Presently first two technologies used widely, but use the third is not gaining popularity in India in this field. The healthy use of standardized Traditional formulation will become generally acceptable in the country and World. Standardization of the products by biological assays will then generate reproducible benefits and the resulting consumer confidence. In addition, in such specific bioassays the same extracts have to be analyzed many times, over and over again, before detecting activities. It would seem more logical to prescreen with general bioassays, and then employ specific bioassays on the actives. Unfortunately, this is not the trend today within the major Traditional formulation manufacturer. The four bioassays that are described below are easily adapted as simplest procedures for standardization of Traditional formulations having crude plant material as ingredients.

They are inexpensive, rapid, and technologically simple, requiring little technical training ^{1, 2}.

1) Rapid General Bioassay for Ensuring Lethality of Traditional formulations: -Bioactive compounds are almost always toxic in high doses. Pharmacology is simply toxicology at higher dose, and toxicology is simply pharmacology at a lower dose. Thus, in vivo lethality in a simple zoologic organism can be used as a convenient monitor for ensuring the lethality of traditional formulations. The eggs of brine shrimp, Artemia salina (Leach), are readily available by supplier at low cost and remain viable for years in the dry state. Upon being placed in seawater, the eggs hatch within 48 hours to pro-vide large numbers of larvae (nauplii) for experimental use. Brine shrimp nauplii have been used pre-viously in a number of bioassay systems. Over 300 novel antitumor and pesticidal natural products have now been isolated in the laboratory using this bioassay as the prescreen . Thus, it is possible to detect and then monitor the fractionation of cytotoxic, (in vivo murine leukemia) active extracts using the brine shrimp lethality bioassay rather than more tedious and expensive in vitro and in vivo antitumor assays. The brine shrimp assay has advantages of being rapid (24 hours), inexpensive, and simple (eg, no aseptic techniques are required). It easily utilizes a large number of organisms for statistical validation and requires no special equipment and a relatively small amount of sample (2–20 mg or less). Furthermore, it does not require animal serum as is needed for cytotoxicities. Animal rights advocates have not yet objected to the use of these invertebrates in experimental work. In laboratories, each researcher conducts his/her own brine shrimp bioassays on his/her own bench. Self-reliance and rapid results are important advantages in this type of work ³.

Materials and Procedures for Brine Shrimp Lethality Bioassay

Materials :-

1. Artemia salina cysts, sea salt, small tank (hatching chamber) to grow shrimp with dividing dam, cover, and lamp to attract shrimp, syringes ( 5 ml, 0.5 ml, 100 mcl, and 10 mcl) and two dram vials (9 per sample + 1 control).

Method:-

1. Prepare seawater (38 g sea salt per liter of water), filter.

2. Put seawater in small tank, add shrimp eggs to one side of the divided tank, and cover this side. The lamp above the other side will attract the hatched shrimp.

3. Allow two days for the shrimp to hatch and mature as nauplii (in warmer climates, hatching may take place sooner).

4. Prepare vials for testing; for each fraction, test initially at 1000, 100, and 10 mcg/ml; pre-pare three vials at each concentration for a total of nine vials; weigh 20 mg of sample and add 2 ml of solvent (20 mg/2 ml); from this solution transfer 500, 50, or 5 mcl to vials corresponding to 1000, 100, or 10 mcg/ml, respectively. Evaporate solvent under nitro-gen and then put under high vacuum for about 30 min.; volatile solvents will evaporate over night. Alternatively, materials may be dissolved in DMSO (dimethylsulfoxide), and up to 50 mcl may be added per 5 ml of brine before DMSO toxicity will affect the results.

5. After two days (when the shrimp larvae are ready), add about 4 ml of seawater to each vial, count 10 shrimp per vial (30 shrimp per dilution), and adjust the volume with seawater to 5 ml/vial. Place the vials, uncovered, under the lamp. Be sure that the lamp does not overheat vials.

