ANNONA SQUAMOSA SEEDS: AS A SOURCE OF NATURAL ANTIMICROBIAL PEPTIDES.
M. PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIVGANDHIUNIVERSITY OF HEALTH
SCIENCES, KARNATAKA,BANGALORE
BY
VAGEESH PANDITHARADHYA B.K.M
B.Pharm.
UNDER THE GUIDANCE OF
Dr.S. RAMACHANDRA SETTY
M.Pharm.,Ph.D
HEAD OF THE DEPARTMENT AND PRINCIPAL
P. G. DEPARTMENT OF PHARMACOLOGY
S. C. S. COLLEGE OF PHARMACY,
HARAPANAHALLI-583131
2008-09.
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
Name and Address of the Candidate / VAGEESH PANDITHARADHYA B.K.M.O.SHADAKHSHARAIH B.K.M.
D.NO.121/135.ASHRAYA BADAVANE.
SRIADIBASAVESHWARANAGARA. HARIHARA ROAD.
HARAPANAHALLI-583131.
02 / Name of the Institution / T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583 131
(Davangere dist.) Karnataka
03 / Course of the Study
Branch / M. Pharm.,
Pharmacology
04 / Date of Admission to course / 30-05-2008.
05 / Title of the Topic / ANNONA SQUAMOSA SEEDS.
AS A SOURCE OF NATURAL ANTIMICROBIAL PEPTIDES
06 /
Brief resume of the intended work
6.1. Need for the Study /Enclosure – I
Enclosure – II6.2 Review of literature.
6.3 Objective of the Study.
/ Enclosure – III07 /
Materials and Methods
7.1. Source of data /Enclosure – IV
7.2. Methods of collection of data
/ Enclosure – V7.3. Does the study require any
Investigations on animals?
If yes give details / NOT REQUIRED
7.4. Has ethical clearance been
obtained form your institution
in case of 7.3. / NOT REQUIRED
08 /
8.1.List of References
8.2.Remarks of guide. / Enclosure – VIEnclosure – VII
09 /
Signature of the Candidate
/ (VAGEESH PANDITHARADHYA B.K.M)10 /
Remarks of the Guide
/ Enclosure – VII11 /
Name and Designation of
(in Block Letters)11.1. Guide
11.2.Signature
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
11.6. Signature / Dr. S. RAMACHANDRA SETTY
M. PHARAM., Ph. D.,
H.O.D. & PRINCIPAL
Dr. S. Ramachandra Setty
12 /
Remarks of the Principal
12.1. Signature / The present study was permitted to work in the institution.(Dr. S. Ramachandra Setty)
ENCLOSURE-I
06. Brief resume of Intended Work
6.1 Need for the study.
New types of antimicrobials are increasingly sought in the field of human infectious disease-care, being necessitated by such factors as emergence of new infectious diseases (e.g. Escherichia coli 0157), re-emergence of highly antibiotic resistant pathogenic microbes (e.g. methicilin resistant- Staphylococcus aureus (MRSA), vancomycin resistant enterococci etc.), etc. Any such new antimicrobials are expected to be able to address the problem of resistance along with the intended antimicrobial action.
Such a need has sparked off searches for novel antimicrobial compounds although on less than optimal scale, which may eliminate the pathogens, either with better efficiency in exerting their actions on already known targets or having altogether new targets in the microbial cells, with novel modes of action arising out of their novel structural frameworks, hitherto not encountered by the science. Quite a few novel strategies that depart radically from the conventional antibiotics have shown promise - Phage therapy using bacteriophages, bacterial cell wall hydrolases, antimicrobial peptides etc.1 Among these, antimicrobial peptides and their derivatives are interesting because of their demonstrated ability to modulate the host immune system in addition to killing microbes (even multi-drug resistant microbes) and evading the resistance mechanisms of pathogens, which is an added advantage for them to be developed and included into therapeutics for infectious diseases in humans.
Antimicrobial peptides (AMPs) arerelatively small (6 to 100 aminoacids), amphipathic molecules of variable length, sequence and structure, forming significant component of innate immunity of host organisms against a wide range of microorganisms including bacteria, protozoa, yeast, fungi, viruses and even tumor cells2. They usually act through relatively non-specific mechanisms resulting in membranolytic activity. They are a highly diverse - in occurrence and structure- group of molecules evident from their discovery in wide variety of organisms – bacteria, fungi, plants, mollusks, insects, amphibians, fishes, birds, mammals etc., since their first description ininsects. There are more than 900 different AMPs described till date3.
