Sugar composition of the moss Rhodobryum ontariense (Kindb.) Kindb.

Article abstract of DOI:10.1080/14786419.2010.535163

Although the second biggest terrestrial group of plants, bryophytes remain poorly known chemically compared to the angiosperms. In this article, the sugars of the moss Rhodobryum ontariense, an unstudied representative of the medicinally known genus, are reported. The chemical analysis revealed the usual plant sugar sucrose, and a new sugar, fructooligosaccharide 1-kestose, which is reported first not only for the genus Rhodobryum, but also for mosses. The trisaccharides have been scantily reported in bryophytes hitherto. This gives more significance to this study for further investigation of its role in the moss species. The health-promoting effect of 1-kestose is also briefly discussed.

Full text of DOI:10.1080/14786419.2010.535163

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Sugar composition of the moss Rhodobryum
ontariense (Kindb.) Kindb.
Boris Pejin , Carmine Iodice , Giuseppina Tommonaro , Marko Sabovljevic ,
Armandodoriano Bianco , Vele Tesevic , Vlatka Vajs & Salvatore De Rosa
To cite this article: Boris Pejin , Carmine Iodice , Giuseppina Tommonaro , Marko Sabovljevic ,
Armandodoriano Bianco , Vele Tesevic , Vlatka Vajs & Salvatore De Rosa (2012) Sugar
composition of the moss Rhodobryum ontariense (Kindb.) Kindb., Natural Product Research,
26:3, 209-215, DOI: 10.1080/14786419.2010.535163
To link to this article: http://dx.doi.org/10.1080/14786419.2010.535163
Published online: 10 Aug 2011.
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Date: 10 November 2015, At: 05:03

Natural Product Research
Vol. 26, No. 3, February 2012, 209–215
Sugar composition of the moss Rhodobryum ontariense (Kindb.)
Kindb.
a
a
d
Boris Pejinabc , Carmine Iodice , Giuseppina Tommonaro , Marko Sabovljevic ,
*
Armandodoriano Biancob, Vele Tesevicc, Vlatka Vajse and Salvatore De Rosaa
b
aInstitute for Biomolecular Chemistry, ICB-CNR, Pozzuoli (Naples), Italy; Department of
Chemistry, Sapienza University of Rome, Rome, Italy; Department of Organic Chemistry,
c
Faculty of Chemistry, University of Belgrade, Serbia; dFaculty of Biology, Institute of Botany
and Garden, University of Belgrade, Serbia; Center of Chemistry, Institute of Chemistry,
Technology and Metallurgy, University of Belgrade, Serbia
e
(Received 3 September 2010; final version received 13 October 2010)
Although the second biggest terrestrial group of plants, bryophytes remain
poorly known chemically compared to the angiosperms. In this article, the
sugars of the moss Rhodobryum ontariense, an unstudied representative of
the medicinally known genus, are reported. The chemical analysis revealed
the usual plant sugar sucrose, and a new sugar, fructooligosaccharide 1-
kestose, which is reported first not only for the genus Rhodobryum, but also
for mosses. The trisaccharides have been scantily reported in bryophytes
hitherto. This gives more significance to this study for further investigation
of its role in the moss species. The health-promoting effect of 1-kestose is
also briefly discussed.
Keywords: mosses; Rhodobryum
fructooligosaccharides; 1-kestose
ontariense;
phytochemistry;
1. Introduction
Bryophyte chemistry is still poorly known compared to that of vascular plants
(Asakawa, 1995, 2007). To date, ca 5% of total bryophytes have been studied
chemically (A. Sabovljevic & M. Sabovljevic, 2008). Among bryophytes, liverworts
have received significant attention due to the presence of oil bodies with terpenoid
substances of high biological activities (Asakawa, 2008). Chemical properties of
mosses are studied very poorly. The reasons for the low amount of bryophyte
chemistry data are due to the problems of lack of availability of clean materials,
taxonomic inconstancy and the problem with taxa recognition.
However, since 1960 interest in the analysis of chemicals isolated from
bryophytes has increased. It has been confirmed that bryophytes possess a great
number of different chemical constituents. Carbohydrates are widely presented as
mono and polysaccharides (Chopra & Kumra, 1988).
*Corresponding author. Email: borispejin@yahoo.com
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2010.535163
http://www.tandfonline.com

