 mL methyl iodide. The methylated sample was then extracted with 1.5 mL methylene chloride. The methylene chloride extract was passed through a sodium sulphate column (0.5 15 cm) to remove water, and evaporated to dry by a stream of nitrogen. The dried methylated sample was hydrolysed in 0.5 mL of 4.0 M trifluoroacetic acid (TFA) in a sealed test tube at 100 C for 6 h and the TFA was removed by evaporation under a stream of nitrogen and the residue was dissolved in 0.3 mL distilled water. The hydrolysate was reduced, using 5 mg sodium borodeuteride and borate was removed by repeated additions and evaporations first of 19:1 methanoleacetic acid then methanol alone. After that, the residue was acetylated with acetic anhydride (0.5 mL) for heating at 100 C for 2 h. The resultant partially methylated alditol acetates (PMAA) were passed through a sodium sulphate column again to remove water. Aliquots
Fig. 3. Illustration of a possible structural fragment of A. senegal gum (Street & Anderson, 1983). R e rhamnose; Um e 4-O-methylglucuronic acid; U e glucuronic acid; Ap e arabinopyranose; A e arabinose; G e galactose.
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Table 1 Methylation analysis and mode of linkage of gum arabic (Acacia senegal). PMAA
Linkage pattern
Peak area percentage (%)
2,3,4-Me3 Rhap 2,3,5-Me3 Araf 2,3,5-Me3 Arap 2,3,4,6-Me4-GlcAp or 2,3,4,6-Me4-GalAp 2,5-Me2 Araf 2,3,4,6-Me4-GalAp or 2,3,4,6-Me4-GlcAp 2,3,6-Me3-GlcAp or 2,3,6-Me3-GalAp 2,3,6-Me3-GalAp or 2,3,6-Me3-GlcAp 2,6-Me2-Galp 3,6-Me2-Galp 2,4-Me2-Galp 2-Me-Galp 4-Me-Galp
T-Rhap1/ T-L-Araf 1/ T-L-Arap 1/ T-UA1/ /3-L-Araf 1/ T-UA1/ /4-UA1/ /4-UA1/ /3,4-Galp1/ /4,6-Galp1/ /3,6-Galp1/ /3,4,6-Galp1/ /2,3,6-Galp1/
7.5 29.1 2.9 2.1 11.5 17.9 0.9 1.5 10.3 1.6 1.6 0.4 12.8
3. Results and discussion 3.1. Methylation analysis of gum arabic
of PMAA were injected on to GCeMS system (ThermoQuest Finnigan, San Diego, CA) fitted with an SP-2330 (Supelco, Bellefonte, Pa) column (30 m 0.25 mm, 0.2 mm film thickness, 160e210 C at 2 C/min, and then 210e240 C at 5 C/min) equipped with an ion trap MS detector (Guo, Cui, Wang, & Young, 2008; Nie et al., 2011).
2.3. NMR analysis Both 1H and 13C spectra were recorded on a Bruker AMX 500FT spectrometer. All samples were dissolved in deuterium oxide at 90 C for 3 h before NMR analysis. The spectra of 1H, 13C, and homonuclear 1H/1H correlation experiments (COSY, TOCSY), and Heteronuclear Multiple-Quantum Correlation (HMQC) and
Fig. 4. The 1H (a) and
13
Heteronuclear Multiple Bond Correlation (HMBC) were measured using a standard Bruker pulse sequence. The experiments were conducted at 35 C.
The presence of uronic acid in gum arabic makes it difficult to carry out the methylation analysis, because uronic acids are liable to b-elimination under alkali conditions. Uronic acids are also generally resistant to acid hydrolysis; so that the linkage between uronic acid and neutral sugars could be lost during methylation. To avoid this, the carboxyl groups of gum arabic were first reduced and then, methylated and analysed by GCeMS to identify the linkage patterns (Cui, 2005, chap. 3). After methylation, the individual peaks of the PMAA and fragmentation patterns were identified by their retention time in GC and by comparison with literature mass spectra patterns (Carpita & Shea, 1989, chap. 9). Based on this analysis of PMAA, the linkage patterns of gum arabic are shown in Table 1. Two signals of terminal uronic acid were identified which had same fragmentation and mass spectra patterns. Both are labelled T-Uronic acids (T-UA) (Table 1). Two signals of 1 / 4 linked uronic acid were found (Table 1). These signals could be derived from glucuronic acid or galacturonic acid and will be the subject of further study. Table 1 shows that the most abundant residues are TL-Araf 1/, T-UA1/, /2,3,6-Galp1/, /3,4-Galp1/, T-Rhap1/ and /3-L-Araf 1/. T-L-Araf is the most important abundant terminal unit, which accounts for 29.1% of the residues. Other nonreducing terminal units are t-UAp, t-Rhap and t-Arap with the molar ratio of 20.0%, 7.5% and 2.9%, respectively. The most branched
C (b) NMR spectrums of gum arabic (Acacia senegal).
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residue is /2,3,6-Galp1/, which accounts for 12.8% of total sugar residues. There were two side chains that could be attached to O-2, O-3 or O-6 position of /2,3,6-Galp1/. The second most abundant branching unit is /3,4-Galp1/ (10.3%). The relative amount of the branched residues, /4,6-Galp1/ and /3,6-Galp1/ and /3,4,6Galp1/, are 1.6%, 1.6% and 0.4%, respectively. The unsubstituted residues were identified as /3-L-Araf 1/ and /4-UAp1/ at 11.5% and 2.4%, respectively. The data from the methylation analysis confirmed that gum arabic is mainly composed of a Galp backbone linked 1 / 3 (25.1%) with the branching points at O-2, O-4 and/or O-6 positions. The following nuclear magnetic resonance spectroscopic analysis (NMR) is also in agreement with these conclusions. 3.2. NMR analysis of gum arabic More than ten peaks can be observed clearly in the anomeric region (4.3e5.8 ppm) in the 1H NMR spectrum of gum arabic (Fig. 4a). The peak at 1.26 ppm can be assigned to the methyl group of rhamnose, which is consistent with the results of methylation analysis (Table 1). Dominant peaks are identified at 5.39, 5.3, 5.0, 4.77 and 4.5 ppm. The high intensity peak at 4.64 ppm is partially due to the presence of H2O. In the 13C NMR spectrum (Fig. 4b), the peak at d 19.3 ppm belongs to the carbon of methyl group of rhamnose, and a peak at d177.9 ppm is a typical C-6 signal of an uronic acid. These results are also consistent with the conclusions from methylation analysis (Table 1). Five significant peaks at d 5.39, 5.32, 5.03, 4.78, and 4.51 ppm can be observed in the anomeric area of the HMQC spectrum, as shown in Fig. 5. Their 13C chemical shifts are identified as 110.8, 112.3, 102.7, 103.4 and 105.7 ppm, respectively, indicating that there are five major sugar residues in this area. A cross-peak can be found at 1.26 ppm of the 1H chemical shift and 19.1 ppm of the 13C chemical shift; these signals are due to the hydrogens and carbon of the methyl group in rhamnose. From methylation analysis, T-Araf is the most abundant residue (Cui, Phillips, Blackwell, & Nikiforuk, 2007; Das et al., 2009; Fischer et al., 2004; Habibi, Mahrouz, Marais, & Vignon, 2004). This peak
Fig. 5. HMQC spectrum of gum arabic (Acacia senegal).
