Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var. azul

Carbohydrate Research 381 (2013) 64–73

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Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var. azul Werner Praznik a,b,⇑, Renate Löppert a,b, Josè M. Cruz Rubio c, Klaus Zangger d, Anton Huber e a

Department of Chemistry, BOKU, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstr. 14, 1090 Vienna, Austria c Nekutli S.A. de C.V., Libramiento Cuquio-Yahualica km 33 S/N, Cuquio, Jalisco, México C.P. 45480, Mexico d Organic and Bioorganic Chemistry, Institute of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria e NAWI Graz, Central Polymer Laboratory/Molecular Characteristics (CePoL/MC), Institute of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria b

a r t i c l e

i n f o

Article history: Received 18 June 2013 Received in revised form 26 August 2013 Accepted 27 August 2013 Available online 6 September 2013 Keywords: Agave tequilana Fructo-oligosaccharides 2D-liquid chromatography Structure analysis Metabolic regulation

a b s t r a c t Fructo-oligosaccharides (FOSs) of a six year old agave plant variety, Agave tequilana, were isolated and fractionated by 2D preparative chromatography (SEC and rpHPLC). Structural analyses of different FOSfractions were performed by reductive methylation analysis connected to GC/FID identification and NMR-analysis. FOSs from leaves (d.p. 3–8) contain single a-D-Glcp residues as well in terminal as internal position, however (2 ? 1)-linked b-D-Fruf residues only. FOSs from stem, however, contain as well (2 ? 1)- and (2 ? 6)-linked b-D-Fruf residues with branched oligomeric repeating units. These characteristics indicate an enzymatically catalyzed metabolic regulation for the biosynthesis and transformation of fructans in A. tequilana which strongly depends on location and transport activities. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Agave tequilana is an important cultivar of Mexican agriculture.1 Traditionally, the stems of A. tequilana are applied in the tequila production; recently, they have been used in the production of highly pure fructans and fructo-oligosaccharides (FOSs) for novel healthy food products.2,3 A. tequilana belongs to the monocotyledons with a Crassulacean Acid Metabolism (CAM) without water transpiration from leaves during the day, optimized for hot climate regions.4 The preferentially accumulated reserve carbohydrates of A. tequilana during the growing period of two to seven years before flowering are fructans in the stem.5 However, for A. deserti it has been shown that synthesis, accumulation, and transportation of FOSs can take place already in vascular tissues and phloem cells of mature leaves.6 Recent information about the composition of agave fructans is available for materials isolated from stems of different agave species and different developing states of agave plants. Fructan preparations from these plants contain a mix of molecules in the range of degrees of polymerization (d.ps.) between 3 and 30,

⇑ Corresponding author. Fax: +48 1 4277 9554. E-mail address: [email protected] (W. Praznik). 0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2013.08.025

almost each with a single a-D-Glcp residue in terminal or internal position due to the initial start-molecule sucrose, and of either strictly (2?1)- or (2?6)-linked fructosyl-residues or of branched mixed-type [(2?1, 2?6)]–linked b-D-Fruf residues.5,7–9 For detailed structural analysis the FOSs from A. tequilana fructans were isolated from a six year old plant as from leaves as from stem for investigations on preparative 2D chromatography (size exclusion chromatography—SEC and reversed phase high performance liquid chromatography—rpHPLC), TLC, reductive methylation analysis combined with GC/FID, and NMR characterization. Analysis of midrange and high d.p. FOSs was performed with particular focus on identification of possible oligomeric repeating units and with respect to specific enzyme activities in the metabolic regulation system managing the formation, transport, and storage of FOSs. 2. Results and discussion Raw material for the investigations was provided by an A. tequilana plantation in the highlands of Mexico, county Jalisco. FOSs were isolated from leaf and stem of a representative single six year old agave plant before flowering. To obtain information on possible transformation of initially formed FOSs during the transport from leaves to the stem samples were taken at distinct locations along the leaf down to the stem.

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65

2.1. Fructo-oligosaccharides from leaves of A. tequilana Due to previous experiences with several fructan crops, FOSs from leaves of A. tequilana were isolated by water extraction at ambient temperature from the inner white part of freeze-dried material. FOS-fractions of different d.ps., sucrose (L8), a mix of glucose, fructose, non-identified carbohydrates, and salts (L9) were obtained on a preparative Biogel P4/P2 SEC system with bi-distilled water as eluent (Fig. 1). Semi-preparative rpHPLC (Fig. 2) of SEC fractions L3–L7 provided the variety of constituting FOSs for subsequent structural analyses by means of reductive methylation analysis with GC/FID identification and NMR. Quantity of each residue and ratio of b-D-Fruf- and a-D-Glcp-residues in the molecules were computed as the molar percentages from response-factor corrected GC/FID peak areas; d.ps. were computed from the ratio of b-D-Fruf- and a-D-Glcp-residues assuming one a-DGlcp residue for each molecule. Results are summarized in Table 1 and include rpHPLC fractions L7_1 and L7_2, identified as d.p. 3 oligomers 1-kestose [a-DGlcp-(1M2)-b-D-Fruf –(1 2)-b-D-Fruf] and neokestose [b-D-Fruf–(2?6)-a-D-Glcp-(1M2)-b-D-Fruf]. rpHPLC fractions L6_1, L6_2, and L6_3 represent the d.p. 4 oligomers nystose [a-D-Glcp-(1M2)-[b-D-Fruf-(1 2)]2 -b-D-Fruf] and two kinds of neo-nystoses with structure 1 (a-D-Glcp in position two) and structure 2 (a-D-Glcp in position three). rpHPLC fractions of SEC fraction L5 primarily consisted of d.p. 5 FOSs: L5_1a predominately molecules with terminal a-D-Glcp, L5_1b predominately 6-linked a-D-Glcp residues in the neo-type position (structure 3 and 4); L5_2 was identified as 6-linked a-D-Glcp residue in a neo-type conformation with a very likely arrangement according structure 5. L5_3, another neo-type FOS was not analyzed in detail. SEC-fraction L4 predominately contained d.p. 5 FOSs (L4_1, L4_2 and L4_3) and d.p. 6 FOSs (L4_4: a terminal a-D-Glcp residue [a-D-Glcp-(1M2)-[b-D-Fruf–(1 2)]4 - b-D-Fruf ], L4_5: a neo-type FOS with suggested structures 6, 7). Verified by TLC-analysis, SEC-fractions L3 and L2 predominately consisted of d.p. 7 and 8 FOSs whereas L3_4 was identified as a neo-type molecule (structure 8). The exclusively found (2?1)-linked b-D-Fruf residues are confirmed by 1H and 13C NMR-analysis (700 MHz) of SEC fractions L4–L7 indicating high purity and homogeneity of the samples. Two varieties of FOS with identical ratios of 1:2 for a-D-Glcp- and b-D-Fruf- residues in a ratio of 1:4 were identified

