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Chemical and immunological modifications of


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International Journal of Biological Macromolecules 43 (2008) 115120

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules


journal homepage: www.elsevier.com/locate/ijbiomac

Chemical and immunological modications of an arabinogalactan present in


tea preparations of Phyllanthus niruri after treatment with gastric uid
Caroline G. Mellinger, Thales R. Cipriani, Guilhermina R. Noleto, Elaine R. Carbonero,
Maria Benigna M. Oliveira, Philip A.J. Gorin, Marcello Iacomini
Departamento de Bioqumica e Biologia Molecular, Universidade Federal do Paran
a, Centro Politecnico 81531-980, Curitiba, PR, Brazil

a r t i c l e

i n f o

Article history:
Received 26 September 2007
Received in revised form 1 April 2008
Accepted 1 April 2008
Available online 8 April 2008
Keywords:
Phylanthus niruri
Arabinogalactan
Immunological properties
Gastric condition
Clinical treatment

a b s t r a c t
An arabinogalactan (AG) obtained from tea preparations of Phyllanthus niruri was previously investigated
and presented immunological properties when tested with peritoneal mice macrophages. AG was now
submitted to acidic and neutral gastric conditions using human gastric uids and aq. HCl solution. Since
the acidic procedures gave rise to the same free monosaccharidic composition, the acid hydrolyzate of
AG at pH 2.00 was treated with ethanol to form insoluble (AG-P) and soluble fractions (AG-S). These
were analyzed using 13 C NMR, HPSEC, and GCMS for monosaccharide composition and methylation
analyses. The results showed an intense partial degradation, including cleavages of the main chain. AG-S
presented the monosaccharides released from the native polymer and some oligosaccharides as shown
by methylation data. AG-P contained larger molecular fragments comprising the internal units from
AG, which were not attacked by the hydrolysis condition. Both fractions were tested in peritoneal mice
macrophages and remained active, promoting an increase of superoxide anion production of 2.0 and 2.3fold, at 250 g/mL, for AG-S and AG-P, respectively. When compared to AG, a slight diminished response
was observed, revealing a structureactivity relation. The signicance of the results is that most plant
extracts are orally ingested and will reach the gastrointestinal tract before performing a biological function, so checking these changes is crucial to propose future clinical therapies based on the rational use of
phytomedicine.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Plants of the genus Phyllanthus are part of the Euphorbiaceae
family, comprising more than 600 species, which are widely distributed throughout South America, Asia, and Africa. Phyllanthus
niruri L., known locally as quebra pedra (stone breaker), is one of
the most common species found in Brazil [1].
A variety of extracts from Phyllanthus spp. and their metabolites
have already been examined to determine their chemical structures
and biological activities. These included secondary metabolites that
inhibited hepatitis B virus, afforded hypoglycemic, hypotensive,
and diuretic effects and functioned as antinociceptive, antitumor,
antioxidative, and anti-inammatory properties [2]. These extracts
also appear to stimulate the immune system by increasing the
natural killer (NK) and antibody dependent cellular cytotoxicity
(ADCC) [3], and nitric oxide (NO) production with mouse peritoneal
macrophages [4].

Corresponding author. Tel.: +55 41 33611655; fax: +55 41 32662042.


E-mail address: iacomini@ufpr.br (M. Iacomini).
0141-8130/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2008.04.001

A class of biologically active molecules that have been in evidence during the last decades, are the carbohydrates, especially
polysaccharides. These macromolecules have already presented
several biological properties including antiviral, leishmanicidal,
and respiratory burst alterations of macrophages and NO, gamma
interferon (IFN-), and tumoral necrosis factor alpha (TNF ) induction. Certain polysaccharides can thus act as Biological Response
Modiers (BRM) in terms of immunomodulatory and antitumor
effects [5,6].
The arabinogalactans (AGs) are, in general terms, biologically
active molecules, and are involved as immunomodulatory agents
such as in Viscum album [7], Plantago major [8], Salvia ofcinalis [9],
and Anadenanthera colubrina [10], among other plant sources. In
a recent study [11], it was demonstrated that an arabinogalactan,
isolated from tea infusion and decoction of P. niruri, stimulated an
intense in vitro peritoneal macrophage response by an augmentation of superoxide anion production.
In our studies, as well as in many other experimental procedures of immunological property evaluations, the tested molecules
were rstly analyzed into in vitro models, without considering any
interference from the digestive tract. But if we assume that people

