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ISSN:2230-7346 Available online http://WWW.JGTPS.COM Review Article Journal of Global Trends in Pharmaceutical Sciences
Vol.2, Issue 1, pp 79-90, January–March 2011
BENCH TOP BIOASSAYS - AN ANIMAL SPARING BIOASSAY
FOR ANTI-TUMOR COMPOUNDS
E. Susithra1, K.V. Ramseshu2, S.Meena2, V.G. Veni1
1. Annamacharya College of Pharmacy, New Boyanapalli, Rajampet-516126, A.P, India
2. K.M Colleges of Pharmacy, Uthangudi, Madurai-625 107, Tamil Nadu, India.
*Corresponding author E-mail: [email protected]
ABSTRACT
Nature’s bioactive compounds are being ignored and are being washed down the
drain by many of today’s phytochemists. The discovery of the useful bioactivities of the natural
product compounds only awaits their detection through screening of the extracts and fractions by
the proper bioassays – the extracts must be screened for biological activity, the “active” extracts
selected, fractionations directed with bioassays and the bioactive compounds identified and then
exploited. Hence the present study emphasizes on the development and application of “bench-
top” bioassays – a simple, yet effective, rapid, inexpensive, safe, animal sparing and statistically
reliable prescreen for isolating numerous novel, bioactive compounds that have potential
applications as anti-tumor agents, pesticides, herbicides and plant growth stimulants or can serve
as lead compounds for synthetic modifications. These bioassays are: brine shrimp lethality, the
inhibition of crown gall tumors on potato discs, monitoring of frond proliferation in Lemna
minor or duckweed and the Ames test for chemical mutagenicity.
KEY WORDS: Bench top bioassay, Anti-tumor compounds, Ames test, Lemna minor
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INTRODUCTION:
Bioassays offer a special advantage in the
standardization and quality control of
heterogeneous botanical products. Physical
analytical methods, such as chromatography,
are useless for this purpose as they are
usually insensitive to the chemical
complexities found in the crude botanical
extracts. Most often a desired biological
response is due to not one but a mixture of
bioactive plant components and the relative
proportions of single bioactive compounds
can vary from batch to batch while the
bioactivity still remains within tolerable
limits. Thus three technologies must be
combined namely:
1. Separation techniques
(chromatography),
2. Structural elucidation methods
(spectrometers, X-ray
crystallography), and
3. Simple bioassays
Today’s natural product chemists are very
familiar with the first two, but usually ignore
the third. Thus the four bioassays which are
described below are easily adapted as
“bench top” procedures for use in natural
product chemistry.
METHODS:
I. BRINE SHRIMP LETHALITY-
A Rapid General Bioassay for Bioactive Compounds
Bioactive compounds are almost
always toxic at high doses. Pharmacology is
simply toxicology at a lower dose and
toxicology is simply pharmacology at a
higher dose. Thus, in vivo lethality in a
simple zoologic organism can be used as a
convenient monitor for screening and
fractionation in the discovery and
monitoring of bioactive natural products.
What is Brine Shrimp?
Brine shrimp Artemia species
(Figure-1), also known as sea monkeys are
marine invertebrates about 1mm in size.
Freeze dried cysts are used in prawn
hatcheries. The common brine shrimp is
included in the phylum Arthropoda, class
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Crustacean. The cysts remain dormant for
several years, they are metabolically active
after incubation in artificial sea water (figure
3) for 20-24 hrs. The equipment to grow
these nauplii (Figure-2) is very simple, a
tank with compartments and a light source.
The most important is water quality and
oxygen content. The eggs of brine shrimp,
Artemia salina (Leach), are readily available
in pet shops at low cost and remain viable
for many years in the dry state.
Figure: 1 -Brine shrimp Figure: 2 -Nauplii
Figure: 3-Brine shrimp in sea water
Purpose: The applications of the assay are
considered useful in preliminary assessment
of toxicity and have been used for the
detection of fungal toxins in plant extract
toxicity, animal and fish feeds. It was first
proposed by Michael et al. (1)
The brine shrimp lethality assay
represents a rapid, inexpensive and simple,
reliable bioassay for assessing the
bioactivity of medicinal plants towards brine
shrimp and in doing so, predicts the
cytotoxic and anti-tumor properties of plant
materials. A brief summary of the
methodology is given below:
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Procedure:
The eggs of brine shrimp, Artemia
salina (Leach), were placed in seawater, the
eggs hatched within 48 hours providing
large numbers of larvae (nauplii) for
experimental use. The natural product
extracts, fractions or pure compounds were
tested. At initial concentrations of 10, 100
and 1000 ppm (or g/ml) in vials containing
5 ml of brine and 10 shrimp in each of 3
triplicates. Survivors were counted after 24
hours. These data were then processed in a
simple program on a personal computer to
estimate LC50 values with 95% confidence
intervals for statistically significant
comparison of potencies. A number of novel
anti-tumor natural products have now been
isolated using this bioassay by the method of
McLaughlin.(2) Recently, the brine shrimp
lethality of extracts of around 120 medicinal
plants used in Indian traditional medicine
was determined using the procedure of
Krishnamraju A.V et al.(3) .Thus, it is
possible to monitor fractionations of
cytotoxic and 3PS active extracts using the
brine shrimp lethality bioassay rather than
the more tedious and expensive in vivo and
in vitro anti-tumor assays (Table No:1).
