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Guilhermina Rodrigues Noleto
Marcello Iacomini
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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].
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
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.
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.
2.8. 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.
117
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
Table 1
Monosaccharide composition of AG, AG-S and AG-P
Fraction
AG
AG-S
AG-P
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).
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
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.
Brazil.
Fundaca o Araucaria,
State of Parana,
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