116

C.G. Mellinger et al. / International Journal of Biological Macromolecules 43 (2008) 115120

drink tea from different medicinal plants to stimulate biological responses against diseases, it is logical to investigate whether
chemical and/or biological alterations in these molecules can occur
when ingested by humans, and what they represent in terms of
availability of biologically active molecules.
The human digestive tract, going from the mouth to the anus,
passes through the stomach and intestines. Human digestion of arabinogalactans occurs mainly in the large intestine due to bacterial
ora, although the gastric phase of digestion is considered to be the
key of the digestive process, since it is in the stomach that chemical and mechanical modications happen, so as to prepare food for
intestinal digestion [12]. Zhang et al. [13] revealed the relation of
potential gastric acidities and the release of arabinosyl units from
arabinoxylans and arabinogalactans of corn hulls, larch wood, and
banana peel. They emphasized that the acidity directly promoted
the release of arabinosyl units from the polysaccharides.
We now investigate not only chemical, but also biological modications of an arabinogalactan present as a main polysaccharide
from tea infusion and decoction of P. niruri after submitting the
molecule to gastric uid and an aqueous, dilute HCl solution.

(2) in the presence of gastric uid collected without this sensorial


stimulus. Experiment (3) was in the presence of aq. HCl solution
at pH 2.00. In all the three different procedures the polysaccharide
(2 mg/mL) was added to the solvent medium and placed in an oven
at 37 C to carry out the partial hydrolyses. Samples were collected
in intervals of 15 min for the rst 120 min. Each collected sample
was immediately frozen. Freeze-drying procedures were carried
out several times, successively, until HCl removal, as monitored by
the solution pH.

2. Materials and methods

Analyses were carried out in a Shimadzu high performance liquid chromatograph, with a refractive index detector
(RID 10A, Shimadzu), using an organic acid column (HPX-87H,
300 mm 780 mm, Aminex ion exclusion, Bio-Rad) eluted with an
8.0 mM aq. solution of H2 SO4 . The temperature was kept at 65 C
and the ow rate at 0.6 mL/min. Samples were dissolved in MilliQ water, ltered through a cellulose acetate Millipore membrane
(0.22 m), and injected (50 L) at a 1 mg/mL concentration.

2.1. Subjects
Two volunteers of 22 and 24 years old, who were healthy, in
terms of a gastric endoscopic evaluation, were selected to provide
their gastric uid. They were well informed and consented to participate in the study, which was approved by the Ethics Committee
of the Hospital do Trabalhador, Curitiba, PR, Brazil.
2.2. Gastric uid collection
The volunteers were submitted to a 12 h overnight fast, then
local anesthesia (oral spray of 10% lidocain) was sprayed, immediately followed by a 30 mL aq. solution of dimeticone (75 mg/mL)
minutes before collection of gastric uid. The procedure was performed using conventional upper endoscopy, with the volunteers
having a tube orally placed in the antrum, and the gastric uid being
drained into a sterile recipient and thereafter kept at 20 C prior
to examination [14].
Before collection of the uid, one of the volunteers was submitted to a sensorial stimulation of food intake, by smelling food until
evident salivation (5 min), just before intubation [15].
2.3. Plant material
P. niruri L. samples were obtained from the entire plant, previously oven-dried and coarsely milled. Its identity was conrmed
in the Botany Department, Federal University of Parana (UFPR),
Curitiba, Brazil, and a sample of the plant was deposited in the
Herbarium of UFPR (UPCB) as voucher no. 42822.

2.6. Fractionation of acid-hydrolyzed polysaccharide


The arabinogalactan (50 mg) was partially hydrolyzed on a
larger scale, using an aq. HCl solution at pH 2.00 for 90 min at 37 C.
After freeze-drying removal of HCl, the residue was dissolved in a
small volume of H2 O, added to ethanol (3 V), resulting in precipitate (AG-P) and supernatant fractions (AG-S). Both were further
chemically analyzed and immunologically tested.
2.7. HPLC

2.8. HPSEC
Alterations in the homogeneity prole of native AG were
monitored by high-performance size-exclusion chromatography
(HPSEC), using a Waters 510 HPLC pump at 0.6 mL/min, with four
gel permeation Ultrahydrogel columns in series (each column with
7.8 mm 300 mm) with exclusion sizes of 1 106 , 4 105 , 8 104 ,
and 5 103 Da, using a refractive index (RI) detector, the eluent
being 0.1 M aq. NaNO2 containing 200 ppm aq. NaN3 . Samples were
dissolved in the eluent solution, ltered through a cellulose acetate
Millipore membrane (0.22 m), and injected (250 L loop) at a
1 mg/mL concentration.
2.9. NMR spectroscopy
13 C NMR spectra were obtained using a 400 MHz Bruker model
DRX Avance spectrometer incorporating Fourier transform (Rheinstetten, Germany). Samples were dissolved in D2 O and examined
at 50 C. Chemical shifts are expressed in ppm (), based on the
resonance of acetone at 30.2.

