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Abstract:
Recent studies have indicated that several plant extracts reputed to
be lactogenic are capable of inducing b-casein synthesis in mammary
epithelial cells. This enhancement is mediated through a stimulation
of prolactin release from lactotropes in anterior pituitary.
Therefore it is admitted that these extracts are capable of
stimulating prolactin secretion from hypophysis. A partial
purification of these extracts, revealed that their active compounds
are pectin in most cases and b-glucan in other cases. In this work,
the effect of various concentrations of both pure pectic acid and b-glucan
on prolactin secretion from ewe hypophysis fragments is
investigated. It is shown that pectic acid in the 50-150 mg/ml range
concentration and b-glucan in the 200-400 mg/ml range concentration
are capable of stimulating prolactin secretion significantly
(P<0.05, P<0.01), from hypophysis explants.
Keywords: Pectic Acid,
b-Glucan, Prolactin Secretion, Ovine Pituitary Explants.
The cell
wall of most fungi is composed of glycoproteins, β-glucan, chitin
andα-glucan. Previously, we showed that in the cell wall of the
ascomycetous fission yeast
Schizosaccharomyces pombe,
α-glucan displays a dimeric structure that is composed of two
covalently-linked building blocks, each consisting of a linear
(1→3)-α-glucan segment
with a
small number of (1→4)-linked residues at its reducing end,
indicating a two-stepbiosynthetic mechanism. In contrast, the
structure of α-glucan in spore walls consists of a single
(1→3)-α-glucan monomer, suggesting an alternative, single-step
biosynthetic mechanism. Here, we examine the chemical structures of
α-glucans from the cell walls of
seven
ascomycetous and basidiomycetous species. We found that the α-glucans
from the cell walls of
Lentinus
edodes
and
Cryptococcus neoformans
Cap67,
similar to α-glucan from fission-yeast cell walls, occur as dimeric
structures, whereas the α-glucans from
Pleurotus
ostreatus,
Piptoporus betulinus,
Neurospora crassa,
and
Schizophyllum commune
occur as
monomeric structures. Interestingly, the fruiting bodies of
Laetiporus sulphureus
contain
both structures of α-glucan. We conclude that for the biosynthesis
of fungal α-glucan, both one-step and two-step biosynthetic
mechanisms are conserved in evolution.
Introduction:
Plant extracts are traditionally used in various societies for their
pharmacological properties. Some plants which are reputed to be
lactogenic have the capacity to enhance milk secretion in lactating
women. Recent studies in this field have indicated that these
extracts induced b-casein synthesis in rat mammary gland in vivo (Sawadogo
& Houdebine, 1988; Sawadogo et al., 1988). It was observed that this
effect is mediated through an effect on prolactin (PRL) secretion (Sawadogo
& Houdebine, 1988) (Sawadogo & Houdebine, 1988; Sawadogo & Houdebine,
1989). The extracts proved to stimulate secretion of growth hormone
and cortisol in addition to PRL, in vivo (Sawadogo & Houdebine,
1988). It is admitted that some plants in Malvaceae, Linaceae,
Euphorbiaceae and Umbelliferae families have the lactogenic
capacity (Sawadogo & Houdebine, 1988). Chemical analysis of these
extracts revealed that active compounds are polysaccharides rich in
pectins in most cases and rich in b-glucan in other cases (Sawadogo
& Houdebine, 1988,1989).
Both pure pectic acid and b-glucan showed a strong capacity to
trigger PRL and GH secretion in experimental animals when injected
intravenously (Sawadogo & Houdebine, 1988). From these data, it was
concluded that pectic substances and b-glucan are the active
lactogenic compounds existing in plants, and that their effects are
mediated through the secretion of PRL and possibly of GH and
cortisol. In vitro experiments have shown that pectic acid is able
to induce the secretion of PRL, GH, LH and b-endorphin from rat
hypophysis (Sawadogo, 1988). Moreover, this compound was shown to
stimulate b-casein secretion from rabbit mammary gland in (Sawadogo
& Houdebine, 1988). The mechanism of action of the plant extracts is
not yet known. Whether the lactogenic substances stimulate PRL
secretion in vivo through a direct action on pituitary cells or
through an indirect effect on hypothalamus hypophysis axis is not
known. To get an answer for this question, the effect of various
concentrations of lactogenic substances on PRL secretion from
incubated ewe hypophysis fragments was investigated. Results confirm
those previously obtained in a preliminary experiment (Sepehri et
al.,1990). In this study, the optimal concentrations of lactogenic
substances which have maximal stimulatory effects were determined.
