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International Journal of Pharmacy and Pharmaceutical Science Research
Universal Research Publications. All rights reserved
ISSN: 2249-0337
Research Note
APPLICATION OF BRINE SHRIMP BIOASSAY FOR SCREENING CYTOTOXIC ACTINOMYCETES
S. SudhaKesavan1*, S .Vijayalakshmi3, S. Usha Nandhini1, M. Bhavani Latha2, M .Masilamani Selvam2.
1
2
Research Scholar, Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Chennai-600 119, India.
Associate Professor, Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Chennai-600 119, India.
3
M.Phil Scholar, Department of Biotechnology, Sathyabama University, Jeppiaar Nagar, Rajiv Gandhi Salai, Chennai-600 119, India.
*E-mail: [email protected]
Received 01 December 2011; accepted 04 December 2011
Abstract
Ethyl acetate extracts of ten Actinomycetes isolates were subjected to a bioscreening study to detect cytotoxic activity by the
brine shrimp lethality bioassay. Brine shrimp lethality test was conducted on each of the extracts at six different concentrations
31.25, 62.5, 125, 250, 500 and 1000 µg.ml1. The extracts of the actinomycete isolates PCL-1, SU1, SU13, and SU4 showed
IC 50 in < 500 µg/ml. The isolates SU2, SU5 and SU3 showed IC 50 in > 500 µg/ml and the isolates SU 6, SU 8, and SU 9
showed the IC 50 in > 1000 µg/ml. These results suggested that more specific bioassays should be encouraged on these
actinomycetes extracts in order to confirm these conclusions.
© 2011 Universal Research Publications. All rights reserved
Key words: Cytotoxic activity, Sreening, Brine Shrimp (Artemia salina)
1.
Introduction:
Microorganisms have shown to be an attractive source of
natural compounds for the pharmaceutical and other
industries. Far from the thousands of microorganisms which
have been found to produce antibiotics, of which 2 / 3 is
produced by the actinomycetes, and some actinomycetes are
also used to produce vitamins, enzymes, and also used for
sewage treatment. Actinomycete from marine environment
produces drugs for cancer, a major problem that people face
today. Chemotherapy is one of the main treatments used to
combat cancer. A great number of antitumor compounds are
natural products or their derivatives, mainly produced by
microorganisms. In particular, actinomycetes are the
producers of a large number of natural products with different
biological activities, including antitumor properties. 60% of
approved drugs are derived from natural compounds (1,2) and
many have been extracted from actinomycetes(3).
The in vivo lethality in a simple zoological organism, such as
the brine shrimp lethality test (BST), developed by(4) might
104
be used as a simple tool to guide screening and fractionation
of physiologically active plant extracts, where one of the
simplest biological responses to monitor is lethality, since
there is only one criterion: either dead or alive. This general
bioassay detects a broad range of biological activities and a
diversity of chemical structures. One basic premise here is
that toxicology is simply pharmacology at a higher dose, thus
if we find toxic compounds, a lower, non-toxic, dose might
elicit a useful, pharmacological, perturbation on a physiologic
system(5). However, it has been demonstrated that BST
correlates reasonably well with cytotoxic and other biological
properties(5). Brine shrimp have been previously utilized in
various bioassay systems. There have been many reports on
the use of this animal for environmental studies(6,7,8) screening
for natural toxins(9,10) and as a general screening for bioactive
substances in plant extracts(11).
Considering that a major challenge today is the discovery of
marine sources with promising activities and the isolation of
active principles, we have applied in this work the brine
shrimp test (BST) for general activity screening of several
extracts of actinomycetes from the costal sediments.
International Journal of Pharmacy and Pharmaceutical Science Research 2011; 1 (3) 104-107
for the assay.
2. Materials and Methods
2.1.Isolation and maintenance
2.5.3. Crude sample production
Thirty six actinomycetes were isolated from 6 marine In the present study, screening of anticancer activity was
sediment sample collected from Ennore, Muttukad, done using 10 isolates among 38 isolates. Crude sample for
Verampattinum For the isolation, starch casein agar with the Artemia lethality assay were produced by adopting the
addition of rifampicin(12) (2.5 mg/ml) and flucanozole (75 submerged fermentation technique in a rotary shaker using
mg/ml) was used to minimize bacterial and fungal ISP-2 medium described by (16). After l3 days of cultivation,
contaminations, respectively. The strain was sub-cultured the mycelia and culture filtrate were separated by
onto starch casein agar slant (medium with 50% sea water) centrifuging at 10,000 rpm for 30 minutes. The culture filtrate
incubated at 28°C for 5 - 7 days to achieve good sporulation was extracted with ethyl acetate at pH 5.
was preserved in 20% glycerol at -80°C.
