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MARINE FINFISH PRODUCTION AND RESEARCH CENTRETANJUNG DEMONG, TERENGGANUTHE ANTIMICROBIAL POTENTIAL OFTHE CRUDE EXTRACTS FROM LEAVESOF SOME LOCAL HERBS ON FISHPATHOGENIC BACTERIANIK HAIHA NIK YUSOFFMOHD-ZAIDI MOHAMEDHUSSIN MAT ALI AND RAHMAH MOHD ZIN

INTRODUCTION

• Aquaculture is a rapidly expanding industry both inMalaysia and world wide• The ultimate goal : To produce the greatest possibleweight per culture unit which leads to intensive fishculture• The incidence of diseases increases as a result ofvarious stressors such as– over crowding– Handling– poor water quality– Others• Many major groups of bacteria have cause diseaseoutbreaks in farmed fish resulting serious economiclosses to the industry

• Estimated economic losses caused by one type ofpathogenic bacteria was RM 20 million (1990)• Bacterial diseases: Prevention disease outbreaks/administration of antibiotics & chemotherapeutant• Indiscriminate use of antibiotics; increased in antibioticresistantbacteria; compromise treatment of bacterialinfections in human• The phasing out of antibiotic affect the aquacultureindustry ; farmers need it for pathogen prevention• Minimize loss, a need to find alternatives

• Non-therapeutic alternatives such as enzymes,probiotics, herbs, immunostimulant and othermanagement practices• Local herbs appears as potential alternatives forprevention & treatment of infectious diseases withoutany side effect• There are many reports concerning the inhibtingactivities from herbs against human pathogens• Reports on the application of medicinal herbs to aquaticanimals is still limited

AIMS OF STUDY

• To determine the antibacterial activity ofextracts of some of the local Malaysianherbs in treatment for fish disease• To identify an inexpensive, simple, andeffective method of preventing andcontrolling fish diseases

PLANTMATERIAL USED

Azadirachta indica• Local name :Semambu/mambu(Neem/Margosa)• Scientific name :Azadirachta indica• Family : Meliaceae• Origin : Burma and India• Evergreen tree native toSoutheast Asia• Constituents :– Nimbidin an essential oil– Fixed oil– Resin,– Traces of a substance ofalkaloidal

Piper betle• Local name : Sireh (Betel)• Scientific name : Piper Betle• Family : Piperaceae• Origin : Malaysia• Evergreen and perennial withglossy heart-shaped leaves• Constituents :– Chavibetol (which gives a smokyaroma)– Chavicol– Cadinene– Methyl eugenol– Tannin

Terminalia catappa• Local name : Ketapang(Catappa)• Scientific name : Terminaliacatappa• Family : Combretaceae• Origin : Malaysia• Elsewhere : Also grown inIndia, Indonesia and SriLanka• Constituents:– Saponin glycosides– Saponins– Steroid– Digitalis glycosides (cardiac)– Tannins– Phenols

MICROBESTARGETED

• Vibrio alginolyticus• Vibrio vulnificus• Photobacterium damsela• Pseudomonas sp

MATERIALS ANDMETHODS

Preparation of Bacteria• Four different fish bacterial pathogens used in thestudy were obtained from stock cultures of NaFisH,Penang.• All organisms were maintained at 4 o C on slantnutrient agar until further use.• Microorganisms were inoculated into nutrient broths& incubated for 18 – 24h at 37 o C.• Bacterial suspensions used was adjusted to 0.5 McFunits (10 5 – 10 6 cfu/ml)

Plant Materials• Herbs utilized were purchased from the localmarket• Fresh healthy leaves were washed underrunning tap water, and air dried under shadedarea• Dried leaves was shredded into small pieces• Ground until powder form in an electric grinder &were kept in sealed plastic bags

PREPARATION OFCRUDE EXTRACT- 2 types of extractionAqueous & Solvent

Aqueous Extract• 100 g of the powdered material was boiled in 1 liter ofdeionised distilled water• Final volume of 100ml• Concentrated mixture was filtered• Clear extract was aliquot in 1-ml volumes tubes• Extracts dried overnight using vacuum concentrator until nofurther changes in weight were observed• Stored at -20 o C until further use

