characterization and chemical composition of...

Original Article

Characterization and chemical composition of epicuticular waxfrom banana leaves grown in Northern Thailand

Suporn Charumanee1 Songwut Yotsawimonwat 1 Panee Sirisa-ard 1 and Kiatisak Pholsongkram2

1 Department of Pharmaceutical Sciences Faculty of PharmacyChiang Mai University Mueang Chiang Mai 50200 Thailand

2Department of Food Science and Technology Faculty of SciencesPayap University Mueang Chiang Mai 50000 Thailand

Received 22 April 2016 Revised 2 July 2016 Accepted 16 July 2010

Abstract

This study aimed to investigate the physicochemical properties and chemical composition of epicuticular waxextracted from leaves of Kluai Namwa a banana cultivar which is widely grown in Northern Thailand Its genotype wasidentified by a botanist The wax was extracted using solvent extraction The fatty acid profiles and physicochemical propertiesof the wax namely melting point congealing point crystal structures and polymorphism hardness color and solubility wereexamined and compared to those of beeswax carnauba wax and paraffin wax The results showed that the genotype of KluaiNamwa was Musa acuminata X M balbisiana (ABB group) cv Pisang Awak The highest amount of wax extracted was 274gcm2 surface area The fatty acid composition and the physicochemical properties of the wax were similar to those ofcarnauba wax It could be suggested that the banana wax could be used as a replacement for carnauba wax in variousutilizing areas

Keywords epicuticular wax banana wax Kluai Namwa Musa spp carnauba wax

Songklanakarin J Sci Technol39 (4) 509-516 Jul - Aug 2017

1 Introduction

Banana (Musa spp) is one of the most widelycultivated plants in Thailand All parts of this plant are usedin daily life (Debabandya et al 2010) Unripe and ripe fruitswhich can be harvested year-round are known as functionalfruits providing nutritional and medicinal values (SampathKumar et al 2013) Banana leaves are commonly used forfood wrapping and decoration because they are large flexibleand waterproof However they are less frequently usednowadays and largely replaced by plastic After harvestingthe crop the mature leaves are considered as agriculturalwaste and they are often destroyed by burning that leads to

air pollution However it is interesting that the outermostpart of banana leaf is covered with lipid substance which isso-called the epicuticular wax This wax is solid mixturescomposed of esters of long chain fatty acids and long chainfatty alcohols free fatty acids fatty dialcohols (diols)aldehydes and n-alkanes (Freeman amp Turner 1985 Yanagidaet al 2005a) This surface wax serves as a mechanical barrierwhich protects the plant tissues against UV radiation andbacterial or fungal attacks and also reduces water loss insummer time (Riederer amp Schreiber 2001) Nowadayscarnauba wax is the most commonly used natural wax invarious industries especially for polishing leathers glasseswooden furniture and automobiles Interestingly it is also acommon ingredient in pharmaceutical and cosmetic productsThis point of view leads us to generate the idea of usingbanana leaves as a natural source of waxy substance Fromthe extensive literature review studies involving extraction

Corresponding authorEmail address suporncharumaneecmuacth

httpwwwsjstpsuacth

S Charumanee et al Songklanakarin J Sci Technol 39 (4) 509-516 2017510

and characterization of the epicuticular wax from bananaleaves have been scarcely reported Those noteworthy oneswere reported by Freeman and Turner (1985) and Yanagidaet al (2003 2005b) According to Freeman and Turner (1985)epicuticular waxes from leaf flower bracts and fruits of 12banana (Musa spp) varieties were extracted and examinedtheir chemical compositions Fruits gave higher wax contentfollowed by bracts and leaf respectively The wax from leaf infield-grown banana was about 230 mgcm2 was 60 higherthan those planted in a glasshouse In addition Thailand hasnumerous cultivars of banana while the yield characteriza-tion and chemical composition of epicuticular wax frombanana leaves grown in Thailand has never been investigatedThe objectives of this study were to investigate andcharacterize the chemical composition and the properties ofwax isolated from leaves of Kluai Namwa a banana cultivarwhich is widely grown in Northern Thailand and to compareits properties to those of other waxy substances includingcarnauba wax

