INTRODUCTION:
Helminth infections are among the most common
infections in man, affecting a large proportion of the world's population. In
developing countries they pose a large threat to public health and contribute
to the prevalence of malnutrition, anaemia, eosinophilia, and pneumonia.
Although the majority of infections due to worms are generally limited to
tropical regions, they can occur to travellers who have visited those areas and
some of them can develop in temperate climates1.
Parasitic diseases cause severe morbidity, including
lymphatic filariasis (a cause of elephantiasis), onchocerciasis (river
blindness), and schistosomiasis. These infections can affect most populations
in endemic areas with major economic and social consequences2.
Trigonella foenum-graecum Linn. (Leguminaceae) (Syn- fenugreek) . It is an annual herb with long velveted
erect stalk, leaves up to 5 cm long stipules triangular, lanceolate, leaflets
about 2.5 cm long, obovate to oblanceolate; flowers 1-2, axillary, sessile,
recemed, whitish yellow; pods 5-7 cm long with persistent beak, hairy with
10-20 quadrilateral seeds.3
The fenugreek seeds are used mainly in treatment of
diabetes. The other activities reported of seeds are hypocholesterolaemic,
anti-inflammatory, antimicrobial and antioxidant.4
According to folk medicinal data, in many villages of India it is remedy for the treatment of dysentery.
The literature survey reveals that no reports were
found on the anthelmintic activity of the seed extracts of Trigonella
foenum-graecum . This prompted us to investigate the anthelmintic activity
of Trigonella foenum-graecum seed extracts.
MATERIALS AND METHODS:
Plant Material:
The seeds of Trigonella foenum-graecum Linn.
(Leguminaceae) were collected from the local market of Amaravati district (India) in December 2007, and were authenticated by taxanomist Dr. Prabha Y. Bhogaonkar,
Director, Vidarbha Institute of Science and Humanities, Amaravati. The seeds
were pulverized in a mechanical grinder and stored in room temperature in a
closed container for further use.
Preparation of Extract:
The powdered seeds (500 g) were repeatedly extracted
in a 2000 mL round bottomed flask with 1500 mL solvents of increasing polarity
starting with petroleum ether, chloroform, ethanol, and hot water. The reflux time
for each solvent was 40 cycles. The extracts were cooled at room temperature,
filtered, and evaporated to dryness under reduced pressure in a rotatory
evaporator.5
Anthelmintic Bioassays:
The earthworm Pheretima posthuma (Annelida,
Megascolecidae) was used for evaluating the anthelmintic activity of crude
extract using the reference substance for comparison. These were procured from
a local supplier and maintained at Govt. College of Pharmacy, Amravati.
Activity
Against Earthworms:
Anthelmintic activity was assessed using earthworms by
the reported methods with slight modification.6 Emulsion of the
crude extracts in Tween-80 (0.1%) containing 5, 10, 50 mg/mL of extracts was
prepared by adding dextrose (6%) solution. Piperazine citrate (3%) containing Tween-80
(0.1%), was prepared using dextrose (6%) solution and used as reference. 25 ml
of each physiological solution was poured into petridishes. The anthelmintic
activity was determined in duplicate. Three worms of about the same size per
petri dish were used. They were observed for their spontaneous motility
and evoked responses. The paralytic score was recorded at different time
intervals. Immediately after inhibition of response to external stimuli, the
worms were placed in fresh water and observed for recovery. Duration required
for final recovery/death was noted; mean paralytic score was tabulated against
time.7 Keeping in the mind of reported method (piperazine 3%
solution) 8, we have chosen piperazine citrate (3%) as reference
standard.
Table No. 1.Paralytic score of earthworms treated with
Pet. Ether extract of seeds of Trigonella foenum-graecum Linn. at different
time intervals.
|
Time in minutes
|
Piperazine Citrate (3%)
|
Extract (50mg/kg)
|
Extract (10mg/Kg)
|
Extract (5mg/kg)
|
|
0
|
Mean
Paralytic
Score
|
-
|
-
|
-
|
-
|
|
50
|
2.2
|
0.6
|
0.1
|
0.1
|
|
100
|
3.0
|
3.1
|
0.3
|
0.3
|
|
150
|
2.3
|
4.3
|
1.8
|
1.4
|
|
200
|
1.5
|
D
|
2.8
|
2.6
|
|
250
|
0.6
|
|
4.4
|
3.4
|
|
300
|
0.2
|
|
D
|
D
|
(Where D shows the death of earthworms)
The death and/or total paralysis time was recorded at
room temperature. The death of the worm was ascertained by transferring it into
a beaker containing hot water (50°C), which stimulated and induced movements if
the worm was live. Two independent experiments were carried out for each
observation to confirm the results.
Table No. 2 Paralytic score of earthworms treated with
Chloroform extract of seeds of Trigonella foenum-graecum Linn. at different
time intervals.
|
Time in minutes
|
Piperazine Citrate (3%)
|
Extract (50mg/kg)
|
Extract (10mg/Kg)
|
Extract
(5mg/kg)
|
|
0
|
Mean
Paralytic
Score
|
-
|
-
|
-
|
-
|
|
50
|
2.4
|
1.6
|
1.3
|
0.2
|
|
100
|
2.8
|
2.2
|
1.8
|
0.8
|
|
150
|
2.5
|
3.0
|
2.5
|
1.8
|
|
200
|
1.7
|
4.2
|
3.6
|
3.0
|
|
250
|
0.6
|
D
|
4.4
|
4.0
|
|
300
|
0.1
|
|
D
|
D
|
(Where D shows the death of earthworms)
RESULTS:
After a brief stimulant
effect, earthworms lost their motility on exposure to crude extracts of seeds
of Trigonella foenum-graecum. Each crude extract containing 5, 10, and
50 mg/mL, produced dose-dependent paralysis ranging from loss of motility to
loss of response to external stimuli, which eventually progressed to death.