2.10. Monosaccharide composition


2.4. Polysaccharide extraction, isolation and chemical
characterization
Purication steps and chemical characterization of the native
polysaccharide has been described by Mellinger et al. [11].
2.5. Partial hydrolyses of the arabinogalactan
The arabinogalactan (2 mg/mL) hydrolyses were carried out at
37 C for 3 h with sample collection intervals of 15 min for the rst
120 min. Experiments were conducted (1) in the presence of gastric uid collected after the sensorial stimulus of food smelling and

Samples (2.0 mg) were hydrolyzed with 1 M TFA at 100 C for


8 h, followed by evaporation to dryness and successive reduction
with NaBH4 and acetylation with Ac2 Opyridine (1:1, v/v; 2 mL) at
room temperature for 12 h [16,17].
The resulting alditol acetates were then examined by GCMS
and identied by their typical retention times and electron impact
proles [18]. Analyses were carried out using a Varian model
3300 gas chromatograph, linked to a Finnigan Ion-Trap, model
810-R12 mass spectrometer, with a DB-225 capillary column
(30 m 0.25 mm i.d.), injector temp. 250 C, programmed from 50
to 220 C at 40 C/min, then hold. Helium was the carrier gas.

C.G. Mellinger et al. / International Journal of Biological Macromolecules 43 (2008) 115120

117

2.11. Uronic acid determination


Uronic acids were estimated using a colorimetric improved mhydroxybiphenyl method in which neutral monosaccharides do not
interfere [19].
2.12. Animals and biological experiments
Swiss mice received a standard laboratory diet (Purine). All recommendations of the Brazilian national law (no. 6638, 05/11/1979)
for scientic management of animals were respected. Methods for
macrophage isolation, cell viability, superoxide anion production,
nitric oxide production, protein determination, and statistical analysis have been previously described [11].
3. Results and discussion
A native arabinogalactan, found as the main polysaccharide liberated in infusions and decoctions of P. niruri, has been chemically
characterized in a previous investigation [11]. It was now submitted
to three different procedures: hydrolyses with (1) aq. HCl solution
at pH 2.00 miming gastric conditions, (2) gastric uid collected after
a sensorial stimulation of food intake by smelling food (pH 1.95),
and (3) gastric uid at pH 7.76, without any sensorial stimulus. The
neutral pH from the gastric uid can be explained as arising from
a physiological buffering reaction during the fast or interdigestive
period, while the acidic pH from the uid arose from HCl release in
the antrum during the cephalic phase of digestion, known to begin
35 min after sham feeding, and which usually lasts for 30120 min
[15].
The procedures for treatment of AG were carried out on samples
at intervals of 15 min, up to 120 min. HPLC proles were similar
for hydrolyzates using aq. HCl, pH 2.00 (Fig. 1A) and that using
gastric uid at pH 1.95 (Fig. 1B), showing the release of arabinose and smaller amounts of galactose, xylose, and mannose which
formed a single peak. It follows that the degree of acidity was
responsible for the cleavage of glycosidic linkages, agreeing with
the ndings of Zhang et al. [13], who showed arabinose release
from polysaccharides under gastric potentials. This was reinforced
by the HPLC prole obtained from gastric uid at pH 7.76 (Fig. 1C),
which contained only traces of peaks, not attributed to any sugar. In
fact, they may correspond to artifacts from the biological material
(ratio).
The similarities found in the proles of all acidic samples
(15120 min), mean that the release of monosaccharides, and even
possibly oligosaccharides, occurred at the beginning of the reaction. This nding is interesting, since these polymers may suffer
alterations by reaching the stomach, no matter how long it takes
them to be conducted to the duodenum.
Comparing the acidic conditions with the chromatogram
obtained with the neutral gastric uid (Fig. 1C), it was possible to
conrm that the chemical alterations in the polysaccharide depend
basically on the acidic strength, since the sample chromatogram
was identical to the negative control prole (Fig. 1C, inset), which
contained only the gastric uid, in the absence of the arabinogalactan.
After the identication of some chemical modications to the
arabinogalactan, we carried out an ethanolic precipitation on a
new hydrolyzed sample with aq. HCl at pH 2.00. The ethanolic
precipitate (AG-P) yielded 34% while the supernatant (AG-S) 54%,
supporting the idea that the gastric fragmentation is more intense
than just a molecular peeling, as thought before. Both fractions
were then examined by HPSEC (Fig. 2) and gave heterogenic proles
showing that the cleavages happened in more than one position

Fig. 1. HPLC chromatograms of AG submitted to hydrolyses using (A) aq. HCl (pH
2.00), (B) gastric uid collected after a sensorial stimulation, and (C) gastric uid
without any sensorial stimulation. Insets: in (B) and (C) superposition of gastric
uid (negative control) and gastric uid plus the sample after 15 min hydrolysis.
*
Lidocain; ** Dimeticone.