Fungal cell morphology and integrity depend on cell-wall
polysaccharides. The cell wallof most fungi consists mainly of
glycoproteins and four types of polymers, namely (1→3)-b-glucan,
(1→6)-b-glucan,
chitin, and (1→3)-b-glucan.
Disruption of enzymes involved in cell-wall synthesis may cause cell
lysis. For instance, mutations in (1→3)-b-glucan
and(1→3)-b-glucan
synthases may lead to cell swelling or cell lysis (Ishiguro et al.
1997;Hochstenbach et al. 1998). Because most fungi possess similar
structural polysaccharides, theenzymes involved in their assembly
form ideal targets for the development of novel antifungal drugs (Georgopapadakou
& Tkaz, 1995; Radding et al., 1998; Onishi et al., 2000; Ohyama et
al.,2000; Feldmesser et al., 2000; De Pauw, 2000; Georgopapadakou,
2001; Kurtz & Rex, 2001).
Recently, we have identified a putative (1→3)-b-glucan
synthase, Ags1p, in fission yeast Schizosaccharomyces pombe (Hochstenbach
et al., 1998). Based on its hydropathy plot and aminoacid sequence
similarities, we identified three Ags1p domains, namely an
intracellular synthase domain, a C-terminal multipass transmembrane
domain and an N-terminal extracellular domain that might act as a
transglycosylase (Hochstenbach et al., 1998). By examining the
chemical structures of ��-glucan
from cell walls of wild-type cells as well as from a (1→3)-b-glucan
synthase mutant that showed an aberrant cell morphology, we proposed
a model for the mechanism of action of Ags1p (Chapter 2). In short,
we found that fission-yeast b-glucan is composed of two (1→3)-b-glucan
polymers that were covalently linked via a short stretch of (1→4)-linked
residues. The b-glucan from themutant strain, however, was composed
of only a single b-glucan monomer, indicating that Ags1p is
essential for both the synthesis and coupling of b-glucan building
blocks, and therefore displaying a two-step biosynthetic mechanism.
In fission yeast, four genes homologous to ags1+, namely mok11+,
mok12+, mok13+, andmok14+, were identified that can be depleted
without forming a noticeable phenotype (Hochstenbach et al., 1998;
Katayama et al.,1999). Based on DNA-microarray analyses, Mata et al.
(2002) showed that
ags1+ was downregulated, whereas the mok+ homologs were upregulated
during meiosis, indicating that these genes may encode b-glucan
synthases required for spore-wall formation. Indeed, recently we
have identified two (1→3)-b-glucans
in fission-yeast spore walls (see Chapter 4). Importantly, unlike b-glucan
from cell walls of vegetatively-grown cells, spore-wall b-glucans
are composed of single (1→3)-b-glucan
monomers, indicating a one-step biosynthetic mechanism. b-Glucan
synthases homologous to fission-yeast Ags1p have been identified in
the fungi Aspergillus fumigatus (with Genbank accession numbers
AAL18964 and AAL28129), Neurospora crassa (with protein
identification numbers NCU02478.1 and NCU08132.1), Schizophyllum
commune (H.A.B. Wösten, personal communication) and Cryptococcus
neoformans.
Interestingly, these homologous enzymes also have a similar
multidomain structure as Ags1p. Therefore, we wondered whether also
the reaction products of these synthases may be conserved, and thus,
whether their b-glucan structures are similar to cell-wall or
spore-wall b-glucan of fission yeast.
