STUDIES ON THE WOUND STUDIES ON THE WOUND HEALING PROPERTY OF HEALING PROPERTY OF

MORINDA CITRIFOLIAMORINDA CITRIFOLIA ON ON CELL LINE CAUSED BY CELL LINE CAUSED BY BACTERIAL INFECTIONBACTERIAL INFECTION

KANNAN.N and SHAJUNA BANU.ZKANNAN.N and SHAJUNA BANU.Z

School of BiotechnologySchool of Biotechnology

K.S.Rangasamy College of Technology,TiruchengodeK.S.Rangasamy College of Technology,Tiruchengode..

BURNSBURNSInfection is a major cause of morbidity and Infection is a major cause of morbidity and mortality in hospitalized burn patients.mortality in hospitalized burn patients.

The infection is due to the combined effect of :The infection is due to the combined effect of :•the impairment of the host natural defense the impairment of the host natural defense system, system, •colonization of the burn wound site, colonization of the burn wound site, •systemic dissemination of the colonizing systemic dissemination of the colonizing organisms.organisms.

Burn wound infections are largely hospital Burn wound infections are largely hospital acquired and infecting pathogens differ from one acquired and infecting pathogens differ from one hospital to another.hospital to another.

PREDOMINANT PATHOGENS IN PREDOMINANT PATHOGENS IN BURN WOUNDS:BURN WOUNDS:

The most commonly prevailing pathogens in The most commonly prevailing pathogens in burn wounds are:burn wounds are:

• Pseudomonas aeruginosaPseudomonas aeruginosa• Staphylococcus aureusStaphylococcus aureus• Klebsiella sp.Klebsiella sp.• Escherichia coliEscherichia coli• Streptococcus pyogenesStreptococcus pyogenes

Of these pathogenes,Of these pathogenes,P.aeruginosaP.aeruginosa(36%) and (36%) and S.aureusS.aureus(29%) accounted more in the burn wound (29%) accounted more in the burn wound infection site.infection site.

Pseudomonas aeruginosa:Pseudomonas aeruginosa:

Characteristics:Characteristics:

• Gram negative rodGram negative rod• Motile, ubiquitous in soil and waterMotile, ubiquitous in soil and water• Never fermentativeNever fermentative• Optimum growth at 37Optimum growth at 37ºc up to 42ºcºc up to 42ºc• Resistant to salts, dyes, weak antiseptics and as Resistant to salts, dyes, weak antiseptics and as

many commonly used antibioticsmany commonly used antibiotics• Produces two types of soluble pigments, the Produces two types of soluble pigments, the

fluorescent pigment fluorescent pigment PyoverdinPyoverdin and blue pigment and blue pigment PyocyaninPyocyanin

•

TOXINOGENESIS:TOXINOGENESIS:Pseudomonas aeruginosaPseudomonas aeruginosa produces two produces two

extracellular protein toxins-Exotoxin A and its extracellular protein toxins-Exotoxin A and its subunit Exoenzyme Ssubunit Exoenzyme S

Exotoxin A catalyses ADP-ribosylation and Exotoxin A catalyses ADP-ribosylation and inactivation of elongation factor 2,leading to inactivation of elongation factor 2,leading to inhibiton of protein biosynthesis and cell death.inhibiton of protein biosynthesis and cell death.

Exoenzyme S, also an ADP-ribosyl transferase. Exoenzyme S, also an ADP-ribosyl transferase. ribosylates GTP binding proteins such as Ras and ribosylates GTP binding proteins such as Ras and Rho. Rho.

Ras and Rho are molecular switches that Ras and Rho are molecular switches that control numerous cellular processes, also control numerous cellular processes, also contribute to wound healing processes and contribute to wound healing processes and tissue regeneration.tissue regeneration.

ANTIBIOTIC SENSITIVITY ANTIBIOTIC SENSITIVITY PATTERNS:PATTERNS:

Pseudomonas aeruginosaPseudomonas aeruginosa is found to be is found to be sensitive tosensitive to….….

• Imepinem & Meropenem-100% Imepinem & Meropenem-100% • Amikacin-68.01%and Amikacin-68.01%and • Gentamycin-33%Gentamycin-33%• Chloramphenical-29% Chloramphenical-29%

Several resistant forms have been Several resistant forms have been developed and it is most resistant to developed and it is most resistant to Netilmicin(70.04%)Netilmicin(70.04%)

Staphylococcus aureus:Staphylococcus aureus:Characteristics:Characteristics:

• Gram positive, cluster forming coccusGram positive, cluster forming coccus• Catalase positiveCatalase positive• Golden yellow colony on agarGolden yellow colony on agar• Pathogens of humans, causes wide range of Pathogens of humans, causes wide range of

supprative (pus-forming) infections and supprative (pus-forming) infections and toxinoses.toxinoses.