Fig. 6. 1H/1H COSY correlation of gum arabic (Acacia senegal).
(5.39 ppm and 110.8 ppm) can be assigned to T-Araf. The chemical shift at 5.39 ppm also indicated that it is an a-linked residue. The chemical shift of H-2 was obtained from the COSY spectrum (Fig. 6), based on the principle that it correlates with H-1. In the same way, H-3 was assigned by its correlation with H-2. Comparison with the chemical shifts of t-a-L-Araf given in the literature (Cui et al., 2007; Das et al., 2009; Fischer et al., 2004; Habibi et al., 2004) and based on the TOCSY spectrum (Fig. 7), H-4, H-5 and H-50 can be assigned to 4.07, 3.80 and 3.70 ppm respectively (Table 2). All the chemical shifts of 13C were obtained from HMQC (Fig. 5). These assignments
Fig. 7. 1H/1H TOCSY correlation of gum arabic (Acacia senegal).
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Table 2 Chemical shift (d) assignments of 1H NMR and Residues A: a-L-Araf-(1/
13
B: T-a-L-Rhap-(1/
13
C: /3)-a-L-Araf-(1/ D: b-D-GalpA-(1/ E: /2,3,6-b-D-Galp1/ F: /3,4-Galp1/ G: /3,6-Galp1/ H: /4)-b-D-GlcpA-(1/ I: /4)-b-D-GalpA-(1/
C H C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 1
13
C NMR spectra of gum arabic (Acacia senegal) on the basis of HMQC, HMBC, COSY and TOCSY. C-1/H-1
C-2/H-2
C-3/H-3
C-4/H-4
C-5/H-5
110.8 5.39 103.4 4.77 112.3 5.32 105.6 4.49 105.5 4.52 105.3 4.51 105.7 4.50 105.2 4.50 102.7 5.03
84.2 4.18 72.4 3.95 82.8 4.39 74.1 3.52 76.4 3.67 71.9 3.70 72.8 3.73 74.6 3.38 73.1 3.76
76.2 3.92 72.1 3.75 87.6 3.94 72.8 3.67 76.1 3.57 78.6 3.75 81.4 3.69 76.4 3.58 71.6 4.05
86.4 4.07 75.2 3.41 85.1 4.26 75.8 3.95 71.4 3.92 74.2 4.03 85.4 4.27 78.4 3.73 82.7 4.38
64.1 3.80 71.1 4.02 64.0 3.93 76.2 3.63 70.9 3.97 73.6 3.78 75.8 3.96 75.9 3.94 72.4 3.86
C-6/H-6 or H-50
H-60
3.70 19.3 1.26 3.74 177.9 64.2 3.82 63.9 3.81 76.1 3.62 177.9
3.69 3.74 4.08
177.9
are in agreement with previous results (Cui et al., 2007; Das et al., 2009; Fischer et al., 2004; Habibi et al., 2004). From the TOCSY spectrum, the 4.77 ppm of the 1H NMR spectrum can be assigned to the H-1 of T-Rhap arising from the signal of the exocyclic eCH3 group (H-6, 1.25 ppm). The literature value (4.62 ppm) is consistent with its assignment as an a-linked residue (Chandra, Ghosh, Ojha, & Islam, 2009; Cui et al., 2007; Potekhina, Shashkov, Evtushenko, Senchenkova, & Naumova, 2003; Roy et al., 2007). From the 1H/1H correlation (COSY) spectrum (Fig. 8), the chemical shifts of the H-5 can be assigned from its correlation with H-6. H-4, H-3, H-2 and H-1 were assigned similarly, and all cross peaks are marked in Fig. 8. These six proton chemical shifts are in agreement with the chemical shifts from the TOCSY spectrum and literature values (Chandra et al., 2009; Cui et al., 2007; Potekhina et al., 2003; Roy et al., 2007). The 13C chemical shifts (Table 2) can be assigned from the 2D 1-bond proton-carbon heteronuclear multiple quantum correlation (HMQC) spectrum (Fig. 5)
and are consistent with previous information (Chandra et al., 2009; Cui et al., 2007; Roy et al., 2007). The H-1 signal at 5.32 ppm indicates that /3)-a-L-Araf-(1/ is an a-linked residue in accordance with literature value (Cardoso, Ferreira, Mafra, Silva, & Coimbra, 2007; Cui et al., 2007; Leon de Pinto, Martinez, & Sanabria, 2001). The H-2, H-30 assignments can be identified by the correlation of the H-1 signals at d5.32 ppm with H-2 at 4.39 ppm, and H-2 with H-3 (3.94 ppm) in the COSY spectrum (Fig. 6). From the TOCSY spectrum and comparison with previous reports (Cardoso et al., 2007; Cui et al., 2007; Kang et al., 2011a, 2011b; Leon de Pinto et al., 2001), the H-4, H-5 and H-50 assignments relate to the signals at 4.26 ppm and 3.93 ppm and 3.74 ppm, respectively, showing, along with the reference values, that the residue is /3)-a-L-Araf-(1/. The corresponding 13C chemical shifts can be obtained from the cross-peaks H1eC1, H2e C2/H6eC6 in the HMQC spectrum (Fig. 5). All these 1H and 13C chemical shifts are assigned in Table 2.
Fig. 8. COSY spectrum of gum arabic (Acacia senegal) (dotted lines are the correlations of protons of the terminal a-L-Rhap).
Fig. 9. 1H/13C HMBC correlation of gum arabic (Acacia senegal).