2 Materials and Methods

The following waxy substances were used to comparetheir physicochemical properties with the banana waxnamely beeswax (S Tong Chemicals Co Ltd Lot P011814Bangkok Thailand) carnauba wax (Union Sciences Co LtdLot 9776 Chiang Mail Thailand) and paraffin wax (B L Hauamp Co Ltd Lot 7117L14 Bangkok Thailand)

21 Sample preparation

Kluai Namwa grown in Northern Thailand particularlyin Chiang Mai Lampang Pitsanulok and Tak provinces werecollected as samples for wax extraction Fresh leaves wereused for wax extraction After measuring their surface areasbanana leaves were hung in the air to keep them partiallydried and dust was removed using an air blower All reagentsand chemicals used were of analytical grade

22 Extraction of epicuticular wax

The epicuticular wax was extracted from bananaleaves by sohxlet apparatus using n-hexane as a solvent andthe solvent was removed by using a rotary evaporator Theextracted wax was purified and decolorized using activatedcharcoal Briefly 1 wv of activated charcoal was added tothe hot hexane extract The mixture was frequently stirred fora few minutes then filtered through filter papers (WhatmanNo 5 Sigma-Aldrich St Louis MO USA) using vacuumfiltration The decolorization process was repeated 3-5 timesdepended on the color intensity of the hexane extract Theresidual of hexane in the wax was quantified by gas chroma-tography The codes were assigned to the extracted waxesaccording to their origins ie BNS BNP BNT BNTk whichrepresented banana leaves cultivated in Chiang MaiPitsanulok Lampang and Tak provinces respectively

23 Identification of banana cultivars

Kluai Namwa from different origins were identifiedand authenticated by the botanist at Queen Sirikit BotanicalGarden Department of Forest Ministry of Natural Resourcesand Environment in Chiang Mai

24 Verification and determination of chemical compositionsin the wax

241 Thin layer chromatography

Thin layer chromatography (TLC) technique was usedto verify the chemical composition of the wax The stationaryphase was Silica Gel 60 F254 and the mobile phase was themixture of hexane ethyl alcohol acetic acid 50501

242 Gas chromatography

Fatty acid composition of the extracted wax wasdetermined by using gas chromatography (GC) ShimadzuGC 2010 equipped with DB-23 column and a flame ionizationdetector The sample was injected in a split mode with a splitratio of 150 and an injector temperature of 250degC Thetemperature program started with an initial temperature of80degC increased at a rate of 10degCmin up to 180degC hold for15 min increased with a ramp rate of 4degCmin to reach a finaltemperature of 220degC with a final hold time of 7 min theduration of the total temperature program was 42 min Thedetector temperature was set at 300degC Helium was used asa carrier gas at a flow rate of 629 mlmin

25 Acid number determination

The acid value of the samples was determined usingthe method previously described (Suzanne 2010) Brieflythe wax was dissolved in ethanol at 50degC The mixture wastitrated with standardized solution of potassium hydroxideusing phenolphthalein as an indicator A blank was alsoundertaken in the same manner Acid value was calculatedfrom the amount of standardized solution of potassiumhydroxide used for wax and blank titration The experimentwas performed in triplicate

26 Saponification number determination

The determination of saponification number wasperformed according to the following procedures (Suzanne2010) the wax was mixed with alcoholic 04 M KOH andrefluxed for 30 min The resultant solution after the additionof phenolphthalein was titrated with standardized solutionof 05 M hydrochloric acid A blank was also carried outSaponification value was calculated from the amount ofstandardized hydrochloric acid solution used for sample andblank titration The experiment was performed in triplicate

511S Charumanee et al Songklanakarin J Sci Technol 39 (4) 509-516 2017

27 Characterization of physical properties

271 Determination of melting point congealing point andpolymorphism by DSC

The melting point and the congealing point of thewax was determined using differential scanning calorimeterDSC 7 (Perkin Elmer USA) The sample weighed 30-35 mgwas placed in an aluminum pan with holes The heatingexperiment was studied from 50degC to 100degC at a heating rateof 5degCmin and followed by the cooling experiment from100degC to 50degC under the same cooling rate Nitrogen wasused as purge gas at a flow rate was 20 mlmin