5 and 10 mg/ml of pet ether extract produced paralysis
within 250-300 min. Mortality was noted with 50 mg/ml of pet ether extract
within 150-200 min [Table 1]. 5 and 10 mg/ml of chloroform extract also
produced paralysis within 250-300 min. [Table 2]. The mortality was also
occurred with 50 mg/ml concentration within 200-250 min.
Ethanol extract produced dose-dependent paralysis at
concentration of 5 and 10 mg/ml, paralysis within 250-300and 200-250 min.
respectively, while concentration 50 mg/ml produced death within 150-200 min
[Table 3]. Aqueous extract also produced dose-dependent paralysis at
concentration of 5 and 10 mg/ml; paralysis was evident between 250-300min.
respectively, while higher concentrations (50 mg mL) produced death within
200-250 min. [Table 4].
The higher concentrations of each crude extract
produced paralytic effect much earlier and the time to death was shorter.
Haemorrhagic and necrotic spots were observed externally on the worms, with the
higher concentrations. The effect of each crude extract was compared with
piperazine citrate (3%), which was found to produce Grade 3 paralyses within 90
min, and this effect could be reversed by placing the worms in fresh water.
DISCUSSION:
To scientifically prove, the
traditional use (anthelmintic) of Trigonella foenum-graecum, the crude
extract from the seeds of this plant was collected. The earthworm Pheretima
posthuma is one of the most important soil invertebrates in promoting soil
fertility. These earthworms are important components of the diets of many
higher animals.9 however; it was used in order to investigate the
activity of Trigonella foenum-graecum against annelids, since there are
human parasitic annelida such as Limnatis sp. and Haemadipsa sp. that primarily
affect the skin.10
.
Table No. 3 Paralytic score of earthworms treated with
Ethanol extract of seeds of Trigonella foenum-graecum Linn. at different
time intervals.
|
Time in minutes
|
Piperazine Citrate (3%)
|
Extract (50mg/kg)
|
Extract (10mg/Kg)
|
Extract (5mg/kg)
|
|
0
|
Mean
Paralytic
Score
|
-
|
-
|
-
|
-
|
|
50
|
2.3
|
1.2
|
0.2
|
0.1
|
|
100
|
3.0
|
1.8
|
1.1
|
0.3
|
|
150
|
2.7
|
4.4
|
1.9
|
1.2
|
|
200
|
1.4
|
D
|
3.4
|
2.5
|
|
250
|
1.0
|
|
D
|
4.4
|
|
300
|
0.2
|
|
|
D
|
(Where D shows the death of earthworms)
Helminthic infections of the gastrointestinal tract of
human beings and animals have been recognized to have adverse effects on health
standards with a consequent lowering of resistance to other diseases. In search
of compounds with anthelmintic activity, a number of substances were screened
using different species of worms, for example, earthworms, ascaris,
nippostrongylus, and heterakis . Of all these species, earthworms have been
used widely for the initial evaluation of anthelmintic compounds in vitro
because they resemble intestinal "worms" in their reaction to anthelmintics
and are easily available. It has been demonstrated that all anthelmintics are
toxic to earthworms and a substance toxic to earthworms is worthy for
investigation as an anthelmintic.11
Although the seeds of Trigonella foenum-graecum
is known to possess various medicinal properties, the chemical composition and
medicinal uses of Trigonella foenum-graecum extract have been reported
widely. It has been reported for its antidiabetic, hypocholesterolaemic,
anti-inflammatory, antimicrobial and antioxidant activity.
Table No. 4 Paralytic score of earthworms treated with
Hot water extract of seeds of Trigonella foenum-graecum Linn. at different
time intervals.
|
Time in minutes
|
Piperazine Citrate (3%)
|
Extract (50mg/kg)
|
Extract (10mg/Kg)
|
Extract (5mg/kg)
|
|
0
|
Mean
Paralytic
Score
|
-
|
-
|
-
|
-
|
|
50
|
2.4
|
1.3
|
0.1
|
0.1
|
|
100
|
3.0
|
2.2
|
1.9
|
0.6
|
|
150
|
2.3
|
2.8
|
2.5
|
1.8
|
|
200
|
1.6
|
4.2
|
3.1
|
3.0
|
|
250
|
0.8
|
D
|
4.2
|
4.0
|
|
300
|
0.2
|
|
D
|
D
|
(Where D shows the death of earthworms)
In this study we have evaluated the effect of Trigonella
foenum-graecum seed extracts on earthworms. Pet ether and ethanol extracts
showed significant wormicidal activity. On the contrary, worms paralyzed by
piperazine citrate remained alive and gained motility when placed in fresh
water. Earthworms have the ability to move by ciliary movement. The outer layer
of the earthworm is a mucilaginous layer and composed of complex
polysaccharides. This layer being slimy enables the earthworm to move freely.
Any damage to the mucopolysaccharide membrane will expose the outer layer and
this restricts its movement and can cause paralysis. This action may lead to
the death of the worm by causing damage to the mucopolysaccharide layer. This
causes irritation leading to paralysis.
Trigonella foenum-graecum has been reported to contain tannins,
saponins, flavonoids, and phenolic glycosides12 .In view of its
safety, the fenugreek is found to be safe for the internal use. The wormicidal
activity of pet ether and ethanol extracts against earthworms suggests that it
is effective against parasitic infections of humans. It is very interesting to
identify the active principle responsible for the anthelmintic activity and to
study its further pharmacological actions.
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