EVALUATION OF PRESERVATIVE EFFECTIVENESS OF FERULIC ACID DERIVATIVES IN ALUMINIUM HYDROXIDE GEL- USP

EVALUATION OF PRESERVATIVE EFFECTIVENESS OF FERULIC ACID DERIVATIVES IN ALUMINIUM HYDROXIDE GEL- USP

Anurag Khatkar, Arun Nanda* and Balasubramanian Narasimhan

Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001, Haryana, India

ABSTRACT: The selected amide and ester derivatives of ferulic acid were subjected to preservative efficacy testing in an official antacid preparation, (Aluminium Hydroxide Gel-USP) against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Candida albicans and Aspergillus niger as representative challenging microorganisms as per USP 2004 guidelines. The selected derivatives were found to be effective against all selected strains and showed preservative efficacy comparable to that of standard and even better in case B. subtilis and C. albicans. The 8- hydroxy quinoline ester derivative showed better preservative efficacy than standard as well as other derivatives and have better potential for use in the pharmaceutical preparations.

Amides, Esters, Preservative, Log CFU/ml

INTRODUCTION: Deterioration of either food or pharmaceutical preparations due to growth of microorganisms is a great challenge and need of preservation becomes very important ¹.

Non-sterile products such as pharmaceuticals, cosmetics, food items etc. with a high degree of water availability may be contaminated with microorganisms which may cause spoilage of the product with loss of therapeutic properties and, if they are pathogenic, serious infections can arise ². To inhibit the growth of contaminating microorganism, antimicrobial preservative systems have been developed and introduced into the pharmaceutical, cosmetic or food products during manufacturing process and/or throughout its use by consumers ³.

In several cases, themicroorganismsbecame resistanttoantimicrobialsandinsomecasesare able todegrademany commonlyusedpreservativesespeciallyp-hydroxybenzoates,e.g.,parabens ⁴. Preservatives to which resistance has been reported includes benzoic acid, benzalkonium chloride, chloramine, chlorhexidine, cholorophenol, dibromodicyanobutane, dimethyl oxazolidine, dimethyl dithiocarbamate, dimethoxy dimethyl hydantoin, formaldehyde, glutaraldehyde, hydrogen peroxide, iodine, mercuric salts, methylene bischlorophenol, methylparaben, propylparaben, phenylmercuric acetate, povidine-iodine, quaternary ammonium compounds and sorbic acid ⁵.

Also, the commonly used chemical preservatives may cause very serious side effects such as the benzalkonium chloride may cause mucosal damage and was also reported as genotoxic and cytotoxic ^{6, 7}. Thiomerosal used in ocular and nasal preparations was reported to be cytotoxic by Liao et al., 2011 ⁸. The use of parabens may cause skin cancer, genotoxicity and breast cancer as reported by the study of Dabre et al., 2008 ⁹.

The United States and Britishpharmacopoeiasdescribe officialmethodsforevaluation of preservativesystem ^{10, 11}. Preservativeefficacy test(challenge test)involvestheartificialintroductionofrepresentative microorganismsincludingGrampositiveandGramnegative bacteria,mouldandyeastintothe productunderstudy,in sufficientamountsfollowedby the collection ofkineticinformation regardingthelossoftheirviability.

The preservative potential of natural organic acids is well established in the literature viz. capryllic acid ¹², veratric acid ¹³, 2, 4 hexadienoic acid ¹⁴ and anacardic acid ¹⁵. Literature reports reveal that the ferulic acid possesses antimicrobial, antioxidant and preservative activities ^{16, 17}.

In view of the potential of microorganisms developing resistance to most common preservatives it became imperative to develop newer and stronger preservatives.

Further, in view of the reported toxicity potential of common synthetic preservatives, it would be quite judicious to develop the preservatives based on the natural sources such as ferulic acid.

In this context, amide and ester derivatives of ferulic acid were investigated for preservative efficacy in the present work. The preservativeefficacyofmost effective amide and esterderivatives of ferulic acid against gram  positive  Staphylococcus  aureus  MTCC  2901,  Bacillus  subtilis  MTCC  2063,  gram negativeEscherichia  coli  MTCC  1652,  fungal  strains  Aspergillus  niger  MTCC  8189  and CandidaalbicansMTCC227 was investigated and comparedthemwiththestandardpreservativesmethyland propylparaben,inAluminium Hydroxide Gel –USP ¹⁸.