S.-P. Nie et al. / Food Hydrocolloids 31 (2013) 42e48 Table 3 The significant 3JH,C connectivities observed in an HMBC spectrum for the anomeric protons/carbons of the sugar residues of the gum arabic (Acacia senegal). Residues
H-1/C-1 dH/dC
A: a-L-Araf-(1/
5.39
B: T-a-L-Rhap-(1/
4.77
C: /3)-a-L-Araf-(1/ D: b-D-GalpA-(1/
5.32 112.3 4.49
E: /2,3,6-b-D-Galp1/
105.6 4.52
105.5 F: /3,4-Galp1/
4.51
105.3 G: /3,6-Galp1/
4.50
105.7 H: /4)-b-D-GlcpA-(1/
4.50
I: /4)-b-D-GalpA-(1/
5.03
Observed connectivities
dH/dC
Residue
Atom
87.6 64.2 82.7 71.9 72.8 85.4 4.52 64.2 76.4 76.1 74.2 3.38 102.7 63.9 64.0 76.1 78.6 3.82 3.81 102.7 63.9 64.0 76.1 78.6 3.82 3.81 63.9 64.0 76.1 78.6 3.82 3.81 63.9 76.1 78.6 87.6 74.2 72.8
C E I F G G E E E E F H I F C E F E F I F C E F E F F C E F E F F E F C F G
C-3 C-6 C-4 C-2 C-2 C-3 H-1 C-6 C-2 C-3 C-4 C-2 C-1 C-6 C-5 C-3 C-3 H-6 H-6 C-1 C-6 C-5 C-3 C-3 H-6 H-6 C-6 C-5 C-3 C-3 H-6 H-6 C-6 C-3 C-3 C-3 C-4 C-2
47
The COSY spectrum (Fig. 6) shows that the proton near 4.5 ppm (H-1) correlates with resonances at 3.38, 3.52, 3.67, 3.70 and 3.73 ppm, respectively, indicating that five residues maybe share the same anomeric proton. Literature data (Cardoso et al., 2007; Cui et al., 2007; Kang et al., 2011a, 2011b), H-2 at 3.52 ppm indicates that the resonances might relate to the terminal b-Galacturonic acid (GalpA) residue. The H-2 at 3.38 ppm can be assigned to the residue /4)-b-D-GlcpA-(1/, the H-2 at 3.73 ppm to the residue /3,6-Galp1/, the H-2 at 3.70 to the residue /3,4-Galp1/ and the H-2 at 3.67 ppm to residue /2,3,6-Galp1/. The TOCSY spectrum and comparison with previous studies (Capek, Matulova, Navarini, & Suggi-Liverani, 2010; Cardoso et al., 2007; Cui et al., 2007; Heiss, Klutts, Wang, Doering, & Azadi, 2009; Kang et al., 2011a, 2011b; Ohta, Lee, Hayashi, & Hayashi, 2009; Shashkov et al., 2007; Yang, Ye, Zhang, Liu, & Tang, 2009), enable these residues to be identified (Table 2). The corresponding 13C chemical shifts were obtained from the cross-peaks of H1eC1, H2eC2/H6e C6 in the HMQC spectrum (Fig. 5). All the 1H and 13C chemical shifts are assigned in Table 2. The signal at 5.03 ppm of the proton spectrum is of relative high intensity; and correlates with H-2 at 3.76 ppm, which might possibly be assigned to the H-1 of /4)-b-D-GalpA-(1/ (Yang et al., 2009). The TOCSY spectrum and literature values (Yang et al., 2009), indicate that the signals at 4.05 ppm, 4.38 ppm and 3.86 ppm can be assigned to H-3, H-4 and H-5 respectively. The corresponding 13 C chemical shifts can be obtained from the cross-peaks of H1eC1, H2eC2/H6-C6 in the HMQC spectrum (Fig. 5). All the 1H and 13C chemical shifts of the /4)-b-D-GalpA-(1/ residue are assigned in Table 2. The linkage sequences between sugar residues can be identified using long-range HMBC, as shown in Fig. 9, and are summarized in Table 3. The correlation between neighbouring sugar units can identify the sequence order of sugar residues in the polysaccharide. The cross-peaks of both anomeric protons and carbons of each sugar residue of gum arabic and both inter- and intra-residual connectivities are identified in Fig. 9. For example, cross-peaks were found between H-1 (d 5.39 ppm) of residue a-L-Araf-(1/ and C-3 (87.6 ppm) of residue /3)-a-L-Araf-(1/, indicating that the terminal arabinofuranosyl residues are linked to 3-a-Araf through 1,3-O-glycosidic bonds. Cross peak between H-1 of residue /2,3,6-b-D-Galp1/ and C-3 of residue /2,3,6-b-D-Galp1/ and
Fig. 10. Proposed structure of gum arabic (Acacia senegal). R is one of these following residues: T-Rhap1/, T-L-Araf 1/, T-L-Arap 1/, T-UA1/, T-UA1/, T-L-Araf 1 / 3-L-Araf 1/, T-UA1 / 4-UA1/. All the galactose and uronic acid are in b-D form, all the arabinose and rhamnose are in a-L form.
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/3,4-Galp1/ showed that the backbone is linked through 1,3-Oglycosidic bonds between /2,3,6-b-D-Galp1/ and /3,4-Galp1/. These results are consistent with the previous results from methylation analysis. Apart from the cross peaks between H-1 of residue /3)-a-L-Araf-(1/ and C-6 of residue /3,6-b-D-Galp1/ there are indications also that some of /3)-a-L-Araf-(1/ residues are linked to 6-b-Galp through 1,6-O-glycosidic bonds. Therefore, on the basis of all the information from the 1H and 13C NMR, HMQC, HMBC, COSY, and TOCSY spectroscopy, a complete assignment of all linkage patterns can be identified as shown in Table 2. 4. Conclusions We, therefore, propose a revised molecular structure for A. senegal (Fig. 10). The backbone is composed of 1,3-linked galactopyransyl (Galp) residues as traditionally described (Anderson, Hirst, & Stoddart, 1966; Cui et al., 2007; Defaye & Wong, 1986; Street & Anderson, 1983; Wang et al., 2008). Now we report that the backbone is substituted at O-2, O-6 or O-4 position, with residues of /2,3,6-b-D-Galp1/, /3,4-Galp1/, /3,4,6-Galp1/ and the substitutions at O-2 and O-4 position which has not previously been described (Anderson et al., 1966; Cui et al., 2007; Defaye & Wong, 1986; Street & Anderson, 1983; Wang et al., 2008). Acknowledgements We are thankful to John Nikiforuk and Dr. Barbara Blackwell of Eastern Cereal and Oilseed Research Centre, AAFC, for providing the NMR analysis. The partial financial support for this study by the Key Program of National Natural Science Foundation of China (No: 31130041), and National Key Technology R & D Program of China (2012BAD33B06), and Training Project of Young Scientists of Jiangxi Province (Stars of Jing gang), is gratefully acknowledged. We also thank Phillips Hydrocolloids Research Ltd for their support. References Al-Assaf, S., Phillips, G. O., & Williams, P. A. (2005a). Studies on Acacia exudate gums. Part I: the molecular weight of Acacia senegal gum exudate. Food Hydrocolloids, 19(4), 647e660. Al-Assaf, S., Phillips, G. O., & Williams, P. A. (2005b). Studies on Acacia exudate gums: part II. Molecular weight comparison of the Vulgares and Gummiferae series of Acacia gums. Food Hydrocolloids, 19(4), 661e667. Anderson, D. M., Hirst, E., & Stoddart, J. F. (1966). Studies on uronic acid materials.17. Some structural features of Acacia senegal gum (gum arabic). Journal of the Chemical Society C: Organic, 21, 1959e1966. Anderson, D. M. W., & Stoddart, J. F. (1966). Studies on uronic acid materialsdpart XV: the use of molecular sieve chromatography in studies on Acacia senegal gum (gum arabic). Carbohydrate Research, 2, 104e114. Capek, P., Matulova, M., Navarini, L., & Suggi-Liverani, F. (2010). Structural features of an arabinogalactan-protein isolated from instant coffee powder of Coffea arabica beans. Carbohydrate Polymers, 80(1), 180e185. Cardoso, S. M., Ferreira, J. A., Mafra, I., Silva, A. M. S., & Coimbra, M. A. (2007). Structural ripening-related changes of the arabinan-rich pectic polysaccharides from olive pulp cell walls. Journal of Agricultural and Food Chemistry, 55(17), 7124e7130. Carpita, N. C., & Shea, E. M. (1989). Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates. In Analysis of carbohydrates by GLC and MS. Boca Raton: CRC Press. Chandra, K., Ghosh, K., Ojha, A. K., & Islam, S. S. (2009). Chemical analysis of a polysaccharide of unripe (green) tomato (Lycopersicon esculentum). Carbohydrate Research, 344(16), 2188e2194. Churms, S. C., Merrifield, E. H., & Stephen, A. M. (1983). Some new aspects of the molecular structure of Acacia senegal gum (gum arabic). Carbohydrate Research, 123, 267e279. Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydrate Research, 131(2), 209e217.
