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Earthworm- a potential source for stable and potent antimicrobial compounds- isolation and purification study

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Yogendra Verma at University of Glasgow
Yogendra Verma
Mahendra Kumar Verma at Indian Institute of Science Education and Research Bhopal
Mahendra Kumar Verma
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In the current work, we have isolated and purified an antimicrobial peptide from celomic fluid of Indian earthworm Pheretima posthumous. The purified peptide was characterized as serine protease and possesses broad range of antimicrobial activity. Another important finding was stability of antimicrobial peptide towards higher temperature, different pH and various inhibitors. The celomic fluid of earthworm was subjected to series of purification step such as ammonium sulphate precipitation, dialysis followed by size exclusion chromatography and ion exchange chromatography. The average molecular weight of peptide was found 20KD by SDS PAGE analysis. The peptide was evaluated for antibacterial and antifungal activity in various temperatures, pH and in the presence of different inhibitors. We have evaluated antibacterial activity of purified peptides among physchrophiles, mesophiles and thermophiles.
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Research Article
EARTHWORM- A POTENTIAL SOURCE FOR STABLE AND POTENT ANTIMICROBIAL
COMPOUNDS- ISOLATION AND PURIFICATION STUDY
1
YOGENDRA KUMAR VERMA,
2*
MAHENDRA KUMAR VERMA
1
Research Fellow, Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110 028, India,
2
Department of
Biotechnology, R.V.R & J.C. College of Engineering, Chowdavaram, Guntur 522019, Andhra Pradesh, India.
Email: mahendra.sonal1983@gmail.com
Received: 1 Aug 2012, Revised and Accepted: 10 Sep 2012
ABSTRACT
In the current work, we have isolated and purified an antimicrobial peptide from celomic fluid of Indian earthworm Pheretima posthumous. The
purified peptide was characterized as serine protease and possesses broad range of antimicrobial activity. Another important finding was stability
of antimicrobial peptide towards higher temperature, different pH and various inhibitors. The celomic fluid of earthworm was subjected to series of
purification step such as ammonium sulphate precipitation, dialysis followed by size exclusion chromatography and ion exchange chromatography.
The average molecular weight of peptide was found 20KD by SDS PAGE analysis. The peptide was evaluated for antibacterial and antifungal activity
in various temperatures, pH and in the presence of different inhibitors. We have evaluated antibacterial activity of purified peptides among
physchrophiles, mesophiles and thermophiles.
Keyword: Celomic fluid, Antimicrobial activity, Purification, Earthworm, Electrophoresis and chromatography.
INTRODUCTION
Since ancient time, earthworm was known and used for its therapeutic
potential
1
. Various therapeutic molecules have been isolated and
characterized such as fibrinolytic, antiviral, antitumor, hepato-
protective, cytotoxic and antioxidant in different species of
earthworms around the world. In last few decades, the fibrinolytic
property has been explored tremendously
2,3.
Earthworm has
recognized as potential source of therapeutics not only because of
availability of various molecules but also stability and potency of these
isolates. The antimicrobial property (antibacterial and antifungal) of
earthworm have been evaluated by many researchers in last
decade
4,5,6
. The entire earthworms as paste has used for treatment of
wounds and tropical ulcer
7
. Such potent molecules naturally reside
predominantly in intestinal fluids and subsequently in tissue fluid of
earthworm. Lumbricin I and II are peptides, which have been reported
in Eisenia. fetida and Lumbricus rubellu
8,9
. These molecules
characterized as glycoprotein, which offers strong antimicrobial
activity
10
. The molecular mechanism and biophysical characterization
of these molecules have not completely explored yet.
The question often arises that why earthworm possesses such
potent molecules in their intestinal fluid and tissue fluid and their
pharmacological significance in earthworm physiology. The most
accepted theory concluded its habitat where earthworm encounters
various microorganism including bacteria fungi and viruses
11
.
Besides advanced animal’s immune system such as humoral and cell
mediated immunity, lower organism’s immunity constituted with
such type of molecules, which offer first line defense mechanism.
Earthworm essentially requires such potent molecules as it feeds on
organic matter with soil
12
. These glycoproteins are characterized as
protease (especially serine protease) offer proteolytic degradation
of microbial population and provide first line defense system
13,14
.
In this study, we have isolated and purified protein offering
antimicrobial property of celomic fluids from Indian Earthworm
Pheretima posthumous. Further, we are looking for the complete
molecular mechanism of these molecules, so these can be used for
treatment of various bacterial and fungal borne diseases.
MATERIALS AND METHODS
The chemicals and consumables used in the following study
purchased form Hi-Media, Sigma and GE healthcare. All the
chemicals were molecular biology grade and prepared freshly at the
time of use. For determination of antimicrobial activity, used
bacterial and fungal strains were purchased from MTCC Chandigarh,
India. Bacterial and fungal strains used were wild type and
nonpathogenic for following study.