Figure 1. SEC-profile for fructo-oligosaccharides (FOSs) isolated from the middle region of leaf of A. tequilana with fractions L1–L9 obtained on Biogel P4/P2 with bidistilled water as eluent.

Figure 2. rpHPLC-profiles of preparative SEC-fractions L7, L6, L5, and L4 (Fig. 1) obtained by isocratic elution on a sequence of two Nucleosil 300-7 C18 columns with 2.2% aqueous MeOH providing a variety of components (fractions with numbers were identified), in particular: (a) L7_1, 2; (b) L6 _1, 2, 3; (c) L5_1a, 1b, 2, 3; (d) L4_1, 2, 3, 4, 5.

in the L7-fraction by the 13C-spectrum. All of these results are in good agreement with results from HPLC and reductive methylation analysis for 1-kestose and neokestose. The shifts for 1-kestose are in good agreement with results for pure 1-kestose (Fig. 7a). The 13C NMR spectrum of SEC-fraction L6 with FOSs of d.p. 4 illustrated in Fig. 3a rather well represents the spectra of FOSs with higher d.p. possessing several signals of a-D-Glcp- and b-D-Fruf-residues; no (2?6) glycosidic linkages could be identified through the lack of significant cross peaks between C6 (H-F6) and C2 (C-F2) of fructosyl residues in the Heteronuclear Multiple Bond Correlation (HMBC) spectrum (Fig. 3b). Contrary to these results the [(2?6)linked b-D-Fruf] fructan from Pucinellia peisonis shows strong correlations between C6 (H-F6) and C2 (C-F2) of fructosyl residues in the HMBC spectrum.10 2.2. Fructo-oligosaccharides from stem of A. tequilana Molar mass analysis of water-extracted fructans from the stem of this six year old A. tequilana plant resulted as well for samples from the center as for the intersection connecting stem and leaves broad d.p.-distributions ranging from 3 to 70 with a mean value of d.p. 30.10 For samples from both locations only approx. 15% of the molar mass distributions was smaller than d.p. 20. As the supposed screening for structural changes in the metabolic transport of FOSs from leaves to the stem requires reasonable quantities, an appropriate procedure to obtain enough material was applied: accumulation of stable methanol extracted FOSs and fructans with d.p. up to 25. Minor low-molecular phenolic compounds due to MeOHextraction did not interfere with subsequent analyses which were performed analogous to those for water-extracted FOSs: preparative Biogel P4/P2 SEC with bi-distilled water as eluent (Fig. 4) followed by semi-preparative rpHPLC fractionations (Figs. 5 and 6). Obtained SEC fractograms indicated at least 14 FOS-fractions (S1–14) with different d.ps. and decreasing resolution for increasing d.ps. (Fig. 4). Carbohydrates of SEC fractions S12, S13, and S14 were identified by TLC as reducing fructobiose, sucrose and a mix of glucose and fructose, respectively. Fractions 13 and 14 additionally contained a high level of unidentified materials, including low molecular phenolic compounds.

Fraction

b-D-Fruf

a-D-Glcp

L 7_1

Terminal 1-Linked Terminal 6-Linked

d.p.

L 7_2

L 6_1

66

Table 1 Percentage (%) and number (n) per molecule of b-D-Fruf and a-D-Glcp residues, d.p. and resulting structure of FOS in the rpHPLC fractions of SEC fractions L3–L7 from leaf of A. tequilana obtained by reductive methylation analysis L 6_2

%

n

%

n

%

n

%

n

32 ± 1.2 37 ± 1.1 28 ± 1.2 3 ± 0.4 3.2

1.0 ± 0.02 1.2 ± 0.03 0.9 ± 0.02 0.1 ± 0.01

68 ± 1.2

2.2 ± 0.03

1.0 ± 0.02 2.2 ± 0.02 1.0 ± 0.01

1.0 ± 0.02

47 ± 1.3 28 ± 0.9 1 ± 0.4 25 ± 1.3 3.8

2. 0 ± 0.05 1.0 ± 0.05

32 ± 1.3 3.1

24 ± 0.8 52 ± 1.1 23 ± 0.6 1 ± 0.2 4.2

1. 0 ± 0.02

HO

OH

O

OH

O

HO OH

OH O

Structure

1-Kestose

Neo-kestose

1-Nystose

HO

O HO O

O

HO

OH OH

HO

O

OH

HO

neonystose A(1) L 6_3

a-D-Glcp d.p.

