Toxicity Evaluation of Abrus precatorius seeds

collected from Bunda District, Tanzania

Candidate name: Peter Urio
Supervisor: Dr. S. Maregesi
Department of Pharmacognosy

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 1.Introduction
 Abrus precatorius commonly known as Rosary pea belongs
to a family Fabaceae.

 It is widely used as an abortifacient, oral contraceptive and

anti-venom.

 Seed contains the toxic poison abrin which inhibit cell

protein synthesis.

 Ingested seeds can affect the gastrointestinal tract, the liver,

spleen, kidney, and the lymphatic system.

 It is a common practice among children to ingest these seeds

in Mara region while in Iringa the seeds are taken as
contraceptives . 2
 2.Statement of the Problem
Tanzanian traditional healers and users are unaware of the
reported toxicity and that there are dangers of toxicity that
takes long to be noticed especially with internal organs. On
the other hand, presence, quantities and proportions of
chemical constituents in plants are controlled by various
factors including the ecozones. Safety of Abrus precatorius of
the locally consumed seeds has never been evaluated
therefore users might be at risk of potential exposure.

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 3.Rationale of the study
 Determination of LC50 and LD50 of Abrus precatorius seeds
growing in Tanzania is essential as an effort to address
safety.

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 4.Objectives
Broad Objective
 To perform preliminary evaluation of Abrus precatorius
seeds growing in Tanzania for safety.

Specific Objectives
1. To carry out acute oral toxicity study (LD50) of
methanol extract of Abrus precatorius seeds

2. Determination of LC50 of methanol extract of Abrus

precatorius seeds

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5.Methodology 1 of 6
Plant identification

 This was done at Botany Department (University Of Dar es Salaam) by a

qualified botanist Mr. Haji Selemani

Seed collection
 Abrus precatorius seeds were obtained from Kung’ombe village in Bunda
district

Extraction

Abrus precatorius seeds

 Defatting with pet ether.
 Extraction with methanol for 18 hours at 60°C using Soxhlet
apparatus

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5.Methodology 2 of 6
Acute oral toxicity (LD50)
Animals
• albino mice
Housing and Diet
• Mice were kept in well ventilated cages and were given
standard feed and water and allowed to acclimatize with
the environment for five days before the start of the
experiment.
Mode of administration
• Abrus precatorius extract was administered orally in a
single dose by gavage using specially designed mice oral
needle

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5.Methodology 3 of 6
Acute oral toxicity
The study was conducted as per Lorke’s Method (A new
approach to practical acute toxicity testing) .
Phase I
Three groups of three mice each were administered with the
extract at respective oral doses of 10mg, 100mg, and 1000mg
per kg body weight.
Phase II
The dose level was stepped up. Three albino mice were
administered higher dose of 1600, 2900 and 5000 mg/kg body
weight respectively.

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5.Methodology 4 of 6
Adverse effects anticipated
Convulsion, Dullness
Paw licking Constipation
Salivation Diarrhea
Tremor Rubbing of nose on floor and wall of the cage
Hyperactivity Death
Paralysis Vocalisation
Gasping piloerection

At the end of observational period, the control and surviving

mice were sacrificed for macroscopic observation.
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5.Methodology 5 of
6Brine Shrimp Test (LC ) 50

Brine shrimp lethality test described by Meyer was employed.

Animals
Artemia salina
Bioassay
• Concentrations used:(1000, 640, 320, 160, 80, 40, 20 and
10μg/ml) were prepared by drawing different volumes from
the stock solutions and then added into petri dishes containing
ten brine shrimps larvae in each of 3 replicates.
• The negative control: Brine shrimp larvae and artificial sea
water.
• The petri dishes were incubated under light and the numbers
of survivors in each concentration after 24 and 48 hours were
counted and the percentage mortality was determined .
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5.Methodology 6 of 6

Data Analysis
 LD50 as geometric mean:

D0 = Highest dose that gave no mortality,

D100 = Lowest dose that produced mortality.

 LC50 was obtained by probit analysis of the brine shrimp test results

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6.Results 1 of 4
Acute Oral Toxicity
Phase I results
Dose
S/No (mg/ kg body Mortality Toxicity signs
weight)

A0I 0 apparently normal

A0II 10 0 apparently normal
A0III 0 apparently normal

B0I 0 Paw licking 1 hour post administration

B0II 100 0 Paw licking 1 hour post administration
B0III 0 Paw licking 1 hour post administration

C0I 0 Paw licking 13 minutes post administration

C0II 1000 0 Paw licking 30 minutes post administration
C0III 0 Paw licking 24 minutes post administration

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6.Results 2 of 4
Acute Oral Toxicity
Phase II results

S/No Dose Mortality Toxicity signs

[mg per kg body
weight]

Paw licking 10 minutes post

P2-0I 1600 0
administration and dullness

Paw licking 7 minutes post

P2-0II 2900 0
administration and dullness

Paw licking 5 minutes post

P2-0III 5000 0
administration and dullness

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6.Results 2 of 4
Acute Oral Toxicity
Macroscopic Observations

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6.Results 2 of 4
Microscopic Observations

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6.Results 3 of 4
Acute Oral Toxicity

 Abrus precatorius extracts did not produce any mortality

throughout the study period even at the limit dose
5000mg/kg body weight.
 The oral LD50 was determined to be more than 5000mg/kg
body weight.