Cui, S. W. (2005). Structural analysis of polysaccharides. In Food carbohydrates: Chemistry, physical properties, and applications. Boca Raton: CRC Press. Cui, S. W., Phillips, G. O., Blackwell, B., & Nikiforuk, J. (2007). Characterisation and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPERGUM (TM)): part 4. Spectroscopic characterisation of Acacia senegal var. senegal and Acacia (sen) SUPERGUM (TM) arabic. Food Hydrocolloids, 21(3), 347e352. Das, D., Mondal, S., Roy, S. K., Maiti, D., Bhunia, B., Maiti, T. K., et al. (2009). Isolation and characterization of a heteropolysaccharide from the corm of Amorphophallus campanulatus. Carbohydrate Research, 344(18), 2581e2585. Defaye, J., & Wong, E. (1986). Structural studies of gum-arabic, the exudate polysaccharide from Acacia-senegal. Carbohydrate Research, 150(1), 221e231. Fischer, M. H., Yu, N. X., Gray, G. R., Ralph, J., Anderson, L., & Marlett, J. A. (2004). The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk). Carbohydrate Research, 339(11), 2009e2017. Guo, Q., Cui, S. W., Wang, Q., & Young, J. C. (2008). Fractionation and physicochemical characterization of psyllium gum. Carbohydrate Polymers, 73(1), 35e43. Habibi, Y., Mahrouz, M., Marais, M. F., & Vignon, M. R. (2004). An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits. Carbohydrate Research, 339(6), 1201e1205. Heiss, C., Klutts, J. S., Wang, Z. R., Doering, T. L., & Azadi, P. (2009). The structure of Cryptococcus neoformans galactoxylomannan contains beta-D-glucuronic acid. Carbohydrate Research, 344(7), 915e920. Kang, J., Cui, S. W., Phillips, G. O., Chen, J., Guo, Q., & Wang, Q. (2011a). New studies on gum ghatti (Anogeissus latifolia) part II. Structure characterization of an arabinogalactan from the gum by 1D, 2D NMR spectroscopy and methylation analysis. Food Hydrocolloids, 25(8), 1991e1998. Kang, J., Cui, S. W., Phillips, G. O., Chen, J., Guo, Q., & Wang, Q. (2011b). New studies on gum ghatti (Anogeissus latifolia) part III: structure characterization of a globular polysaccharide fraction by 1D, 2D NMR spectroscopy and methylation analysis. Food Hydrocolloids, 25(8), 1999e2007. Leon de Pinto, G., Martinez, M., & Sanabria, L. (2001). Structural features of the polysaccharide gum from Acacia glomerosa. Food Hydrocolloids, 15(4e6), 461e467. Mahendran, T., Williams, P. A., Phillips, G. O., Al-Assaf, S., & Baldwin, T. C. (2008). New insights into the structural characteristics of the Arabinogalactan-Protein (AGP) fraction of gum arabic. Journal of Agricultural and Food Chemistry, 56(19), 9269e9276. Nie, S.-P., Cui, S. W., Phillips, A. O., Xie, M.-Y., Phillips, G. O., Al-Assaf, S., et al. (2011). Elucidation of the structure of a bioactive hydrophilic polysaccharide from Cordyceps sinensis by methylation analysis and NMR spectroscopy. Carbohydrate Polymers, 84(3), 894e898. Ohta, Y., Lee, J. B., Hayashi, K., & Hayashi, T. (2009). Isolation of sulfated galactan from Codium fragile and its antiviral effect. Biological & Pharmaceutical Bulletin, 32(5), 892e898. Phillips, G. O., & Williams, P. A. (2009). Handbook of hydrocolloids (2nd and enlarged ed.). Cambridge: Woodhead Publishing Limited. Potekhina, N. V., Shashkov, A. S., Evtushenko, L. I., Senchenkova, S. N., & Naumova, I. B. (2003). A mannitol teichoic acid containing rhamnose and pyruvic acid acetal from the cell wall of Brevibacterium permense VKM Ac-2280. Carbohydrate Research, 338(23), 2745e2749. Roy, S. K., Chandra, K., Ghosh, K., Mondal, S., Maiti, D., Ojha, A. K., et al. (2007). Structural investigation of a heteropolysaccharide isolated from the pods (fruits) of Moringa oleifera (Sajina). Carbohydrate Research, 342(16), 2380e2389. Sanchez, C. (2010). Personal communication. Sanchez, C., Schmitt, C., Kolodziejczyk, E., Lapp, A., Gaillard, C., & Renard, D. (2008). The acacia gum arabinogalactan fraction is a thin oblate ellipsoid: a new model based on small-angle neutron scattering and Ab Initio. Biophysical Journal, 94, 629e639. Shashkov, A. S., Kocharova, N. A., Zatonsky, G. V., Blaszczyk, A., Knirel, Y. A., & Rozalski, A. (2007). Structure of the O-antigen of Providencia stuartii O20, a new polysaccharide containing 5,7-diacetamido3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid. Carbohydrate Research, 342(3e4), 653e658. Street, C. A., & Anderson, D. M. W. (1983). Refinement of structures previously proposed for gum arabic and other acacia gum exudates. Talanta, 30(11), 887e 893. Taylor, R. L., & Conrad, H. E. (1972). Stiochiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Biochemistry, 11(8), 1383e1388. Wang, Q., Burchard, W., Cui, S. W., Huang, X. Q., & Phillips, G. O. (2008). Solution properties of conventional gum arabic and a matured gum arabic (Acacia (sen) SUPER GUM). Biomacromolecules, 9(4), 1163e1169. Yang, Y., Ye, L. B., Zhang, J. S., Liu, Y. F., & Tang, Q. J. (2009). Structural analysis of a bioactive polysaccharide, PISP1, from the medicinal mushroom Phellinus igniarius. Bioscience Biotechnology and Biochemistry, 73(1), 134e139. York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T., & Albersheim, P. (1986). Isolation and characterization of plant-cell walls and cell-wall components. Methods in Enzymology, 118, 3e40.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
A further amendment to the classical core structure of gum arabic (Acacia senegal) Shao-Ping Nie a, b, Cathy Wang b, Steve W. Cui a, b, *, Qi Wang b, Ming-Yong Xie a, Glyn O. Phillips c, d a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 c Gyln O. Hydrocolloid Research Centre, Glyndwr University, Wrexham LL11 2AW, Wales, UK d Phillips Hydrocolloids Research Ltd, 45 Old Bond Street, London W1S 4QT, UK b
ˇ
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2012 Accepted 24 September 2012
Using the more recently available techniques such as methylationeGCeMS, 1D (1H, 13C) and 2D (COSY, TOCSY, HMQC and HMBC) NMR spectral analysis, we have revisited the classical structure of gum arabic (Acacia senegal). Methylation and GCeMS analysis confirmed that gum arabic (A. senegal) is a highly branched polysaccharide with the backbone composed of 1,3-linked galactopyransyl (Galp) residues substituted at O-2, O-6 or O-4 positions. The terminal sugar residues are 59.5% of the total sugars. The residues of /2,3,6-b-D-Galp1/, /3,4-Galp1/, /3,4,6-Galp1/ and substitutions at O-2 and O-4 position were not identified in previous studies. Ó 2012 Published by Elsevier Ltd.