Solvent Extraction• 100 gm dried powdered plant were immersed separately in 500ml of methanol (80 %) & ethanol (90%) for 72 h at roomtemperature• Extract filtered through a Buchner funnel with Whatan No.1 filterpaper• Filtrate was evaporated to dryness with a rotary evaporator at60 °C• Extracts re-suspended in Methanol/Ethanol to achieve the stockconcentration 100 mg/ml• Plants stored in dark vials/bottles at 4 o C until further use for theantimicrobial test.

CrudeExtractPreparationFresh leafDried undershaded areaImmersed insolventkept in sealedplastic bagGriound untilpowderedfromFilteredthroughBuchner funelEvaporated usingrotary evaporatorKept in darkbottles untilfurther use

ANTIBACTERIAL SUSCEPTIBILITY TESTPaper discs were soakedin plant extract and left to dryZone inhibition were measuredand data were analysedA loop of bacteria wasinoculated in TSB until reachesMc Farland standard need100ul bacteria was spreadon the surface of Mueller-Hintonagar (MHA) platesThe plates were incubated at37 o C for 24 hoursOxytetracycline (OTC) as positive controland solvent as negative controlPaper discs containing the plant extractwere placed on the surface ofeach plate

Screening for antibacterial activityExtractantSterile disc soaked ineaxtractantBacterial preparationZone ofinhibitionDISCDIFFUSIONMETHOD(Baeur, 1966;Anderson,1974)Incubatingat 37 o CPlacing paperdisc on plateMc Farland unitSwabbingbacteria onMHA

Grading of result (MacKeen et al, 1997)- No inhibition zone+ Zone of inhibition < 10 mm in diameter(weak activity)++ Zone of inhibition between 10 – 14 mm in diameter(moderate activity)+++ Zone of inhibition between 15 – 19 mm in diameter(strong activity)++++ Zone of inhibition ≥ 20 mm in diameter(moderate activity)

DILUTION IN MIC DETERMINATION50 25 12.5 6.25 3.13 1.56 0.78 0.39 0.20 (mg/ml)% concentration of Antimicrobial AgentsDark tubes = growthLight tubes = no growth

METHODS TO DETERMINE MBC• 100µl of each test tube showing no turbidity(no growth) were sub-cultured onto sterilenutrient agar plate and re-incubated foranother 24 hours• Control samples (positive and negative) wereincubated under the same conditions• The lowest concentration from which themicroorganisms did not recover was taken asMBC value

STATISTICAL ANALYSIS• Since the readings of control in invitroantibacterial studies of medicinal herbswere zero, the data was analyzed bysimple arithmentic means of thedifferent extracts and standard errorcompare to the control (Mohan, 2004)

RESULT

Azadiracta indicaInhibition zone (mm)3025201510Antibacterial activity of Azadirachta indicaAqueousMethanolEthanol• A. indica extractsshowed weak/noactivity against allbacteria tested50Va Vv Ph sBacterial strain

Azadiracta indica

Piper betleAntibacterial activity of Piper betle• The MeoH & EtoHextract of P. betleextract exhibitedprominentantibacterialactivity against allbacteria testedInhibitionzone (mm)30252015105AqueousMethanolEthanol0Va Vv Ph PsBacterial strain

Piper betle

Terminalia catappa• MeOh & EtOH T.catappa extractsalso showed goodantibacterial activityAntibacterial activity of Terminalia catappa302520Inhibitionzone (mm)1510AqueousMethanolEthanol50Va Vv Ph PsBacterial strain

Terminalia catappa

• V. vulnificus and P. damsela were inhibited by almost allthe extracts• T. catappa and A. indica extracts showed weak/noactivity against V. alginolyticus and Pseudomonas sp• Effect of different solvent :– MeOH and EtOH extract > antibacterial activity than Aqueous• Negative control : No antibacterial activity; Solvent usedfor solubilisation of drug had no anti bacterial activity

