An Investigation into the Anti-microbial and Anti-fungal Properties of Earthworm Powder Obtained from Eisenia fetida
Abdullah Adil Ansari
Present study was carried out during the year 2006-2007 on dried earthworm powder collected from culture of Eisenia fetida in vermicomposting units and focused on the effect of the dried earthworm on microbes determining the anti-microbial and anti-fungal properties of the worms, as well as the chemical composition of worms obtain from vermicomposting units. The earthworm powder was also subjected to analysis of nitrogen and potassium using standard procedures. Antimicrobial disc diffusion suspecting tests were carried out against Bacteria (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) as well as the fungus Candida albicans with the following treatments: Control, earthworm powder in water (1:1), earthworm powder in water (1:2) and earthworm powder in acetone (1:2). The technique involves using disk diffusion susceptibility testing where disks from both the pure and diluted honey as well as the antibiotic disk erythromycin (control) were impregnated onto the surface of the Mueller Hinton agar. The study conclusively proved that the earthworm powder (all dilutions) has antifungal properties which would be effective in treating fungal infections, such as candidosis whereas did not indicate the anti-microbial properties which may be attributed to different composition of elements in earthworm powder obtained from vermicomposting unit rather than garden soil. ANOVA analysis (Single factor) at p = 0.05, proved that differences between the different concentrations of earthworm powder treatment containing colonies of C. albicans was significant, indicating the effect of earthworm powder by inhibiting the growth of the fungus C. albicans.
Received: April 27, 2010;
Accepted: July 13, 2010;
Published: August 23, 2010
There are approximately 4500 species of worms in the world. Of those about
2500 are earthworm species. In the classification system, earthworms are the
larger members of the Oligochaeta in the phylum Annelida. They are placed in
the order Opisthopora, on the basis of the male pores opening to the outside
of the body posterior to the female pores, even though the male segments are
anterior to the female (Ismail, 1997;
Edwards and Bohlen, 1996). Earthworms are found virtually worldwide and
live in almost any type of soil that contains the right amounts of moisture
and organic particles. Earthworms are of various sizes and colors. The color
ranges from brownish-black tinge to purple with the exception of some being
green. However the most common color is reddish-brown, which result from the
pigment hemoglobin in the blood. The dorsal side of the worm is darker while
the ventral side in paler (Mihara et al., 1996).
Earthworm has been known for many centuries as a therapeutic drug source for
various diseases in China and other parts of the Far East (Ismail,
2005). However, practical pharmacological studies have not been performed
except on lumbrofebrin as an antifebrile (Mihara et al.,
1996). In 1983, it was reported that very strong and novel fibrinolytic
enzymes could be extracted from the earthworm, Lumbricus rubellus. These
enzymes were fractionated and purified as six novel fibrinolytic enzymes and
named collectively as Lumbrokinase (Mihara et al.,
1996; Tang et al., 2000; Li
et al., 2008). It was also found that earthworm powder contains two
kinds of inhibitory substances for the platelet aggregation induced by collagen
and ADP. One of these inhibitors of platelet aggregation was identified with
adenosine. However, the other was a novel substance of MW 260. The structure
of this substance was decided on the basis of NMR, mass spectra and infrared
spectra. This novel substance also displays a relaxation effect for the canine
saphenous vein induced by prostaglandin F in vitro and an inhibitory
effect on the Active Partial Thromboplastin Time (APTT) (Mihara
et al., 1996). Earthworm powder can be given orally, thus for this
reason, earthworm powder has a potential application as a thrombolytic and also
exerts an inhibitory effect on platelet aggregation, an anticoagulation effect
and a relaxation effect for the vascular system, which are all effective for
thrombotic therapy (Kim et al., 1998). It can
be concluded therefore that earthworm powder represents a very promising agent
for the treatment of thrombosis (Mihara et al., 1996).
Its tonic properties also make it beneficial support for the liver and other
organ systems. In China, Korea, Vietnam and most of Southeast Asia, Lumbricus
has been used for their therapeutic benefits for thousands of years and referred
to as Earth Dragons. In Korea, these earthworms are believed to promote general
health and prevent a wide variety of diseases (El-Kamali,
2000). Furthermore, the earthworm is a primary ingredient in the traditional
Vietnamese remedy known as, Miracle Medicine that can Save lives in 60 min.
Several Vietnamese-based studies have demonstrated the safety and effectiveness
of the earthworm for supporting immunity and cardiovascular health. Because
it originates from the soil, the earthworm has dense nutritional content as
well as anti-oxidant properties (Mihara et al., 1996).