Preparation of Extract
For the current work, Indian Earthworm Pheretima posthumous was
selected as source of antimicrobial molecules. The earthworms were
collected from vermicomposting unit at Vijayawada, Andhra Pradesh,
India. Fully-grown healthy Earthworms were subjected to autolysis
and further homogenization in 20mM phosphate buffer pH 7.5 for one
week at 50
0
C with 0.02% sodium azide as bacteriostatic. Autolysed
earthworms were further subjected to high-speed centrifugation at
20,000rpm for 30 minute and soup was collected in separate sterile
tubes. The clear soup was filtered by membrane filters (0.4µm, Hi-
Media) and stored at 4
0
C for purification cascade
15,16
.
Purification of Peptide
The crude celomic filtrate was further processed by series of
chromatographic step for purification of desired peptide.
Ammonium Sulphate Precipitation
The salt precipitation is ideal method to recover total protein
content of crude extract. The precipitation of proteins via
ammonium sulphate does not hamper protein activity. Ammonium
sulphate was used to precipitate total protein from crude filtrate.
More than 90% of total protein was precipitated at 60% of
ammonium sulphate. The precipitate was separated from soup by
high speed centrifugation 20,000 rpm at 4
0
C for 30 minute. The
precipitate was suspended in 20mM phosphate buffer pH 7.5 and
further filtered with membrane filter of pore size 0.4µm.
Dialysis
To remove the salt proportion from protein approximately 12 hour
Dialysis was performed to remove salt from filtrate. Dialysis
membrane possessing Molecular Weight Cut Off (MWCO) of 12KD
purchased from Hi Media was preferred for desalting. The 2ml of
ammonium sulphate precipitate was loaded into dialysis bag and
desalted against 20mM phosphate buffer pH 7.5. The buffer was
replaced in each 4 hour and dialysis was run for 12 hour. After 12
hour, soup was collected in sterile tube and preserved at 4
0
C.
Size exclusion chromatography
The soup collected after dialysis was subjected to size based
separation of proteins and peptides from crude filtrate. In this study,
we have used Sephadex G 50. The Sephadex G 50 (GE Healthcare)
beads were allowed for complete swelling in 20mM phosphate
buffer pH 7.5 for 48 hours at room temperature. The swelled beads
were sonicated to remove traces of entrapped air. Further complete
swelled beads were loaded in glass column (60cm*1.5cm) up to
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 4, Issue 4, 2012
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Verma et al.
Int J Pharm Pharm Sci, Vol 4, Issue 4, 540-543
541
paced bed length 30cm. 2 ml of dialyzed sample was loaded in the
Sephadex G-50 column and eluted with excess of 20mM phosphate
buffer pH 7.5 in automated fraction collector with 2ml per minute
speed
17
. Total 30 fractions were collected (3ml each) and each of
fractions was assessed for antimicrobial and protease activity. The
fractions shown maximum activity were pooled together and
subjected to further purification by charge-based separation. The
total protein estimation of each fraction was carried out by Lowery
method to calculate concentration.
Ion Exchange Chromatography
Pooled fractions possessing higher antimicrobial activity were
further fractionated by ion exchange chromatography where DEAE
Cellulose (GE Healthcare) was used for column preparation. The
DEAE resin was charged by treatment with acid (HCl) pH (2-3),
alkali (NaOH) (pH10-11), distilled water and finally in phosphate
buffer 20mM, pH 7.5. The charged DEAE Cellulose resin was loaded
on the glass column (60cm*1.5cm) with 20 cm packed bed length.
2ml of pooled fraction was loaded in column and eluted with excess
of phosphate buffer and different concentration of Sodium Chloride
(0.1% - 0.5%)
18
. A total 20 fractions were collected and each fraction
was subjected to the antimicrobial and protease activity analysis.
SDSPAGE Analysis
The average molecular weight of purified peptide possessing
antimicrobial activity was determined by SDS-PAGE. A 15% of
acrylamide gel was prepared, 15µl of purified sample was loaded
and electrophoresis was run for 3 hour at 50 milli-volts with 0.5 X
tris glycine electrophoresis buffer. With purified sample protein
marker was loaded to determine average molecular weight. After
completion of electrophoresis gel was stained with
Coomassie Brilliant Blue R-250 and destained subsequently with
excess of methanol in destaining solution.
Determination of Caseinolytic Activity
The purified fractions were analyzed for the proteolytic activity by using
casein as substrate. Casein agar plates were prepared with 2% casein
(Hi-Media) with agar and phosphate buffer 20mM pH 7.5. The
caseinolytic activity of purified fractions was assessed by well diffusion
method where casein agar plates incubated overnight at 37
0
C
19
.