6. Twenty-four hours later count and record the number of survivors.

7. Analyze the data and determine LC50 values and 95% confidence intervals.

8. Additional dilutions at less than 10 mcg/ml may be needed to determine the LC50 values for potent materials; also, intermediate concentrations, eg, at 750, 500, and 250 mcg/ml can be prepared and tested to narrow the confidence intervals. By starting with 2 mg/ml (step 4 above) dilutions at 100, 10, and 1 mcg/ml are easily prepared for more potent materials ^{4, 5.}.

2) Yellow Fever Mosquito (YFM) Test: A Bioassay for Pesticides content

The yellow fever mosquito larvae micro plate assay (YFM) is a simple “bioassay can evaluate the pesticidal content of Traditional formulations and crude plant material. The eggs of the yellow fever mosquitoes, Aedes aegypti (Linnaeus), are stored at room tem-perature in a sealed container. Another small open jar with saturated zinc sulfate solution can be put in the container to maintain a relative humidity between 80–90%. After being hatched in warm water over-night, the larvae are allowed to develop in bovine liver powder solution for four days. The surviving larvae are then transferred to MES (2[N-morpholino] ethanesulfonic acid) buffer solution and used for the tests. Traditional formulations and crude plant and isolate tested in five, 1: 10, dilutions starting at 5000 mg/ml. After four days of incubation with test material, the surviving larvae are counted, and estimate LC50 values with 95% confidence intervals for statistically significant comparisons of potencies. The amounts of samples used in this assay are fairly small, 5–25 mg (corresponding to testing concentrations of 1000 mg/ml to 5000 mg/ml). Rotenone can be used as a positive control for the assay. Pure substances with LC50 values £1.0 mg/ml are worthy of commercial development ⁶.

Materials and method for the Yellow Fever Mosquito (YFM) Test

Materials:-

Aedes aegypti (yellow fever mosquito) eggs, MES (2-[N-morpholino] ethanesulfonic acid), bovine liver powder, 96 U and F microwell plates and lids, syringes( 10 ml, 50 ml, 100 ml), pipette ,Vials: 4 ml glass vials with septums (five for each sample tested), A glass vessel, a jar, or a 500 ml beaker for storing developed mosquito larvae

Method:-

1. Put YFM eggs in a small vial with 5–10 ml of warm water, let stand for 2–3 hours

2. Transfer the above contents into a jar or a beaker containing bovine liver powder solution at a concentration of ,4 mg/ml

3. Allow developing for four days

4. Harvest the live larvae with a pipette, transfer them into a jar or beaker that contains 5 mM MES pH 6.5 solution

5. Use a Vaccu-Pette/96 to fill 96-well microwell plate with 240 ml 5 mM MES solution per well

6. Prepare solutions for testing: to start with a concentration of testing material of 5000 ml/ ml, 25 mg of the test material is weighed and dissolved in 100 ml of methanol in a sealed vial; this testing solution (250 ml/ml) is then injected into eight wells on the micro liter plate with 5 ml each. The final concentration of the testing material in these eight wells will be 5000 mg/ml. Another 5 ml of this solution is injected into another vial and diluted with 45 ml of methanol to make a solution with a concentration of testing material of 500 mg/ml of methanol; further diluted solutions can be made in the same way. Each 96 well plate can hold two testing materials as shown below. The contents of the control wells are the same as the other wells except that 5 ml of pure methanol should be used instead of 5 ml of testing material in methanol

7. Use the motorized microliter pipette to add larvae in the wells, one larvae per well in as close to 10 ml as possible

8. Cover the plate and incubate in the dark at room temperature for four days. (Keep the environment humid to avoid drying the plate)

9. To score for dead larvae, tap the plates sharply with a pencil to induce movement

10. Analyze the mortality data and determine LC50 values and 95% confidence intervals.

3) The Potato Disc Bioassay for ant tumor compound

Materials:-

A). Preparation of Agrobacterium tumefaciens-Prepare growth medium by adding 0.5 g sucrose, 0.8 g nutrient broth, and 0.1 g yeast extract to 100 ml of water in a 250 ml flask. Plug the flask with cotton, cover with aluminum foil, and sterilize in an autoclave for 12 minutes. Allow the medium to cool and add 1 loop of Agrobacterium tumefaciens, from a storage culture on an agar slant, using sterile technique. Place the flask on a shaker for 48 hours below 30°C.

