Biological Methods for Estimation of Botanicals: An Overview

 

Akshay D. Sapakal*, Shweta B. Surve, R.S. Adnaik

Rajarambapu College of Pharmacy, Kasegaon Tal:-Walva Dist:-Sangli M.S. India

*Corresponding Author E-mail:- akshay.sapakal@gmail.com

 

 

ABSTRACT:

Botanical products are “heterogeneous” due to the presence of mixture of bioactive plant components. Physical analytical are usually insensitive to the chemical complexities found in crude botanical extracts. Also physical or chemical analysis of a single component in such mixtures is not completely satisfactory.  The goal of many phytochemists has been simply to isolate, characterize, and publish a plethora of novel botanically-derived chemical substances without regard to bioactivities. To achieve applied meaning and significance, today’s work in natural product chemistry must incorporate bioassays. Extracts must be screened for biological activity, the “active” extracts selected, fractionations directed with bioassays, and the bioactive compounds identified and then exploited. We have made an attempt to summarize different bioassays (Brine Shrimp Lethality, Crown Gall Tumors on Potato Discs, Yellow Fever Mosquito Test, The Lemna Bioassay) which are useful in the detection of biologically active components of botanical extracts.

 

KEYWORDS: Bioassay, Crown gall tumours; Duckweed; Mosquito larvae; Annonaceous,Brine shrimp

 

 


INTRODUCTION:

Bioassay offers a special advantage in the standardization and quality control of heterogeneous botanical products. They are inexpensive, rapid and technologically simple, requiring less technical training as compared to the various physical and chemical analytical methods. Physical analytical methods, such as chromatography, are useless for this purpose as they are insensitive to the chemical complexities found in 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, physical or chemical analysis of a single component in such mixtures is not completely satisfactory. The authors have adopted four “bench top” bioassays to aid “drug discovery” work with botanicals. They have used these methods over the past 15 years, and they appear to be adaptable to the purpose of standardization or quality control of bioactive components in such heterogeneous botanicals.

 

Unfortunately the role of phytochemists has been simply to isolate, characterize and publish plethora on novel botanically derived chemical substances without regard to bioactivities. To achieve applied meaning and significance, today’s work in natural product chemistry must incorporate bioassay. The four bioassays which are described below are easily adapted as “bench top” procedures for use in natural product. They are inexpensive, rapid, and  simple, requiring little technical training.

 

1.     Brine Shrimp Lethality: A Rapid General Bioassay for Bioactive Compounds1,2

Bioactive compounds are almost always toxic in 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 zoo logic organism can be used as a convenient monitor for screening and fractionation in the discovery and monitoring of bioactive natural products. The eggs of brine shrimp, Artemia salina (Leach), are readily available in pet shops at low cost and remain viable for years in the dry state. Upon being placed in sea water, the eggs hatch within 48 hours to provide large numbers of larvae (nauplii) for experimental use ( Fig.1). Brine shrimp nauplii have been used previously in a number of bioassay systems, but, the authors have developed a method wherewith mechanism-based assays3.

 

Where by natural product extracts, fractions, or pure compounds are tested at initial concentrations of 10, 100, and 1000 ppm (or mcg/ ml) in vials containing 5 ml of brine and 10 shrimp in each of three replicates (1,2). Survivors are counted after 24 hours. These data are processed in a simple program for probit analysis on a personal computer to estimate LC50 values with 95% confidence that transforms normal, wounded, plant cells intervals for statistically significant comparisons of potencies. The authors initially found positive correlation between brine shrimp toxicity and 9KB (human nasopharyngeal carcinoma) cytotoxicity (p =0.036 and kappa=L.) and now use the brine shrimp test as a pre-screen for a panel of six human solid be active in the cell lines2,3.

 

Applications:-

·      Brine shrimp lethality assay used as a pre screen for a panel of six human solid tumour cell lines at cell culture laboratory.

·      For detection and then monitoring fractionation of cytotoxicity.

 

Brine shrimp lethality assay having advantages such as inexpensive, rapid (24 hrs), simple and it does not requires any special instrument.

