their pharmacological properties an

their pharmacological properties and therapeutic
features
(
3
)
. Many of these compounds are found in
marine organisms. The sea cucumber is an important
marin
e organism with commercial, pharmaceutical,
and food value. Sea cucumbers have traditionally
been used in China and Malaysia to treat
hypertension, eczema, and cancer
(
4
)
. The health
benefits of these animals are associated with the
presence of bioactive components such as saponins,
glycosaminoglycans, sterols, cerberosides, peptides,
sul
fated polysaccharides, and essential fatty acids
(
3
)
.
Saponins are important bioactive compounds in sea
cucumbers. Saponins are composed of triterpene or
steroid aglyco
nes (sapogenins) and sugar side chains,
and because of their amphiphilic nature have the
ability to generate stable foam and lyse blood cells
(
5
)
. Numerous studies have shown that saponins
have hemolytic
(
6
)
, antiproliferative
(
7
)
,
antimicrobial
(
8
)
, and antitumor
(
9
)
activities. Hu et
al. (2010), showed that saponins isolated from the sea
cucumber,
Pearsonothuria graeffei
can help to
alleviate fatty liver
(
10
)
. Tian et al. (2007), extracted a
previously unknown sulfated saponin, philin
opside
E, from sea cucumber and investigated its role in
angiogenesis and its effect on a breast cancer cell line
(
7
)
. Many studies have investigated the cytotoxic
effect of saponins extract
ed from sea cucumbers on
different cell lines. The cytotoxic effects of five
saponins: fuscocinerosides A, B, and C, pervicoside
C, and holothurin A, extracted from
Holothuria
fuscocinerea Jaeger
, on human leukemia HL
-
60 and
human hepatoma BEL
-
7402 cells,
were examined
and the results demonstrated that all these saponins
are strongly cytotoxic on both these cell lines
(
11
)
.
Despite numerous studies on the biological effects of
secondary metabolites extracted from various
species
of sea cucumbers, there is a little information about
the bioactive compounds isolated from sea
cucumbers in Iran. The aim of this investigation was
to extract saponins from the Persian Gulf sea
cucumber
H.leucospilota
and evaluate their
hemolytic
and cytotoxic properties.
Materials and Methods
Sampling
Sea cucumbers
)
H. leucospilota
(
were collected from
the coast of Qeshm
, Bandar Abbas, Iran. After
transfer to the laboratory, their body walls were
isolated and stored at
-
70 °C for further analysis.
Chemicals and cells
HP
-
20 resin and 3
-
(4, 5
-
dimethylthiazol
-
2
-
yl)
-
2, 5
-
diphenyltetrazolium bromide (MTT were purchased
from S
igma
-
Aldrich (USA) Company. Thin
-
layer
chromatography (TLC plates (silica
-
gel
-
60
-
F254),
ethanol, n
-
butanol, dichloromethane, and acetic acid
were purchased from Merck (Darmstadt, Germany).
All cell culture reagents were obtained from Gibco
(USA). A549 cell
s, a human lung cancer cell line,
were purchased from the Pasteur Institute of Iran,
Tehran.
Extraction of saponins from H.leucospilota
Saponins of
H.leucospilota
were extracted according
to the method described by Hu et al
(
10
)
. The body
walls were air
-
dried, powdered by grinding, and
stored in 70% ethanol at room temperature for two
days to release the temperature
-
sensitive compounds.
Then the saponins were refluxed in ethanol three
times for six hours. In next step, obtained e
xtract was
filtered by watman paper 1 μm and evaporated on a
rotary evaporator (Heidolph, Germany). The dry
extract was diluted in dichloromethane/water for 24
hours. Then, the water phase was extracted using n
-
butanol. Finally, the organic layer was evapo
rated,
dissolved in water, and loaded onto a Diaion HP
-
20
resin column. The column was washed with distilled
water to remove inorganic salts and eluted, first with
80% and then 100% ethanol, to separate saponin
compounds. The elutions were air
-
dried and
ly
ophilized to obtain dried, crude saponin extracts
(
10
)
.
Phytochemical screening
To analyze the n
-
butanol fraction for saponins, the
foam test was used. For this purpose, 1 mg of extract
was diluted in 10 ml of distilled water and shaken for
10 min.
Quantitative hem
olytic activity assay
The hemolysis test was performed using the method
described by Bondoc et al. (2013) (12). This test was
performed using blood from an adult woman with
O+ blood group. The blood was collected in tubes
containing ethylenediaminetetraace
tic acid as an
anticoagulant and pelleted by centrifugation at 800 ×
g for 15 min to remove the plasma. The pellet
containing erythrocytes was washed three times in
phosphate
-
buffered saline (pH=7) (PBS) and