B). Items to prepare for experiment: Strips of parafilm, Four (4) mg of sample in a 2 dram (10 ml) vial, 1.5 g bacto-agar for each sample including control, Cover with aluminum foil: tweezers, cutter, 1.8 mm cork borer (size 13), and tray, One (1)-tube rack with 1 screw cap culture tube per sample including control , For each sample, 1 screw cap culture tubes containing 2 ml distilled water, Red-skinned potatoes (Solanum tuberosum) and bleach, Ethanol for disinfection, Three (3)-Petri dishes per sample and three (3) for the control ^{7, 8, 9}.

Method :-

1. Prepare 1.5% agar by adding 100 ml water per sample to step B.3

2. Sterilize by autoclave all things in steps B. 3–6 for 15 minutes

3. Wash potatoes with water and soak in bleach

4. Clean laminar flow hood with ethanol

5. Pour 20 ml sterilized agar solution per Petri dish and let cool

6. Dissolve 4 mg sample in 1 ml DMSO

7. Use 1 ml of DMSO as blank standard

8. Preparation of inoculums

a. add 1.5 ml water, 2.0 ml of the 48 hr bacterial culture, and 0.5 ml sample in DMSO to tube

b. Prepare control by replacing sample with 0.5 ml DMSO

c. Use sterile technique (flame and cap).

9. Take potato out of bleach, cut away ends, and bore out cylinders onto the sterile tray

10. Cut cylinders into discs and place 5 discs per Petri dish by gently pushing the discs into the agar using aseptic technique

11. Prepare at least 3 Petri dishes per sample and control

12. Add one drop (0.05 ml) of the prepared innoculum per disc

13. Seal the edge of each Petri dish with parafilm strips to prevent moisture loss during the incubation period; keep the dish level at all times to keep the inoculum on the tops of the discs

14. Keep in the dark at 27°C and count the tumors after 12 to 21 days

15. Calculate the percent inhibition of crown gall tumors as follows:

% Inhibition = (100 - Average number tumors of sample) x100/ Average number tumors of control

4) Frond Inhibition of Lemna (duckweed): A Bioassay for Plant Growth Stimulants and Inhibitors 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 filamentious root. Under normal conditions, the plants reproduce exponentially with buddings of daughter fronds from pouches on the sides of the mother fronds. A previous report by Einhellig et al. provided general guide-lines for developing a Lemna bioassay to screen a large number of plant extracts and chemical substances for their effects on plant growth. Single Lemna plants, consisting of three fronds (1 mother and 2 daughter fronds), are placed into two dram vials containing 2 ml of a special medium (E medium). By evaporation of volatile solvents, appropriate dilutions of test substances have been previously evaporated in the vials to deliver initial con-centrations 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 a plant growth chamber at 27– 29°C with 24 hours of fluorescent and incandescent light. After seven days the number of fronds are counted, and FI50 values (concentrations necessary to inhibit 50% of frond poliferation) or FP50 values (concentrations using 50% increase in proliferation of fronds) are determined 95% confidence intervals are also determined to ovide statistical relevance. The commercial need for such natural, biodegradable, herbicides and plant growth stimulants may meday be filled with natural products detected by this simple and convenient Lemna bioassay ¹⁰.

The material and method:- The Lemna Bioassay Modified for Plant Extracts and Compounds

Materials:-

1. Lemna minor prepared by aseptic technique described below, if contaminated, E Medium (about 80 ml per compound), syringes( 10 ml, 100 ml,1ml,2ml ), 2 dram vials (40 per compound), large glass container to hold vials; translucent plate glass to form a lid; stopcock grease to form a seal to avoid moisture loss, Growth chamber with temperature range of 27 to 29°C and 24 hours of fluorescent and incandescent lights

Method:-

1. Prepare inorganic medium E add KOH pellets to pH 5.5–6.0

2. Prepare vials for testing: 10 vials per dose (500, 50, 5 ppm, control)

a. Weigh 15 mg of compound and dissolve in 15 ml solvent

b. Add 1000, 100, and 10 ml solutions to vials for 500, 50 and 5 ppm. Allow solvent to evaporate overnight

c. Add 2 ml of E Medium and then a single plant containing a rosette of three fronds to each vial