Table 2. Minimum inhibitory concentration and minimumbactericidal concentrationMicroorganismPlantsV.alginolyticusMICMBCV. vulnificusMICMBCPhotobacterium damselaMICMBCPseudomonasspMICMBCPiper betel(Methanolicextract)12.525.06.2512.56.2512.5NTNTPiper betel(Ethanolicextract)6.2512.53.156.253.156.25NTNTTerminaliacatappa(Methanolicextract)NTNT12.512.56.2512.5NTNTTerminaliacatappa(Ethanolicextract)* NT : Not testedNTNT252512.525NTNT

MIC• A wide range of activities of thedifferent herbs and extract againstthe bacterial tested.• The MIC values indicated that P.betle was more efficient than T.catappa (MIC values ranging from3.25 -12.5mg/ml and MIC valuesranging from 6.25 – 25 mg/mlrespectively.• The MIC values of EtOH extracts ofP. betle is lower in P.damsela andV. vulnificus in comparison to V.alginolyticus; suggests that this twobacteria species showed greatersensitivity towards the extracts.

MBC• The MIC of the plant extractsis < the MBC against the testorganism• No colony growth wasobserved on the solid mediumafter the incubation periodwhen the MBC of the plantextracts was used against testorganisms

DISCUSSION

• MeOH & EtOH extracts of P.betle & T. catappa showedremarkable antibacterialactivities• They produce antibacterialactivity against almost all testorganisms(Ramji et al, 2002; Pauli, 2002;Pawar and Pal, 2002; Liu etal,1996; Burapadaja,1997).

Azadirachta indica• No/weak antibacterial activityin extracts of against all thetested pathogens• Results are contradictory withsome researchers whoreported antibacterial activityof the above herbs (Das et al,1999; Hasna-Banu, 2005 ;Haniffa et al, 2006; Mahfuzul-Hoque et al, 2007,)• Variation due to:– Dose used– Method of extraction– Age– Parts of herbs used

• Crude extracts of all herbs tested havenegligible effect on growth of Pseudomonas sp.(Babayi, 2003)• MIC value for V. alginolyticus > V. vulnificus andP. damsela sp for P. betle; higher dosesrequired in infections where V. alginolyticus isthe aetiologic agent• The MIC < MBC value of four active plantextracts ; Extracts were bacteriostatic at lowerconcentration & bactericidal at higherconcentration.

• Crude extracts of all the herbs studiedcontain one or more of the phytochemicalcompounds (saponins, saponinglycosides, steroid, cardiac glycosides,tannins, volatile oils and phenols)• Phytochemical components have inhibitoryeffects on the bacteria (Punita et al,2008 ; Direkbusarakom et al, 1995, 1997).

• EtOH & MeOH efficient in extracting the activecompounds for these species (Durmaz et al , 2006).• Components with antimicrobial activity extracted fromherbs are aromatic or saturated organic compound ; andthey are more soluble in methanol and ethanol.• Aqueous extracts showed no/weak antibacterial activity(Martin, 1995; Paz et al, 1995; Vlientinck et al, 1995).• Solvent > Aqueous : 2 reasons;– The nature of biological active components (saponins, tannins,alkaloids and anthraquinone) enhanced in the presence ofsolvent– Stronger extraction capacity of solvent produced greater numberof active constituents responsible for the antibacterial activity.

CONCLUSION

• EtOH & MeOH of P.betle and T.catappacontains positive effects for treatment of fishbacteria.• They may become alternative sources ofantimicrobial drugs to complement existingantibiotics and/or provide novel/or leadcompounds employed in controlling the fishpathogen.• Decrease production costs, minimize theamount of antibiotics entering the environment,and help ensure a safer, healthier food supplyfor the world.

• Greater ecological & economicsustainability of the aquacultureindustry.• Plants are locally available & cultivable• However, further investigations onToxicity, stability & metabolism of theplants & plants component

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