272 Examination of internal crystal structure

X-ray diffractometer (Siemens-D 500 Germany) wasused to examine the internal structure of the wax crystals atroom temperature using the following conditions voltage20 kV current 20 mA time constant 05 second scan speed4 qmin and scattering angle range (2q) of 5-30deg

273 Examination of crystal modifications (Habits)

The crystal modification of wax crystals was examinedusing scanning electron microscope (JSM-5910LV JeolJapan)

274 Hardness measurement

The hardness of wax was determined using TextureAnalyzer TAXT2i (stable micro system) This method wasmodified from the standard method used for testing thehardness of waxes in industry (American Society for Testingand Materials International [ASTM] D1321 2015) Thehardness was presented in terms of the force (Newton) actingagainst the penetration of the needle into the wax matrix atthe definite distance (in mm) at a fixed temperature Theexperiment was performed in triplicate

275 Color measurement

The color of the wax was examined using MinoltaColor Reader CR-10 The CIE color parameters of L a andb were recorded

276 Density and specific gravity determination

The density of the wax was determined using thedensity factor ie a ratio by weight of wax to water of anexactly known volume The wax was melted and poured intothe suppository mold After solidified the excessive waxabove the mold surface was removed The solid wax in themold was taken out and weighed The volume inside eachsuppository mold was determined using purified water Thespecific gravity was calculated by dividing the density value

obtained by the density of water at same temperature Theexperiment was performed in triplicate

277 Solubility determination

The solubility of the extracted waxes in varioussolvents was determined using the method previouslydescribed by Yanagida et al (2003a) with some modificationsThe solvents used were hexane dichloromethane chloroformethanol and methanol The excess amount of wax sample wasplaced in a well-closed flask containing a specific solventAfter the mixture was stirred using a small magnetic bar for12 hours at room temperature it was centrifuged to obtainclear supernatant The known volume of the supernatant wassampling and the solvent was evaporated The solid residueof the wax was weighed The experiment was performed intriplicate

3 Results and Discussion


All banana samples collected from different originswere identified as Kluai Namwa (Musa acuminata X Mbalbisiana (ABB group) cv Pisang Awak The samples werekept as a voucher specimen at Queen Sirikit Botanical Gardenin Chiang Mai Kluai Namwa is a diploid cultivar and acommon edible banana widely grown in Northern Thailandas well as all parts of Thailand

32 Extraction of banana wax

The yield of the wax extracted from Kluai Namwarsquosleaves depended on cultivating areas They were 2738 gcm2 2126 gcm2 1674 gcm2 and 1291 gcm2 fromChiang Mai Lampang Pitsanulok and Tak provinces respec-tively The yield value of BNS (2738 gcm2) was higher thanthe values reported by Freeman and Turner (1985) with thehighest one found in field-grown banana (230 gcm2) whichwas 60 higher than those planted in a glasshouse Yanagidaet al (2003b) reported the yields of wax extracted frombanana leaves of different species on dried weight basis ieIto bashou (Japanese name) or M liukiuensis (058)kluai Pa or Macuminata (105 ) and kluai Roi Wee or Mchiliocarpa (141) Based on the percentage of moisturecontent reported the maximum yield of 141 from Mchiliocarpa can be calculated as only 015 g100 g of freshleaves which was significantly lower than the result of ourstudy (092 g100 g of fresh leaves) Thus Kluai Namwarsquosleaves are the most abundant source of banana wax reporteduntil now According to the higher yield percentage the BNSand BNT were selected to be the samples for characterizationand physicochemical properties determination

In order to obtain purified white-colored wax theextracted wax was subjected to decolorization process usingactivated charcoal After decolorization the color of banana


wax became whiter as shown in Figure 1 Although loss ofwax content was observed its main composition as revealedby TLC analysis did not change as shown in Figure 2 andthe melting point and congealing point determined by DSCdid not altered (data not shown)