Terminal 1-Linked Terminal 6-Linked

n 2.0 ± 0.05 1.0 ± 0.05 1.0 ± 0.02

HO

L 5_1b

% 27 ± 1.5 53 ± 0.8 15 ± 0.5 6 ± 0.8 4.8

n 1.3 ± 0.03 2.6 ± 0.05 0.7 ± 0.03 0.3 ± 0,04 HO

OH

O

1.0 ± 0.02

O HO

HO

OH

O

OH

O

O

OH

O

HO

O

OH

O

OH

a-D-Glcp d.p.

Terminal 1-Linked Terminal 6-Linked

HO

HO

n 1.1 ± 0.05 3.8 ± 0.10 1.0 ± 0.04

HO

neokestopentaose A(4) n 1.9 ± 0.10 3.3 ± 0.05

16 ± 0.8 6.2

1.0 ± 0.03

HO

HO

HO

HO HO

O

OH

HO O

O

OH OH

OH

3

HO HO

HO

kestohexaose (6)

OH

O OH

O

O O

HO O

OH OH

HO OH

O

HO

OH OH O

HO

O

OH

O

OH

O

OH

HO

OH

O OH

O

OH

O

OH OH

Structure

n 2.0 ± 0.05 3.7 ± 0.06 0.1 ± 0.03 0.9 ± 0.04

OH

O O

% 30 ± 1.9 55 ± 2.1 1 ± 0.4 14 ± 1.2 6.7

OH

O

OH

HO

neokestopentaose B(5) L 3_4

% 31 ± 2.0 53 ± 2.1

OH

O

OH OH

O

OH

L 4_5

% 19 ± 1.7 64 ± 1.9 16 ± 1.0 1 ± 0.4 5.9

HO O

O

HO

O

HO

L 4_4 b-D-Fruf

HO

OH OH

O

kestopentaose (3)

OH O

HO

HO O

OH

O

OH

O

HO

OH

OH

HO O

OH

HO O

O

HO

OH

HO

neonystose B (2)

OH

O

HO

2

OH

HO

HO OH

HO O

O

HO

OH

OH

O

OH

O OH

HO

HO

O

OH

O

1.0 ± 0.02

OH

OH

HO

n 2.1 ± 0.05 2.2 ± 0.05

O

OH

Structure

% 41 ± 2.0 41 ± 1.5 1 ± 0.5 18 ± 1.4 5.3

OH

O

HO

OH

OH

O

n 1.9 ± 0.04 2.1 ± 0.05

OH

O

OH O HO

L 5_2

% 38 ± 2.0 42 ± 1.5 1 ± 0.2 19 ± 1.1 5.0

2

HO HO

O O

HO

neokestohexaose (7)

HO O

OH OH

HO OH

O

O HO

neokestoheptaose (8)

3

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

b-D-Fruf

L 5_1a

% 49 ± 1.2 27 ± 1.3 1 ± 0.3 24 ± 1.0 4.0

67

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

Figure 3. NMR analysis of preparative SEC-fraction L6 (Fig. 1)—FOSs of d.p. 4 (1:1-ratio of nystose and neo-nystose): (a) Correlation (HMBC) spectrum (13C, 1H).

13

C NMR; (b) Heteronuclear Multiple Bond

Figure 4. SEC-profile (fractionation on Biogel P4/P2) of FOSs isolated from the stem close to the leaf; eluent: bi-distilled water; S1–S14 indicate the SEC-fractions for subsequent semi-preparative HPLC.

Figure 5. rpHPLC-profile of preparative SEC fractions S11–S9 (Fig. 4) from the stem close to the leaf on two columns Nucleosil 300-7 C18 with bi-distilled water isocratically eluted delivered a variety of identified components, in particular: (a) S11_2,5,7; (b) S10_2a-b, 3a-b, 5a-b, 6, 7a-b, 8a-b; (c) S9_2, 3, 4, 8, 9a-b, 14.

The rather small quantities of FOSs in the fractions S10 and S11 were identified by rpHPLC fractionation and subsequent reductive methylation analysis: S11 contained the trisaccharides 6-kestose [a-D-Glcp-(1M2)-b-D-Fruf -(2?6)-b-D-Fruf ] (S11_2), 1-kestose (S11_5), and neokestose (S11_7) (Table 2); S10 contained a mix of structurally different d.p.4 FOSs, indicated by double-peaks

(a,b). This split is in particular obvious for S10_2a,b and S10_3a,b with (2?6)- linked b-D-Fruf residues and a-D-Glcp residues with suggested structures 9, 10, and 11; S10_5a,b contain terminal and (2?1)- linked b-D-Fruf residues in a ratio of 1:3 - a fructotetraose with a terminal reducing hemiacetal group according to structure 12. This structure was identified by analogous alditols