Here we examine the chemical structures of cell-wall b-glucans from
seven fungal species and compare the chemical structures with those
of ��b-glucans
from fission-yeast cell walls and spore walls. By using
high-performance size-exclusion chromatography (HPSEC) in
combination with chemical analysis and NMR spectroscopy, we found
that in two out of seven fungi an b-glucan is present as dimers
similar to cell-wall b-glucan from fission yeast, indicating a
two-component biosynthetic mechanism. In four species the b-glucan
is present as monomers similar to b-glucan from fission-yeast spore
walls,indicating a one-component biosynthetic mechanism. One
species, Laetiporus sulphureus,bears both structures. These
one-component and two-component biosynthetic mechanisms have been
found in both Ascomycetes and Basidiomycetes, indicating that both
mechanisms are conserved in evolution.
Materials and Methods:
Chemical Pectic acid (poly-D-galacturonic acid) was from Fluka and
b-glucan from Sigma. These materials were dissolved in 0.7% NaCl
(pH=7.8) after stirring for 1 hour at 30°C. The pH of these
solutions (1 mg/ml) which was 3.2 and 5.6 respectively, was
neutralized by 0.01M NaOH. Since above the mentioned compounds are
only partially soluble in water, the solutions were centrifuged at
3.000 g for 10 minutes and only the supernatant was added to the
incubation Medium 199 at pH 8.2.
l
Animals: Mature ewes which have not been pregnant were uses (5.5-6.5
months old, 13.5-16.5kg).
Incubation of hypophysis fragments:
hypophysis were harvested immediately after the sacrifice and
transported to the laboratory in culture medium. The gland was cut
into fragments of 1 mm3 with a razor blade. About 20 mg of tissue
were spread on each stainless grids and incubated in Medium 1991 ml/
grid in 35 mm culture dishes at 37°C and under 95% O2, 5% CO2
atmosphere. In this experiment, the samples were first incubated in
a medium with none of the compounds for 30 minutes (preincubation)
to allow the discharge of lactotropic cells, and to determine the
basal level of PRL secretion in each dish. The medium was collected
and a fresh medium was then added without (control) or with the
above-mentioned compounds at various concentrations and incubation
was pursued for one additional hour (first incubation). After
collecting the media, the samples were incubated for one additional
hour in conditions similar to the first incubation (second
incubation). The medium was then collected and kept frozen at 20°C
until prolactin measurements. Sterile condition was not necessary in
this work since incubation time was short. Nevertheless, all the
culture steps were carried out in a culture room under O2 flux to
minimize contamination.
Prolactin measurement:
PRL was measured in the culture medium using a radioimmunoassay (RIA)
in duplicate samples. In all cases, the hormone concentration was
measured in incubation media of each dish after the pre-, the first
and the second incubation. The hormonal content of the preincubation
medium reflects the basal secretion level of each hypophysis, and it
depends on the amount and the quality of the tissue present in each
dish. The PRL content of the first and second incubation media
reflects of the added compounds. In each experiment, ten dishes were
used as control and ten dishes with added compounds for each assay.
Each result is the mean SD of ten dishes. As expected, the
concentration of PRL in the first and second incubation media of the
control dishes was lower than in the preincubation medium. This drop
in the second incubation was slightly more intensive. Therefor, the
PRL level in the first and second incubation media of control dishes
was taken as a reference (100% secretion) and PRL levels in each
group of dishes were compared to it.
Results:
Effect
of b-glucan on PRL Secretion
Various concentrations of b-glucan (25-500 mg/ml) added to the
incubation media of hypophysis fragments showed a clear effect on
PRL secretion (Figs. 3 & 4). The maximum stimulation was in the
200-400 mg/ml range of concentration. At concentrations more than
400 mg/ml, b-glucan had less stimulatory effect. These results were
observed in the first and second incubations in a similar way.
Results in Figure 3 shows the stimulatory effect of b-glucan on PRL
secretion, which is significant (P<0.05 or P<0.01) at concentrations
of 300 to 350 mg/ml. Figure 4 which shows the percentage of PRL
secretion in various concentrations of b-glucan, indicates that PRL
secretion was strongly stimulated (from 103% to 230%).
Conclusion:
The mechanism of action of lactogenic plant extracts on PRL
secretion is unknown. Pectic substances and b-glucan were shown to
stimulate, in vivo, PRL, GH and possibly cortisol secretion, to
induce b-casein synthesis in mammary gland. The data reported here
clearly show that these compounds stimulate PRL secretion when added
to ewe hypophysis fragments. It was therefore concluded from these
data that these compounds act as unspecific secretagogues through a
direct effect on PRL secretion on lactotropic cells of pituitary.