33 Chemical compositions of banana wax

331 Chemical compositions of by TLC and GC

TLC chromatogram shown in Figure 3 demonstratedthat the major compositions of the wax extracted from bananaleaves and carnauba wax were similar This was alsosupported by the fatty acid profiles obtained by gaschromatographic technique after saponification as shown inTable 1 The saturated fatty acids presented in both waxeswere behenic acid (C-22) lignoceric acid palmitic acid andstearic acid For unsaturated fatty acids linolenic acidlinoleic acid and oleic acid were found predominantly inbanana wax These results were in agreement with thosereported by Yanagida et al (2003a) which demonstrated thatC-22 fatty acid was the most abundant component of bananawax

Table 1 Composition of wax from banana leaves and the reference waxes

composition Compounds

BNT BNS Carnauba wax Beeswax

Myristic acid (C140) ND ND 2875 NDPalmitic acid (C160) 1219 2214 1023 3403Stearic acid (C180) 214 369 1172 5349Oleic acid (C181) 217 584 142 NDLinoleic acid (C182) 859 451 077 020Linolenic acid (C183) 3379 865 024 256Arachidic acid (C200) ND ND 2875 NDEPA(C205) 786 739 ND 0185Behenic acid (C220) 1430 2355 1675 0144Lignoceric acid (C240) 945 1393 2866 086

Note ND (undetected) EPA Eicosapentaenoic acid

Figure 1 Epicuticular wax extracted from banana leaves BNT andBNS (A) before decolorization (B) after decolorization

Figure 2 TLC chromatograms of wax from banana leaves (A) (B)- BNT before and after decolorization (C) (D) BNSbefore and after decolorization respectively

Figure 3 TLC chromatograms of (A) carnauba wax (B) BNT (C)BNS

332 Acid number and saponification number

The acid number andor saponification number ismeaningful parameters providing information about thechemical functionality of the wax that contains carboxylicgroups As shown in Table 2 the acid number and saponi-


fication number of banana waxes BNT and BNS weresignificantly different however they were in the range ofcarnauba waxrsquos values specified in Pharmacopeias Thissupported the similarity in terms of the componentscontaining carboxylic acids of the two natural waxes

34 Characterization of the physicochemical properties ofbanana wax

341 Melting point congealing point and polymorphism

The DSC thermogram of banana wax obtained fromdifferent sources BNS and BNT were compared to that ofcarnauba wax as shown in Figure 4 The melting point (peaktemperature) and congealing point of BNT were 8138degCand 7381degC and those of BNS were 7901degC and 7411degCrespectively They were not much different Whereas carnaubawax exhibited the major melting peak at 7898degC and thecongealing point peak at 7391degC They were more similar tothose of BNS However these properties are more vital forwax utilization than chemical compositions Despite of somedifferences in their chemical composition observed thecomparable melting points of both waxes revealed the identi-cal internal strengths of wax crystals The measured meltingpoint of carnauba wax was in agreement with that specifiedin USP30 NF30 ie 80-86degC The extracted banana wax was ofhigh purity as evidenced by a single sharp and approachingsymmetrical melting peak whereas the thermogram ofcarnauba wax showed two distinct melting peaks Moreoverthe thermogram of banana wax did not signify the existenceof polymorphism


The X-ray powdered diffractograms in Figure 5illustrated the internal crystal structure of banana wax ascompared to carnauba wax The sharp peaks demonstratedthat banana waxes as well as carnauba wax were in crystallinestate It also indicated that the internal structure of bananawax and carnauba wax were similar which denoted by thesame peak positions of Braggrsquos angle (2q) at of 21deg 23deg and19deg respectively These observations supported the similarity

Table 2 Physicochemical parameters (mean plusmn SD n = 3) of wax from banana leaves and those ofreference waxes

Wax samples Acid number Saponification number Hardness (N) Specific gravity

BNT 168 plusmn 026a 8166 plusmn 439a 3917 plusmn 436b 09266 plusmn 00173d

BNS 358 plusmn 092a 9472 plusmn 518a 2930 plusmn 342 bc 09025 plusmn 00101de

Beeswax 17-24 87-104 3535 plusmn 216 09543 plusmn 00022de

Carnauba wax 2-7 78-95 3868 plusmn 347c 09373 plusmn 00010e

Paraffin wax NS NS 2524 plusmn 747 b 09308 plusmn 00039e

Note Reference values specified in PhEur USP NS ndashnot specified in USP Significant differenceswere tested at p lt 005