68

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

as obtained from reducing FOSs isolated from a commercial sample of Raftilose. S10_6 was identified as 1-nystose, S10_7a,b and S10_8a,b with similar molar ratio of b-D-Fruf and a-D-Glcp residues were found to be neo-nystoses according to structures 1 and 2 (Table 1). The rpHPLC profile of the SEC-fraction S9 contained a variety of components: S9_2 was identified as a tetrasaccharide with a terminal a-D-Glcp residue and b-D-Fruf residues linked in (2?1), (2?6) and in terminal positions (structure 13 and 14). S9_3 represents a branched penta-saccharide containing a starter a-D-Glcp residue, a branched and a (2?6)-linked b-D-Fruf residue and two b-D-Fruf residues in terminal positions (structure 15). S9_4 is similar to S9_3 with an additional b-D-Fruf residue linked in 1-position (structure 16); the 6-position of b-D Fruf residues in the side- or in the main-chain (S9_3 and S9_4), however, could not be assigned. S9_8 was identified as nystose, S9_9a, b as a neonystose type and S9_14 as a neo-type of (2?1)- linked FOS with 5 fructosyl-residues according to structure 7 (Table 1). The rpHPLC profiles of the SEC-fractions S5–S8 obtained by elution with 2.5% aqueous MeOH contained a wide variety of FOSs (Fig. 6), however, the resolution was too low to pool materials for reliable structural analyses of individual FOS-fractions. Table 3 lists the average molar percentages, the number of a-DGlcp and b-D-Fruf residues and the resulting mean d.p. for the compounds in SEC-fractions S1–S11. Reductive methylation analysis of a pool of S7_5, 6,7 proved higher quantities of a-D-Glcp residues in terminal and b-D-Fruf residues in 1- substituted positions compared to the pool of S7_8, 9, 10,11. However, the ratio of 6- and 1,6- substituted b-D-Fruf residues were in the same range for both pools. Distinct from fraction S7, a pool of fractions S6_5, 6, 7, and 8 was rather similar to the full mix of S6. Nevertheless, there is good reason that even these fractions contain a mix of different structures, which, however, could not be resolved. For an estimation of oligomeric repeating units the ratio of various residues in different fractions was screened. As a result, the ratio of a-D-Glcp residues in linked and terminal position shifted toward neo-type series with increasing d.p. - 7:3 for fractions S7–S4(d.p. 7-10), 8:2 for the fractions S2 (d.p.20) and S1(d.p. 25); the ratio of 1:1 for 6- and 1,6-substituted b-D-Fruf residues, however, was rather constant and 1-substituted b-D-Fruf residues were found in approx. double the quantity than the 6-substituted b-DFruf residues (Table 3). These ratios strongly indicate oligomeric repeating units in the investigated FOSs. NMR-analysis proves these indications for the high-d.p. stemderived FOSs with rather inhomogeneous signals in the 13C NMR spectrum of SEC fraction S1 (d.p.25, Fig. 7b). Assignment of the carbon signals was achieved by comparison with the NMR spectra of pure 1-kestose (Fig. 7a). A simple explanation for the large number of carbon signals is the wide spectrum of chemical environment for the individual b-D-Fruf residues which even are linked by various glycosidic linkages. For the carbon C-5 of b-D-Fruf residues, for instance, chemical shifts of ?6)-Fruf-(2?, ?1)-Fruf -(2?, Fruf -(2? and ?1,6)- Fruf-(2? could be present. Additionally, the carbon signals of the a-D-Glcp residues are rather weak indicating the high d.p. of these compounds. Obtained spectra in general are in good agreement with reported NMR data for sinistrin11 and garlic-extracted fructans.12,13 2.3. Composition and enzymatic regulation of FOSs in A. tequilana FOSs isolated from mature leaves of A. tequilana contained b-DFruf residues in terminal and (2?1)-linked configuration and an either terminal or 6-linked a-D-Glcp residue. FOSs with d.p. 3-7 were identified as inulin type fructans with terminal a-D-Glcp

Figure 6. rpHPLC-profile of preparative SEC fraction S8–S5 (Fig. 4) on two columns Nucleosil 300-7 C18, eluent: aqueous 2.5% MeOH (isocratically eluted); apolar peak (ap) downscaled in all fractograms; in particular: (a) S8; (b) S7with identified pools of fractions 5,6,7 and 8,9,10,11; (c) S6 with identified pool 5,6,7,8; (d) S5.

and as neo series FOSs with fructosyl-linked a-D-Glcp residue on both terminals. Based on these results there is good reason for the assumption that only (2?1) linked inulin-type and neo-series type FOSs are synthesized by fructosyl-transferases in leaves of A. tequilana. According to reports on the biosynthesis in A. deserti accumulation of FOS takes place in the vascular tissues of leaves followed by subsequent transport within the phloem to the stem.6 Obtained results for A. tequilana are in agreement with this report, however, only rather low concentrations of sucrose could be detected as well in the center as in the bottom of leaves and the maximum level was found to be d.p.4 FOSs. The predominant transport-sugars in mature leaves of A. tequilana were FOSs in the range of d.p. 3–6, whereas a high level of sucrose and neokestose, 1-kestose, nystose, and an unidentified pentafructan are reported for vascular tissues and in phloem sap of mature leaves of A. deserti.6 The FOSs isolated from the stem of A. tequilana were identified as a mix of various structures with a-D-Glcp residue in terminal and 6-linked positions, b-D-Fruf residues in (2?1), (2?6) linked, terminal and branching positions. Several structures of FOSs in the range of d.p. 3–5 were identical with those in the leaves. By means of rpHPLC fractionation and TLC analysis three stemderived FOSs of d.p. 3 were identified as a mix of 6-kestose, 1-kestose, and neokestose. Additionally, d.p. 4 FOSs with as well strictly (2?6)-linked b-D-Fruf residues as with molecules including both, (2?1) and (2?6) linkages of b-D-Fruf residues without branching (structure 13 and 14, Table 2) and molecules lacking a a-D-Glcp residue but with a terminal reducing hemiacetal fructosyl group (fructo-tetraose) were found. Obviously the metabolic process rather soon forms and transforms FOSs with and without a-D-Glcp residues. Furthermore, d.p. 5 and 6 FOSs were identified as inulin-type compounds including neo series, as molecules with primarily (2?6)-linked b-D-Fruf and branching residues and as molecules with (2?6)- and (2?1)-linked b-D-Fruf and branching residues (structure 15 and 16, Table 2). The expected smallest branched molecule, however, bifurcose with a b-D-Fruf residue linked in both 1- and 6-positions, could not be identified. Obtained structures for d.p. 3, 4, and 5 FOSs are in good accordance for most compounds with recently published data for A. tequilana achieved by HPAEC-PAD (Dionex) analysis referring to pure standards, leaving, however, branched and non-branched higher oligomers due to the lack of standard compounds.5

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

Figure 7.