• It is a major cause of nosocomial infection in It is a major cause of nosocomial infection in surgical wounds and infections are associated surgical wounds and infections are associated with indwelling medical devices.with indwelling medical devices.

TOXINOGENESIS:TOXINOGENESIS:The virulence factors of The virulence factors of S. aureus S. aureus are found to be are found to be due to due to • Surface proteins that promote colonization of host Surface proteins that promote colonization of host

tissues.tissues.• Invasins that promote bacterial spread in tissues Invasins that promote bacterial spread in tissues

(leukocidin, hyaluronidase, kinases)(leukocidin, hyaluronidase, kinases)• Surface factors that inhibit phagocytic engulfment Surface factors that inhibit phagocytic engulfment

(capsule, protein A).(capsule, protein A).• Biochemical properties that enhance their survival Biochemical properties that enhance their survival

in phagocytes (carotenoids, catalase production)in phagocytes (carotenoids, catalase production)• Membrane damaging toxinsMembrane damaging toxins

S.aureus produces enterotoxin A, B, S.aureus produces enterotoxin A, B, leukocidin, Exfoliatins, leukocidin, Exfoliatins,

Heamolysins (Heamolysins (αα,,ββ,,γγ,,δδ) lyse erythrocytes. ) lyse erythrocytes. αα--lysin is most important in pathogenicity and is lysin is most important in pathogenicity and is thought to facilitate the tissue destruction thought to facilitate the tissue destruction associated with staphylococcal growth.associated with staphylococcal growth.

Leukocidin kills polynuclear leukocytes and Leukocidin kills polynuclear leukocytes and macrophages. macrophages.

ANTIBIOTIC SENSITIVITY PATTERNS:ANTIBIOTIC SENSITIVITY PATTERNS:

Staphylococcus aureusStaphylococcus aureus is found to be resistant to is found to be resistant to • Ampicillin and Tobramycin -99.3% eachAmpicillin and Tobramycin -99.3% each• ErythromycinErythromycin -25.3%-25.3%• Trimethoprim-sulphamethoxazole-13.9%Trimethoprim-sulphamethoxazole-13.9%

The most effective antibiotics against The most effective antibiotics against S. aureus aS. aureus are,re,• Chloramphenical-78%Chloramphenical-78%• GentamycinGentamycin -63% -63%• Tetracycline Tetracycline -57% -57%

The treatment against bacterial pathogens in burn patients The treatment against bacterial pathogens in burn patients are often difficult due to antibiotic resistance. are often difficult due to antibiotic resistance.

The mechanism of resistance to drugs includeThe mechanism of resistance to drugs include• Reduced cell wall permeabilityReduced cell wall permeability• Production of chromosomal and plasmid mediated Production of chromosomal and plasmid mediated

ββ-lactamase, aminoglycoside modifying enzymes -lactamase, aminoglycoside modifying enzymes • The active multi-drug efflux mechanism.The active multi-drug efflux mechanism.

Several types of vaccines are being tested but none is Several types of vaccines are being tested but none is currently available for general use.currently available for general use.

So, now the researchers are striving to provide an insight So, now the researchers are striving to provide an insight into the use of plant extracts against these pathogens in into the use of plant extracts against these pathogens in in vitroin vitro studies.studies.

The objective of the present work is to study the wound The objective of the present work is to study the wound healing property of healing property of Morinda citrifoliaMorinda citrifolia on mouse cell line infected on mouse cell line infected with pathogens.with pathogens.

DEVELOPMENT OF MOUSE CELL LINE:DEVELOPMENT OF MOUSE CELL LINE:Requirements:Requirements:

• 13.5 day pregnant mouse13.5 day pregnant mouse• Two sets sterile instruments, one containing a Two sets sterile instruments, one containing a

pair of curved forceps and a pair of iris scissors pair of curved forceps and a pair of iris scissors and the other containing two pairs of curved and the other containing two pairs of curved forceps, one pair iris scissors and no.3 sized forceps, one pair iris scissors and no.3 sized scalpel handle.scalpel handle.