Hasil (Bahasa Indonesia) 1:

sifat Farmakologi dan terapeutikFitur(3). Banyak senyawa ini ditemukan diorganisme laut. Laut mentimun adalah pentingMarine organisme dengan komersial, farmasi,dan nilai makanan. Teripang secara tradisionaltelah digunakan di Cina dan Malaysia untuk mengobatihipertensi, eksim, dan kanker(4). Kesehatanmanfaat dari hewan-hewan ini terkait dengankehadiran komponen bioaktif seperti saponin,glikosaminoglikan, sterol, cerberosides, peptida,Sulditakdirkan polisakarida dan asam lemak esensial(3).Saponin yang senyawa bioaktif yang penting di lautmentimun. Saponin yang terdiri dari triterpin atausteroid aglycoSPN (sapogenins) dan gula sisi rantai,dan karena mereka ambifilik alam memilikikemampuan untuk menghasilkan busa stabil dan melisiskan sel-sel darah(5). Sejumlah penelitian telah menunjukkan bahwa saponinmemiliki hemolitik(6), antiproliferative(7),antimikroba(8), dan antitumor(9)kegiatan. Hu etAl. (2010), menunjukkan bahwa saponin terisolasi dari lautmentimun,Pearsonothuria graeffeidapat membantumengurangi lemak hati(10). Tian et al. (2007), diambilsebelumnya tidak diketahui sulfated saponin, philinopsideE, dari Laut mentimun dan diselidiki perannya dalamangiogenesis dan efeknya pada garis sel kanker payudara(7). Banyak studi telah diselidiki sitotoksikEfek ekstrak saponinEd dari Teripang padabaris sel yang berbeda. Efek sitotoksik limasaponin: fuscocinerosides A, B, dan C, pervicosideC, dan holothurin A, diekstrak dariHolothuriafuscocinerea Jaeger, pada manusia leukemia HL-60 danmanusia hepatoma BEL-sel-sel 7402,Diperiksadan hasilnya menunjukkan bahwa semua saponin inisangat sitotoksik pada kedua baris sel ini(11).Meskipun banyak penelitian tentang efek biologisMetabolit sekunder yang diambil dari berbagaispesiesLaut mentimun, ada sedikit informasi tentangsenyawa bioaktif yang terisolasi dari lautmentimun di Iran. Tujuan penyelidikan ini adalahuntuk mengekstrak saponin dari laut Teluk PersiamentimunH.leucospilotadan mengevaluasi merekahemolitikdan sifat sitotoksik.Bahan dan metodeSamplingTeripang)H. leucospilota(dikumpulkan dariPantai Qeshm, Bandar Abbas, Iran. Setelahtransfer ke laboratorium, tubuh mereka dindingnyaterisolasi dan disimpan di-70 ° C untuk analisa lebih lanjut.Bahan kimia dan selHP-20 resin dan 3-(4, 5-dimethylthiazol-2-YL)-2, 5-diphenyltetrazolium bromida (MTT dibelidari Sigma-Perusahaan Aldrich (USA). Tipis-lapisanKromatografi (TLC piring (silika-gel-60-F254),etanol, n-butanol, diklorometana dan asam asetatdibeli dari Merck (Darmstadt, Jerman).Semua sel budaya reagen Diperoleh dari Gibco(USA). A549 sels, garis sel kanker paru-paru manusia,dibeli dari Institut Pasteur di Iran,Teheran.Ekstraksi Saponins dari H.leucospilotaSaponinH.leucospilotadiambil menurutuntuk metode yang dijelaskan oleh Hu et al(10). Tubuhdindingnya udara-kering, bubuk dengan menggiling, dandisimpan dalam 70% etanol pada suhu kamar untuk duahari untuk melepaskan suhu-senyawa-senyawa yang sensitif.Kemudian saponin yang refluxed di etanol tigakali selama enam jam. Pada langkah berikutnya, memperoleh extract adalahdisaring oleh watman kertas 1 μm dan menguap padarotavapor (Heidolph, Jerman). Keringekstrak diencerkan dalam air diklorometana 24jam. Kemudian, fase air diambil menggunakan n-butanol. Akhirnya, lapisan organik adalah evapodinilai,dilarutkan dalam air, dan dimuat ke Diaion HP-20kolom resin. Kolom dicuci dengan sulingair untuk menghilangkan anorganik garam dan eluted, pertama dengan80% dan kemudian 100% etanol, untuk memisahkan saponinsenyawa. Elutions adalah udara-kering danTCophilized untuk mendapatkan kering, kasar saponin ekstrak(10).Skrining fitokimiaUntuk menganalisis n-Fraksi butanol untuk saponin,tes busa digunakan. Untuk tujuan ini, 1 mg ekstrakdiencerkan dalam 10 ml air suling dan terguncang untuk10 min.Kuantitatif hemolytic aktivitas assayTes hemolisis dilakukan menggunakan metodedijelaskan oleh Fals et al. (2013) (12). Tes inidilakukan dengan menggunakan darah dari wanita dewasa denganO + darah group. Darah dikumpulkan dalam tabungyang mengandung ethylenediaminetetraaceTic asam sebagaiantikoagulan dan pelleted oleh sentrifugasi di 800 ×g untuk 15 menit untuk menghapus plasma. Peletyang mengandung eritrosit dicuci tiga kalifosfat-buffered saline (pH = 7) (PBS) dan