STUDIES ON THE WOUND HEALING PROPERTY OF MORINDA CITRIFOLIA ON CELL LINE CAUSED BY BACTERIAL INFECTI

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STUDIES ON THE WOUND HEALING PROPERTY OF MORINDA
CITRIFOLIA ON CELL LINE CAUSED BY BACTERIAL
INFECTION

• KANNAN.N and SHAJUNA BANU.Z
• School of Biotechnology
• K.S.Rangasamy College of Technology,Tiruchengode.

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BURNS

• Infection is a major cause of morbidity and
  mortality in hospitalized burn patients.
• The infection is due to the combined effect of
  
• the impairment of the host natural defense
  system,
• colonization of the burn wound site,
• systemic dissemination of the colonizing
  organisms.
• Burn wound infections are largely hospital
  acquired and infecting pathogens differ from one
  hospital to another.

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PREDOMINANT PATHOGENS IN BURN WOUNDS

• The most commonly prevailing pathogens in burn
  wounds are
• Pseudomonas aeruginosa
• Staphylococcus aureus
• Klebsiella sp.
• Escherichia coli
• Streptococcus pyogenes
• Of these pathogenes,P.aeruginosa(36) and
  S.aureus(29) accounted more in the burn wound
  infection site.

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Pseudomonas aeruginosa

• Characteristics
• Gram negative rod
• Motile, ubiquitous in soil and water
• Never fermentative
• Optimum growth at 37ºc up to 42ºc
• Resistant to salts, dyes, weak antiseptics and as
  many commonly used antibiotics
• Produces two types of soluble pigments, the
  fluorescent pigment Pyoverdin and blue pigment
  Pyocyanin

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TOXINOGENESIS

• Pseudomonas aeruginosa produces two
  extracellular protein toxins-Exotoxin A and its
  subunit Exoenzyme S
• Exotoxin A catalyses ADP-ribosylation and
  inactivation of elongation factor 2,leading to
  inhibiton of protein biosynthesis and cell death.
• Exoenzyme S, also an ADP-ribosyl transferase.
  ribosylates GTP binding proteins such as Ras and
  Rho. Ras and Rho are molecular switches that
  control numerous cellular processes, also
• contribute to wound healing processes and
• tissue regeneration.

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ANTIBIOTIC SENSITIVITY PATTERNS

• Pseudomonas aeruginosa is found to be sensitive
  to.
• Imepinem Meropenem-100
• Amikacin-68.01and
• Gentamycin-33
• Chloramphenical-29
• Several resistant forms have been developed and
  it is most resistant to Netilmicin(70.04)

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Staphylococcus aureus

• Characteristics
• Gram positive, cluster forming coccus
• Catalase positive
• Golden yellow colony on agar
• Pathogens of humans, causes wide range of
  supprative (pus-forming) infections and
  toxinoses.
• It is a major cause of nosocomial infection in
  surgical wounds and infections are associated
  with indwelling medical devices.

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TOXINOGENESIS

• The virulence factors of S. aureus are found to
  be due to
• Surface proteins that promote colonization of
  host tissues.
• Invasins that promote bacterial spread in tissues
  (leukocidin, hyaluronidase, kinases)
• Surface factors that inhibit phagocytic
  engulfment (capsule, protein A).
• Biochemical properties that enhance their
  survival in phagocytes (carotenoids, catalase
  production)
• Membrane damaging toxins

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• S.aureus produces enterotoxin A, B, leukocidin,
  Exfoliatins,
• Heamolysins (a,ß,?,d) lyse erythrocytes. a-lysin
  is most important in pathogenicity and is thought
  to facilitate the tissue destruction associated
  with staphylococcal growth.
• Leukocidin kills polynuclear leukocytes and
  macrophages.

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ANTIBIOTIC SENSITIVITY PATTERNS

• Staphylococcus aureus is found to be resistant
  to
• Ampicillin and Tobramycin -99.3 each
• Erythromycin -25.3
• Trimethoprim-sulphamethoxazole-13.9
• The most effective antibiotics against S.
  aureus are,
• Chloramphenical-78
• Gentamycin -63
• Tetracycline -57

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• The treatment against bacterial pathogens in
  burn patients are often difficult due to
  antibiotic resistance.
• The mechanism of resistance to drugs include
• Reduced cell wall permeability
• Production of chromosomal and plasmid mediated
  ß-lactamase, aminoglycoside modifying enzymes
• The active multi-drug efflux mechanism.
• Several types of vaccines are being tested but
  none is currently available for general use.
• So, now the researchers are striving to provide
  an insight into the use of plant extracts against
  these pathogens in in vitro studies.
• The objective of the present work is to study
  the wound healing property of Morinda citrifolia
  on mouse cell line infected with pathogens.

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DEVELOPMENT OF MOUSE CELL LINE

• Requirements
• 13.5 day pregnant mouse
• Two sets sterile instruments, one containing a
  pair of curved forceps and a pair of iris
  scissors and the other containing two pairs of
  curved forceps, one pair iris scissors and no.3
  sized scalpel handle.
• Sterile medium sized Petri dishes (tissue culture
  standard)
• 18 gauge needle
• Luer lock syringe
• No.11 sized flat-edged scalpel blade
• Large flasks ( tissue culture standard
• about 154 sq.cm area)

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Media and Reagents

• Phosphate buffer saline (PBS)
• Trypsin /EDTA
• Dulbeccos modification of Eagles medium (DMEM)
• 10 fetal calf serum,
• 1 penicillin/ streptomycin,
• 1 L-glutamine
• 0.2 of 0.1mBME

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ISOLATION OF PRIMARY MOUSE EMBRYO FIBROBLAST

• Embryo from 13 or 14 day pregnant mouse is
  removed .
• Washed with PBS
• Finely minced the tissue with iris scissors or 2
  ml Trypsin/EDTA was added and incubated at
  37ºc(20 min).
• Neutralize the Trypsin/EDTA with 20 ml culture
  medium.

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• Mix the contents and add to T75 culture flask
  containing 20 ml culture medium with
  approximately three embryos per T75.
• Incubate the flasks overnight at 37ºc
• The next day , change the medium to remove debris
  and toxic cell death products.
• Upon confluence, harvest and freeze the primary
  fibroblasts at a freezing density of 3.0 x 107
  cells/ml.
• To the developed cell line, infection is induced
  with the pathogens isolated from burn patients.

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CELL LINE CHARACTERIZATION

• The cell line has to be characterized every
  30min from the time of infection to observe
• Changes in the cell morphology
• The cell viability by MTT3-(4,5-Dimethyl
  thiazol-2-yl)-2-5,-diphenyl Tetrazolium bromide
  assay.
• The cell count using heamocytometer.

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TREATMENT OF INFECTED CELL LINE

• The active ingredients in plant extracts are the
  alternatives for pathogens that developed
  resistance agains conventional and synthetic
  antibiotics.
• In this way Morinda citrifolia is found to
  possess antibacterial and other antimicrobial
  activities.
• The compounds responsible for the antibacterial
  properties are
• Acubin,
• L-asperuloside,
• Alizarin, and
• Other anthroquinone compounds

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• The pure extracts from the various parts (roots,
  leaves) of the plant Morinda citrifolia can be
  obtained by solvent extraction.
• These extracts will be injected to the infected
  cell line at various concentrations and the
  subsequent changes/improvement in the cell
  regeneration process could be noted.

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THANK YOU.