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BÁO CÁO KHOA HỌC: "Nghiên cứu độc tính và tác dụng kìm hãm tăng sinh dịch chiết nước và cồn của nấm đa niên lên tế bào invitro của người và khỉ" ppt

Nghiên cứu độc tính và tác dụng kìm hãm tăng sinh
dịch chiết nước và cồn của nấm đa niên lên tế bào
invitro của người và khỉ

Dịch chiết nước va cồn của một số loai nấm lỗ đa nien
thuộc các chi Ganoderma ( duối chi Elfwingia),Phellinus,
Nigrofomes, Perenniporia cũng như hỗn hợp của chúng
không thể hiện ảnh hưởng độc tế bào đối với các dòng tế
bào đã được thử nghiệm bao gồm Hella( tế bào ung thư cổ
tử cung),Vero-B4 ( tế bào thận của loài khỉ xanh Châu Phi),
Hep-G2 (Tế bào ung thư gan), CLS-354 (tế bào ung thư
hình vảy trong miệng) và MDA-MB 436 ( tế bào ung thư
vú của người). Sau khi sử lýyý với dịch chiết nước và cồn,
chúng ta quan sát thấy sự ức chế phụ thuộc nồng độ dịch
chiết lên quá trình tăng sinh của tế bào ở nồng độ cao; một
số dịch chiết biểu hiện tác dụng kìm hãm quá trình tăng
sinh tế bào. Các tế bào thể hiện nhiều đặc tính quan trọng
của quá trình appoptosis.
INTRODUCTION

For millennia, mushrooms have been valued by humankind

as an edible and medical resource. A number of bioactive
molecules, including antitumor substances, have been
identified in many mushroom species. Polysaccharides are
the best known and most potent mushroom-derived
substances with antitumor and immunomodulateing
properties (Wasser, 2002). Moreover, the small-weighed
molecules such as terpens are also proved that having
pharmaceutical properties.

In recent decades, people have studied much about fungi
classification, biology, cultivation techniques and about
bioactive compounds, medical properties, etc. of some
important annual fungal species. However, the perennial
polypore species are still not studied carefully.

In the present study, we investigated the effects ethanolic
and aqueous extracts of some wood-inhabiting polypores o-
n the growth of human cervix carcinoma (Hela), African
green monkey kidney (Vero-B4), human hepatocellular
carcinoma (Hep-G2), squamous carcinoma (mouth) (CLS-
354) and human breast carcinoma (MDA-MB 436) cells.
(Strains are signed E1, H1, H3, N1, and P1).



METHODS

1. Preparation the polypore extractions

We have 5 samples of 5 Vietnamese fungal species, they
are: Phellinus sps (H3, H1), Nigrofomes sp (N1),
Perenniporia sp (P1), Ganoderma sp, sub genus Elfwingia
(E1). To extraction, we take 300g each sample. With each
sample, we boil it with water 3 times, each in 2 hours.
Then, mix 3 solutions and boil down until we have about
25-30ml. The solutions are lyophilized. Then we dissolve
them again with water and store in the refrigerator.

The rest materials are dried by sunlight. All 5 samples are
again extracted in Ethanol 96%, 98% in 24 hours. After 24


hours, we distill them and then dissolve again with EtOH
and store in the refrigerator for later experiments.


2. Cells and culture conditions

Cells of adherent cell lines were cultured in RPMI 1640
medium (GIBCO BRL g/ml42402-016), supplemented
with 100 U/ml penicillin G-sodium salt/100 streptomycin-
sulfate (GIBCO BRL 15140-114), 10% heat-inactivated
fetal bovine serum (GIBCO BRL 10500-064), and L-
glutamine (GIBCO BRL 25030-024) at 37ºC in high
density polyethylene flasks (NUNC 156340). Hela cells
were grown in RPMI 1640 culture medium (GIBCO BRL
21875-034) g/mlsupplemented with 100 U/ml penicillin
G-sodium salt/100 streptomycin-sulfate (GIBCO BRL
15140-114), 10% heat-inactivated fetal bovine serum
(GIBCO BRL 10500-064), NS 10 ml/l non-essential
amino-acid (GIBCO BRL 1140-035) at 37ºC in high
density polyethylene flasks (NUNC 156340). The cells
were harvested at the logarithmic growth phase after soft
trypsinization, using 0,25% trypsin in HBSS containing
0,038% EDTA (GIBCO BRL 25200-056).

Antiproliferative and cytotoxic assays

The cells were incubated with ten concentrations of the test
compounds. For eacg assay 10000 cells were seeded with
0,1 ml RPMI 1640 culture medium (GIBCO BRL 21875-
034), containing 10% heat-inactivated fetal bovine
g/mlserum (GIBCO BRL 10500-064), 100 U/ml penicillin
G-sodium salt/100 streptomycin-sulfate (GIBCO BRL
15140-114) into 96-well microplates. The plates were
previously prepared with ten dilutions of test substances in
0,1 ml RPMI 1640 medium. Hela cells were preincubated
for 48 h without the test substances. Cells were incubated
for 72h at 37ºC in a humidified atmosphere and 5% CO
2
.