2.5.4. Sample preparation
2.2. Taxonomy
Different concentrations of stock solutions were prepared by
Taxonomic characteristics of the isolate were determined by dilution with dimethyl sulphoxide (DMSO) so as to obtain six
cultivation on various media as described by(13). different concentrations 31.25, 62.5, 125, 250, 500 and 1000
Morphological characteristics were observed after incubation µg.ml1 of actinomycetes extracts. 10 Artemia naupli were
of the culture at 28°C for 14 days on Oatmeal agar (ISP 3). added into each with different concentration of extracts and
The carbon source utilization, NaCl tolerance, optimum pH,
0.2% of DMSO instead of extract as control. After 24 hrs,
optimum temperature for growth was determined as per the dead shrimp was counted using microscope. The percentage
ISP protocol (14).
of mortality was calculated as follows
2.3. Fermentation
% Mortality = Percentage of survival in the control A full grown slant culture of the isolates on starch casein
Percentage of survival in the treatment
agar (composition: 1.0% soluble starch, 0.03% Casein, 0.2%
The percentage of mortality was transferred to profit analysis
KNO3,0.2% NaCl, 0.005% K2HPO4, 0.002% CaCO3, software and the IC 50 was calculated.
0.001% FeSO4.7H2O and 2.0% agar) was transferred into 3. Results
Erlenmeyer flasks (2 x 250 ml) containing 50 ml of seed In the course of screening for cytotoxic actinomycetes, 36
medium (composition: 1.0% Soyabean meal, 1.0% corn steep actinomycetes were isolated from 6 different marine sediment
solids, 0.5% glucose and 0.5% CaCO3 with pH 7.0) and samples of Ennore, Muttukadu, Verampattinum. Fig: 1
incubated for 3 days at 28°C on a rotary shaker (220 rpm). A shows some of the isolated actinomycetes.
10% level of this inoculum was transferred into 200 ml of
production medium in 1 L EM flasks. The medium
composition is: 1.0% soyabean meal, 0.5% corn steep solids,
1.0% soluble starch, 0.5% glucose and 0.7% calcium
carbonate with pH 7.0. The inoculated production flasks were
incubated for 72 h at 28°C on a rotary shaker (220 rpm).
2.4. Extraction of cell free crude extracts
The culture broth was centrifuged at 4000 rpm for 10 min, at
10°C and clear culture supernatant was separated. It was
extracted twice with ethyl acetate and washed with 500ml
water at pH 7.0. The ethyl acetate layer was concentrated in
vacuum at 35°C to get the crude ethyl acetate extract.
2.5. Screening for anticancer activity
2.5.1. Artemia lethality assay
Figure: 1 Isolated Actinomycetes
The Artemia lethality assay was conducted according to(15)
with minor modifications. This procedure determines lethal The cultural characteristics of the strain are summarized in
concentrations of active compounds in brine medium. The Table 1. The isolates PCL-1 exhibits grey colored aerial
activities of a broad range of active compounds are mycelium, pale yellow substrate mycelium with extended
manifested as toxicity to the shrimp. This method is rapid, spiral spores on the aerial mycelium. The isolates SU-1 and
reliable and has been used for over thirty years in SU2 are with white aerial and butter white substrate
toxicological studies. A positive correlation exists between mycelium, the spore of the former is spiral and compact
spiral for the later. SU-3 and SU-4 shows grey colored aerial
brine shrimp lethality and human carcinoma.
mycelium with white colored substrate mycelium with spiral
2.5.2. Hatching the shrimp
Dried cysts of Artemia salina was incubated in natural sea and compact spiral spores respectively. The isolate SU5,
water (1g.1-1) at 28-300C under constant aeration for 48 hrs. SU6, and SU 8 shows grey colored aerial mycelium and
After 48 hours, the phototropic napulii were collected by yellow, grey, black colored substrate mycelium respectively.
