Brine Shrimp Lethality Test of Andrographis paniculata

 

Mamatha A.*

 

ABSTRACT:

Brine Shrimp Lethality Test is a simple, reliable, rapid, cost effective bioassay for screening of biological activity and also to determine the cytotoxicity of sample under study. The test determines LC₅₀ values in µg/ml for the samples. Ten napulii were added into three replicates of each concentration of the plant extracts. Surviving brine shrimp larvae were counted after 24 hours and LC₅₀ was assessed. Results of five extracts of Andrographis paniculata subjected to electron beam irradiation at doses of 0, 2.5, 5, 7.5 and 10kGy showed that all samples possess significant biological activity and there was no cytotoxicity. This clearly indicates that, the doses at which the samples were irradiated are themselves not causing any further toxicity to the samples and thus is safe.

 

 

1. INTRODUCTION:

Andrographis paniculata, commonly known as Kalmegh is widely used in Indian and Chinese system of medicine for its wide range of pharmacological activity¹. The whole plant is used for its  hepatoprotective, anti-inflammatory, febrifuge, anthelmentic, antidiaarrhoeal, antimalarial, antiviral, antipyretic, antiatherosclerotic, hypotensive, immunomodulator , antifertility activity ^2-3. As these naturally obtained herbs are more prone to microbial contamination, herbal formulations prepared from them also end up being a low quality, microbially contaminated, less efficacious product^4-9. In order to improve the quality of such products, several techniques are used and irradiation by electron beam is one among them. Ionizing radiation, preferably treating with electron beam is considered to be most suitable technique for microbial decontamination of medicinal plants. Samples of Andrographis paniculata are irradiated at different doses of 0, 2.5, 5, 7.5 and 10kGy in order to find out the suitable dose required to make the product microbially free. But the skepticism which prevails in using this type of irradiation, is the toxicity caused due to them.

Brine Shrimp Lethality Test (BSLT) is a simple, sensitive, reliable, economical and easy method for determining the cytotoxicity of plant extracts, fractions or pure compounds.

 

This method also helps to determine, if there is any additional cytotoxicity caused due to electron beam irradiation. Samples are tested at concentrations of 10, 100 and 1000 µg/ml in brine solution. Shrimps are put and survivors are counted after 24 hours. The data obtained is statistically analyzed and LC₅₀ is determined to find out the extent of cytotoxicity. In this study, samples of Andrographis paniculata are subjected to BSLT to ensure that the biological activity is retained after irradiation and also to confirm that there is no cytotoxicity caused due to irradiation.

 

2.   MATERIALS AND METHODS:

2.1. Plant materials:

Whole plant of Andrographis paniculata was procured from Natural remedies, Bangalore.  The herb was identified and authenticated at Department of Horticulture GKVK, Bangalore and voucher specimen deposited.  They were further ground to powder and kept at ambient temperature.

 

2.2     Preparation of extracts and electron beam irradiation:

100 g of coarsely powdered samples were packed in 5 different sterile polythene bags – 6x6cm and subjected to electron beam irradiation at Microtron Centre, Mangalagangotri, Mangalore University, Mangalore.  The applied doses were 0, 2.5, 5, 7.5 and 10 kGy and labeled as (K1- non-irradiated), K2, K3, K4 and K5 respectively. The samples were then subjected to aqueous extraction by soxhlet method. Extracts were concentrated and stored properly.

2.3 Brine Shrimp Lethality Test (BSL Bioassay)^10-14:

BSL Bioassay was carried out according to the method given by Mclaughlin et al¹⁰. It helps in determination of LC₅₀ that is Lethal dose or Lethal Concentration 50, which means the concentration that kills 50% of the test animals during the observation period. LC₅₀ of the extracts is calculated using SPSS software.

 

2.3.1 Materials:

   i.      Artemia salina eggs: Brine shrimp’s eggs were collected from KLEU’s College of Pharmacy, Belgaum, which were stored in an air tight opaque container at room temperature. This storage condition is ideal for maintaining viability across several years. The eggs are small and brown colored. 110mg of eggs corresponds to 2500-3000 eggs.

  ii.      Sea water: for hatching of the shrimps.

iii.      Hatching chamber: consists of a glass chamber with aluminium or Bakelite lid. The dimension of the chamber is 40x20x20cm. A laminated plywood divider with numerous 2mm holes is clamped in the chamber which divides the chamber into two unequal parts. The smaller compartment was illuminated with a half shaded lamp and the larger compartment was aerated with an aerator.