Figure 4 DSC thermograms wax from banana waxes BNT-upperBNS-middle and carnauba wax-lower


of the two waxes as previously demonstrated by thecomposition and DSC studies

343 Examination of crystal modifications (crystal habits)

The SEM micrograph in Figure 6 illustrated the crystalhabits of banana waxes obtained from different sourcesBNT and BNS They habits were quite similar exhibited theaggregated plate-liked crystals of varying sizes Despite ofthe irregular habits the internal structure exhibited highdegree of crystallinity as shown by X-ray diffractogramThe thin and uniform thickness of these plate-liked habitscontributes to sharp melting point peak as demonstrated byDSC study


According to the modified method by using Textureanalyzer TAXT2i the hardness of the wax presented in termsof the resistant forces (Newton) against the penetration of theneedle into the solid wax was shown in Table 2 The hardnessof BNT and carnauba were insignificantly different whereasBNS was softer than BNT beeswax and carnauba wax thelower hardness value of BNS was significantly different fromthose waxes at p lt 005


The color of the wax was determined according toCIE (International Commission on Illumination 1976) colorparameters (L a b) The L value represents thelightness the maximum L value of 100 represents pure whitecolor whereas zero value indicates black color Each a andb represents two color components The positive value ofa indicates red color whereas the negative value representsfor green color For b value its positive value representsyellow color and the negative one represents blue colorAs shown in Table 3 paraffin wax had the highest L valuefollowed by banana wax (non-decolorized) and carnauba waxrespectively This demonstrated the intensity of white colorof waxes in descending order As banana wax was whiterthan carnauba wax the decolorization process might not benecessary for some cases of utilization In addition when aand b values are considered altogether the color of bananawax can be summarized as white with greenish yellow colorwhereas carnauba wax had white color with slightly red-yellow color tone


The specific gravity of banana wax and those of thereference waxes were shown in Table 2 Although the methodused was adopted from density factor determination theresults obtained from the reference beeswax was in agree-ment with that specified in the reference book (Rowe 2012)The measured values of the samples were then reliable

Figure 5 X-ray powdered diffractograms of wax from bananaleaves BNT BNS and carnauba wax

Figure 6 SEM micrograph of banana waxes BNT- left and BNS-right


The banana waxes from different origins had significantlydifferent specific gravity and they were significantly lowerthan that of carnauba wax (p lt 005)


The solubility of wax from banana leaves BNT andBNS in various solvents was shown in Table 4 The wax wassoluble in chloroform hexane dichloromethane ethyl alcoholand methyl alcohol in descending order According to USPdescriptive terms of solubility the solubility of banana waxin all solvents can be classified as slightly soluble ie 100-1000 parts of solvent are needed to dissolve one part of thewax (USP 35NF30) However the significant differences inthe solubility of BNT and BNS in hexane and methanol wereobserved at p lt 005

4 Conclusions

All Kluai Namwa samples collected from differentplanting areas in Northern Thailand were Musa acuminata XM balbisiana (ABB group) cv Pisang Awak The percentageyield and the physicochemical properties of the extractedwax depended on planting area The sample which gave thehighest yield of wax was Kluai Namwa grown in Chiang Maiprovince (092 g100 g of fresh leaves) whereas that grown

in Lampang province was more similar to carnauba waxHowever the reported yield of unpurified carnauba waxobtained from natural source was approximately five percent(Duke amp duCellier 1993) From extensive investigation byusing different characterized methods we could confirm thatthe wax extracted from Kluai Namwarsquos leaves grown inNorthern Thailand was similar to carnauba wax in terms ofchemical composition and physicochemical properties Thiscould bring about the idea of replacing banana wax forcarnauba wax in pharmaceutical cosmetic and the other waxutilizing areas

Acknowledgements

The authors thank the National Research Council ofThailand (NRCT) for financial support We also thankFaculty of Pharmacy Chiang Mai University for providingfacilities and equipment to conduct the study