Fig. 2. HPSEC elution prole of native AG, and their fractions after ethanol precipitation: (AG-S) and (AG-P).

118

C.G. Mellinger et al. / International Journal of Biological Macromolecules 43 (2008) 115120

Table 1
Monosaccharide composition of AG, AG-S and AG-P
Fraction

AG
AG-S
AG-P

Monosaccharide composition (mol%)a


Rha

Ara

Xyl

Man

Gal

Glc

UAb

8
7
7

35
26
6

9
8
2

4
9
15

21
34
28

8
12
23

15
4
19

a
Native arabinogalactan (AG) and its digested fractions: low (AG-S) and high (AGP) molar mass fragments.
b
Uronic acid.

since many peaks were observed for each fraction, and that the
stomach digestion may play an important role in molecular biological activity after oral ingestion of arabinogalactans. Comparing
the chromatographic prole of AG-P and AG-S it was also possible
to observe a great similarity between them, except by the fact that
the supernatant gave rise to more intense peaks with a delayed
elution time, which means that this fraction is composed of fragments of smaller molar masses when compared to the precipitate.
The opposite is true when analyzing the precipitate fraction.
The monosaccharide components of AG-P and AG-S were
present in different proportions (Table 1), the main differences
being due to the removal of arabinose from the arabinogalactan,
and the retention of uronic acid. Also, arabinose was present in AG
as nonreducing end-units residues, as shown in a previous methylation analysis [11]. Higher contents of galactose and galacturonic
acid in AG-P were expected, since they are more resistant to hydrolysis and occur in more internal positions of AG.
Methylation analyses (Table 2) agreed with these presented
data, showing higher amounts of 2,3,5-Me3 -Ara in AG-S, and high
amounts of 2,3,4,6-Me4 -Gal and 2,3,6-Me3 -Gal in both AG-P and
AG-S from the main chain peeling.
The NMR spectrum of AG-S (Fig. 3B) also contained many
differences from the native molecule of AG (Fig. 3A) [11]. Signals corresponding to -Araf from 109.5 to 107.1 [20,21] are not observed
in AG-S as well as the signal at 102.0 from -galacturonic acid [22],
which means that the non-reducing end residues of arabinose are
now observed as reducing units either in - ( 97.5) or - ( 93.8)
congurations, not observed in the native polysaccharide. Reducing Galp residues can also be present in this same spectrum region
(-Galp 92.593.5; -Galp 9798), but were also shown as

Fig. 3. 13 C NMR spectra of (A) native AG, and (B) AG-S. Solvent D2 O at 50 C, numerical values in (ppm).

non-reducing units about 103.2, meaning that larger fragments


also compounds the fraction. Reducing Gal residues can also be
identied by an intense peak at 62.5 (C-6) [23]. In a general view of
the spectra, a greater complexity was also observed after digestion,
showing that different small chains and units were now forming
the fraction. The fraction of AG-P became partially insoluble and a
spectrum could not be obtained but the idea that the main chain
was cleaved is supported by the other chemical analyses.
Since the chemical data showed signicant alterations in the
molecule when submitted to gastric conditions, the next question was: Are these molecular fragments still biologically active in

Table 2
Partially O-methylalditol acetates formed on methylation analyses of AG-S and AG-P
Alditol acetatea

Rt b

2,3,5-Me3 -Ara
2,3,4-Me3 -Rha
2,3,4-Me3 -Ara
2,3,4-Me3 -Xyl
3,4-Me2 -Rha
2,3,4,6-Me4 -Man
2,3,4,6-Me4 -Glc
2,3-Me2 -Ara
2,3,4,6-Me4 -Gal
3-Me-Rha
3,4,6-Me3 -Man
3,4,6-Me3 -Glc
2,4,6-Me3 -Glc
2,3,6-Me3 -Man
2,3,4-Me3 -Man
2,3,6-Me3 -Gal
2,3,4-Me3 -Gal

0.7818
0.7824
0.83
0.84
0.95
0.99
1.00
1.04
1.06
1.24
1.28
1.29
1.31
1.39
1.40
1.42
1.61

Mol (%)
AG-S

AG-P

10

6
9
6
7
14
6
11

4
4

17
6

6
6
7
9
3
9

14
4

11
3
4
2
17
5

Obtained using a DB-225 column at 215 C.