In Guyana, there have been experiments carried out isolating enzymes from the
earthworm powder and converting it into dietary supplement, such as Lumbrokinase
(Gao and Qin, 1999). Glycolipoprotein (G-90), a mixture
obtains from tissue homogenate of Eisenia foetida exhibits anti-bacterial
properties (Popovic et al., 2005). Present study
focused on anti-bacterial properties along with the anti-fungal properties in
the powder of Eisenia foetida which may have applications indirectly
implicated in treatment of diseases related to different microbes and fungi.
MATERIALS AND METHODS
The present work was carried out during the year 2006-2007 at University of
Guyana, Georgetown. Approximately 2500 of the cultured earthworm, Eisenia
fetida, were collected. The earthworms were soaked in distilled water for
5 h to allow the soil that was in its tract to be excreted. They were then washed
thoroughly with distilled water and place in a Petri dish and place in an incubator
for 24 h at a temperature of 55°C. After the 24 h period, the earthworms
were removed and pounded to make it into a powder. The powder was stored in
a refrigerator at normal temperature (Yegnanarayan et
Sulphuric acid and nitric acid digestion method (Homer, 2003)
was used for the estimation of various trace elements in the earthworm powder.
The trace elements tested for, included the following:
The earthworm powder was also subjected to analysis of nitrogen and potassium
using standard procedures (Homer, 2003). The analysis
of the samples was done at Central Laboratory, Research Center, Agriculture
Department, LBI Compound, GuySuCo.
Antimicrobial disc diffusion suspecting tests were carried out. The technique
involves using disk diffusion susceptibility testing where disks from both the
pure and diluted honey as well as the antibiotic disk erythromycin (control)
were impregnated onto the surface of the Mueller Hinton agar. At areas where
the concentration of both the diluted and undiluted honey were sufficient to
prevent bacterial growth, a distinct margin known as the inhibition zone (Bauer
et al., 1959) can be seen. Similarly, if the organism tested has
no resistance to the antibiotic disk impregnated on agar surface then as the
antibiotic diffused from the disk an inhibition zone is formed around the disk.
Bacteria (Staphylococcus aureus, Escherichia coli and Pseudomonas
aeruginosa) as well as the fungus Candida albicans were administered
the following treatments: Control, earthworm powder in water (1:1), earthworm
powder in water (1:2) and earthworm powder in acetone (1:2).
All the petri dishes (with replications) were inoculated with respective bacterium and fungus which were isolated from pure cultures. The disks soaked with various concentrations [Control, earthworm powder in water (1:1), earthworm powder in water (1:2) and earthworm powder in acetone (1:2)] were placed on the surface of agar. An antibiotic control disk, erythromycin was also inserted onto the surface of agar. The inoculated plates were incubated for 24 h at 37°C. Each plate was then observed accordingly for any inhibition zones. Inhibition zones present were measured in diameters. The growth of colonies was also estimated for each plate. One of each petri-dish with growing microorganism was left untreated and was used as a control. The treatment was done in triplicate. Before each treatment, the colonies of all of the tested organisms were counted using population estimation charts.
In dried earthworm (Eisenia fetida) powder iron is found in the greatest
concentration, while magnesium is in the lowest concentration (Table
1). The concentration from highest to lowest is iron, manganese, zinc, cupper,
calcium, potassium, phosphorus and magnesium, respectively.
|| The concentration of various elements present in dried earthworm
The concentration of the elements analyzed in the earthworm powder differs
from that seen in previous experiments (Mihara et al., 1996).
Antimicrobial disc diffusion suspecting tests conducted showed that the plates that were inoculated with disks soaked in acetone and dried earthworm powder showed less number of colonies as compared to water at different concentration (Table 2). This small number of colonies is as a result bactericidal effect (cause the death of the organisms). The number of colonies in the plates inoculated with disks soaked in water and earthworm powder was similar to the number seen in the control which is due to bacteriostatic effect (cause temporary inhibition of growth). Dried earthworm (Eisenia fetida) powder is only effective against fungus, which was determined by the presence of an inhibition zone (Table 3). The less concentrated the medium in which the disks were soaked, the greater the size of the inhibition zone. Disks soaked in water also had a larger inhibition zone than disks soaked in acetone, because of bactericidal effect while in water the effect was bacteriostatic (Table 4). The ANOVA Single Factor analysis at p = 0.05, showed that the F value (27.04) is greater than the F-critical value (5.14), therefore there is significant differences between the different concentration of earthworm powder treatment containing colonies of C. albicans which indicate that the earthworm powder is inhibiting the growth of the fungus C. albicans (Table 5). However, the dried earthworm powder that was tested had no anti-bacterial properties which may be attributed to difference in composition of elements in earthworm powder obtained from vermicomposting unit. Anova analysis (Single factor) at p = 0.05, the F = 27.04 is greater than F-crit. = 5.14. Therefore, there is significant differences between the different concentrations of earthworm powder treatment containing colonies of C. albicans indicating the effect of earthworm powder by inhibiting the growth of the fungus C. albicans. The presence of fungal proteases which act in conjunction with other proteolytic enzyme in the earthworm powder, against the fungus Candida albicans.