Although plants are constantly exposed to various microbial pathogens, infections that lead to death of whole plant remain rare events. This is because plants have evolved several quick, effective defense mechanisms to inhibit pathogen spread after initialinfection and thus limit disease4. A wide array of antimicrobial peptides and proteins are believed to be involved in such mechanisms. Within plants AMPs have most commonly been discovered in seeds where they accumulate to high levels5. This may be rationalized as the deployment of AMPs against possible microbial attack on the seeds (with significant amounts of seed storage carbohydrates and proteins), which most often have to germinate on natural substrate (soil) that is rich in microorganisms. Although, low water content of the seed and the hard seed coat in many seeds provide effective physical barriers against bacterial and fungal invasion, imbibition phase preceding seed germination gradually disrupts these barriers, whereupon protection of the germinating seed mainly relies upon antimicrobial compounds including peptides. Keeping this in view it is postulated that few peptides with antimicrobial properties may be present in the seeds. The present study is useful in exploring the possibility one more abundantly available source for antimicrobial peptides. Therefore the present study is justifiable and needed.
ENCLOSURE -II
6.2. REVIEW OF LITERATURE:
Natural Antimicrobial peptides;
Antimicrobial peptides are typically relatively short (12 to 100 amino acids), are positively charged (net charge of _2 to _9), are amphiphilic, and have been isolated from single-celled microorganisms, insects and other invertebrates, plants, amphibians, birds, fish, and mammals, including humans etc2. Thionins, defensins, Lipid transfer proteins and cyclotides are the major groups of proteins in plants that have been demonstrated to possess antimicrobial activities. Among these, defensins are small (45–54 amino acids) highly basic cysteine-rich peptides have been mainly reported from seeds of plants – Radish4, 4 o Clock plant6, Passion fruit 7, Chilli8, Melon 9, Wheat , etc10. belonging to diverse taxonomic groups11. The seeds of the edible plant Annona squamosa are reported to contain 1–4% proteins12. The leaves of the this plants are reported to possess antimicrobial13 hypoglycaemic and antidiabetic14, insecticidal properties15.The bark, leaves and roots of different species of this genera are used in folk medicines. It has been shown that the leaves and bark of this plant possessantifungal, bacteriostatic, and especially cytostatic capability. A large number of chemical compounds, including flavonoids, alkaloids and acetogenins, have been extracted from the seeds and many other parts of these plants. Flavonoids and alkaloids have shown antibacterial properties, and have been used for treatment of medical conditions, such as skin disease, intestinal worms12.
However there are no specific reports on the antimicrobial peptides from the seeds of the study plant i.e. Annona squamosa.
Annona squamosa Linn. belonging to family Annonaceae which is known as custard apple, sugar apple in English, Sitaphala in Kannada, Sitaphal in Hindi, Sitappalam in Malayalam, Sitaphalam in Sanskrit, Sitappalam in Tamil and Sitaapandu in telugu.
The plant is traditionally used for the treatment of epilepsy, dysentery, cardiac problems, worm infestation, constipation, haemorrhage, antibacterial infection, dysuria,fever, and ulcer. It also has antifertility, antitumour and abortifacient properties.16-21
Ethanolic extracts of leaves and stem are reported to possess anticancerous activity.22
The aqueous leaf extract reported to ameliorate hyperthyroidism,23 which is often considered as a causative factor of diabetes mellitus.24
The tribes and villagers of the Aligarh district 17 and Chotanagpur division 18 in India extensively use the young leaves of A. squamosa along with the seeds of Piper nigrum for the managementofdiabetes mellitus.
ENCLOSURE -III
6.3 OBJECTIVE OF THE STUDY:
The following objectives of my thesis work.
- To prepare crude extract of antimicrobial peptides from A. squamosa seeds and to test the antimicrobial activity of extract against a set of microbes
- To purify defensins from crude extract
- To fractionate and characterize purified defensins fraction
ENCLOSURE – IV
7. MATERIALS AND METHODS :
7.1 SOURCES OF DATA:
This study is planned to generate data by conducting laboratory-based research on set of microbes. To prepare crude extract of antimicrobial peptides from A. squamosa seeds and to test the antimicrobial activity of extract against a set of microbes and to purify and to quantify defensins from crude extractwhich will further fractionate and characterize purified defensins fraction.
Further an attempt will be made to determine the primary structure of the peptide exhibiting potent antimicrobial activity.
ENCLOSURE – V
7.2 METHODS OF COLLECTION OF DATA:
The whole study is divided into three phases to generate data. Following are three phases.