Keywords: Gum arabic (Acacia senegal) Structural characterization Methylation analysis 2D NMR
1. Introduction Gum arabic is a tree exudate, mainly from African Sahelian countries, and is an important additive and ingredient used in the food and pharmaceutical industries (Phillips & Williams, 2009, chap. 11). Some 93% of the structure is associated with the core carbohydrate. It was the classical work of the late Douglas Anderson in the UK and Alistair Stephens in South Africa that led to the structure that we are currently using for this core arabinogalactan carbohydrate component (Anderson & Stoddart, 1966; Churms, Merrifield, & Stephen, 1983). Subsequently, we have seen, of course, that gum arabic (Acacia senegal) is a polydisperse molecule which includes morphological structures in the form of a compact arabinogalactan protein (AGP) (Mahendran, Williams, Phillips, AlAssaf, & Baldwin, 2008) and a disk arabinogalactan (AG) (Mahendran et al., 2008). The AGP consists of a polypeptide chain possibly containing w250 amino acids with short arabinose side chains and much larger blocks of carbohydrate of molecular mass w4.0 104 Da attached. The carbohydrate is highly branched. The molecule adopts a very compact conformation with Rg of w36 nm (Fig. 1) (Mahendran et al., 2008). Sanchez (2010) has also proposed a structure for the AGP which is consistent with this model. A thin
oblate ellipsoid structure has been proposed for the AG component (Fig. 2). The branched structure of this disc-like structure is mainly composed of 1,3-linked b-D-galactopyranosyl units with 1,6-linked b-D-galactopyranosyl side chains to which there are linked many aarabinosyl, uronic acid and rhamnose residues (Anderson & Stoddart, 1966; Churms et al., 1983; Sanchez et al., 2008). The classical AndersoneStephen structure has stood the test of time (Fig. 3) (Street & Anderson, 1983; Wang, Burchard, Cui, Huang, & Phillips, 2008). Here we revisit the structure of these carbohydrate blocks of A. senegal using more recently available techniques. The methods used were methylation analysis and 2D NMR spectroscopy including homonuclear 1H/1H correlation spectroscopy (COSY, TOCSY), and heteronuclear 13C/1H multiple quantum coherence experiments (HMQC, HMBC). 2. Materials and methods 2.1. Materials The A. senegal var. senegal samples used in this investigation were from the Gum Arabic Company, Sudan and had been well authenticated (Al-Assaf, Phillips, & Williams, 2005a, 2005b). 2.2. Methylation and GCeMS for gum arabic
* Corresponding author. Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9. Tel.: þ1 519 780 8028; fax: þ1 519 829 2600. E-mail addresses: [email protected] (S.-P. Nie), [email protected] (C. Wang), [email protected] (S.W. Cui), [email protected] (Q. Wang), [email protected] (M.-Y. Xie), [email protected] (G.O. Phillips). 0268-005X/$ e see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodhyd.2012.09.014
Uronic acids were reduced to neutral polysaccharide before methylation following the previous procedure (Taylor & Conrad, 1972; York, Darvill, McNeil, Stevenson, & Albersheim, 1986), with minor modifications. Duplicated samples of gum arabic (5 mg)
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43
Fig. 2. Different views of the thin oblate ellipsoid AG structural component of gum arabic (Sanchez et al., 2008).
Fig. 1. Schematic illustration of the structure of the gum arabic arabinogalactan protein complex (Mahendran et al., 2008).
were dissolved in deuterium oxide D2O (2 mL). To the solution, 50 mg of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide methyl-p-toluenesulfonate was added while using 0.1 mol/L HCl in D2O to keep the pH at 4.75. The solution was left for 1 h while stirring, and followed by adding 5 mL of sodium borodeuteride (160 mg/mL) drop wise over a period of 0.5 h, and the reaction mixture pH was maintained at 7.0, using 2.0 mol/L HCl in D2O during the reduction reaction. The reaction continued with constant stirring for 0.5 h at pH 7.0 after the addition of sodium borodeuteride. The solution pH was then brought back to pH 4.0. The reduced polysaccharide was separated from salts by dialysis against distilled water overnight at 25 C (3500 Da molecular weight cut off), then lyophilized. The polysaccharide was redissolved in 0.5 mL distilled water and 0.5 mL 10% acetic acid in methanol was added. The mixture was dried under a stream of nitrogen to remove boric acid. Another 0.5 mL of 10% acetic acid in methanol was added to the residue and evaporated under nitrogen stream. This process was repeated 3e4 times to ensure that most of the boric acid was removed. Finally, a few drops of methanol were added and the solution evaporated (two times) to remove any boric acid remaining. Methylation analysis of gum arabic was conducted according to the method of Ciucanu and Kerek (1984) with slight modification. The dried samples (about 2e3 mg) were dissolved in anhydrous
DMSO (0.5 mL), sonicated at 50 C for 6.5 h, then heated at 85 C for 2 h with constant stirring. The sample in DMSO solution was stirred at room temperature overnight to obtain a clear solution. Dry sodium hydroxide (20 mg) was added, and the mixture was stirred for 3 h at room temperature before introducing methyl iodide. The mixture was stirred for another 2.5 h after adding 0.3 mL methyl iodide. The methylated sample was then extracted with 1.5 mL methylene chloride. The methylene chloride extract was passed through a sodium sulphate column (0.5 15 cm) to remove water, and evaporated to dry by a stream of nitrogen. The dried methylated sample was hydrolysed in 0.5 mL of 4.0 M trifluoroacetic acid (TFA) in a sealed test tube at 100 C for 6 h and the TFA was removed by evaporation under a stream of nitrogen and the residue was dissolved in 0.3 mL distilled water. The hydrolysate was reduced, using 5 mg sodium borodeuteride and borate was removed by repeated additions and evaporations first of 19:1 methanoleacetic acid then methanol alone. After that, the residue was acetylated with acetic anhydride (0.5 mL) for heating at 100 C for 2 h. The resultant partially methylated alditol acetates (PMAA) were passed through a sodium sulphate column again to remove water. Aliquots
Fig. 3. Illustration of a possible structural fragment of A. senegal gum (Street & Anderson, 1983). R e rhamnose; Um e 4-O-methylglucuronic acid; U e glucuronic acid; Ap e arabinopyranose; A e arabinose; G e galactose.