Assessment of antimicrobial activity
Purified fractions were evalu ated for antib acter ial and
antifungal activity by well diffusion method. Different wild type
bacterial and fu ngal strai ns including phy schro philes,
mesophiles and thermophi les were used for assessing the
antimicrobial activity. Both Gram positive a nd Gram negative
bacterial strains were used in th e study included Escherichia
coli, Pseudomona s putida, Streptococcus aureus, Azotobactor,
Bacillus streothermophilous and Pectobacteri um carotovorum.
For the determination of antibacteria l activity, well diffusion
metho d was used with sli ght modifications where n utrie nt Broth
used for growth of bacterial cultures and nutrient agar for plate
preparation. The 6 mm diamete r wells were punched into
nutrient agar plates and plates were left for solidification. The
purified fraction of celomic fluid was filled (20µl) in each well
with ampicillin (100µg/µl) positive control. The plates were
incubated at 37oC for 14hours and antibacterial activit y was
deter mined by measuring the diameter of zone of inhibition.
Further, we have incubated purified fractio ns in various
tempe rature 200C-500C and variou s pH 4- 12 fur ther then
analy zed for t heir antimicrobial acti vity.
For the determination of antifungal activity, we have used
Sabouraud’s dextrose/agar (SDA) for the growth of fungal culture.
The protocol runs same as that for assaying, the antibacterial
activity was evaluated and fungal cultures were kept for 48 h to
determine the diameter of zone of inhibition. Here we have used
ketoconazole (1mg/ml) as positive control. For the accessing
antifungal activity Penicillium and candida, species have been used
20
.
RESULT
Isolation and Purification
The crude celomic fluid was purified by successive
chromatographic matrix. The total protein content of celomic fluid
was precipitated by 60% ammonium sulphate and recovered more
than 90% of total protein. Further precipitate was dialyzed against
20mM phosphate buffer pH 7.5 with dialysis membrane molecular
weight cut off (MWCO) 12KD procured from HiMedia. In Gel
filtration chromatography, we have collected 30 fractions and
fraction number 12,13,14,15 shown maximum antimicrobial
activities, which were further, pooled and loaded in run in ion
exchange chromatography using DEAE Cellulose column. In 20
eluted fractions from Ion exchange chromatography 9 and 10
fraction were selected for antimicrobial activity as shown
enormous activity for antibacterial and antifungal property and
SDSPAGE study.
Graph 1: Sephadex G 50 eluted fractions and their absorbance at 280nm for dialyzed celomic fluid
Graph 2: DEAE Cellulose eluted fractions and their absorbance at 280nm for Sephadex G 50 fractions have shown maximum antimicrobial activity
Verma et al.
Int J Pharm Pharm Sci, Vol 4, Issue 4, 540-543
542
Proteolytic Activity
The crude as well as purified fractions have shown proteolytic
activity on casein agar plate as zone of clearance after incubation for
14 hour at 20
0
C-50
0
C. The activity was accessed in different
temperature and various pH. The proteolytic activity was
constrained for purified fraction, which was found maximum at 40
0
C
at pH 8.0 while least in 20
0
C at pH 4.0.
Fig. 1: Caseinolytic activity of celomic fluid while purification
Dialyzed (1 & 2), Sephadex G 50 fraction (3, 4, 5 & 6) and
DEAE Cellulose fractions (7 & 8), while A, B and C are Phosphate
Buffer, distilled water and native Protease
Molecular weight determination
The average molecular weight for purified fraction was determined
by SDS-PAGE. The 15% acrylamide gel was prepared and purified
fraction was resolved with standard protein marker. After staining
with Coomassie Brilliant Blue R-250 in both two fractions after
DEAE Cellulose column separation a thick band was observed
corresponding to 20KD of standard protein marker.
Fig. 2: SDSPAGE analysis of purified fraction on 15% acrylamide
with standard protein ladder
Antimicrobial Activity
The antimicrobial activity of purified fractions was evaluated in the
context with zone of inhibition on nutrient agar plate after overnight
incubation. The following data is collected while accessing activity
with different microbial population, which is shown in table-
Table 1: Assessment of antimicrobial activity of purified
peptide by well diffusion method
Bacterial strain
s
Zone of Inhibition
(mm)
Escherichia
coli
19
P
actobacterium
.
carotovorum
14
P
seudomonas
putida
16
S
treptoc
occus.
aureus
17
B
acillus
streothermophilous
14
Azotobactor
16
Penicillium sp
.
14
Candida sp
.