69

13

C NMR-analysis of preparative SEC fraction S1 (Fig. 4): (a) reference spectrum of pure 1-kestose; (b) spectrum of S1 (d.p. 25).

Convergence of structural variety of stem-derived d.p. 6-9 FOSs of A. tequilana is a good indicator for repeating units in FOS of higher d.ps. (structure 17, Table 3), a fact which becomes particularly evident for FOSs with strictly (2?1)-linked b-D-Fruf residues in the main chain, for branching b-D Fruf residues and (2?6)-linked b-D-Fruf residues in the side chains. a-D Glcp residues are predominantly found in the middle section of these FOSs indicating neo series structure. Recently reported structures of the fructan from stem of mature A. tequilana5,8 support the presence of oligomeric repeating units in high d.p. fructan as also in fructan from red squill–sinistrin (U. maritime),11 and in fructan isolated from garlic (A. sativum).12,13 The results for identified FOSs from leaf and stem of A. tequilana strongly support the assumption of two sets of enzymatic systems: An enzymatic pattern of fructosyl-transferases generates inulintpye and neo-series FOS up to d.p. 8–9 in the leaves; these compounds then are transported within the phloem to the storage cells in the stem, where they were transformed into higher FOSs by a second set of fructosyl-transferases capable to form branches and (2?6)-linked b-D-Fruf residues in side chains and performing oligomeric repeating units. The same set of enzyme systems for fructan biosynthesis in stem of A. tequilana was proposed recently by Mellado-Mojica and López,5 however, assuming solely sucrose as transport sugar from leaves to stem and assuming the structural variety of identified FOSs as a result of aging of the plant. Different to this approach, the FOSs found in leaf and stem of a mature agave A. tequilana indicate an early, most probably immediate start of formation of different structures after initial carbohydrate synthesis. The development of structurally different FOSs rather depends on biosynthesis at different locations, transport activities, and general environmental impacts than representing an aging phenomenon. Details about the regulation of fructan metabolism in the A. tequilana are under investigation.

3. Experimental 3.1. Plant material Samples were taken from a single six year old plant, a cultivar of Agave tequilana from a plantation in the highlands of Mexico near Tequila grown on red clay ground, with sun light all day, and appropriate local agricultural treatment including temporary ground irrigation and manual and mechanical weed control every six months. The samples from one leaf (middle and bottom region) and stem (close to the bottom of leaf and from the center) were cut in small pieces, freeze dried, and stored at 6 °C for subsequent analyses immediately after harvesting. 3.2. Extraction of FOSs from leaf According to previous experiences with various fructan crops, 2.5 g ground freeze dried white inner core leaf-material got dispersed in 30 mL of distilled water with NaN3-addition (0.005%) at room temperature to avoid hydrolysis of glycosidic linkages by microbial affection. After stirring for 5 h the dispersion got centrifuged at 3800 rpm for 30 min, and, after washing the solid residue twice with 20 mL of water the pooled supernatants of several attempts containing the extracted FOSs were pooled, freeze dried, and stored at dry conditions. 3.2.1. Extraction of FOSs from stem 10 g of ground white freeze dried pieces of stem-material was suspended in 80 mL MeOH at room temperature and stirred for 14 h; subsequently, the FOSs-containing MeOH was sucked off and the residue got washed twice with 20 mL of MeOH. MeOH got evaporated from stable FOS/MeOH-solutions at elevated

Fraction

b-D-Fruf

a-D-Glcp

S11_2

Terminal 1-Linked 6-Linked 1,6-Linked Terminal 6-linked

d.p. Structure

b-D-Fruf

d.p.

S11_7

%

n

%

n

%

n

33 ± 0.3

1.0 ± 0.05

33 ± 0.4 35 ± 1.3

1.0 ± 0.02 1.1 ± 0.04

66 ± 0.7

1.9 ± 0.03

32 ± 1.2

0.9 ± 0.05

31 ± 1.1 4 ± 0.5 2.9

0.9 ± 0.05 0.1 ± 0.02

29 ± 0.8 3 ± 1.2 3.1

0.9 ± 0.02 0.1 ± 0.01

1 ± 0.02 34 ± 1.2 2.9

1.0 ± 0.02

6-Kestose

1-Kestose

Neo-kestose

S10_2a,b

S10_3a,b

S10_5a,b

% 26 ± 0.6 3 ± 0.2 43 ± 0.8 4 ± 0.3 23 ± 0.7 1 ± 0.05 4.2

n 1.1 ± 0.03 0.1 ± 0.01 1.8 ± 002 0.1 ± 0.01 0.9 ± 0.02 0.1 ± 0.01

HO

% 52 ± 1.0 7 ± 0.5 17 ± 1.3

n 2.2 ± 0.06 0.3 ± 0.02 0.7 ± 0.02

5 ± 0.4 19 ± 0.8 4.3

0.2 ± 0.01 0.8 ± 0.02

OH

O

HO

HO

OH O

O O

HO

OH

O HO

O

OH

O

HO

HO

OH

OH

HO

OH

HO

OH

O

OH O

HO

OH

O

OH

O

HO

HO HO

HO OH

O

HO

OH

O

n 1.0 ± 0.04 2.9 ± 0.06 0.1 ± 0.01 0.1 ± 0.01 0.1 ± 0.01 0 .1 ± 0.01

OH

O

OH O

HO

% 23 ± 1.1 67 ± 1.5 2 ± 0.2 3 ± 0.2 2 ± 0.2 3 ± 0.2 4.3

O

OH

O

O HO

HO

HO

or

6-nystose (9)