Applications:
Table: 1 -Brine shrimp bioassay Results of plant extract of Meliaceace Plant Part used Fraction LC (µg/ml)Azadirachta indica Stem bark Crude extract 158.18
Hexane fraction >1000 90%Methanol fraction 181.5 Water fraction >1000
Leaf Crude extract >1000
Azadirachta indica var. siamensis Bark Crude extracts 133.28 Hexane fraction 741.74
90%Methanol fraction 29.99 Water fraction 729.16
Crude extract >1000Sandoricum indicum Stem bark Crude extract 234.08
Hexane fraction 48.89 90%Methanol fraction 56.03 Water fraction 796.99
Crude extract 724.28
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II. CROWN GALL TUMORS ON POTATO DISCS –
An Animal-Sparing Bioassay for Anti-Tumor Compounds :
Crown gall is a neoplastic disease of plants
(Figure 4) induced by specific strains of
Gram negative bacterium, Agrobacterium
tumefaciens containing Ti plasmids which
transforms normal, wounded plants to
autonomous tumor cells.
Figure: 4-Crown gall disease in plant Figure: 5-Agrobacterium tumefaciens
Agrobacterium tumefaciens (Figure
5) is a Gram negative soil bacterium(4) has
worldwide distribution by Furuya et al.,
2004 (5). It causes crown gall tumors on a
wide range of plants including most dicots,
some monocots and some gymnosperms by
Matthysse, 2006(6). The crown gall tumor-
inducing capability of A. tumefaciens
requires the presence of a large plasmid,
designated the Ti plasmid by Onyesom,
2006(7). During the course of infection, the
bacterium transfers a specific portion (T-
DNA) of Ti plasmid to a host cell, which
then integrates itself into the host genome by
Winans, 1992(8). After its integration into the
plant genome, the T-DNA genes encode
enzymes responsible for the uncontrolled
synthesis of the plant hormones auxin and
cytokinin which account for the appearance
of abnormal tissue proliferation and gall
formation on the crown, roots and in some
cases on stems by Rhouma et al., 2006(9).
The DNA transmission capabilities of
Agrobacterium have been discovered by
Schell and Van Montagu., 1977(10) and
development of methods to alter
Agrobacterium into an efficient delivery
system for gene engineering in plants and
makes it of great concern to the agriculture
by Moore et al., 1997(11). Under laboratory
conditions the T-DNA has also been
transferred to human cells, demonstrating
the diversity of insertion application by
Kunik et al., 2001(12). The mechanism by
which Agrobacterium inserts materials into
the host cell by a type IV secretion system,
is very similar to mechanisms used by
animal pathogens to insert materials (usually
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proteins) into human cells also by type IV secretion by Lai and Kado, 2000(13).
Procedure:
The crown gall potato disc bioassay (Figure
6) is a simple bioassay which involves
inhibition of crown gall tumors on discs of
potato tubers – materials that inhibit these
plant tumors have a high predictability of
showing activity against the P388 (3PS)
leukemia in mice. A modified procedure of
the Galsky et al was given by Crocker(14)
tested on the ethanol and hexane extracts of
41 Euphorbiaceae seeds. The noculums was
prepared under sterile conditions using
strains of Agrobacterium tumefaciens two
days before the assay. Potatoes were taken
and cylinders bored into trays. The cylinders
were cut into discs and 5 discs placed per
petridish. A drop of Inoculums was added to
each disc, the discs wrapped and kept in
dark at 270C. The tumors were counted after
12 to 21 days and the percentage inhibition
of crown gall tumors calculated. Statistical
relationships were assessed by (1) four-fold
tables (2) the Fisher-Irwin test for
significance of association and (3)
calculation of kappa values to indicate the
degree of data agreement.
Figure: 6-Crown Gall Potato Disc
Applications:
Thus Agrobacterium has become an
important topic of medical research as well.
Besides, it plays a vital role in aspect of anti-
tumor studies (15, 16).A variety of sample
(figure 7, 8, 9, 10) consisting of purified
compounds (of various origins) and ethanol
extracts from plants were analyzed for their
activity on the growth and initiation of
crown-gall tumors on potato discs (Galsky et
al16) ). The results demonstrated a high
correlation between the ability of these
compounds to inhibit the initiation and
growth of crown gall tumors on potato discs
and their corresponding activity on the
mouse P388 leukemia protocol.