13
Fig. 3: Zone of clearance after incubation of purified fraction on
nutrient agar plate plated with Escherichia coli and
Pseudomonas putida
CONCLUSION
In the current work, we have collected Indian earthworms and
processed mature worms for collection of intestinal fluid. The
intestinal fluid was further processed and purified by different
chromatographic techniques including size based and charged based
separation
21,22
. The purified fractions were analyzed for apparent
molecular weight of purified protein was found 20KD. Further
purified fractions were accessed proteolytic activity and found
serine protease by casein agar plate as zone of clearance after
overnight incubation with casein as substrate. Further purified
fractions were evaluated for antimicrobial activity (antibacterial and
antifungal activity) by well diffusion method and zone of inhibition
was measured
31,24&25
.
Many of facts about the molecules possessing antimicrobial property
of earthworm’s intestinal and tissue fluids need more attention in
order to explore at molecular level
26,27
The biophysical
characterization of such potent molecules led to understand precise
molecular mechanisms and mode of action that further can be tuned
to cure for various microbes borne diseases. Further, molecular
characterization can lead to identification of DNA coding sequence of
these molecules in earthworm genome
28
. That can provide a
platform for production of antimicrobial peptides by r-DNA
technology, which is economical and does not violate bioethics.
Currently our team is working for finding exact molecular weight
and N-terminal protein sequence
29
. That will be useful not only to
understand molecular properties of antimicrobial molecules but also
to predict evolution and biological inter-relation of biomolecules
after generating phylogenetic tree in various species of earthworm
.We are also looking for crystallography of purified peptide in order
to generate pdb, essentially useful for insilico analysis and system
biology for new generation antimicrobial compounds.
ACKNOWLEDGEMENT
I would like to thank Management and Principal, R.V.R. & J.C. College
of Engineering, Chowdavaram, Guntur, Andhra Pradesh, India for
providing laboratory facility to carry out research and manuscript
preparation.
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... Cooper et al. [46] illustrated the presence of antimicrobial and anticancer molecules in the earthworms. Similarly, several species of earthworms were screened for antimicrobial activities by Kathireswari et al. [47], Istiqumah et al. [48], Verma and Verma [49], and Chauhan et al. [50]. ...
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... This is related to the activity of several types of bacteria in the digestive tract of the earthworm (endosymbiont). Besides, according to Verma and Verma (2012), earthworms coexist with various types of viruses and bacteria that are abundant in the ground. Therefore, earthworms secrete certain compounds (secondary metabolites) to be used by pathogenic bacteria or viruses (Gopikrishnan et al. 2021). ...
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Full-text available
  • Sep 2019
... The comparison between the immune system of Vertebrates versus in-vertebrates should be on the structural level but must be on its functionality. For example, our immune system is a complete collapse in the case of novel SARS-CoV-2 infections that raise a question immediately do we are really superior over lower animal invertebrates encounter daily such infections [1][2][3][4][5][6]. ...
Mapping Immunity; Vertebrates Versus Invertebrates
Article
Full-text available
  • Jul 2020
Humans evolved with a unique immune system consist of innate and adaptive immunity. Both innate and adaptive immunity comprised of different types of cells, immune players, and associated with different functions. In higher animals, including humans, innate immunity derived from birth hence called inbuilt immunity. On the contrary, adaptive immunity called acquired immunity is gain due to the environment. Vertebrates are ranked as superior over invertebrates on the classification table due to many reasons, including the presence of vertebra and spinal cord. Invertebrates often considered as lower animals due to poorly developed organs, lack of vertebra and spinal cord. Similarly, the immune system and set of immune organs in a lower organism are entirely different and primitive on the basis of the organization. Like humans and other vertebrates, the invertebrates do not have a distinct clas-sification/setup of innate and adaptive immunity. In human and higher vertebrates, innate and adaptive immune systems are consisting of different cells; immune players such as cytokines, anti-bodies, and chemokines, etc. enable crosstalk between innate and adaptive immune systems. On the contrary, invertebrates' immune system is consists of unique cells and potent biomolecules; enzyme , peptides, and proteins. These molecules provide a complete defense to the animal via several mechanisms. The question we raise here does the classification superiority of humans, and other vertebrates over in vertebrates are justified in terms of immunity. How a complex and evolved immunity system mostly in human often fail to fight against infections while lower animals with the primitive immune set can survive even in hearse conditions. Apart from organization setup in humans and higher verte-brate's immunity can be further classified based on function such as cell-mediated and Humoral