 

2.3.2 Procedure:

   i.      Sample preparation: Water soluble extracts K1, K2, K3, K4 and K5 were dissolved in sea water directly. Samples were prepared by dissolving 50mg of extracts in 5ml sea water (Solution A). 0.5ml of solution A was diluted to 10ml with the same solvent (Solution B). Appropriate amount of solutions which corresponds to 10ppm, 100ppm and 1000ppm were transferred from each dose level. Control was prepared by adding same amount of solvent in control vials.

  ii.      Sea water: Fresh sea water can be used or artificial sea water can be prepared as per table 1. All major, minor and trace salts are weighed correspondingly and dissolved in 500ml of fresh distilled water and the final volume adjusted to 5L with distilled water. This is filtered through Whatman filter paper before using.

iii.      Hatching brine shrimps: 5L of sea water was added to the special chamber. 50 mg of the eggs were washed with distilled water and sprinkled into the larger compartment, provided with aeration. The napulii after hatching being phototropic moves towards smaller compartment. They are pipetted out against light background and used for bioassay.

iv.      Bioassay: Napulii drawn from the hatching chamber was counted and 10 shrimps were transferred to each vial. Sea water is added to make it upto 5ml. A drop of yeast suspension (3mg in 5ml sea water) was added to vial as food for shrimps. The vials were maintained under illumination. After 24 hours, survived shrimps were counted and LC₅₀ values were calculated by means of SPSS software.         

 

Table 1 Artificial sea water composition

 

3. RESULTS AND DISCUSSION:

Brine Shrimp nauplii are used to determine the toxicity of the samples of Andrographis paniculata which were subjected to electron beam irradiation at doses of 0, 2.5, 5, 7.5 and 10kGy. Aqueous extracts of Kalmegh samples irradiated at different doses were tested at 10, 100 and 1000µg/ml. After 24 hours, surviving shrimps were counted and % death caused by each dose was recorded Results of BSLT of electron beam irradiated Andrographis paniculata samples were evaluated using SPSS software to arrive at LC₅₀.

 

Results of BSLT are given in Table 2.

LC₅₀ for the samples K1, K2, K3, K4 and K5 were 283.49, 267.33, 194.95, 221.40 and 199.88 respectively. LC₅₀ obtained for all the samples was greater than 100 and less than 1000, which means that the all samples have retained their biological activity and there is no cytotoxicity observed.

 

 

Table 2 Brine Shrimp Lethality Test for Microtron irradiated samples of Kalmegh

Sl No.	Sample	No. of dead shrimps			Avg. % death			LC50
		10µg	100µg	1000µg	10μg	100μg	1000μg
1.	K1	0,1,0	1,1,2	9,7,10	3.33	13.33	26.67	283.49
2.	K2	1,3,0	2,1,3	9,10,8	1.33	20.00	90.00	267.33
3.	K3	0,1,1	1,1,2	10,9,10	6.66	13.33	96.77	194.95
4.	K4	0,2,1	1,2,1	8,10,9	10.00	13.33	90.00	221.40
5.	K5	1,0,2	2,1,1	10,9,10	10.00	13.33	96.66	199.88

Note: LC₅₀ > 1000– Samples are biologically inactive

LC₅₀ < 1000– Samples are biologically active

LC₅₀ < 100  – Samples are cytotoxic

LC₅₀ < 10    – Samples are highly cytotoxic

4. CONCLUSION: 

Results obtained in BSLT clearly indicate that the samples have LC₅₀ values between 100 and 1000, which means that all the samples have biological activity and also that they are not cytotoxic. This indicates that electron beam irradiation at the doses employed is not causing any toxicity in any samples. Thus, on the basis of these results, it may be concluded that even the highest irradiation dose used for the study does not exhibit significant toxicity.

 

5. ACKNOWLEDGEMENT:

BRNS, Dept. of Atomic Energy is gratefully acknowledged for financial support for this research work. Special thanks to KLEUCOP, Bangalore for providing necessary facilities for the research and also to Dr. Kiran, KLEUCOP, Belgaum for providing assistance.

 

6. REFERENCES:

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Received on 18.04.2014          Modified on 25.04.2014

Accepted on 30.04.2014          © RJPT All right reserved