• Sterile medium sized Petri dishes (tissue culture Sterile medium sized Petri dishes (tissue culture standard)standard)

• 18 gauge needle 18 gauge needle • Luer lock syringe Luer lock syringe • No.11 sized flat-edged scalpel blade No.11 sized flat-edged scalpel blade • Large flasks ( tissue culture standardLarge flasks ( tissue culture standard

about 154 sq.cm area) about 154 sq.cm area)

Media and Reagents:Media and Reagents:

• Phosphate buffer saline (PBS)Phosphate buffer saline (PBS)• Trypsin /EDTATrypsin /EDTA• Dulbecco’s modification of Eagles medium Dulbecco’s modification of Eagles medium

(DMEM)(DMEM)• 10% fetal calf serum,10% fetal calf serum,• 1% penicillin/ streptomycin,1% penicillin/ streptomycin,• 1% L-glutamine 1% L-glutamine • 0.2% of 0.1mBME0.2% of 0.1mBME

ISOLATION OF PRIMARY MOUSE EMBRYO ISOLATION OF PRIMARY MOUSE EMBRYO FIBROBLAST:FIBROBLAST:

• Embryo from 13 or 14 day pregnant mouse Embryo from 13 or 14 day pregnant mouse is removed .is removed .

• Washed with PBS Washed with PBS • Finely minced the tissue with iris scissors or Finely minced the tissue with iris scissors or

2 ml Trypsin/EDTA was added and 2 ml Trypsin/EDTA was added and incubated at 37incubated at 37ºc(20 min).ºc(20 min).

• Neutralize the Trypsin/EDTA with 20 ml Neutralize the Trypsin/EDTA with 20 ml culture medium.culture medium.

• Mix the contents and add to T75 culture flask Mix the contents and add to T75 culture flask containing 20 ml culture medium with containing 20 ml culture medium with approximately three embryos per T75.approximately three embryos per T75.

• Incubate the flasks overnight at 37Incubate the flasks overnight at 37ºcºc• The next day , change the medium to remove The next day , change the medium to remove

debris and toxic cell death products.debris and toxic cell death products.• Upon confluence, harvest and freeze the primary Upon confluence, harvest and freeze the primary

fibroblasts at a freezing density of 3.0 x 10^7 fibroblasts at a freezing density of 3.0 x 10^7 cells/ml.cells/ml.

• To the developed cell line, infection is induced with To the developed cell line, infection is induced with the pathogens isolated from burn patients.the pathogens isolated from burn patients.

CELL LINE CHARACTERIZATION:CELL LINE CHARACTERIZATION:

The cell line has to be characterized every 30min The cell line has to be characterized every 30min from the time of infection to observe :from the time of infection to observe :

• Changes in the cell morphologyChanges in the cell morphology• The cell viability by MTT{3-(4,5-Dimethyl The cell viability by MTT{3-(4,5-Dimethyl

thiazol-2-yl)-2-5,-diphenyl Tetrazolium thiazol-2-yl)-2-5,-diphenyl Tetrazolium bromide} assay.bromide} assay.

• The cell count using heamocytometer.The cell count using heamocytometer.

TREATMENT OF INFECTED CELL TREATMENT OF INFECTED CELL LINE:LINE:

The active ingredients in plant extracts are the The active ingredients in plant extracts are the alternatives for pathogens that developed resistance alternatives for pathogens that developed resistance agains conventional and synthetic antibiotics.agains conventional and synthetic antibiotics.

In this way In this way Morinda citrifoliaMorinda citrifolia is found to possess is found to possess antibacterial and other antimicrobial activities.antibacterial and other antimicrobial activities.

The compounds responsible for the antibacterial The compounds responsible for the antibacterial properties are…properties are…

• Acubin,Acubin,• L-asperuloside,L-asperuloside,• Alizarin, andAlizarin, and• Other anthroquinone compoundsOther anthroquinone compounds

The pure extracts from the various parts (roots, The pure extracts from the various parts (roots, leaves) of the plant leaves) of the plant Morinda citrifoliaMorinda citrifolia can be obtained can be obtained by solvent extraction.by solvent extraction.

These extracts will be injected to the infected These extracts will be injected to the infected cell line at various concentrations and the subsequent cell line at various concentrations and the subsequent changes/improvement in the cell regeneration process changes/improvement in the cell regeneration process could be noted.could be noted.

THANK YOU….THANK YOU….