Rt : Relative retention time compared with that of the acetate of 2,3,4,6-tetra-Omethylglucitol.
a

Fig. 4. Effect of AG-S and AG-P on macrophage viability. Adherent macrophages


were incubated at different concentrations of these fractions. The medium was
then removed and MTT added, followed by incubation for 3 h. Excess 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was removed and
formazan crystals were dissolved by addition of DMSO. The absorbance was measured at 550 nm. Values are means S.D. of three experiments, each one in triplicate.
Samples were not signicantly different from the control, p < 0.05. Control (100%)
corresponds to the medium in the absence of arabinogalactan complexes: time of
exposure to the AG fractions was 48 h.

C.G. Mellinger et al. / International Journal of Biological Macromolecules 43 (2008) 115120

119

Fig. 5. Effect of AG-S and AG-P on superoxide anion and nitric oxide production by macrophages. (A) Effect of AG fractions on superoxide anion production by macrophages.
Adherent macrophages were incubated with HBSS containing ferricytochrome c (80 M) and different concentrations of the fractions. PMA (1 g/mL) was used as positive
control. After 2 h the supernatant was removed and the absorbance measured at 550 nm. Results are expressed as nmol of superoxide anion/mg of cell protein. Values are
means S.D. of three experiments, each one in triplicate. * Signicantly different from experiments performed in the absence of AG samples (negative control), ** positive
control. (B) Effect of AG fractions on nitric oxide production by macrophages. Adherent macrophages were incubated for 48 h in the absence (negative control) or presence
of samples. LPS (50 g/mL) plus IFN- (26 U/mL) was used as a positive control. NO accumulation was measured in the supernatant using the Griess reaction and calculated
as nmol nitrite/mg cell protein. Values are means S.D. of four experiments, each one in triplicate. * Signicantly different from control, p < 0.05; ** positive control.

macrophage functions after stomach digestion? As an answer, the


same previous biological experiments were carried out on AG-S and
AG-P. There was no signicant loss of cell viability on macrophages
after 48 h incubation with each fraction. These results were somewhat different from those obtained with the native polymer AG,
in which a small loss could be observed after 48 h incubation for
concentrations higher than 50 g/mL (Fig. 4).
Fig. 5A shows macrophage activation by AG-S and AG-P via
O2 production after 2 h of cell exposure to them. Both fractions
were able to activate this pathway in a dose-dependent manner,
just as observed for the parent AG. AG-S produced an activation of 0.9-fold at 100 g/mL, reaching 2.0-fold for the highest
tested concentration (250 g/mL). AG-P caused an activation of
2.0-fold at 100 g/mL, reaching 2.3-fold at 250 g/mL. Both results
showed a diminished activation when compared to AG, namely a
3.0-fold activation at 250 g/mL, meaning that there is a relation
between structure and macrophage stimulation of arabinogalactans from P. niruri teas, and the size of the molecules must be
involved since the native AG and AG-P were more active than AGS. The chemical arrangement of the units in the molecule and its
conformation in aqueous systems could also be involved in such
activity.
Macrophage activation can also occur via augmentation of NO
production. To investigate this possibility, AG-S and AG-P were also
tested (Fig. 5B), but both gave rise to negative results in activating
this pathway, just as observed for the native arabinogalactan.
The present results are of importance for establishing a future
rational use of phytomedicines, since they demonstrated that
a biological system might interfere in chemical and biological
availability of molecules. An arabinogalactan from P. niruri tea
preparations was partially hydrolyzed in the stomach, but the
resulting fragments were still active on macrophage functions with
a signicant augmentation of O2 production in an in vitro system.
There was a relation between structure and biological activities,
albeit not still identied, since the biological response of the native
arabinogalactan was higher than that of its fragments. Further studies will be carried out to investigate possible biological response

mechanisms to these molecular fragments when they reach the


small intestine.
Acknowledgements
The authors would like to thank Dr. Alfreli Arruda do Amaral
for collecting the gastric uid, Ms. Nathieli Keila Takemori and
Prof. Dr. Cleusa Bona for conrming the plant identity, the Brazilian Pharmaceutical Industry As Ervas Curam Ltda.1 for supplying
the sample of P. niruri, the Istituto di Chimiche e Biochimiche
G. Ronzoni, Milan, Italy, for preparation of 13 C NMR spectra, and
the Brazilian funding agencies Conselho Nacional de Desenvolvi
mento Cientco e Tecnologico
(CNPq), PRONEX-Carboidratos, and

Brazil.
Fundaca o Araucaria,
State of Parana,
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