Previous work carried out in vivo has shown that GH secretion was
also stimulated by these compounds, whereas it was never stimulated
in vitro. One possible explanation is that GH releasing factor (GHRH)
rather than GH secretion is directly affected by the plant extracts
in vivo (Sepehri,1991). Alternatively, GH secretion may be too low
in vitro to be significantly stimulated by the added compounds.
Pectic substances and b-glucan exhibit rather limited homologies of
structure and it seems that they act through different mechanisms.
However, one point remains striking. Both polymeric compounds
studied here are direct precursors of substances named elicitors
which recognize specific receptors on plant cells, act as vegetal
hormones and induce the expression of certain genes and defensive
responses (Ryan, 1987). No receptor has been identified in the cells
of higher vertebrates for these compounds. A possible hypothesis is
that pectic substances and b-glucan have some structural homologies
with extracellular matrix of mammalian cells and the active
compounds of plant extracts might affect cellular secretion by
binding to these receptors. It remains of find which cellular
receptors are involved in which part of cell and by which mechanism
of action the stimulatory effects of active compounds of plant
extracts on PRL secretion are mediated.
Biological Activities:
Water-extracted polysaccharides from Coriolus vesicolorare
considered to act as "biological response modifiers." These
compounds exhist various biological activities, directed mostly
towards, although not limited to, the immune system.
The β-glucans
from Coriolus vesicolor pass into the bloodstream unchanged
by the digestive process. Receptor(s) for bets-glucans have been
found on neutrophils, monocytes/macrophages,natural killer cells,and
T and B lymphocytes.
Japanese
studies with theCoriolus polysaccharides have shown them to
stimulate the antigen-presenting cell function of macrophages and,
consequently, to stimulate overal immune function.Several Japanese
studies have also reported the ability ofCoriolus
polysaccharides to enhance the in vitro proliferation of T
and B lymphocytes,as well as the cytotoxic activity of T and NK
cells.
Recent US
research has confirmed the significant immuno-modulating properties
of these unique protein-bound polysaccharides. These polysaccharides
acted as a potent inducer of proliferation, tumor cytotoxicity, and
lymphokine production by human lymphocytes in in vitro
studies.
Figure 1 – Effect of
various concentrations of petctic acid on PRL secretion from ewe
pituity fragments in the first and second incubations. Each result
is the mean ±SEM of ten dishes. *p<0.05
Figure 2 – Effect of
various concentrations of b-glucan on PRL secretion from ewe
pituitary fragments in the first o and second o incubations. Each
result is the mean ±SEM of ten dishes. *p<0.05; **p<0.01
Figure 4 – Effect of b-glucan
on PRL secretion in the first o and second o incubations. Results
are expressed in percentage in respect to controls.
Beta Glucans are currently available in two broad types of product:
Insoluble bran and Soluble:
Insoluble bran
products consist primarily of oat bran which has been purified by
extensive milling and/or extraction with organic solvents. In any
case the end-product contains low levels of beta glucans (12 to
22%), high fibre and starch contents. Therefore, it is still a
bran-type (fibrous) product which has several limitations in food
applications, hence the relatively low price and market penetration.
The largest drawbacks to such a product is the “gritty aftertaste”
due to the insoluble milled bran components, the settling out and
sedimentation of these components in liquid preparations, and to a
relatively strong and distinctive oat cereal taste.
Soluble products can
be produced from a variety of processes and are much more
interesting from the application point of view. For example they may
have functionalities which are quite ideal for beverages, baked
goods, dairy products, etc. The level of beta glucan may vary from 1
to 70%, but without any exception the price per kilo of beta glucan
has been far too high and only affordable in rather niche areas.
It can be produced
with very high levels of beta glucans, but it can be marketed at
considerably lower price.
Up to the present
date, beta glucan have been sold into neutraceutical, functional
food and cosmetic markets whereas microbial-derived beta glucans
almost exclusively go into the high end neutraceutical and cosmetics
market. |