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Table 1 Methylation analysis and mode of linkage of gum arabic (Acacia senegal). PMAA
Linkage pattern
Peak area percentage (%)
2,3,4-Me3 Rhap 2,3,5-Me3 Araf 2,3,5-Me3 Arap 2,3,4,6-Me4-GlcAp or 2,3,4,6-Me4-GalAp 2,5-Me2 Araf 2,3,4,6-Me4-GalAp or 2,3,4,6-Me4-GlcAp 2,3,6-Me3-GlcAp or 2,3,6-Me3-GalAp 2,3,6-Me3-GalAp or 2,3,6-Me3-GlcAp 2,6-Me2-Galp 3,6-Me2-Galp 2,4-Me2-Galp 2-Me-Galp 4-Me-Galp
T-Rhap1/ T-L-Araf 1/ T-L-Arap 1/ T-UA1/ /3-L-Araf 1/ T-UA1/ /4-UA1/ /4-UA1/ /3,4-Galp1/ /4,6-Galp1/ /3,6-Galp1/ /3,4,6-Galp1/ /2,3,6-Galp1/
7.5 29.1 2.9 2.1 11.5 17.9 0.9 1.5 10.3 1.6 1.6 0.4 12.8
3. Results and discussion 3.1. Methylation analysis of gum arabic
of PMAA were injected on to GCeMS system (ThermoQuest Finnigan, San Diego, CA) fitted with an SP-2330 (Supelco, Bellefonte, Pa) column (30 m 0.25 mm, 0.2 mm film thickness, 160e210 C at 2 C/min, and then 210e240 C at 5 C/min) equipped with an ion trap MS detector (Guo, Cui, Wang, & Young, 2008; Nie et al., 2011).
2.3. NMR analysis Both 1H and 13C spectra were recorded on a Bruker AMX 500FT spectrometer. All samples were dissolved in deuterium oxide at 90 C for 3 h before NMR analysis. The spectra of 1H, 13C, and homonuclear 1H/1H correlation experiments (COSY, TOCSY), and Heteronuclear Multiple-Quantum Correlation (HMQC) and
Fig. 4. The 1H (a) and
13
Heteronuclear Multiple Bond Correlation (HMBC) were measured using a standard Bruker pulse sequence. The experiments were conducted at 35 C.
The presence of uronic acid in gum arabic makes it difficult to carry out the methylation analysis, because uronic acids are liable to b-elimination under alkali conditions. Uronic acids are also generally resistant to acid hydrolysis; so that the linkage between uronic acid and neutral sugars could be lost during methylation. To avoid this, the carboxyl groups of gum arabic were first reduced and then, methylated and analysed by GCeMS to identify the linkage patterns (Cui, 2005, chap. 3). After methylation, the individual peaks of the PMAA and fragmentation patterns were identified by their retention time in GC and by comparison with literature mass spectra patterns (Carpita & Shea, 1989, chap. 9). Based on this analysis of PMAA, the linkage patterns of gum arabic are shown in Table 1. Two signals of terminal uronic acid were identified which had same fragmentation and mass spectra patterns. Both are labelled T-Uronic acids (T-UA) (Table 1). Two signals of 1 / 4 linked uronic acid were found (Table 1). These signals could be derived from glucuronic acid or galacturonic acid and will be the subject of further study. Table 1 shows that the most abundant residues are TL-Araf 1/, T-UA1/, /2,3,6-Galp1/, /3,4-Galp1/, T-Rhap1/ and /3-L-Araf 1/. T-L-Araf is the most important abundant terminal unit, which accounts for 29.1% of the residues. Other nonreducing terminal units are t-UAp, t-Rhap and t-Arap with the molar ratio of 20.0%, 7.5% and 2.9%, respectively. The most branched
C (b) NMR spectrums of gum arabic (Acacia senegal).
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45
residue is /2,3,6-Galp1/, which accounts for 12.8% of total sugar residues. There were two side chains that could be attached to O-2, O-3 or O-6 position of /2,3,6-Galp1/. The second most abundant branching unit is /3,4-Galp1/ (10.3%). The relative amount of the branched residues, /4,6-Galp1/ and /3,6-Galp1/ and /3,4,6Galp1/, are 1.6%, 1.6% and 0.4%, respectively. The unsubstituted residues were identified as /3-L-Araf 1/ and /4-UAp1/ at 11.5% and 2.4%, respectively. The data from the methylation analysis confirmed that gum arabic is mainly composed of a Galp backbone linked 1 / 3 (25.1%) with the branching points at O-2, O-4 and/or O-6 positions. The following nuclear magnetic resonance spectroscopic analysis (NMR) is also in agreement with these conclusions. 3.2. NMR analysis of gum arabic More than ten peaks can be observed clearly in the anomeric region (4.3e5.8 ppm) in the 1H NMR spectrum of gum arabic (Fig. 4a). The peak at 1.26 ppm can be assigned to the methyl group of rhamnose, which is consistent with the results of methylation analysis (Table 1). Dominant peaks are identified at 5.39, 5.3, 5.0, 4.77 and 4.5 ppm. The high intensity peak at 4.64 ppm is partially due to the presence of H2O. In the 13C NMR spectrum (Fig. 4b), the peak at d 19.3 ppm belongs to the carbon of methyl group of rhamnose, and a peak at d177.9 ppm is a typical C-6 signal of an uronic acid. These results are also consistent with the conclusions from methylation analysis (Table 1). Five significant peaks at d 5.39, 5.32, 5.03, 4.78, and 4.51 ppm can be observed in the anomeric area of the HMQC spectrum, as shown in Fig. 5. Their 13C chemical shifts are identified as 110.8, 112.3, 102.7, 103.4 and 105.7 ppm, respectively, indicating that there are five major sugar residues in this area. A cross-peak can be found at 1.26 ppm of the 1H chemical shift and 19.1 ppm of the 13C chemical shift; these signals are due to the hydrogens and carbon of the methyl group in rhamnose. From methylation analysis, T-Araf is the most abundant residue (Cui, Phillips, Blackwell, & Nikiforuk, 2007; Das et al., 2009; Fischer et al., 2004; Habibi, Mahrouz, Marais, & Vignon, 2004). This peak
Fig. 5. HMQC spectrum of gum arabic (Acacia senegal).
Fig. 6. 1H/1H COSY correlation of gum arabic (Acacia senegal).
(5.39 ppm and 110.8 ppm) can be assigned to T-Araf. The chemical shift at 5.39 ppm also indicated that it is an a-linked residue. The chemical shift of H-2 was obtained from the COSY spectrum (Fig. 6), based on the principle that it correlates with H-1. In the same way, H-3 was assigned by its correlation with H-2. Comparison with the chemical shifts of t-a-L-Araf given in the literature (Cui et al., 2007; Das et al., 2009; Fischer et al., 2004; Habibi et al., 2004) and based on the TOCSY spectrum (Fig. 7), H-4, H-5 and H-50 can be assigned to 4.07, 3.80 and 3.70 ppm respectively (Table 2). All the chemical shifts of 13C were obtained from HMQC (Fig. 5). These assignments
Fig. 7. 1H/1H TOCSY correlation of gum arabic (Acacia senegal).