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Figure : 7-Pictorial representation of Fagonia cretica with its leaves, fruits and inflorescence
Figure: 8-Effect of the extract of aerial parts of Fagonia cretica on three tumor-producing strains of Agrobacterium tumefaciens
Figure: 9-Effect of the extract of aerial parts of Fagonia cretica on the viability of Agrobacterium
tumefaciens (At10) strain. C = Cefixime-USP; R = Roxycithromycin
Figure: 10 shows tumor forming ability of selected isolates on potato disc.
III. FROND INHIBITION of Lemna (duckweed) – A SIMPLE BIOASSAY FOR
INHIBITORS AND PROMOTORS OF PLANT GROWTH
Lemna minor.Leach - Duckweed
(figure 11) is a miniature aquatic monocot
consisting of mother frond with two
daughter fronds and a filamentous root
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which under normal conditions reproduce
exponentially with budding of daughter
fronds from punches on the sides of mother
fronds. Lemna minor.Leach - Duckweed
(figure 11) is a miniature aquatic monocot
consisting of mother frond with two
daughter fronds and a filamentous root
which under normal conditions reproduce
exponentially with budding of daughter
fronds from punches on the sides of mother
fronds. Monitoring of the frond proliferation
in Lemna minor provides a basis for
herbicides (including allelochemicals) and
plant growth stimulants.
Figure: 11-Lemna minor.Leach (Duckweed)
The general guidelines for developing Lemna bioassay has been provided from the earlier reports
of Einhellig et al(18) and Nishitoba et al(19). A brief summary of the assay is given below:
Procedure:
Single Lemna plants consisting of three
fronds were placed into vials containing E
medium. Test substances at initial
concentrations of 500, 50 and 5 ppm were
delivered into the medium and the vials
placed in plant growth chamber at 270C-
290C with 24 hour of fluorescent and
incandescent light. After seven days, the
number of fronds counted and FI50 values or
FP50 values determined using a Finney
program on an IBM personal computer, 95%
confidence intervals also determined to
provide statistical relevance.
Applications:
Using the Lemna assay, a blind study
with ten compounds supplied by NCI
(National Cancer Institute) was performed
by McLaughlin group(20) and the results
showed potent herbicidal and growth
stimulating properties, thus fulfilling the
commercial need for such natural,
biodegradable herbicides and plant growth
stimulants.
IV. THE AMES TEST
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The test was developed in 1975 by Bruce
Ames and his colleagues at The University
of California at Berkeley (21). The Ames
method is based on inducing growth in
genetically altered strains of the bacterium
Salmonella typhimurium. To grow, the
special strains need the amino acid histidine.
However, when the chemical agent
(mutagen) that is being studied is given to
bacteria, some of the altered Salmonella
undergo mutations. Following a particular
type of mutation, the bacteria can grow like
the original “wild” (unaltered) strains
without histidine. Because the mutant
bacteria revert to their original character
with regard to the nutrient histidine, they are
called “revertants”(22).Reverse mutants to
histidine independence are scored by
growing Salmonella typhimurium on plates
deficient in histidine in the presence of the
chemical (test) and in its absencen (control).
The Ames test is an exquisitely sensitive
biological method for measuring the
mutagenic potency of chemical substances.
The Ames test by itself does not
demonstrate cancer risk; however,
mutagenic potency in this test does correlate
with the carcinogenic potency for many
chemicals in rodents.The more mutations the
agent caused in the bacteria, the more likely
it is for some of them to reverse the original
mutation, that is, the mutation occurs exactly
in the right place to allow the bacteria to
produce histidine again by themselves. The
number of surviving bacteria colonies thus
corresponds with the strength of the
mutagen. (23) A large number of compounds
have been screened and their carcinogenicity
or lack of it was established.
CONCLUSION:
Four simple “bench top” bioassays are
described which are useful in the detection
of biologically active components of
botanical extracts. The bioassays permit the
convenient and rapid evaluation of various
plant parts, ontogenic and seasonal
variations within individual plants and
highly bioactive genotypes within the
infraspecific variations. In cases where
crude botanical extracts containing mixtures
of bioactive compounds, will be effective,
these simple procedures may permit their
standardization. Subsequent analyses with
liquid chromatography/mass spectrometry
(LC/MS/MS) can quickly confirm that the
activities are due to desired components.
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FUTURE PERSPECTIVES:
It is our attempt to bring these novel assays
into focus of biotechnologists for
improvements and also to fund in training of
scientists. Refinements to the techniques and
development of more responsive models can
widen the scope of drug discovery
particularly anti-proliferative diseases. Some
of the suggested refinements can be in
creating transgenic brine shrimp to be more
specific to mammalian systems. Recently it
has reported the DNA dependent RNA
polymerase A and Ouabain sensitive
ATPase of brine shrimp nauplii are similar
to mammalian type. Compounds or extracts
related to diseases associated with enzyme
can be screened. Finally, further refinements
may provide more reliability and evidence in
extrapolating animal data to human systems,
which, at present, is a major concern for the
regulatory agencies in granting drug
approvals.
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