2

OH

O

HO

OH

O OH

HO HO

Structure

OH HO

O

O

HO

reducing fructotetraose (12)

OH O HO O

HO

HO

OH

O OH

HO OH

O HO

neo-6-nystoses A, B (10, 11) S10_6 b-D-Fruf

a-D-Glcp

terminal 1-linked 6-linked 1,6-linked terminal 6-linked

d.p. Structure

b-D-Fruf

Terminal 1-Linked 6-Linked 1,6-Linked

S10_7a,b

% 28 ± 1.2 43 ± 2.0 5 ± 0.5

n 1.1 ± 0.05 1.7 ± 0.04 0.2 ± 0.01

23 ± 0.6 3 ± 0.3 4.0

0.9 ± 0.02 0.1 ± 0.01

% 45 ± 1.0 21 ± 0.7 7 ± 0.2 3 ± 0.2 4 ± 0.1 21 ± 0.5 4.2

S10_8a,b n 1.9 ± 0.05 0.9 ± 0.04 0.3 ± 0.01 0.1 ± 0.01 0.1 ± 0.01 0.9 ± 0.01

% 41 ± 1.3 23 ± 1.1 10 ± 0.7 5 ± 0.5 3 ± 0.4 19 ± 0.8 4.4

1-Nystose

Neonystose

Neonystose

S9_2

S9_3

S9_4

% 26 ± 1.1 22 ± 1.0 28 ± 1.3

n 1.1 ± 0.05 0.9 ± 0.02 1.1 ± 0.02

% 42 ± 1.5 2 ± 0.2 18 ± 1.2 19 ± 1.3

n 2.1 ± 0.08 0.1 ± 0.02 0.9 ± 0.02 1.0 ± 0.02

% 34 ± 1.2 18 ± 1.3 16 ± 1.1 16 ± 1.3

n 1.8 ± 0.05 1.0 ± 0.03 0.4 ± 0.02 0.2 ± 0.01 0.1 ± 0.01 0.9 ± 0.02

n 2.1 ± 0.04 1.1 ± 0.04 1.0 ± 0.05 0.9 ± 0.03

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

a-D-Glcp

Terminal 1-Linked 6-Linked 1,6-Linked Terminal 6-linked

S11_5

70

Table 2 Percentage (%) and number (n) per molecule of b-D-Fruf and a-D-Glcp residues, d.p. and resulting structure of FOS in the rpHPLC fractions of SEC fractions S9, S10, and S11 from stem of A. tequilana obtained by reductive methylation analysis

Table 2 (continued) Fraction

a-D-Glcp

S11_2

Terminal 6-Linked

d.p.

S11_5

S11_7

%

n

%

n

%

n

25 ± 1.0

1.0 ± 0.01

20 ± 0.5

1.0 ± 0.02

15 ± 0.8 2 ± 0.2 6.1

0.9 ± 0.03 0.1 ± 0.01

4.1

5.1 HO

OH

O

HO

OH

HO

HO OH O

HO

OH

O

HO

OH O

HO

O

O

HO OH

HO OH

HO OH

HO

OH O

HO

HO

OH

OH

O HO

OH

OH

O

HO OH

HO

OH

O

O OH

O

O

O

HO

HO

HO

or HO

OH

O OH

HO

HO

O H

O OH

HO

O O

HO

HO

HO

OH O

O HO

O

Structure

OH

O

O O

OH

OH

O

OH

O OH

OH HO

O

O

O

O

kestopentaose, branched A (15)

HO

kestohexaose, branched B (16)

OH O

OH

HO

O

HO

OH

O OH

OH

O HO

1,6-nystose A, B (13, 14) S9_8 b-D-Fruf

a-D-Glcp

Terminal 1-Linked 6-Linked 1,6-Linked Terminal 6-linked

S9_9a,b

% 27 ± 1.8 47 ± 1.9

n 1.0 ± 0.03 1.8 ± 0.03

26 ± 1.0

1.0 ± 0.02

S9_14

% 44 ± 1.2 26 ± 1.1 7 ± 0.7

n 1.8 ± 0.05 1.1 ± 0.03 0.3 ± 0.02

% 35 ± 1.3 48 ± 1.0 2 ± 0.3

n 2.2 ± 0.05 3.1 ± 0.04 0.1 ± 0.01

1.0 ± 0.01

16 ± 1.1 6.4

1.0 ± 0.02

dp

3.8

24 ± 0.6 4.2

Structure

1-Nystose

Neonystose

Neokestohexaose

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

HO

71

72

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

Table 3 Percentage (%) and number (n) per molecule of b-D-Fruf and a-D-Glcp residues, d.p. in the SEC fractions S1-S11 and combined rpHPLC fractions of SEC fractions S6 and S7 from stem of A. tequilana obtained by reductive methylation analysis Fraction

b-D-Fruf

a-D-Glcp

S11

Terminal 1-Linked 6-Linked 1,6-Linked Terminal 6-Linked

d.p.