Phase-I: To prepare crude extract of antimicrobial peptides from A. squamosa seeds and to test the antimicrobial activity of extract against a set of microbes
The crude extracts for antimicrobial peptides from A. squamosa will be prepared by using the method .6&4
The antimicrobial activity against selected microbes will be assessed by determining minimum inhibitory concentration by using disc diffusion method25
Phase II:To purify and to quantify defensins from crude extract
The crude extract is purified by adopting the methods as described 4&6
Peptide concentration in the fraction will be determined using Bicinchoninic acid method26
Phase III: To fractionate and characterize purified defensins fraction
Defensin fraction of A.squamosa seeds will be further fractionated by electrophoresing a known volume of it in the sample buffer (62.5mM Tris-Hcl, 10% glycerol, 0.1% bromophenol blue) alongside suitable molecular weight marker, on polyacrylamide gels (formed by polymerization of 16% acrylamide in Trizma polymerization buffer – 1 M Trizma base, 10.6% glycerol, 10 µl TEMED, 0.033% ammonium persulfate) while supplying DC power (constant power) through cathode (0.1M Trizma, 0.1M Tricine), anode (0.2 M Trizma) buffers under non-denaturing conditions, in a vertical electrophoresis apparatus placed in an ice bath 27&28
Gels after completion of electrophoresis will be stained by copper staining29&30. For this native gel will be carefully released into shallow trough of water from glass plates and allowed to stand for a while. Later the gel will be transferred and immersed under sufficient volume of 0.3M CuCl2 taken in another shallow trough and rocked for 30 minutes at room temperature, then gel will again be transferred into trough of water, for washing (about 2-3 minutes).
Each of the bands which may appear as cleared regions against semi-opaque whitish-blue polyacrylamide gel will be excised and taken in a eppendorf vial (identified by molecular weight with reference to proximal band of molecular weight marker) and destained with 25mM Tris- Glycine buffer (PH 8.3) for 10 minutes followed by 12.5mM Tris-Glycine buffer (PH 8.3) for 10 minutes. Later the bands will b rinsed with water and immersed separately in 1 ml or more of extraction buffer (50 mM Tris-HCl, 50mM EDTA, PH 8.8). Peptides in the gel bands will be extracted by end-on-end rotation of tubes for 12-24 hours at room temperature. Supernatant will later be collected and dialysed against PBS to remove Cu2+ and EDTA in a dialysis bag of molecular weight cut-off of 1-2 kDa29.
Peptides in the dialysis residue (from each gel band) will be recovered by lyophilization and dissolved in minimum volume of suitable buffer (with appropriate concentrations of protease inhibitors)andtheir concentration will be determined by bicinchoninic acid method mentioned above and stored at 4oC until further use.
Series of 10 fold dilutions prepared from peptide released and purified from each gel band will be used for Quantitative disc diffusion growth inhibition assays against the above mentioned microorganisms to determine the Minimum inhibitory concentration (which is the highest dilution or corresponding lowest concentration of the peptide required to inhibit the growth of microorganism when compared to discs impregnated with plain buffer used to dissolve lyophilized peptide) of the peptide, if at all it exhibits antimicrobial activity. Appropriate statistical tests will be employed to determine the significance of inhibition.
Further an attempt will be made to determine the primary structure of the peptide exhibiting potent antimicrobial activity.
ENCLOSURE -VI
References:
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bacteriophages, bacterialcell wall hydrolases, and antimicrobial peptides J Appl
Microbiol 104: 1–13.
2.Jenssen , H., Hamill, P., and Hancock, R.E.W. (2006) Peptide Antimicrobial Agents
Clin Microbiol Rev 19(3): 491–511.
3.Zaiou, M. (2007) Multifunctional antimicrobial peptides: therapeutic targets in
several human diseases. J Mol Med 85: 317–329.
4.Terras F.R.G., and Schoofs H.M.E. (1992) Analysis of Two Novel Classes of Plant
Antifungal Proteins from Radish (Raphanus sativus L.) Seeds. J Biol Chem 267
(22): 15301-15309.
5.Last, D.I., and Llewellyn, D. J. (1997) Antifungal proteins from seeds of Australian
native plants and isolation of an antifungal peptide from Atriplex nummularia. N Z J
Bot35: 385-394.
6.Cammue B. P.A., De Bolle M.F.C. and others (1992) Isolation and Characterization
of a Novel Class of Plant Antimicrobial Peptides from Mirabilis jalapaL. Seeds. J
Biol Chem 267(4): 2228-2233.
7.Pelegrini, P.B., Noronha, E.F. and others (2006) An antifungal peptide from passion
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Biophys Acta 1764: 1141–1146.
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novel peptides from chillipepper seeds: Antimicrobial activities against pathogenic
yeasts.Toxicon 50: 600–611.
9.Ribeiro, S.F.F., Agizzio, A.P. and others (2007b)A new peptide of melon seeds
which shows sequence homology with vicilin:Partial characterization and antifungal
activity Scient Horticult 111 (2007) 399–405.
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605-612.
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bicinchoninic acid. Analyt. Biochem. 150:76–85.
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ENCLOSURE -VII
Remarks of the guide;
The study is highly justifiable & is feasible to work in the institution.
.