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Table 2 Chemical shift (d) assignments of 1H NMR and Residues A: a-L-Araf-(1/
13
B: T-a-L-Rhap-(1/
13
C: /3)-a-L-Araf-(1/ D: b-D-GalpA-(1/ E: /2,3,6-b-D-Galp1/ F: /3,4-Galp1/ G: /3,6-Galp1/ H: /4)-b-D-GlcpA-(1/ I: /4)-b-D-GalpA-(1/
C H C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 13 C 1 H 1
13
C NMR spectra of gum arabic (Acacia senegal) on the basis of HMQC, HMBC, COSY and TOCSY. C-1/H-1
C-2/H-2
C-3/H-3
C-4/H-4
C-5/H-5
110.8 5.39 103.4 4.77 112.3 5.32 105.6 4.49 105.5 4.52 105.3 4.51 105.7 4.50 105.2 4.50 102.7 5.03
84.2 4.18 72.4 3.95 82.8 4.39 74.1 3.52 76.4 3.67 71.9 3.70 72.8 3.73 74.6 3.38 73.1 3.76
76.2 3.92 72.1 3.75 87.6 3.94 72.8 3.67 76.1 3.57 78.6 3.75 81.4 3.69 76.4 3.58 71.6 4.05
86.4 4.07 75.2 3.41 85.1 4.26 75.8 3.95 71.4 3.92 74.2 4.03 85.4 4.27 78.4 3.73 82.7 4.38
64.1 3.80 71.1 4.02 64.0 3.93 76.2 3.63 70.9 3.97 73.6 3.78 75.8 3.96 75.9 3.94 72.4 3.86
C-6/H-6 or H-50
H-60
3.70 19.3 1.26 3.74 177.9 64.2 3.82 63.9 3.81 76.1 3.62 177.9
3.69 3.74 4.08
177.9
are in agreement with previous results (Cui et al., 2007; Das et al., 2009; Fischer et al., 2004; Habibi et al., 2004). From the TOCSY spectrum, the 4.77 ppm of the 1H NMR spectrum can be assigned to the H-1 of T-Rhap arising from the signal of the exocyclic eCH3 group (H-6, 1.25 ppm). The literature value (4.62 ppm) is consistent with its assignment as an a-linked residue (Chandra, Ghosh, Ojha, & Islam, 2009; Cui et al., 2007; Potekhina, Shashkov, Evtushenko, Senchenkova, & Naumova, 2003; Roy et al., 2007). From the 1H/1H correlation (COSY) spectrum (Fig. 8), the chemical shifts of the H-5 can be assigned from its correlation with H-6. H-4, H-3, H-2 and H-1 were assigned similarly, and all cross peaks are marked in Fig. 8. These six proton chemical shifts are in agreement with the chemical shifts from the TOCSY spectrum and literature values (Chandra et al., 2009; Cui et al., 2007; Potekhina et al., 2003; Roy et al., 2007). The 13C chemical shifts (Table 2) can be assigned from the 2D 1-bond proton-carbon heteronuclear multiple quantum correlation (HMQC) spectrum (Fig. 5)
and are consistent with previous information (Chandra et al., 2009; Cui et al., 2007; Roy et al., 2007). The H-1 signal at 5.32 ppm indicates that /3)-a-L-Araf-(1/ is an a-linked residue in accordance with literature value (Cardoso, Ferreira, Mafra, Silva, & Coimbra, 2007; Cui et al., 2007; Leon de Pinto, Martinez, & Sanabria, 2001). The H-2, H-30 assignments can be identified by the correlation of the H-1 signals at d5.32 ppm with H-2 at 4.39 ppm, and H-2 with H-3 (3.94 ppm) in the COSY spectrum (Fig. 6). From the TOCSY spectrum and comparison with previous reports (Cardoso et al., 2007; Cui et al., 2007; Kang et al., 2011a, 2011b; Leon de Pinto et al., 2001), the H-4, H-5 and H-50 assignments relate to the signals at 4.26 ppm and 3.93 ppm and 3.74 ppm, respectively, showing, along with the reference values, that the residue is /3)-a-L-Araf-(1/. The corresponding 13C chemical shifts can be obtained from the cross-peaks H1eC1, H2e C2/H6eC6 in the HMQC spectrum (Fig. 5). All these 1H and 13C chemical shifts are assigned in Table 2.
Fig. 8. COSY spectrum of gum arabic (Acacia senegal) (dotted lines are the correlations of protons of the terminal a-L-Rhap).
Fig. 9. 1H/13C HMBC correlation of gum arabic (Acacia senegal).
S.-P. Nie et al. / Food Hydrocolloids 31 (2013) 42e48 Table 3 The significant 3JH,C connectivities observed in an HMBC spectrum for the anomeric protons/carbons of the sugar residues of the gum arabic (Acacia senegal). Residues
H-1/C-1 dH/dC
A: a-L-Araf-(1/
5.39
B: T-a-L-Rhap-(1/
4.77
C: /3)-a-L-Araf-(1/ D: b-D-GalpA-(1/
5.32 112.3 4.49
E: /2,3,6-b-D-Galp1/
105.6 4.52
105.5 F: /3,4-Galp1/
4.51
105.3 G: /3,6-Galp1/
4.50
105.7 H: /4)-b-D-GlcpA-(1/
4.50
I: /4)-b-D-GalpA-(1/
5.03
Observed connectivities
dH/dC
Residue
Atom
87.6 64.2 82.7 71.9 72.8 85.4 4.52 64.2 76.4 76.1 74.2 3.38 102.7 63.9 64.0 76.1 78.6 3.82 3.81 102.7 63.9 64.0 76.1 78.6 3.82 3.81 63.9 64.0 76.1 78.6 3.82 3.81 63.9 76.1 78.6 87.6 74.2 72.8
C E I F G G E E E E F H I F C E F E F I F C E F E F F C E F E F F E F C F G
C-3 C-6 C-4 C-2 C-2 C-3 H-1 C-6 C-2 C-3 C-4 C-2 C-1 C-6 C-5 C-3 C-3 H-6 H-6 C-1 C-6 C-5 C-3 C-3 H-6 H-6 C-6 C-5 C-3 C-3 H-6 H-6 C-6 C-3 C-3 C-3 C-4 C-2
47
The COSY spectrum (Fig. 6) shows that the proton near 4.5 ppm (H-1) correlates with resonances at 3.38, 3.52, 3.67, 3.70 and 3.73 ppm, respectively, indicating that five residues maybe share the same anomeric proton. Literature data (Cardoso et al., 2007; Cui et al., 2007; Kang et al., 2011a, 2011b), H-2 at 3.52 ppm indicates that the resonances might relate to the terminal b-Galacturonic acid (GalpA) residue. The H-2 at 3.38 ppm can be assigned to the residue /4)-b-D-GlcpA-(1/, the H-2 at 3.73 ppm to the residue /3,6-Galp1/, the H-2 at 3.70 to the residue /3,4-Galp1/ and the H-2 at 3.67 ppm to residue /2,3,6-Galp1/. The TOCSY spectrum and comparison with previous studies (Capek, Matulova, Navarini, & Suggi-Liverani, 2010; Cardoso et al., 2007; Cui et al., 2007; Heiss, Klutts, Wang, Doering, & Azadi, 2009; Kang et al., 2011a, 2011b; Ohta, Lee, Hayashi, & Hayashi, 2009; Shashkov et al., 2007; Yang, Ye, Zhang, Liu, & Tang, 2009), enable these residues to be identified (Table 2). The corresponding 13C chemical shifts were obtained from the cross-peaks of H1eC1, H2eC2/H6e C6 in the HMQC spectrum (Fig. 5). All the 1H and 13C chemical shifts are assigned in Table 2. The signal at 