immunity. Cell-mediated immunity provides protection using different cells such as white blood cells (WBC), Macrophage, Phagocytes, Dendritic cells and T cell (CD4 and CD8), etc. Humoral immunity is a function of B cells, memory cells and antibodies. The immune response in human and other higher vertebrates consist of activity of different cells and immune players for identification of the foreign substance, presentation to immune system followed by killing/removal. The effective identification and clearance of foreign objects depend on synchronization between immune cells and players of both innate and adaptive immunity. On the contrary, lower animals, i.e. invertebrates, do have a more straightforward mechanism of identification and clearance of foreign objects that ultimately depends on endogenous short peptides, proteins, and enzymes. The more straightforward sets often provide ease in synchronization of immune players in lower animals reported more effective and robust. There is growing evidence that enzyme and protein as part of the immune system in the lower animal are highly dynamic and do possess promiscuity at the substrate and catalytic level. Enzyme promiscuity is capacity often describe for broad substrate affinity and catalytic diversity. The short peptides, proteins, and enzymes present in lower animals; invertebrates are highly promiscuous and capable of catalyzing multiple biochemical reactions. Earthworm, a classic invertebrate model for immune comparison , studied tremendously in the last couple of decades. The animal habitat is an environment rich in infections and invading elements. The presence of promiscuous and potent biomolecules including enzyme; serine protease provides a complete defense to the animal. The coelomic cavity of animals is rich in many short peptides, and other bioactive molecules offer extended protection to a wide range of infections. The key molecules are Fetidin 1, Fetidin 2, Lysenin, Eiseniapore, Coelomic Cytolytic Factor (CCF), and Lumbricin I. These molecules exist in isoforms and vary in size among different species of earthworm. The isoforms of these molecules provide extended support in defense mechanisms. There are growing research findings suggested protease present in coelome of earthworm are generally exist in isoforms. Each isoforms possesses a broad substrate affinity and catalytic diversity. This can be compared with VDJ recombination in B cell for the diversity of anti-bodies. There is another advantage in having promiscuous and iso-forms of series protease in coelome of animal, i.e. a large number Citation: Mahendra Kumar Verma and MV Raghavendra Rao. "Mapping Immunity; Vertebrates Versus Invertebrates". Acta Scientific Biotechnology 1.8 (2020): 42-43.
... Natural extracts are advantageous in this regard as their therapeutic properties can be exploited with minimal adverse effects. The medicinal benefits of earthworm extracts have been explored by researchers in the past few decades with notable antimicrobial, antipyretic, antiinflammatory, fibrinolytic and antioxidant properties [15][16][17]. The earthworms possess innate and adaptive immunity properties as a wide range of immunoprotective leukocytes are synthesized and secreted [18,19]. ...
In vitro Cytotoxic and Apoptotic induction effect of Earthworm Coelomic Fluid of Eudrilus Eugeniae, Eisenia Foetida, and Perionyx Excavatus on Human Oral Squamous Cell Carcinoma‑9 Cell Line
Article
  • Apr 2019
The current protocol of cancer management includes surgery, radiotherapy and chemotherapy. However, these modalities have significant adverse effects and affect the quality of life. Further intensification of treatment is hindered as maximal toxicity levels are reached impeding improvement. Hence researchers are in the quest for adjunctive naturally available therapies that can alter tumor proliferation without causing significant adverse reactions. The present study aims to explore the cytotoxic potential of earthworm coelomic fluid (ECF)of Eudrilus eugeniae (EE), Eisenia foetida (EF), and Perionyx excavatus (PE)on oral cancer cell line SCC-9. The effect of ECF on cell cycle analysis and mechanism of cell death have also been investigated. All experiments reported in this paper were performed as 3 replicates per experiment. The results indicated that ECF of EE, EF and PE have potent variable cytotoxic effect on SCC-9 cells demonstrated through LDH, clonogenic and comet assay. An effective cell cycle arrest was observed at the G2M phase of cell cycle with apoptotic induction that was observed through an Annexin V – FITC/PI assay. ECF of EE was found to be superior in its cytotoxic action closely followed by ECF of PE. The present findings provide evidence for the first time that ECF of EE, EF and PE have potent cytotoxic effect on oral cancer cells in vitro. They significantly induce G2M cell cycle arrest and promote apoptosis in SCC-9 cell line. Gene expression studies have been planned to ascertain the pathways of cell death.
... These invertebrates can be considered as one of the rich sources of biologically and pharmacologically active compounds for use in the treatment of various diseases, including cancers (5,6). Earthworm coelomic fluid and extracts showed antibacterial and antifungal activities (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) as well as antiviral action (18). Earthworm tissues, extracts, or isolated fractions and compounds exhibited anticancer activity toward different cancer cell lines (19)(20)(21)(22)(23)(24)(25)(26). ...