S10

a-D-Glcp

Terminal 1-Linked 6-Linked 1,6-Linked Terminal 6-linked

d.p.

%

n

%

n

%

n

37 ± 0.8 18 ± 1.2 12 ± 1.2 1 ± 0.2 19 ± 2.1 13 ± 1.5 3.2

1.2 ± 0.05 0.6 ± 0.03 0.4 ± 0.05

35 ± 1.4 22 ± 1.8 15 ± 1.2 6 ± 0.6 11 ± 12 11 ± 1.2 4.3

1.5 ± 0.04 0.9 ± 0.03 0.6 ± 002 0.3 ± 0.01 0.5 ± 0.02 0.5 ± 0.01

39 ± 1.6 24 ± 1.2 11 ± 0.7 7 ± 0.3 10 ± 0.5 9 ± 0.3 5.3

2.0 ± 0.03 1.3 ± 0.01 0.6 ± 002 0.4 ± 0.01 0.5 ± 0.02 0.5 ± 0.01

34 ± 1.2 29 ± 1.2 13 ± 0.7 8 ± 0.5 6 ± 0.7 10 ± 0.6 6.2

2.1 ± 0.05 1.8 ± 0.05 0.8 ± 002 0.5 ± 0.02 0.4 ± 0.03 0.6 ± 0.04

0.6 ± 0.04 0.4 ± 0.02

S6

% 35 ± 1.2 24 ± 1.5 14 ± 1.2 13 ± 1.3 4 ± 0.4 10 ± 0.8 7.2

n 2.5 ± 0.06 1.7 ± 0.03 1.0 ± 0.02 0.9 ± 0.02 0.3 ± 0.04 0.7 ± 0.03

% 34 ± 1.1 29 ± 1.5 13 ± 1.0 12 ± 0.7 4 ± 0.2 8 ± 0.3 8.3

S2

b-D-Fruf

a-D-Glcp

Terminal 1-Linked 6-Linked 1,6-Linked Terminal 6-Linked

d.p.

S8

n

S7 b-D-Fruf

S9

%

S5 n 2.8 ± 0.10 2.4 ± 0.08 1.1 ± 0.03 1.0 ± 0.03 0.3 ± 0.03 0.7 ± 0.03

S4

% 34 ± 1.2 29 ± 2.0 13 ± 0.5 13 ± 2.0 3 ± 0.6 8 ± 0.3 9.0

n 3.0 ± 0.05 2.6 ± 0.04 1.2 ± 0.05 1.2 ± 0.04 0.3 ± 0.04 0.7 ± 0.02

% 34 ± 1.0 29 ± 1.5 13 ± 0.8 14 ± 0.8 3 ± 0.2 7 ± 0.5 10.0

n 3.4 ± 0.05 2.9 ± 0.04 1.3 ± 0.01 1.4 ± 0.01 0.3 ± 0.01 0.7 ± 0.01

S1

%

n

%

n

28 ± 1.3 33 ± 1.4 17 ± 0.7 17 ± 1.2 1 ± 0.1 4 ± 0.4 20.0

5.6 ± 0.05 6.6 ± 0.06 3.4 ± 0.02 3.4 ± 0.04 0.2 ± 0.01 0.8 ± 0.03

26 ± 1.3 34 ± 1.5 18 ± 1.2 18 ± 1.4 1 ± 0.2 3 ± 0.3 25.0

6.5 ± 0.05 8.5 ± 0.05 4.5 ± 0.06 4.5 ± 0.07 0.2 ± 0.03 0.8 ± 0.03

O

HO

OH

HO

OH

HO O

O OH

O

OH

O

OH

O

O

HO

O OH

HO

O HO

O

OH OH

O HO

n

structure (17) repeating unit S7_5,6,7

b-D-Fruf

a-D-Glcp

Terminal 1-Linked 6-Linked 1,6-Linked Terminal 6-Linked

d.p.

S7_8,9,10,11

S6_5,6,7,8

%

n

%

n

%

n

22 ± 1.5 31 ± 1.2 17 ± 1.1 16 ± 1.3 9 ± 0.5 5 ± 0.5 7.1

1.6 ± 0.08 2.2 ± 0.02 1.2 ± 0.02 1.1 ± 0.02 0.6 ± 0.02 0.4 ± 0.03

39 ± 1.4 20 ± 1.3 13 ± 1.1 14 ± 1.5 1 ± 0.2 13 ± 0.4 7.1

2.8 ± 0.05 1.4 ± 0.04 0.9 ± 0.05 1.0 ± 0.03 0.1 ± 0.04 0.9 ± 0.02

31 ± 1.8 27 ± 1.9 15 ± 1.2 15 ± 1.3 4 ± 0.5 8 ± 0.3 8.3

2.6 ± 0.03 2.2 ± 0.03 1.2 ± 0.05 1.2 ± 0.03 0.3 ± 0.02 0.7 ± 0.02

temperatures of 50 °C in vacuum of 150 mbar to leave the extracted FOSs which then were dissolved in 20 mL water, freeze dried, and stored at dry conditions. 3.3. Preparative SEC Column system: Biogel P-4 (extra fine, <45 lm, MW range 800– 4000; column 89  2.5 cm) and Biogel P-2 (fine, 45–90 lm, MW range 100–1800; column 90  2.5 cm) in series (gel: Bio Rad Laboratories, Inc.); eluent: bi-distilled water; flow rate of elution: 30– 36 mL/h by peristaltic pump; mass detection: DRI-detector (Knauer, Co.); fraction collector and recorder (Healthcare, Co). Injected volume: 300 mg carbohydrate in 3 mL bi-distilled water (centrifuged at 13000 rpm, 10 min); elution volume: 900 mL; fraction volume: 8–9 mL; to obtain appropriate quantities for structural analyses the fractions of identical peak maxima from several runs were accumulated, freeze dried, and stored under dry conditions.