The monolayer of the cells were fixed by glutaraldehyde
and stained with a 0,05% solution of methylene blue for 15
min. After gently washing the stain was eluted by 0,2 ml of
0,33 N HCl in the wells. The optical densities were
measured at 630 nm in a Dynatech MR 7000 microplate
reader. Comparisons of the different values were performed
with Microsoft Excel. From the dose response curves the
IC50 values (concentration which inhibited cell growth by
50%) were calculated with CASYSTAT (Schọfe), software
for data evaluation. The IC50 was defined as being where
the concentration-response curve intersected the 50% line,
determined by means of the cell counts/ml, compared to
control.


RESULTS AND DISCUSSION

The reseach data are demonstrated in table 1 and differend
figures:



The IC50 values were found and shown in the table above.
The IC50 values of the perenial polypores extracts in these
cell lines are completely different. In some cases, the IC50
values are higher than even the first concentrations used,
such as in Hela with all aqueous extracts or in Vero-B4
with aqueous extracts of N1, E1, P1 and mixture. However,
in g/ml.some other cases, the IC50 values are smaller than
10

The dose-resonse curves of the perenial polypore extracts
on cell proliferation after 72 hours of incubation are
depicted in the following figures.









All extracts and their mixtures do not exhibit cytotoxic
effect against the tested cell lines. Upon treatment with
ethanolic and aqueous extracts, a concentration-dependent
inhibition of cell proliferation was observed and cells
developed many of the hallmark features of apoptosis. At
high concentration, some of them express antiproliferative
effect.

Some authors confirm that many, if not all, Basidiomycetes
contain biologically active polysaccharides in fruit bodies,
cultured mycelium, culture broth. Their polysaccharides are
of different chemical composition, most of which belong to
the group of ò-glucans, these have ò-(1 >3) linkages in the
main chain of the glucan and additional ò-(1 >6) branch
points that are needed for antitumor actions. Mushroom
polysaccharides prevent oncogenesis, show direct
antitumor activity against various allogeneic and syngeneic
tumors, and prevent tumor metastasis. Polysaccharides
from mushrooms do not attack cancer cells directly, but
produce their antitumor effects by activating different
immune responses in the host. The antiumor action of
polysaccharides requires a T-cell component, their activity
is mediated through a thymus-dependent immune
mechanism. These conclusions are given after many
satisfactory results obtained by many research groups on
Grifolia frondosa, Trametes versicolor, Flamulina
velutipes, Lentinula edodes, Wolfiporia cocos, Agaricus
blazei …. Ganoderma lucidum is the most concerned and
studied fungi among medicinal mushrooms and its
commercial products are common all over the world. So
that we can completely trust that its related species, the
perennial species of subgenus Elfvingia and some other
perennial polypore groups, also have pharmaceutical
properties and can be used to treat many diseases, even
cancers.



MAIN REFERENCES

1. Bao, T. T. T. (2005), “Research on taxonomy of the
main perennial polypores and biological characteristics on
some imporortant species in Vietnam.” Graduation
dissertation honor programme biology. Hanoi – Jena.
2. Dahse, H. –M., Schlegel, B., Graefe, U. (2001),
„Differentiation between inducers of apoptosis and
nonspecific cytotoxic drugs by means of cell analyzer and
immunoassay”, Pharmazie 56 (6), 489-491, Germany.
3. Hawksworth, D. L. (1993), „The tropical fungal biota:
census, pertinence, prophylaxis and prognosis“. In: Isaac,
S. Frankland, J. C., Watling, T., Whalley, A. J. S. (eds)
Aspects of tropical mycology, Cambridge University Press,
Cambridge.
4. Kiet, T. T., Doerfelt H., Graefe U. (2004), ”New and
interesting macrofungi of Vietnam and their bioactive
compounds”, In: Makoto, M. W., Ken-ichiro, S., Tatsuji,
S., Innovative Roles of Biological Resource Centers, 169-
172, Chiba.
5. Kleinwaechter P., Anh, N., Kiet, T. T., Schlegel B.,
Dahse H. –M., Họrtel, A., Graefe U. (2001),
„Colossolactones, new triterpenoid metabolites from a
Vietnamese mushroom Ganoderma colossum“, J. Nat.
Prod. 64(2), 236-239.
6. Mizuno, T (1999), “The extraction and development
of antitumor-active polysaccharides from medicinal
mushrooms in Japan”, Int. J. Med . Mushrooms 1, 9-29.
7. Wasser, S. P. (2002), “Medicinal mushrooms as a
source of antitumor and immunomodulating
polysaccharides”, Appl. Microbiol. Biotechnol. 60, 258-
274.

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