pipette from the lighter side of the hatching chamber and used The isolate SU 13 shows flexuous spores on greenish white
International Journal of Pharmacy and Pharmaceutical Science Research 2011; 1 (3) 104-107
105
Table 1: The cultural characteristics of the selected actinomycetes isolated from marine sediments
ISOLATES
NUMBER
PCL 1
SU1
SU 2
SU 3
SU 4
SU 5
SU 6
SU 8
SU 9
SU 13
CULTURAL CHARACTERISTICS
ISP-3
Aerial
Substrate
Diffusible
mycelium
Mycelium
pigment
Pale Grey
Pale yellow
White
Butter White
White
Butter white
Grey
White
Grey
White
Grey
yellow
Grey
Grey
Grey
Black
Black
White
yellow
Greenish white Pale yellow
-
Melanin
Pigment
Black
-
Characteristics
Extended spiral spores on substrate mycelium
Spiral spores on substrate mycelium
Compact Spiral spores on substrate mycelium
Spiral s spores on substrate mycelium
Compact Spiral spores on substrate mycelium
Extended Spiral spores on substrate mycelium
Spiral spores on substrate mycelium
Compact spiral spores on substrate mycelium
Compact spiral spores on substrate mycelium
Flexuous spores on substrate mycelium
Table 2: The biochemical characteristics of the selected actinomycetes isolated from marine sediments
Test
Carbon
Utilization:
Glucose
Sucrose
Lactose
Fractose
D-Maltose
D-Galactose
Mannitol
Starch
PCL-1
SU1
SU2
SU3
SU4
SU5
SU6
SU8
SU9
SU 13
+
+/+
+
+/+
+
+
+
+/+
+
+/+
+
+/+/+
+
+
+
+
+
+
+/+/+
+/+
+/+
+/+
+
+
+
+
+
+
+
+
+
+
+/+/+/+/+
+
+
+/+
+/+
+
+
+
+
+
+/+
+
+
+/+
+
+/+
7--11
7--11
7--11
7--11
7--11
7—11
7-20
7-20
7-20
7-20
7-20
7-20
20-40
20-40
20-40
20-40
20-40
20-50
Optimum pH:
7--11
7-11
7--11
7--11
3,5,7,9,11
NaCl tolerance
7-20
7-20
7-10
7-20
(%)
0,2,5,7,10,20
Optimum
20-40
20-50
20-40
20-40
temperature:
10-50oC
+: presence of growth, +/-: partial growth, -: absence of growth
Table: 3 Brine Shrimp lethality assay of different concentrations of cell free extracts of actinomycetes
95 % CONFIDENCE LIMIT
ISOLATES
IC/LC 50
LOWER
UPPER
PCL 1
310.554
229.269
420.728
SU1
353.980
256.018
493.292
SU 2
613.383
416.349
1165.658
SU 3
912.893
576.561
3615.459
SU 4
377.679
271.089
534.695
SU 5
939.752
510.430
5890.654
SU 6
1145.285
785.169
176488.531
SU 8
1405.413
807.803
5749716.500
SU 9
1121.184
730.041
17431.066
SU 13
450.281
312.567
706.437
colored aerial mycelium with pale yellow substrate
mycelium. The melanin pigment was produced only by the
isolate SU4. A diffusible pigment was produced by the isolate
SU 8 only.
106
The biochemical characteristics of the strain are summarized
in Table 2. Starch was utilized by all the isolates. Mannitol
was utilized well by the isolate SU1, SU2, SU4, SU5, the
isolates SU2, SU6, SU8 and SU9 were not utilizing lactose.
International Journal of Pharmacy and Pharmaceutical Science Research 2011; 1 (3) 104-107
All the isolates were growing well at the optimum pH of 7-11
and the temperature of 20-400C. The isolates SU 1 and SU 13
were shows presence of growth at 500 C and all are tolerate 7
to 20 % of NaCl.
The extracts studied in this work showed significant lethality
against brine shrimp, the LC50 results of the ten
actinomycetes evaluated in this screening are listed in Table
3. The ethyl acetate extract of the isolates PCL-1, SU1, SU13,
and SU4 shows IC 50 in < 500 µg/ml. The isolates SU2, SU5
and SU3 showed IC 50 in > 500 µg/ml.
4. Discussion
The brine shrimp lethality assay is considered to be one of the
most useful tools for the preliminary assessment of
biotoxicity and the bioassay with cytotoxic activity against
some human solid tumors. Meyer (1982) reported that, if the
brine shrimp lethality assay displayed LC50 1000 μg/ml of
natural derived products was known to contain
physiologically active principles. The extracts studied in this
work showed significant lethality against brine shrimp, which
has been successfully used as a simple biological test to guide
the fractionation process of biological extracts in order to
detect antitumor compounds. The extracts of PCL-1, SU1,
SU13, SU4, SU2, SU5 and SU3 were showed LC50 values
less than <1000 μg.ml-1 than the other extracts, so this could
be used as a potential anticancer drug. These extracts can be
regarded as a promising candidate for a actinomycetes
derived antitumor compound. This bioassay has good
correlation with the human solid tumors cell lines (17).
However, further and more specific bioassays are necessary
in order to confirm these conclusions.
5. Conclusion
The biological product which showed > 1000 µg/ml
cytotoxicity can be regarded as a promising antitumor
compound. The present study reveals seven cytotoxic
actinomycetes, which showed > 1000 µg/ml cytotoxicity
against artemia salina among the ten tested isolates. These
organisms must be studied further for getting new antitumor
compounds.
Acknowledgment
The authors are grateful to the authorities of Sathyabama
University for providing necessary facilities.
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Source of support: Nil; Conflict of interest: None declared
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International Journal of Pharmacy and Pharmaceutical Science Research 2011; 1 (3) 104-107