MATERIALSAND METHODS:

Materials: Nutrientagar,nutrientbroth,sabourauddextroseagarandsabourauddextrosebrothwereobtainedfrom Himedia,Mumbai.Mannitol,methylandpropylparabenwereobtainedfromCDH,Mumbai.

Methods: Aluminium Hydroxide Gel USPwasusedasthepharmaceuticalproductforevaluationof preservative efficacytesting.

Formula for preparation of Aluminium Hydroxide Gel USP2004: AluminiumHydroxide Gel,36g;Mannitol,7g;Methylparaben,0.2g;Propylparaben,0.02g; Saccharin,0.05g;Peppermintoil,0.005ml;Alcohol,1ml; Purifiedwaterq.s., 100ml. Theweighedquantity ofaluminum hydroxidegelandmannitolweretrituratedwith50 mlofwaterina mortar.Methylparaben,propylparaben,saccharinandpeppermintoilweredissolvedinalcoholand addedto abovemixtureandtrituratedwell.Thevolumewasmadeupto 100mlwithpurifiedwater followedbyitssterilizationbyautoclaving.

Forpreservativeefficacytesting,theAluminium Hydroxide Gel waspreparedusing thepreservatives mentionedinTable1by replacingmethylparabenandpropylparabenfromtheaboveformula.The equimolaramountofselectedpreservatives (Fig. 1) werecalculatedwithreferenceto theamountof methyl paraben(0.0013mol)andaddedintoaluminumhydroxide gel ¹⁹.

TABLE1:AMOUNTOF SELECTEDPRESERVATIVESADDEDINALUMINUMHYDROXIDE GEL–USP

FIG. 1:   STRUCTURES OF SELECTED FERULIC ACID DERIVATIVES

Strains: StaphylococcusaureusMTCC2901,BacillussubtilisMTCC2063,EscherichiacoliMTCC 1652,CandidaalbicansMTCC227andAspergillusniger MTCC8189wereusedinthisstudywerecommoncontaminantsandprescribedinUSPforpreservativeefficacytesting inpharmaceuticalpreparations.

Preservativeefficacytesting inAluminium Hydroxide Gel USP2004: Thepreservativeefficacytestwasperformedessentiallyfollowingthestandardprotocoldescribedin USP-2004.InallcasesthepreservativeefficacytestwasdoneinAluminiumhydroxidegel-USPwith andwithoutthepreservativesystem.Theunpreservedproductwasusedasacontrolto evaluatethe viabilityoftheinoculatedcellsandtheirabilitytogrowintheproduct.

Preparationof inoculum: TherepresentativemicroorganismswereinoculatedinnutrientagarI.P.(S.aureus,B.subtilis,E.coli) andsabouraudagarI.P. (C. albicans,A.niger).The seededplateswere incubatedat37^oCfor24h(S. aureus, B.subtilisand E.coli),37^oCfor48h(C. albicans)and25^oCfor7d(A.niger).Aftertheincubation period,suspensionsofmicroorganismswerepreparedinsterilesalinesolution(0.9%w/vNaCl) togivea microbialcount of1x10⁴CFU/ml¹³.

TestProcedure: Aluminiumhydroxidegel-USPintheirfinalcontainerwasusedinthechallengetest.Thepreparation wasinoculatedwiththemicrobialcellsuspensionwithacellcountof1x10⁴CFU/ml.Theinoculum neverexceeded1%ofthevolumeoftheproductsample.Inoculatedsamplesweremixedthoroughly to ensurehomogeneousmicroorganismdistributionandincubated.TheCFU/mlof theproductwas determinedatanintervalof 0,7,14,21 and28donagarplate.Thelogvaluesof numberof CFU/ml (Table2 toTable6)ofAluminium Hydroxide Gel wascalculatedandcomparedaspertheguidelinesof USP2004.

Criteria of acceptance for preservative system: As per USP 2004 requirement for antacid made with an aqueous base, preservative effectiveness is met if there is no increase from initial calculated count at 14and 28d in case of bacteria, yeast and moulds and where, no increase is defined as not more than 0.5 log₁₀ higher than previous value measured (USP 2004).