 

Fig.1:-Brine shrimp bioassay

 

2.     Crown Gall Tumors on Potato Discs: An Animal-Sparing Bioassay for Antitumor Compounds4.

Crown gall is a neoplastic disease of plants induced by specific strains of the Gram negative bacterium, Agrobacterium tumefaciens (Smith and Townsend) Conn. The bacteria contain large Ti (tumour-inducing) plasmids which carry genetic information (T-DNA) that transforms normal, wounded, plant cells into autonomous tumour cells. Inhibition of crown gall tumor on discs of potato (Solanum tuberosum L.) tubers showed an apparent correlation with compounds and plant extracts known to be active in the 3PS (in vivo, murine leukaemia) antitumor assay4. The modified assay was initially performed on a series on natural antitumor compounds, on plant extracts suspected to have 3PS activity and on ethanol, hexane extract seeds of Euphoriacea species; inhibition of 20% or more of the tumours are considered significant (Fig.2)

 

Application:-

·      Pre screen for in vivo 3PS antitumor activity

 

Crown gall tumor assay generally used as it is inexpensive, simple, safe, animal sparing and statistically reliable.

 

Fig.2:- Crown Gall Tumors on Potato Discs

 

3.     Frond Inhibition of Lemna (duckweed): A Bioassay for Plant Growth Stimulants and Inhibitors3

Lemna minor L. (duckweed) is a miniature aquatic monocot. Lemna plants consist of a central oval frond or mother frond with two attached daughter fronds and a filamentous root. Under normal conditions, the plants reproduce exponentially with buddings of daughter fronds from pouches on the sides of the mother fronds.

 

Single lemna plants consisting of 3 fronds (1 mother and 2 daughter fronds) are placed in to two dram vials containing 2 ml of special medium (E medium). By evaporation of volatile solvents, appropriate dilutions of test substances have been previously evaporated in the vials to deliver initial concentration of 500, 50 and 5 ppm in the medium. The vials are placed in translucent, glass-covered dishes, to avoid moisture loss, and placed in plant growth camber at 27-290c with 24 hours of fluorescent and incandescent light. After 7 days the no. of fronds are counted and FI50 values (concentrations necessary to inhibit 50% of fronds proliferation) or FI50 value (concentrations causing 50% increase in proliferation of fronds) are determined using FENNY program.

 

Application:-

Various natural, biodegradable herbicides and plant growth stimulants may be filled with natural products detected by this bioassay.

                

4.     Yellow Fever Mosquito (YFM) Test: A bioassay for pesticides1,2

The yellow fever mosquito larvae microtiter plate assay (YFM) is a simple “bench top” bioassay that can be used to determine the pesticidal activities of botanical extracts, fractions, and isolated compounds. The eggs of the yellow fever mosquitoes, Aedes aegypti (Linnaeus), are stored at room temperature in sealed container .Another open jar with saturated zinc sulphate solution can be put in the container to maintain relative humidity between 80-90%.

 

After being hatched in warm water overnight, the larvae allowed to develop in bovine liver powder solution for four days. The surviving larvae then transferred to MES (2-[N-morpholino] ethanesulphonic acid) buffer solution and used for test. Natural product extracts, fractions or pure compounds are tested in five, 1:10, dilutions starting at 5000µg/ml. After four days of incubation with test material surviving larvae are counted and  the data can be processed in simple probit analysis program to estimate LC50 values.

 

CONCLUSION:

We have made an attempt to summarize different bioassays (Brine Shrimp Lethality, Crown Gall Tumors on Potato Discs, Yellow Fever Mosquito Test, The Lemna Bioassay) 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.

 

Fig.3:- Frond Inhibition of Lemna (duckweed)


 

REFERENCES:

1.     Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med. 1982; 45:31–34.

2.     McLaughlin JL. Crown-gall tumours in potato discs and brine shrimp lethality: Two simple bioassays for higher plant screening and fractionation. In: Hostettmann K, Ed Methods in plant biochemistry: London Academic Press; 1991; 6:1–31.

3.     McLaughlin JL, Chang C-J, Smith DL. Simple bench-top bioassays (brine shrimp and potato discs) for the discovery of plant antitumor compounds Human Medicinal Agents from Plants. Washington, DC; ACS Symposium Series 534, American Chemical Society; 1993:112–134.

4.     Galsky AB, Kozimor R, Piotrowski D, Powell RG. The crown-gall potato disc bioassay as a preliminary screen for compounds with antitumor activity. J Nat Cancer Inst. 1981; 67:689–692.

 

 

 

 

Received on 11.02.2013          Modified on 28.02.2013

Accepted on 13.04.2013         © RJPT All right reserved

Research J. Pharm. and Tech 6(6): June 2013; Page 603-606