Antitumor activity and apoptotic action of coelomic fluid from the earthworm Dendrobaena veneta against A549 human lung cancer cells
Article
Full-text available
  • Feb 2019
M. J. Fiołka, J. Rzymowska, S. Bilska, K. Lewtak, M. Dmoszyńska‐Graniczka, K. Grzywnowicz, W. Kaźmierski, T. Urbanik‐Sypniewska Antitumor activity and apoptotic action of coelomic fluid from the earthworm Dendrobaena veneta against A549 human lung cancer cells It is known that earthworm coelomic fluid (CF) can affect not only cancer but also normal cells. The study demonstrated that the CF of the earthworm Dendrobaena veneta exhibited cytotoxicity against A549 lung cancer cells but did not toward the bronchial epithelial cell line BEAS‐2B. The selective effect on the tumor cells was achieved after a short‐term CF heat pre‐treatment at 70°C. The cytotoxic effect of the CF was time‐ and concentration‐dependent. The CF noticeably decreased the viability and affected the morphology of the A549 cells. Scanning electron microscopy revealed a different degree of destruction of the nucleus and cytoplasm of A549 cells. As determined by atomic force microscopy, the cell surface roughness increased while the cell stiffness was reduced upon the CF treatment. A two‐fold increase in the caspase 3, 4, 5, and 10 levels was observed in the A549 cells after the incubation with the CF. The results obtained by flow cytometry using Annexin V confirmed the proapoptotic effect of the earthworm CF on A549 lung cancer cells. The D. veneta CF and active fraction obtained with cytotoxicity toward A549 lung cancer is an interesting and promising preparation for further biological, chemical, and biomedical research. This article is protected by copyright. All rights reserved.
... Serine protease has been produced by many organisms, including plants, animals and microbes (Liu et al. 2010;Sakanari et al. 1989). Verma and Verma (2012) reported the isolation and purification of antimicrobial peptide from celomic fluid of Indian earthworm Pheretima posthumous. They identified the isolated peptide as serine protease that possesses a broad range of antimicrobial activity. ...
Differential-display reverse transcription-PCR (DDRT-PCR): a new technology for molecular detection and studying one of the antagonistic factors of Bacillus endophyticus strain SA against Staphylococcus aureus (MRSA)
Article
Full-text available
  • Dec 2016
Differential Display (DDRT-PCR) is a powerful technique for analyzing differences in gene expression. In-vivo expression technologies and differential display RT-PCR are providing new approaches to further examine a microbe’s response to experimental conditions which more closely resemble natural microbial associations and habitats. In this study, Bacillus endophyticus strain SA isolated from the inner tissue of the stem of the cultivated plant (Salvadora persica, Asir, Kingdom of Saudi Arabia) produces an antagonistic factor. This factor has a broad spectrum of activity against Gram-positive and specifically against Staphylococcus aureus (MRSA). The antagonistic factor was isolated from the bacterial culture medium and purified by thin layer chromatography technique, then analyzed by GC–MS analysis. Identification of the producer strain was performed using the partial nucleotide sequence of 16S rRNA gene, which indicated that this strain is identical to B. endophyticus with 99 % similarity. The sequence of this strain was deposited at NCBI GenBank under accession number KF011545. Application of differential display RT-PCR revealed that the isolate was able to up-regulate a gene with serine protease like protein. The protein is well known as antimicrobial agent and was reported to be produced by plants, animals and insects. Serine protease is also known to be produced by bacteria for purposes oth er than bacterial–bacterial antagonistic effect, which has been confirmed by this study.
... Has been done fractionation of the intestinal fluid Pheretima posthuma, then processed with a different chromatographic techniques. The results showed that the purified protein fraction having a molecular weight of 20 kDa 36 . Futher more, multiple SDS-PAGES were run to determine average molecular weight of candidate protease from Indian earthworm P posthuma. ...
Fractionation and characterization of proteins in Lumbricus rubellus powders
Article
Full-text available
  • Jan 2016
In order to find drugs from natural materials, earthworm powders Lumbricus rubellus can be used as a source of protein which can be developed into various types of drugs. The purpose of this study do fractionation and characterization of protein in L rubellus powders based on molecular weight. Protein in L rubellus powders extracted gradually to obtain a crude extract, precipitate and dialysate as final extract. Measurement of proteins levels in the crude extract, precipitate and dialysate is done by spectrophotometer, while electrophoresis was done to proteins characterization base on molecular weight. We conclude that 5 grams powder L rubellus in the dialysate fractions containing 2487 ug/mL proteins that show four dominant types of proteins with a molecular weight of 12.2, 13.3, 14.6 and 29.2 kiloDalton (kDa). © 2016, International Journal of Pharmaceutical and Clinical Research. All Rights Reserved.
... The salt water in which the earthworms are soaked is often given to manage postpartum complications in women [4] . Additionally, earthworm paste, since ancient times, is used clinically for wound heal and as antimicrobial in surgeries [5] . In Chinese Materia Medica earthworm is considered as complete medicine and used for various therapeutic purposes such as blood purification, blood disorders, against jaundice, wound healing, and as antimicrobial, anti-inflammatory and antioxidant agent [6] . ...
