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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: suchitra303@yahoo.com

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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