RESULTSAND DISCUSSION: The results of preservative efficacy testing performed in triplicate were reported as mean values in Table 2 to Table 6. In case of B. subtilis, among the esters of ferulic acid, the p-amino and 8-hydroxy quinoline esters showed less than 0.5 log  values of increment of CFU/ml at 14 dand 28d and hence, both passes the preservative efficacy test. These results of 8-hydroxy quinoline were also supported by the study of Judge et al., 2008 ¹⁴.

Among the amide derivatives of ferulic acid, naphthyl amide was active on 14 and 28 d but the morpholine amide fails to meet the required limit on 14 d. Also, the standard passes the preservative efficacy test on 14 d but fails on 28 d as the change was more than 0.5log CFU/ml (Table 2).

TABLE2:BACTERIALCOUNTOFB.SUBTILISINALUMINIUMHYDROXIDEGEL-USP SUPPLEMENTEDWITH PRESERVATIVES

In case of S. aureus all the esters and amide derivatives of ferulic acid and standard meets USP 2004 guidelines for preservative effectiveness testing, but the naphthyl amide derivative of ferulic acid showed more than 0.5 log values on 14 d and hence failed to meet the required limit but on 28 d the same derivative showed the slight increase in log values and hence was less potent as compared to others (Table 3).

 TABLE3:BACTERIALCOUNTOFS.AUREUSINALUMINIUMHYDROXIDEGEL-USPSUPPLEMENTEDWITH PRESERVATIVES

As per the result shown in Table 4, all the ester and amide derivatives of ferulic acid were found to be active against E. coli on 14as well as on 28d and met the requirement for preservative efficacy testing as per USP 2004.

IncaseofC. albicans,the amide and ester derivatives of ferulic acid showedless than 0.5 logCFU/mlfrom7to 28 d,hencetheypassesthepreservative effectivenesstest.There wasdecrease inlog CFU/ml from7to14d in case of p-amino ester derivative of ferulic acid that was more than 0.5 log values and hence its efficacy was less as compared to other derivatives against C. albicans. Also, the log CFU/ml values of standard exceeded the prescribed USP 2004 criteria on 28 d and hence the standard was less effective preservative as compared to the synthesized esters and amide derivatives of ferulic acid  against C. albicans (Table5).

TABLE4: BACTERIALCOUNTOFE.COLIINALUMINIUM HYDROXIDE GEL –USPSUPPLEMENTEDWITH PRESERVATIVES

TABLE5:FUNGALCOUNTOFC. ALBICANSINALUMINIUM HYDROXIDE GEL -USPSUPPLEMENTEDWITH PRESERVATIVES

As shown in Table6, the change in log CFU/mlon14aswell ason28dforp-amino ester, morpholine amide and 8-hydroxy quinoline ester derivatives was within the limits prescribed in USP 2004 but the naphthyl amide derivative showed more than 0.5 log value from 7 to 14 d and in case of standard also the change was more than the prescribed limit and hence it was less active preservative than the other esters and amide derivatives against A.niger. Also, these results are in accordance with the study of Ohlan et al., 2008¹³.

 TABLE6:  FUNGAL COUNT OFA.  NIGERIN ALUMINIUM HYDROXIDEGEL-USP SUPPLEMENTEDWITH PRESERVATIVES

CONCLUSION: Thestudyhas shownthe preservative potentialofp-amino ester and 8-hydroxy quinoline ester, naphthyl amide, morpholine amide of ferulic acid inthepharmaceuticalpreparation.The selectedamide and ester derivatives of ferulic acid were foundeffectiveagainstallselectedstrainsand showedpreservativeefficacycomparabletothatofstandardandevenbetterincase of B. subtilis and C. albicans. The8- hydroxy quinoline ester derivative showed better preservative efficacy than standard as well as other derivatives and it can be a better alternative to the existing preservatives for use in the pharmaceutical preparations.

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How to cite this article:

Khatkar A, Nanda A and Narasimhan B: Evaluation of Preservative effectiveness of Ferulic acid derivatives in Aluminium hydroxide Gel- USP. Int J Pharm Sci Res 2013: 4(7); 2721-2725. doi: 10.13040/IJPSR. 0975-8232.4(7).2721-25

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