Evaluation of cytotoxic and anti-tumor activity apjtb-03-11-896
Data
  • Nov 2015
... The salt water in which the earthworms are soaked is often given to manage postpartum complications in women [4] . Additionally, earthworm paste, since ancient times, is used clinically for wound heal and as antimicrobial in surgeries [5] . In Chinese Materia Medica earthworm is considered as complete medicine and used for various therapeutic purposes such as blood purification, blood disorders, against jaundice, wound healing, and as antimicrobial, anti-inflammatory and antioxidant agent [6] . ...
Evaluation of cytotoxic and anti-tumor activity of partially purified serine protease isolate from the Indian earthworm Pheretima posthuma
Article
  • Nov 2013
eCAM: A closer look at clinical analyses
Article
Full-text available
  • Oct 2009
eCAM continues as it began in its quest for providing linkages and revealing the interdependence of basic science and clinical analyses. If one examines the 50 most cited papers for the month of June 2009, it is difficult to determine what is purely clinical and what is basic science; there is significant overlap and blurring. To clarify, one way is to define as clinical any investigations that use exclusively humans in trials. However, using human cells in culture could be classified as an overlap between basic science and clinical investigations. One would not argue that animal models constitute basic science, or that this category would include products from animals or plants and their role in modulating disease. Taking this stance has provided me, the biologist/immunologist, with a chance to explore those areas that (i) overlap, (ii) are biological, (iii) are immunological, and (iv) have a certain “orientation” or trajectory that renders them intimately pertinent to complementary and alternative medicine (CAM). Clinical evidence-based approaches present unique problems primarily due to human nature and are often difficult to structure, support, plan and complete. We must also examine ethical implications and what may emerge as reputable. It is not always easy to move past single-case reports, which is the base of the hierarchical evidence-based pyramid suggested by Goldrosen and Strauss (1), a paper that has been emphasized as a model as it relates to CAM and immunology and evidence-based analyses (2).
Fibrinolytic enzyme extracted from the earthworm
Article
  • Jan 1983
Animal-based medicines: biological prospection and the sustainable use of zootherapeutic resources
Article
Animals have been used as medicinal resources for the treatment and relieve of a myriad of illnesses and diseases in practically every human culture. Although considered by many as superstition, the pertinence of traditional medicine based on animals cannot be denied since they have been methodically tested by pharmaceutical companies as sources of drugs to the modem medical science. The phenomenon of zootherapy represents a strong evidence of the medicinal use of animal resources. Indeed. drug companies and agribusiness firms have been evaluating animals for decades without paying anything to the countries from where these genetic resources are found. The use of animals' body parts as folk medicines is relevant because it implies additional pressure over critical wild populations. It is argued that many animal species have been overexploited as sources of medicines for the traditional trade. Additionally, animal populations have become depleted or endangered as a result of their use as experimental subjects or animal models. Research on zootherapy should be compatible with the welfare of the medicinal animals. and the use of their by-products should be done in a sustainable way. It is discussed that sustainability is now required as the guiding principle for biological conservation.
Purification and characterization of two antibacterial peptides from Eisenia fetida
Article
Two novel antibacterial peptides were isolated and characterized from the earthworm Eisenia fetida. The antibacterial peptides were purified to homogeneity by means of cation-exchange chromatography and reverse FPLC and named F-1 and F-2. By means of ESI-MS, F-1 and F-2 were determined to be two peptides with the relative molecular mass of 535.27 and 519.27. By means of MS/MS, the amino acid sequence of F-1 and F-2 were determined to be Ac-Ala-Met-Val-Ser-Ser and Ac-Ala-Met-Val-Gly-Thr respectively. F-1 and F-2 exibited an antibacterial activity not only to bacteria but also to fungi. Minimal inhibitory concentration (MIC) of F-1 and F-2 against several Gram-positive and Gram-negative bacteria were determined by incubating approximately 10 4-10 5 CFU/ml of cell with serial dilutions of peptide in a 96-well microtiler plate. The MIC of F-1 and F-2 against Enterococcus gallinarum, Pseudomonas pyocyanea, Acinetobacter baumanii, Klebsiella terrigena1 were 11.4 mg/L and 12.85 mg/L. The MIC of F-1 and F-2 against Enterococcus faecalis was 22.8 mg/L and 25.68 mg/L.
The Biology and Ecology of Earthworms
Book
Stable Earthworm Serine Proteases. Application of the Protease Function and Usefulness of the Earthworm Autolysate.
Article
  • Feb 2000
The fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N. et al., Biosci. Biotechnol. Biochem., 57, 1726–1730 (1993), 60, 293–300 (1996), and 63, 2031–2033 (1999)] were further characterized to exploit their catalytic functions. These enzymes are stable in solution for long periods at room temperature and strongly resistant to organic solvents, even toluene and n-hexane. The serine proteases can act on various protein substrates such as elastin and hemoglobin as well as fibrin, and also catalyzed the hydrolysis of esters such as ethyl acetate and a bioplastic, poly[(R)-3-hydroxybutyrate] film. The enzymes, in the absence of microbial degradation, contributed to the production of the earthworm autolysate possessing antioxidant ability and protease activity, whose components were similar to those of soy sauce. The extract of the earthworm autolysate could be used as a peptone substitute in media for the cultivation of microorganisms.