References

American Society for Testing and Materials International(2015) Standard test method for needle penetrationof petroleum waxes ASTM D1321-10 WestConshohocken PA Author Retrieved from httpswwwastmorgStandardsD1321htm

International Commission on Illumination (1976) Colorimetry- Part 4 1976 L a b colour space Retrieved fromhttpwwwciecoatindexphpi_ca_id=485

Debabandya M Sabyasachi M amp Namrata S (2010)Banana and its by-product utilization an overviewJournal of Scientific and Industrial Research 69323-329 Retrieved from httpwwwresearchgatenetpublication313177939

Duke J A amp duCellier J L (1993) CRC Handbook ofalternative cash crops London England CRC Press

Freeman B amp Turner D W (1985) The epicultcular waxeson the organs of different varieties of banana (Musaspp) Differ in Form Chemistry and ConcentrationAustralian Journal of Botany 33(4) 393-408 doi101071bt9850393

Table 3 CIE Color parameters (L a b) of wax from banana leaves(before decolorization) and the reference waxes

Color parameters (L a b)(mean plusmn SD)

Wax samplesL (white-black) a (red-green) b (yellow-blue)

BNT 882 plusmn056 -28 plusmn019 +290 plusmn087BNS 859 plusmn063 -38 plusmn040 +235 plusmn071Beeswax 857 plusmn170 -24 plusmn029 +27 plusmn048Carnauba wax 779 plusmn034 +17 plusmn016 +17 plusmn016Paraffin wax 934 plusmn114 -17 plusmn015 +05 plusmn026

Table 4 Solubility of wax from banana leaves in varioussolvents

(g100 ml) (mean plusmn SD n = 3)SolventsSolubility

BNT BNS

Chloroform 064 plusmn018 069 plusmn010Dichloromethane 034 plusmn003 038 plusmn008Ethyl alcohol 015 plusmn003 014 plusmn004Hexane 050 plusmn011 080 plusmn005Methyl alcohol 010 plusmn002 015 plusmn001

Significant difference at plt 005 (SPSS version 17)


Riederer M amp Schreiber L (2001) Protecting against waterloss Analysis of the barrier properties of plantcuticles Journal of Experimental Botany 52(363)2023-2032 Retrieved from httpjstororgstable23696406

Rowe R C Sheskey P J amp Quinn M E (2009) Handbookof pharmaceutical excipients (6th ed) (pp779-780)London England Pharmaceutical Press

Suzanne S N (2010) Food analysis laboratory manual (pp105-108) New York NY Springer

Sampath Kumar K P Debjit B Duraivel S amp UmadeviM (2013) Traditional and medicinal uses of bananaJournal of Pharmacognosy and Phytochemistry 1(3)51-63 Retrieved from httpwwwphytojournalcom

United States Pharmacopeial Convention (2011) USP35NF30 2012 US Pharmacopoeia National FormularyRockville MD Author

Yanagida T Shimizu N amp Kimura T 2003 Properties ofwax extracted from banana leaves Proceedings of theAmerican Society Agricultural Engineering (ASAE)Annual International Meeting doi 10130312013141212013

Yanagida T Shimizu N amp Kimura T (2005a) Extractionof wax and functional compounds from fresh and drybanana leaves Japan Journal of Food Engineering6(1) 79-87 doi 1011301jsfe200679

Yanagida T Shimizu N amp Kimura T (2005b) Extractionof wax from banana as an alternative way of utilizingagricultural residues Japan Journal of Food Engi-neering 6(1) 29-35 doi 1011301jsfe200629


and characterization of the epicuticular wax from bananaleaves have been scarcely reported Those noteworthy oneswere reported by Freeman and Turner (1985) and Yanagidaet al (2003 2005b) According to Freeman and Turner (1985)epicuticular waxes from leaf flower bracts and fruits of 12banana (Musa spp) varieties were extracted and examinedtheir chemical compositions Fruits gave higher wax contentfollowed by bracts and leaf respectively The wax from leaf infield-grown banana was about 230 mgcm2 was 60 higherthan those planted in a glasshouse In addition Thailand hasnumerous cultivars of banana while the yield characteriza-tion and chemical composition of epicuticular wax frombanana leaves grown in Thailand has never been investigatedThe objectives of this study were to investigate andcharacterize the chemical composition and the properties ofwax isolated from leaves of Kluai Namwa a banana cultivarwhich is widely grown in Northern Thailand and to compareits properties to those of other waxy substances includingcarnauba wax

