5 Kannan

							 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.
                 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.
  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.
        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
•
           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.
     ANTIBIOTIC SENSITIVITY
           PATTERNS:
      Pseudomonas aeruginosa is found to be
sensitive to….
       •   Imepinem & Meropenem-100%
       •   Amikacin-68.01%and
       •   Gentamycin-33%
       •   Chloramphenical-29%
     Several resistant forms have been
developed and it is most resistant to
Netilmicin(70.04%)
     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.
     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
       S.aureus produces enterotoxin A, B,
leukocidin, Exfoliatins,
      Heamolysins (α,β,γ,δ) lyse erythrocytes. α-
lysin is most important in pathogenicity and is
thought to facilitate the tissue destruction
associated with staphylococcal growth.
      Leukocidin kills polynuclear leukocytes and
macrophages.
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%
       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
                 β-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.
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)
             Media and Reagents:
•   Phosphate buffer saline (PBS)
•   Trypsin /EDTA
•   Dulbecco’s modification of Eagles medium
    (DMEM)
        • 10% fetal calf serum,

        • 1% penicillin/ streptomycin,

        • 1% L-glutamine

        • 0.2% of 0.1mBME
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.
•   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 10^7
    cells/ml.
•   To the developed cell line, infection is induced with
    the pathogens isolated from burn patients.
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 MTT{3-(4,5-Dimethyl
        thiazol-2-yl)-2-5,-diphenyl Tetrazolium
        bromide} assay.
      • The cell count using heamocytometer.
   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
        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.
THANK YOU….