3. Place vials in glass dish filled with about 2 cm water, seal container with stopcock grease and glass plate

4. Place dish with vials in growth chamber for seven days

5. Count and record number of fronds per vial on days 3 and 7

6. Analyze data as percent of control with ED50 (software),

500 ppm = 15 mg compound/15 ml solvent X1000 ml solution/2 ml E Medium

50 ppm = 15 mg compound/15 ml solvent X100 ml solution/2 ml E Medium

5 ppm = 15 mg compound/15 ml solvent X10 ml solution/2 ml E Medium

E MEDIUM

S.No.	Content	Quantity (mg/ liter)
01	KH₂PO₄	680
02	KNO₃	1515
03	Ca(NO₃)₂₄H₂O	1180
04	MgSO₄7H₂O	492
06	H₃BO₃	286
07	MnCl₂4H₂O	3.62
08	FeCl₃6H₂O	5.40
09	ZnSO₄7H₂O	0.22
10	CuSO₄5H₂O	0.22
11	EDTA	11.2

CONCLUSIONS: -

Now a days Traditional formulations are gaining popularity because of rigorous side effects and high cost of modern formulations. But due to the lack of scientific studies on traditional formulations the physicians do not recommend them freely. Such requirements attracted the researchers to develop the standardization parameters for traditional formulations. The current techniques like TLC, UV spectrophotometery, HPLC, HPTLC, and GC are employed for qualitative and quantitative determination of the chemical entities present in the formulation, but on the basis of these results, it is not possible to evaluate the biological efficacy and safety of the formulation. For assuring the safety of the traditional formulations use of the animal based models are widely used but these models are time consuming, tedious, costly with maintenance cost of animal house, feed and opposed now a days by various regulatory bodies for welfare of animals. With a lot of drawbacks manufacturers ignored to performing these type of studies, results in scientific destabilization of traditional formulations. Bioassay gives a unique remedy for assuring the safety by determining lethality and assurance for absence of pesticide residue from the traditional formulations by a economic and time saving methodology.

REFERENCES:-

1. Bhanu P.S. Sagar, R. Zafar, Rajiv Panwar, Vipin Kumar and Arun Mangla, Herbal drug standardization, The Indian Pharmacist, 2005, 19-22

2. Jerry l. Mclaughlin, and Lingling l. Rogers, The Use Of Biological Assays To Evaluate Botanicals, Drug Information Journal, 1998 Vol. 32, pp. 513–524,

3. 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.

4. McLaughlin JL, Chang C-J, Smith DL. Bench top bioassays for the discovery of bioactive natural products:An update. In: Atta-ur-Rahman, ed. Studies in Natural Products Chemistry. Amsterdam: Elsevier;1991;9:388–409.

5. McLaughlin JL. Crown-gall tumours in potato discs and brine shrimp lethality: Two simple bioassays for higher plant screening and fractionation. In: HostettmannK, ed. Methods in Plant Biochemistry. London:Academic Press; 1991;6:1–31.

6. Anonymous. Instructions for determining the susceptibility or resistance of mosquito larvae to insecticides. Wld Hlth Org Rep Ser. 1970;443:66–79.

7. 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.

8. Galsky AB, Wilsey JP, Powell RG. Crown-gall tumor disc bioassay: A possible aid in the detection of compounds with antitumor activity. Plant Physiol 1980;65:184–185.

9. Ferrigni NR, Putnam JE, Anderson B, Jacobsen LB, Nichols DE, Moore DS, McLaughlin JL. Modifica-tion and evaluation of the potato disc assay and antitumor screening of euphorbiaceae seeds. J NatProd. 1982;45:679–686

10. Einhellig FA, Leather GR, Hobbs LT. Use of Lemna minor L. as a bioassay on allelopathy. J Chem Ecol.1985;11:65–92.

Received on 23.04.2009

Accepted on 11.05.2009

Research Journal of Pharmacognosy and Phytochemistry. 1(1): July.-Aug. 2009, 01-04