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6.Results 4 of 4
Brine Shrimp Test
 LC50 of methanol extract was found to be 169.36µg/ml (95%
Confidence interval of 157.85 – 179.02).
 LC50 values above 100µg/ml, implies that the extract is non toxic.

Mortality Vs Concentration after 48 hours

120% Highest
mortality
100%

80%
% Mortality

60%

40%

20%

0%
10 20 40 80 160 320 640 1000
Concentration µg/ml
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7.Discussion 1 of 2
 No mortality was recorded in any of the test group within
24 h which imply that the LD50 is greater than 5000mg/kg

 According to Hodge and Sterner toxicity scale, oral LD50

of 5000mg/kg or more is considered as practically
harmless.
 This could be attributed by the fact that abrin being a
protein is poorly absorbed from the gastro intestinal system
of the mice or
 Abrin is present in trace amount in the seeds from Bunda
to have a significant adverse effect after single dose
administration

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7.Discussion 2 of 2
 The amount of abrin present in the seeds is highly
dependent on the geographical location and climatic
factors particularly temperature, rainfall, humidity and
sunlight as well as the mineral contents of the soil.

 The observations from Brine Shrimp Lethality Test also

suggest methanol extract of Abrus precatorius is non toxic.

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9.Conclusion 1 of 1
 The results from both tests indicates that Abrus precatorius
methanol extract is non toxic.
 Despite the fact these results are crucial in judging the
safety of Abrus precatorius but there is a problem
regarding extrapolating animals’ data to humans.
 Therefore extremely high doses are not be advisable until
further studies on sub-chronic and chronic toxicity,
reproductive toxicity, immune toxicity (tests for allergic
reactions) and teratogenicity have been carried out so as to
rule out the long term effects of the extract. 20
10.Recommendations 1 of 1
 I do recommend quantification of Abrin presents in the
seeds by High Performance Liquid Chromatography.

 Also chronic toxicity studies have to be carried out to rule

out long term effects of the extract.

 Analysis of soil composition in the areas where the seeds

are used must be carried out so as to establish the effect of
plant nutrients on the level of abrin.

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 11. References 1 of 1
• Dickers, K.J. et al., 2003. Abrin poisoning. Toxicological Reviews, 22,
pp.137–142.
• Lorke, D., 1983. A New Aproach to Practical Acute Toxicity Testing.
• Meyer, B.. et al., 1982. Brine Shrimp: A Convenient General Bioassay for
Active Plant Constituents. , pp.31–34.
• Moshi, M. et al., 2007. Brine Shrimp Toxicity Evaluation of Some
Tanzanian Plants Used Traditionally for the Treatment of Fungal
Infenctions. African. J. Traditional,, 4, pp.219–225.
• Moshi, M.J. et al., 2010. Brine shrimp toxicity of some plants used as
traditional medicines in Kagera Region, north western Tanzania. Tanzan J
Health Res, 12(1), pp.63–67.
• Senin, R.(2006).Acute toxicity study. Retrieved from
(http://www.ccohs.ca/oshanswers/chemicals/Id50.html) on 27/5/2015.
• Amberger, A., 1975. Protein Biosythesis and Effect of Plant Nutrients on the
Process of Protein Formation.

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THANK YOU

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 Weight Dose
S/No Mortality Toxicity signs
[gm] [mg]
Dose: 10mg per kg body weight
A0I 29.6 0.296 0 apparently normal
A0II 27.0 0.270 0 apparently normal
A0III 30.0 0.300 0 apparently normal
Dose: 100mg per kg body weight
B0I 21.0 2.1 0 Paw licking 1 hour post administration
B0II 30.0 3.0 0 Paw licking 1 hour post administration
B0III 22.5 2.25 0 Paw licking 1 hour post administration
Dose: 1000mg per kg body weight
C0I 23.7 23.7 0 Paw licking 13 minutes post administration
C0II 26.7 26.7 0 Paw licking 30 minutes post administration
C0III 29.2 29.2 0 Paw licking 24 minutes post administration 24
 S/No Dose Weight Dose
[mg per kg body Mortality Toxicity signs
  weight]
[gm] [mg]

Paw licking 10 minutes post

P2-0I 1600 22.1 35.36 0
administration and dullness
Paw licking 7 minutes post
P2-0II 2900 22.0 63.80 0
administration and dullness
Paw licking 5 minutes post
P2-0III 5000 25.0 125.00 0
administration and dullness

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