210
B. Pejin et al.
Up till now, only two species from the genus Rhodobryum, namely, Rhodobryum
roseum and Rhodobryum giganteum, have been studied for their chemical and
bioactive components (Dai, P. Liu, C. Liu, Wang, & Chen, 2006; Hu et al., 2009; Lei
et al., 2001; Qiao, Ma, Lin, & Kong, 2004; Tan et al., 1983; Wang et al., 2006). For
saccharides, the isolation and identification of fructose from R. roseum have been
recently reported (Wang, P. Liu, & H. Liu, 2008). Data on Rhodobryum ontariense
chemistry are lacking.
Rhodobryum ontariense is a widely distributed moss (Dierssen, 2001). In Serbia,
R. ontariense is present in Deliblatska sands, in open steppic-woody dry areas, where
the yearly temperature amplitude goes up to 60ꢀC.
With the aim of studying the chemistry of R. ontariense, adequate screening of
this moss was performed.
2. Results and discussion
The n-butanol extract obtained from the water residue of methanol extract of
R. ontariense (Kindb.) Kindb., upon repeated reverse-phase chromatography
afforded two pure compounds, which were classified as oligosaccharides by means
of their spectroscopic data and typical chromatographic profile. The less polar
compound was obtained as a colourless solid with a m.p. of 185–187ꢀC on
decomposition. Its molecular formula was determined to be C12H22O11 by TOFMS
and NMR data. The 13C-NMR and DEPT spectra exhibited 12 carbon signals
(1 quaternary, 8 methine and 3 methylene). These data suggested that it was sucrose,
which was confirmed by comparison with an authentic sample.
The most polar compound was obtained as amorphous solid. Its molecular
formula was determined to be C18H32O16 from TOFMS and NMR data. The
13C-NMR and DEPT spectra exhibited 18 carbon signals (2 quaternary, 11 methine
and 5 methylene). These data suggested that it was a trisaccharide with one pyranose
and two furanose moieties, which was confirmed by the presence of only one anomeric
1
proton at ꢀ 5.41 (d, J ¼ 3.8 Hz) in its H-NMR spectrum. Its acid hydrolysis gave
D-glucose and D-fructose (ratio 1 : 2). A combination of 2-D NMR experiments
(COSY, TOCSY, HMQC and HMBC) allowed us to assign all signals in the 1H- and
13C-NMR spectra and to define the spin system of the glucose unit. The HMBC
showed the correlation between the anomeric proton at ꢀ 5.41 and the quaternary
carbon at ꢀ 104.9, which allowed for the connection between the glucose and a fructose
moiety, like sucrose. It was difficult to establish the connection of the second fructose
unit, because the chemical shifts of both quaternary carbons were very close. In order
to establish this connection, the isolated trisaccharide was acetylated. The correlation
between the anomeric proton of glucose unit (ꢀ 5.71) and the quaternary carbon at
ꢀ 104.3, observed in the HMBC experiment of the full acetylated derivative, identified
the C-2 of the fructose moiety linked to glucose. Furthermore, the difference of
chemical shifts of the quaternary carbon atoms (ꢀ 104.3 and 103.1), observed in the
13C-NMR spectrum of the full acetylated trisaccharide, suggested that the second
fructose was (2!1)-linked to fructose 1, because only this connection was in
accordance with all 13C chemical shifts (assigned by 2-D NMR), and the less high field
shift of C-20 due to only one ꢁ-acetylation shift. Thus, the structure of iso-
lated trisaccharide was elucidated as ꢁ-D-Fruf-(2!1)-ꢁ-D-Fruf-(2$1)-ꢂ-D-Glcp

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Figure 1. 1-Kestose.
(1-kestose) (Figure 1). The obtained NMR data for 1-kestose compare well with those
from literature (Calub, Waterhouse, & Chatterton, 1990).
The fructooligosaccharide 1-kestose is first recorded in the genus Rhodobryum
and mosses as a whole. Marschall, Proctor, and Smirnoff (1998) found this
trisaccharide in the liverwort Porella platyphylla (L.) Lindb.
In nature, about 15% of flowering plant species belonging to selected families of
both monocots and dicots synthesise fructooligosaccharides (FOS) from sucrose.
These FOS are generally shorter and have diverse structures (Banguela &
Hernandez, 2006).
´
1-Kestose is potentially attractive from an ecological point of view: there is strong
evidence that FOS protect the plant against drought and freezing (Hisano et al.,
2004), most likely by stabilising cell membranes (Hincha, Zuther, Hellewege, &
Heyer, 2002; Vereyken, Chupin, Demel, Smeekens, & De Kruijff, 2001).
In addition, FOS of short and medium sizes are prebiotics that are now in
increasing demand in the functional market. According to the concepts, functional
foods have an added health value above their nutritive properties (Roberfroid, 2000).
They are attractive because of their health-promoting effect. Although the simple
sugars fructose and glucose are quickly absorbed into the body by the intestines,
FOS are indigestible for the most part and therefore act as non-digestible fibres in the
diet. Actually, these sugars have a low caloric value and dietary fibre like properties
due to the fact that the ꢁ-fructosyl linkages cannot be hydrolysed by the digestive
enzyme in the upper part of the human gastrointestinal tract. The commercially
available FOS are the typical examples of prebiotics for bifidobacteria, the
predominant group of the colonic micro flora. Short-chain FOS are more convenient
substrates for the rapid growth of bifidobacteria (Van der Meulen, Avonts, & De