|| The number of colonies in each plate for the various treatments
||Presence or absence of inhibition zone in the plates studied
|+: Presence of inhibition zone, -: No inhibition zone
|| The length (mm) of inhibition zone seen in the Candida
albicans inoculated plates
|| The statistical analysis of the zone of inhibition seen in
the fungus Candida albicans
Sherman-Huntoon reported that the magnesium content of vermicompost is relatively
low (Hashemimajd et al., 1998). This is supported
by the analysis carried out on the dried earthworm powder where magnesium was
in the lowest concentration. Hisashi Mihara and his collaborators objective
was to produce dried earthworm powder by which a pharmaceutically acceptable
and highly safe dried earthworm powder exhibiting excellent antihyperlipemic,
antidiabetic or hypoglycemic, antihypertensive and/or antihypotensive activities
without producing any side effects. The dried garden earthworm powder (Eisena
fetida) that was produced had approximately 11% soluble nitrogen. In their
analysis the element with highest to lowest concentration present in the dried
powder was zinc, cupper, manganese, potassium, phosphorus, calcium, magnesium
and iron, respectively (Mihara et al., 1996). Dickerson
(1994) reported that the elements present in worms obtained from vermicomposting
unit differ from worms obtain from garden soil. The concentration from highest
to lowest is iron, manganese, zinc, cupper, calcium, potassium, phosphorus and
magnesium, respectively. The concentration of the elements analyzed in the earthworm
powder differs from that seen in previous experiments (Mihara et al., 1996).
Mitchell pointed out that 18% of total N from composting material entered earthworm
bodies (Hashemimajd et al., 1998). This can also
be supported by the analysis carried out, where the powder showed relative amount
of nitrogen (0.965 mg L-1) present. Especially in the Oriental countries,
earthworms (also called dilong) have been used as a drug from remote antiquity.
It has been reported that earthworms have a variety of pharmacological activities,
i.e., they show gastro protective effect (Prakash and Gunasekaran,
2010), they are effective in reducing the size of vesical calculi and eliminating
them from the body, in the treatment of jaundice and as a parturifacient, restrative,
hair grower, tonic and an antipyretic. On the other hand, they have also reported
some toxic actions of earthworms. That is, earthworm poison injures the nervous
systems and causes hemolysis (or the destruction of red blood cells) (Clarke,
1997). Earthworm tincture, that is an ethyl alcohol extract of the earthworm,
has a hypotensive effect (Yakugo, 1997). The dried earthworm
powder when administered orally to rats and human beings, has an antihyperlipemic
effect, a blood sugar lowering effect and a blood pressure regulating effect
(i.e., an antihypertensive and/or antihypotensive effect), but the reason for
this has not been fully understood. Nevertheless, these effects are believed
to be due to the action of the proteolytic enzymes (proteins) contained in the
dried earthworm powder, precursors (proteins) of these enzymes, other proteins,
lipids or unknown compounds, or a combination thereof (Mihara
et al., 1996).
Japanese scholars like Mihara et al. (1996) succeeded
in extracting a fibrin-dissolving enzyme from Lumbricus rubellus and
found that this enzyme consists of a few proteolytic sub-enzymes, which are
collectively named lumbrokinase. G-90 is a glycolipoprotein mixture obtained
from the tissue homogenate of earthworm Eisenia foetida (Annelida Concentrations
of 10 and 10 μg mL-1 of G-90 exhibited an inhibitory effect
on in vitro growth of non-pathogenic and facultative-pathogenic microbes
(Popovic et al., 2005; Matausijc-Pisl
et al., 2010).
The most prevalent trace element present in the dried earthworm powder is iron
since its composition is 282.7195 mg L-1 as compare to magnesium,
the least concentrated element in the powder with 0.0465 mg L-1.
The dried earthworm powder obtained from vermicomposting unit had appropriate
composition of elements as reported in earlier reports. The presence of fungal
proteases which act in conjunction with other proteolytic enzyme in the earthworm
powder, against the fungus Candida albicans (Popovic
et al., 2005). It was determined that Eisenia foetida have
anti-fungal properties. Earthworm powder tested with water is more effective
than when tested with acetone.
The authors express gratitude to Faculty of Natural Sciences (University of Guyana), St. Joseph Mercy Hospital and Central Laboratory, Guyana Sugar Corporation Inc for the facilities and support rendered.
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