Anti-inflammatory and anti-pyretic activities of earthworm extract-Lampito mauritii (Kinberg)
Article
Experiments were conducted to understand the therapeutic properties such as anti-inflammatory and anti-pyretic activities of biologically active extract isolated from whole earthworm (Lampito mauritii, Kinberg). Inflammation in the hind paw of Wistar albino rat, Rattus norvegicus, was induced by histamine, granuloma pouch was induced by turpentine and pyrexia induced by Brewer's yeast in rats were followed as earlier studies. Anti-inflammatory drug-indomethacin and anti-pyretic drug-paracetamol were used as standard drug for comparison. Administration of indomethacin (10mg/kg), paracetamol (150 mg/kg) and/or different doses of earthworm extract (EE) (50, 100 and 200mg/kg) reduced and restored to normal conditions in a dose-dependent manner of histamine and turpentine induced inflammation, and Brewer's yeast induced pyretic in rats. The most significant inhibition of paw oedema and granuloma and also the significant reduction in hyperpyrexia in rats when treated with standard drugs as well as different doses of EE, reflect the presence of anti-inflammatory and anti-pyretic properties of EE similar to glycoprotein complex (G-90).
A new source of biologically active compounds-earthworm tissue (Eisenia foetida, Lumbricus rubelus)
Article
1. Earthworms possess immunological recognition and memory as well as high regenerative abilities. Coelomic fluid was used as a source of biologically active compounds. 2. A biologically active glycolipoprotein extract from a whole earthworm tissue homogenate was isolated and named G-90. 3. G-90 forms precipitation arcs in gel with different animal and human sera. 4. It alters murine cell growth rate in vitro in serum in a dose-dependent manner and slows murine tumor growth in vivo. 5. G-90 does not contain mutagens or carcinogens.
Resistance of Enterococcus faecium to neutrophil-mediated phagocytosis
Article
  • Dec 1994
During a previous study of the opsonic requirements for neutrophil (polymorphonuclear leukocyte [PMN])-mediated killing of enterococci, we identified two strains of Enterococcus faecium (TX0015 and TX0016) that were resistant to PMN-mediated killing. To better define the mechanism of this resistance, we examined phagocytosis with a fluorescence assay and found that TX0016 was completely resistant to phagocytosis by PMNs; this finding was confirmed by electron microscopy. Examination of multiple strains of enterococci revealed that all 20 strains of Enterococcus faecalis tested were readily phagocytosed (mean, 18 intracellular organisms per PMN; range, 7 to 28). In contrast, only 13 (50%) of 26 strains of E. faecium tested were susceptible to phagocytosis (> or = 7 organisms per PMN); the other 13 strains showed < or = 3 organisms per PMN. Enterococcus casseliflavus ATCC 25788 and one strain of Enterococcus hirae were also resistant to phagocytosis, while two strains of Enterococcus durans, Enterococcus mundtii ATCC 43186, and one strain each of Enterococcus raffinosus and Enterococcus solitarius were readily phagocytosed. Exposure of E. faecium TX0016 to sodium periodate, but not to the protease trypsin or pronase or to phospholipase C, eliminated resistance to phagocytosis. Sialic acid, a common periodate-sensitive structure used by microorganisms to resist opsonization, could not be demonstrated in E. faecium TX0016 by the thiobarbituric acid method, nor was phagocytosis of TX0016 altered by neuraminidase treatment. This study suggests that there is a difference in susceptibility to phagocytosis by PMNs between different species of enterococci and that a carbohydrate-containing moiety which is not sialic acid may be involved in the resistance of E. faecium TX0016 to phagocytosis.
Antimicrobial Activity and Conformation of Tachyplesin I and Its Analogs.
Article
  • Jun 1993
We investigated the structure-antimicrobial activity relationship of tachyplesin I (T-I). Even when Lys1 and Trp2 were both deleted from the N-terminal end of T-I, the antimicrobial activity against gram-negative bacteria was not decreased. But as Lys1 and Trp2 were deleted one by one, the antimicrobial activity against gram-positive bacteria and antiviral activity were gradually decreased. Deletion of two disulfide bridges caused a significant decrease in all activities. The circular dichroism (CD) spectra revealed that the analogs containing the two disulfide bridges took a beta-sheet structure and that the analogs without the disulfide bridges took a random coil conformation. These results suggest that the beta-sheet structure maintained by two disulfide bridges plays an important role in the antimicrobial activity of T-I.