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B. Pejin et al.
Vuyst, 2004), whereas branched FOS are claimed to provide for a long-lasting source
of energy (Weyens et al., 2004).
Although FOS with low and medium degrees of polymerisation are important
primarily because of their functional properties, they have additional applications in
´
the food industry (Banguela & Hernandez, 2006). The trisaccharide 1-kestose has a
natural sweet taste, and in blend with other low calorie sweeteners, it can replace
sucrose in certain specific uses.
3. Experimental
3.1. General
Melting points were measured on a Kofler apparatus and are uncorrected. Optical
rotations were measured on a Jasco DIP 370 polarimeter, using a 10-cm microcell.
HRESI-MS spectra were recorded with Micromass Q-TOF Micro Mass
Spectrometer (Waters) in the electrospray-ionisation mode. 1H- and 13C-NMR
spectra were recorded at 400 and 100 MHz, respectively, on a Bruker Avance-400
spectrometer, using an inverse probe fitted with a gradient along the z-axis, using the
solvent signal as an internal standard. The 2-D NMR spectra were obtained using
Bruker’s microprograms. Si gel chromatography was performed using pre-coated
Merck F254 plates and Merck Kieselgel 60 powder. Reverse-phase chromatography
was performed using Lichroprep RP-18 (Merck) and Lobar C-18 column (Merck).
3.2. Plant material
The R. ontariense sample was collected from Deliblatska sands, NE Serbia in March
2007. A voucher specimen has been deposited in the Herbarium of the Institute of
Botany, University of Belgrade, Serbia (bryophyte collection BEOU4708).
3.3. Extraction and isolation
Before extraction, the moss was carefully inspected for contaminants: soil and other
plant materials were completely removed. The gametophyte tips were used for the
extraction. Air-dried parts of R. ontariense (12 g) were ground and extracted with
90% methanol at room temperature. The extract was filtered and concentrated under
reduced pressure to give 0.92 g of residue, which was dissolved in water and extracted
with petroleum ether, ethyl acetate and n-butanol. The crude n-butanol extract
(0.39 g) obtained after evaporation of the solvent in vacuo was fractioned by RP-18
flash chromatography, eluting with increased gradient of methanol in water.
Sugar-rich fractions were then purified on Lobar RP-18 columns (water/methanol
gradient) to yield 1-kestose (92 mg; 0.77% of dry weight of plant) and sucrose (62 mg;
0.57% of dry weight).
3.4. Sucrose
Colourless solid; m.p. 185–187ꢀC with decomposition; [ꢂ]D þ 66.5ꢀ (c 0.02, MeOH);
HRESIMS (positive) m/z: 365.1095 [M þ Na]þ, calculated for C12H22O11Na,
365.1072; NMR data were identical to those of an authentic sample.