5.03 ppm of the proton spectrum is of relative high intensity; and correlates with H-2 at 3.76 ppm, which might possibly be assigned to the H-1 of /4)-b-D-GalpA-(1/ (Yang et al., 2009). The TOCSY spectrum and literature values (Yang et al., 2009), indicate that the signals at 4.05 ppm, 4.38 ppm and 3.86 ppm can be assigned to H-3, H-4 and H-5 respectively. The corresponding 13 C chemical shifts can be obtained from the cross-peaks of H1eC1, H2eC2/H6-C6 in the HMQC spectrum (Fig. 5). All the 1H and 13C chemical shifts of the /4)-b-D-GalpA-(1/ residue are assigned in Table 2. The linkage sequences between sugar residues can be identified using long-range HMBC, as shown in Fig. 9, and are summarized in Table 3. The correlation between neighbouring sugar units can identify the sequence order of sugar residues in the polysaccharide. The cross-peaks of both anomeric protons and carbons of each sugar residue of gum arabic and both inter- and intra-residual connectivities are identified in Fig. 9. For example, cross-peaks were found between H-1 (d 5.39 ppm) of residue a-L-Araf-(1/ and C-3 (87.6 ppm) of residue /3)-a-L-Araf-(1/, indicating that the terminal arabinofuranosyl residues are linked to 3-a-Araf through 1,3-O-glycosidic bonds. Cross peak between H-1 of residue /2,3,6-b-D-Galp1/ and C-3 of residue /2,3,6-b-D-Galp1/ and
Fig. 10. Proposed structure of gum arabic (Acacia senegal). R is one of these following residues: T-Rhap1/, T-L-Araf 1/, T-L-Arap 1/, T-UA1/, T-UA1/, T-L-Araf 1 / 3-L-Araf 1/, T-UA1 / 4-UA1/. All the galactose and uronic acid are in b-D form, all the arabinose and rhamnose are in a-L form.
48
S.-P. Nie et al. / Food Hydrocolloids 31 (2013) 42e48
/3,4-Galp1/ showed that the backbone is linked through 1,3-Oglycosidic bonds between /2,3,6-b-D-Galp1/ and /3,4-Galp1/. These results are consistent with the previous results from methylation analysis. Apart from the cross peaks between H-1 of residue /3)-a-L-Araf-(1/ and C-6 of residue /3,6-b-D-Galp1/ there are indications also that some of /3)-a-L-Araf-(1/ residues are linked to 6-b-Galp through 1,6-O-glycosidic bonds. Therefore, on the basis of all the information from the 1H and 13C NMR, HMQC, HMBC, COSY, and TOCSY spectroscopy, a complete assignment of all linkage patterns can be identified as shown in Table 2. 4. Conclusions We, therefore, propose a revised molecular structure for A. senegal (Fig. 10). The backbone is composed of 1,3-linked galactopyransyl (Galp) residues as traditionally described (Anderson, Hirst, & Stoddart, 1966; Cui et al., 2007; Defaye & Wong, 1986; Street & Anderson, 1983; Wang et al., 2008). Now we report that the backbone is substituted at O-2, O-6 or O-4 position, with residues of /2,3,6-b-D-Galp1/, /3,4-Galp1/, /3,4,6-Galp1/ and the substitutions at O-2 and O-4 position which has not previously been described (Anderson et al., 1966; Cui et al., 2007; Defaye & Wong, 1986; Street & Anderson, 1983; Wang et al., 2008). Acknowledgements We are thankful to John Nikiforuk and Dr. Barbara Blackwell of Eastern Cereal and Oilseed Research Centre, AAFC, for providing the NMR analysis. The partial financial support for this study by the Key Program of National Natural Science Foundation of China (No: 31130041), and National Key Technology R & D Program of China (2012BAD33B06), and Training Project of Young Scientists of Jiangxi Province (Stars of Jing gang), is gratefully acknowledged. We also thank Phillips Hydrocolloids Research Ltd for their support. References Al-Assaf, S., Phillips, G. O., & Williams, P. A. (2005a). Studies on Acacia exudate gums. Part I: the molecular weight of Acacia senegal gum exudate. Food Hydrocolloids, 19(4), 647e660. Al-Assaf, S., Phillips, G. O., & Williams, P. A. (2005b). Studies on Acacia exudate gums: part II. Molecular weight comparison of the Vulgares and Gummiferae series of Acacia gums. Food Hydrocolloids, 19(4), 661e667. Anderson, D. M., Hirst, E., & Stoddart, J. F. (1966). Studies on uronic acid materials.17. Some structural features of Acacia senegal gum (gum arabic). Journal of the Chemical Society C: Organic, 21, 1959e1966. Anderson, D. M. W., & Stoddart, J. F. (1966). Studies on uronic acid materialsdpart XV: the use of molecular sieve chromatography in studies on Acacia senegal gum (gum arabic). Carbohydrate Research, 2, 104e114. Capek, P., Matulova, M., Navarini, L., & Suggi-Liverani, F. (2010). Structural features of an arabinogalactan-protein isolated from instant coffee powder of Coffea arabica beans. Carbohydrate Polymers, 80(1), 180e185. Cardoso, S. M., Ferreira, J. A., Mafra, I., Silva, A. M. S., & Coimbra, M. A. (2007). Structural ripening-related changes of the arabinan-rich pectic polysaccharides from olive pulp cell walls. Journal of Agricultural and Food Chemistry, 55(17), 7124e7130. Carpita, N. C., & Shea, E. M. (1989). Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates. In Analysis of carbohydrates by GLC and MS. Boca Raton: CRC Press. Chandra, K., Ghosh, K., Ojha, A. K., & Islam, S. S. (2009). Chemical analysis of a polysaccharide of unripe (green) tomato (Lycopersicon esculentum). Carbohydrate Research, 344(16), 2188e2194. Churms, S. C., Merrifield, E. H., & Stephen, A. M. (1983). Some new aspects of the molecular structure of Acacia senegal gum (gum arabic). Carbohydrate Research, 123, 267e279. Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the permethylation of carbohydrates. Carbohydrate Research, 131(2), 209e217.
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