2 Materials and Methods

The following waxy substances were used to comparetheir physicochemical properties with the banana waxnamely beeswax (S Tong Chemicals Co Ltd Lot P011814Bangkok Thailand) carnauba wax (Union Sciences Co LtdLot 9776 Chiang Mail Thailand) and paraffin wax (B L Hauamp Co Ltd Lot 7117L14 Bangkok Thailand)

21 Sample preparation

Kluai Namwa grown in Northern Thailand particularlyin Chiang Mai Lampang Pitsanulok and Tak provinces werecollected as samples for wax extraction Fresh leaves wereused for wax extraction After measuring their surface areasbanana leaves were hung in the air to keep them partiallydried and dust was removed using an air blower All reagentsand chemicals used were of analytical grade

22 Extraction of epicuticular wax

The epicuticular wax was extracted from bananaleaves by sohxlet apparatus using n-hexane as a solvent andthe solvent was removed by using a rotary evaporator Theextracted wax was purified and decolorized using activatedcharcoal Briefly 1 wv of activated charcoal was added tothe hot hexane extract The mixture was frequently stirred fora few minutes then filtered through filter papers (WhatmanNo 5 Sigma-Aldrich St Louis MO USA) using vacuumfiltration The decolorization process was repeated 3-5 timesdepended on the color intensity of the hexane extract Theresidual of hexane in the wax was quantified by gas chroma-tography The codes were assigned to the extracted waxesaccording to their origins ie BNS BNP BNT BNTk whichrepresented banana leaves cultivated in Chiang MaiPitsanulok Lampang and Tak provinces respectively


Kluai Namwa from different origins were identifiedand authenticated by the botanist at Queen Sirikit BotanicalGarden Department of Forest Ministry of Natural Resourcesand Environment in Chiang Mai

24 Verification and determination of chemical compositionsin the wax

241 Thin layer chromatography

Thin layer chromatography (TLC) technique was usedto verify the chemical composition of the wax The stationaryphase was Silica Gel 60 F254 and the mobile phase was themixture of hexane ethyl alcohol acetic acid 50501

242 Gas chromatography

Fatty acid composition of the extracted wax wasdetermined by using gas chromatography (GC) ShimadzuGC 2010 equipped with DB-23 column and a flame ionizationdetector The sample was injected in a split mode with a splitratio of 150 and an injector temperature of 250degC Thetemperature program started with an initial temperature of80degC increased at a rate of 10degCmin up to 180degC hold for15 min increased with a ramp rate of 4degCmin to reach a finaltemperature of 220degC with a final hold time of 7 min theduration of the total temperature program was 42 min Thedetector temperature was set at 300degC Helium was used asa carrier gas at a flow rate of 629 mlmin

25 Acid number determination

The acid value of the samples was determined usingthe method previously described (Suzanne 2010) Brieflythe wax was dissolved in ethanol at 50degC The mixture wastitrated with standardized solution of potassium hydroxideusing phenolphthalein as an indicator A blank was alsoundertaken in the same manner Acid value was calculatedfrom the amount of standardized solution of potassiumhydroxide used for wax and blank titration The experimentwas performed in triplicate

26 Saponification number determination

The determination of saponification number wasperformed according to the following procedures (Suzanne2010) the wax was mixed with alcoholic 04 M KOH andrefluxed for 30 min The resultant solution after the additionof phenolphthalein was titrated with standardized solutionof 05 M hydrochloric acid A blank was also carried outSaponification value was calculated from the amount ofstandardized hydrochloric acid solution used for sample andblank titration The experiment was performed in triplicate







































































4 Conclusions



Acknowledgements


References












BNT BNS


















































































4 Conclusions



Acknowledgements


References












BNT BNS












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