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3.5. 1-Kestose
Amorphous solid, [ꢂ]D þ 13.6ꢀ (c 0.02, MeOH); 1H-NMR (400 MHz, CD3OD):
ꢀ 5.41 (1H, d, J ¼ 3.8 Hz, H-1), 4.17 (1H, d, J ¼ 8.5 Hz, H-30), 4.15 (1H, d, J ¼ 8.5 Hz,
H-300), 4.12 (1H, t, J ¼ 8.5 Hz, H-40), 4.10 (1H, t, J ¼ 8.5 Hz, H-400), 4.00 (2H, d,
J ¼ 10.8 Hz, H-10), 3.95 (1H, dd, J ¼ 9.7 and 4.7 Hz, H-5), 3.87–3.62
(11H, overlapped signals), 3.51 (1H, dd, J ¼ 9.7 and 3.8 Hz, H-2) and 3.46 (1H, t,
13
J ¼ 9.7 Hz, H-4); C-NMR (100.6 MHz, CD3OD): ꢀ 104.95 (s, C-200), 104.90
(s, C-20), 93.1 (d, C-1), 82.8 (d, C-500), 82.7 (d, C-50), 78.3 (d, C-300), 78.1 (d, C-30),
75.9 (d, C-400), 75.1 (d, C-40), 73.9 (d, C-3), 73.1 (d, C-5), 72.5 (d, C-2), 70.8 (d, C-4),
63.8 (t, C-100), 63.3 (t, C-600), 63.2 (t, C-60), 61.8 (t, C-6) and 61.5 (t, C-10); HRESIMS
(positive) m/z: 527.1625 [M þ Na]þ, calculated for C18H32O16Na, 527.1600.
3.6. Acetylation of 1-kestose
The trisaccharide (20 mg) was dissolved in 5 mL of pyridine and 1 mL of acetic
anhydride. The mixture was refluxed at 160ꢀC for 2 h. After evaporating to dryness,
the residue was chromatographed on a Si gel column and eluted with chloroform to
yield 1-kestose hendecaacetate (18 mg), as colourless syrup, [ꢂ]D ꢁ 13.5ꢀ (c 0.018,
1
MeOH); H-NMR (CDCl3): ꢀ 5.71 (1H, d, J ¼ 3.4 Hz, H-1), 5.46 (1H, d, J ¼ 8.6 Hz,
H-300), 5.42 (1H, t, J ¼ 10.2 Hz, H-3), 5.37 (1H, d, J ¼ 8.6 Hz, H-30), 5.22 (1H, t,
J ¼ 10.2 Hz, H-4), 4.88 (1H, dd, J ¼ 10.2 and 3.4 Hz, H-2), 4.44–4.20 (13H,
overlapped signals), 2.16 (6H, s, COCH3), 2.12 (3H, s, COCH3), 2.11 (3H, s,
COCH3), 2.10 (3H, s, COCH3), 2.09 (3H, s, COCH3), 2.08 (3H, s, COCH3), 2.07
(3H, s, COCH3), 2.05 (3H, s, COCH3), 2.02 (3H, s, COCH3) and 2.00 (3H, s,
COCH3); 13C-NMR (100.6 MHz, CDCl3): ꢀ 170.4–169.7 (11 s, COCH3), 104.3
(s, C-20), 103.1 (s, C-200) 90.3 (d, C-1), 79.1 (d, C-500), 78.1 (d, C-50), 76.6 (d, C-300),
76.2 (d, C-30), 76.0 (d, C-400), 75.3 (d, C-40), 70.3 (d, C-2), 70.1 (d, C-3), 69.0 (d, C-5),
68.0 (d, C-4), 64.4 (t, C-100), 63.7 (t, C-10), 62.9 (t, C-600), 62.5 (t, C-60), 59.7 (t, C-6)
and 21.5–20.8 (11 q, COCH3); HRESIMS (positive) m/z: 989.2695 [M þ Na]þ,
calculated for C40H54O27Na, 989.2662.
3.7. Hydrolysis of 1-kestose
1-Kestose (5 mg) dissolved in 0.5 mL of 2N HCl was refluxed for 1 h. The reaction
mixture was neutralised, and analysed by TLC, using acetone–butanol–H2O (8 : 1 : 1)
as the solvent, in comparison with glucose and fructose, which are used as standards.
The sugar spots were visualised by reaction with ꢂ-naphthol. Afterwards, monosac-
charide composition was established by high-pressure anion exchange-pulsed
amperometric detector (HPAE-PAD, Dionex) equipped with a Carbopac PA1
column eluted with 15 mM NaOH (1 ml minꢁ1), giving D-glucose and D-fructose
(ratio 1 : 2) identified by their retention times in comparison with those of authentic
samples.
4. Conclusion
The saccharide 1-kestose was isolated for the first time from mosses. It is the first
record for FOS in this biggest group of bryophytes. The compound is particularly

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B. Pejin et al.
interesting regarding the investigation of moss physiological adaptations to drought
and freezing as well as for its health value.
However, more studies should be done for obtaining a better understanding of
the R. ontariense sugar chemistry. A possible way of producing large amounts is to
develop adequate micropropagation technology, in vitro culture. This method can
enable easier and larger production of the plant material that can be used for new
investigations.
Acknowledgements
One author (BP) is particularly grateful to the European Commission for the 10-month
scholarship in the scope of the Basileus project. MS acknowledges the support grant
No. 143015 (Ministry of Science, Republic of Serbia). The assistance of Mr V. Mirra (ICB-
CNR) is gratefully acknowledged.
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