1.5 mL/min; mass-detection: RI-detector (Thermo Fisher, Co); HPLC pump (K 500, Knauer, Co.), pressure 7.4 bar; elution time: 140 min; injected volume: 3–4 mg carbohydrate/100 lL (sample loop); data acquisition and processing software package CODAwin32 and CPCwin32, a.h. group, Graz; manual sampling of fraction by visual elution control; fractions were freeze dried and stored under dry conditions. 3.5. TLC analysis SEC- and rp-HPLC fractions were analyzed on HPTLC Silica gel 60 (Merck, Germany) with oligosaccharide eluent system (1-butanol/1-propanol/EtOH/H2O - 2/3/3/2), two times developed and with monosaccharide eluent system (AcCN/H2O—17/3), three times developed and detected with thymol reagent. Commercial (Megazyme) and home-prepared plant-derived carbohydratestandards were applied as references.

3.4. Semi-preparative rpHPLC

3.6. Reductive methylation analysis (reductive cleavage method)

Column system: Two columns of Nucleosil 300-7 C18 (250  10 mm), Macherey-Nagel, Co., in series; eluent: water HPLC grade, 2.2% and 2.5% MeOH in water HPLC grade; flow rate:

Methylation, reductive cleavage, and acetylation were performed according to established procedures.14,15 Well defined [(2?1)-linked b-D-Fruf] linkage-types of inulin from chicory

W. Praznik et al. / Carbohydrate Research 381 (2013) 64–73

(Orafti,Co; house prepared samples) and of house-prepared [(2?6)-linked b-D-Fruf] fructan from Pucinellia peisonis were applied as standards. GC identification was performed on Shimadzu GC 2010 with a capillary column (DB-1701; 30 m, 0.25 mm i.d.: 0.25 lm film thickness; Agilent, Co.), He as carrier gas and FID (flame ionization detection) with temperature program: 80 ? 135 °C at 10 °C/min,135 ? 155 °C at 2 °C/min, 155 ? 200 °C at 3 °C/min, 200 ? 260 °C at 5 °C/min, injector temperature of 230 °C and detector temperature 270 °C. Measurements were performed with a split ratio of 1:5 in three duplicates. 3.7. NMR analysis The NMR- spectra were recorded on a Bruker Avance III 700 MHz. NMR spectrometer equipped with a 5 mm TCl cryoprobe at 298 K. 5–50 mg of each compound was dissolved in 600 ll of 50 mM KPi, pH 6.5 in 100% D2O (Eurisotop, Saarbrücken, Germany). The 1H dimension was referenced to the residual non-deuterated solvent peak. Acknowledgments We would like to thank the company of Nekutli S.A. de C.V., Mexico, for financial support of the work and for the provision of

73

agave samples. Further we are grateful to Frank M. Unger for critical review of the manuscript. References 1. Ruiz-Corral, J. A.; Pimienta-Barrios, E.; Zanudo-Herandes, J. Agrociencia 2002, 36, 41–53. 2. Santos-Zea, L.; Leal-Díaz, A. M.; Cortés-Ceballos, E.; Gutiérrez-Uribe, J. A. Curr. Bioact. Compd. 2012, 8, 218–231. 3. Valenzuela, A. GCB Bioenergy 2011, 3, 15–24. 4. Nobel, P.S. Desert Wisdom/Agaves and Cacti: CO2, Water, Climate Changes; ISBN: 978-1-4401-9151-0, iUniverse, Inc., Bloomington, USA, 2010, pp. 161. 5. Mellado-Mojica, E.; López, M. G. J. Agric. Food Chem. 2012, 60, 11704–11713. 6. Wang, N.; Nobel, P. S. Plant Physiol. 1998, 116, 709–714. 7. Lopez, M. G.; Mancilla-Margalli, N. A.; Mendoza-Diaz, G. J. Agric. Food Chem. 2003, 51, 7835–7840. 8. Mancilla-Margalli, N. A.; Lopez, M. G. J. Agric. Food Chem. 2006, 54, 7832–7839. 9. Arrizon, J.; Morel, S.; Gschaedler, A.; Monsan, P. Food Chem. 2010, 122, 123–130. 10. Article in preparation. 11. Spies, T.; Praznik, W.; Hofinger, A.; Altmann, F.; Nitsch, E.; Wutka, R. Carbohydr. Res. 1992, 235, 221–230. 12. Baumgartner, S.; Dax, T. G.; Praznik, W.; Falk, H. Carbohydr. Res. 2000, 328, 177– 183. 13. Ernst, M. K.; Chatterton, N. J.; Harrison, D. A.; Matitschka, G. J. Plant Physiol. 1998, 153, 53–60. 14. Praznik, W.; Löppert, R.; Huber, A. Analysis and molecular composition of fructans from different plant sources. In Recent Advances in Fructooligosaccharide Research; Shiomi, N., Benkeblia, N., Onodera, S., Eds.; India: Research Signpost, Kerala, 2007; pp 93–117. 15. Rolf, D. G.; Gray, R. Carbohydr. Res. 1984, 131, 17–28.