Pectin was extracted from unshiu orange (Citrus unshiu) peels and was subjected to chemical modification using hydroxamic acid. The structural and physical properties of the resulting derivatives were investigated as a function of hydroxamic acid content (4.68-9.58%). The extracted unshiu orange pectin showed 66.8% degree of esterification, 787.5 mg/g galacturonic acid, and 92 mg/g neutral sugars, which were composed of arabinose (53%), galactose (35%), glucose (5%), rhamnose (5%), and fructose (2%). Compared to the native
pectin, the FT-IR spectra of the hydroxamic acid derivatives showed two new absorption bands at 1,646 cm-1 (C=O) and 1,568 cm-1 (N-H). Specifically, the pectin derivatives with more hydroxamic acids were shown to have enhanced water solubility, upto two-fold higher than that of the native pectin. Thus, the introduction of hydroxamic acid into the pectin structure appears to be a useful tool for improving the solubility of pectin

Hyeon Gyu Lee

Hyun Jae Rha

In Young Bae

Suyong Lee

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Technical Note Preparation and Characterization of Pectin Hydroxamates fromCitrus Unshiu Peels.In Young Baea, Hyun Jae Rhaa, Suyong Leeb, Hyeon Gyu Leea,*a Department of Food and Nutrition, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Koreab Department of Food Science & Technology and Carbohydrate Bioproduct Research Center, Sejong University, 98 Kunja-dong,Kwangjin-gu, Seoul, 143-747, KoreaReceived: 12 July, 2011;  Accepted: 11 August 2011ABSTRACTPectin was extracted from unshiu orange (Citrus unshiu) peels and was subjected to chemical modificationusing hydroxamic acid. The structural and physical properties of the resulting derivatives were investigated asa function of hydroxamic acid content (4.68-9.58%). The extracted unshiu orange pectin showed 66.8%degree of esterification, 787.5 mg/g galacturonic acid, and 92 mg/g neutral sugars, which were composed ofarabinose (53%), galactose (35%), glucose (5%), rhamnose (5%), and fructose (2%). Compared to the nativepectin, the FT-IR spectra of the hydroxamic acid derivatives showed two new absorption bands at 1,646 cm-1(C=O) and 1,568 cm-1 (N-H). Specifically, the pectin derivatives with more hydroxamic acids were shown tohave enhanced water solubility, upto two-fold higher than that of the native pectin. Thus, the introduction ofhydroxamic acid into the pectin structure appears to be a useful tool for improving the solubility of pectin.KEY WORDS: unshiu orange peel, pectin, hydroxamic acid, FT-IR, water solubilityINTRODUCTIONJeju native citrus fruits, such as unshiu orange(Citrus unshiu), jigak (Citrus aur-antitum),sadoogam (Citrus pseudogulgul ), and yooja (Citrusjunos) are the most abundantly produced fruitsin Korea (1). According to the annual reportsof statistics (2), citrus-cultivated areas werereported to be approximately 21,400 hectarsproducing approximately 662,000 tons of fruitin 2006. Citrus fruits are widely consumed asfresh fruit and in the form of processedproducts such as juice, canned fruit andmarmalade. The citrus fruit peels are major by-products of citrus fruit processing and aregenerally treated as waste (3). It would thus beworthwhile to look for a way to utilize thesecitrus fruit peels. Pectin exists abundantly in fruit peels andpomace such as citrus and apple, and isextensively used in the food industry as agelling agent and stabilizer (4-5). Recently,pectin has received significant attention due toits diverse biological effects, such as loweringcholesterol (6) and ability to delay the emptyingof the gastric content (7). Additionally, it has* Corresponding author: Department of Food and Nutrition, HanyangUniversity, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Korea,Tel: 82-2-2220-1202, Fax: 82-2-2292-1226E-mail: hyeonlee@hanyang.ac.krThis Journal is © IPEC-Americas Inc J. Excipients and Food Chem. 2 (3) 2011 -  89 Technical Notebeen reported that chemical modification ofpectin, such as sulfation and hydroxamationincreased, or produced biological activity,including anticoagulant and antimicrobialeffects (8-11). Previous studies focused onhydroxamic acid derivatives because of theirprotective effects against cellular oxidation (12-14). Thus, galacturonic acid (15) and alginicacids (16) as well as apple pectin (17) weresubjected to hydroxamation to increase and/orproduce anti-oxidative effects. However, noefforts to prepare and characterize pectinhydroxamates from orange peels have beenreported, especially none on the influence onvarieties in their structures or theirphysicochemical properties. A study of thepectin hydroxamates from different plantsources would thus be useful to expand theiruse in a variety of scientific and commercialapplications.MATERIALS AND METHODSExtraction of pectin from unshiu orange peelsPectins were extracted from the domesticunshiu orange peels according to the method ofKoubala et al. (18). Unshiu orange peels werekindly provided by the First Fruits Company(Seoul, Korea). They were dried in an airconvection oven (J-300M, Jisico Co., Ltd.,Seoul, Korea) at 80EC and ground using a labblender (FM 680T, Hanil Co., Seoul, Korea)and passed through a 350 mesh sieve (TestingSieve, Chung Gye Sang Gong Sa, Seoul,Korea). The dried peel powder was mixed with85% ethanol at 80EC for 20 minutes. The residue (100 grams), that was insoluble ina l coho l ,  was  t r ea ted  wi th  oxa l i cacid/ammonium oxalate (0.25%, pH 4.6, 4 L)agitating at 80EC for 1 hour. After pressurefiltration, the filtrate was washed with ethanol(96% 6 70% 6 96%), desolvated using anevaporator (N-1000, Eyela, Tokyo, Japan), andthen freeze-dried (FD-8508, Ilsin Lab Co.,Seoul, Korea).Composition analysis of pectin extractThe degree of esterification (%) of theextracted pectin was calculated from the molarratio of methanol to galacturonic acid.Anhydrogalacturonic acid content wasmeasured by colorimetric method at 524 nm(DU 650, Beckman Coulter Inc., USA) (19) andthe methanol content was determined by anenzymatic method with alcohol oxidase (20).The free sugar content of the pectin sampleswas measured using a liquid chromatography(HPAEC-PAD system, Dionex DX500,Sunnyvale, CA, USA) equipped with aCarboPac™ PA1 (4 x 250 mm). The pectin washydrolyzed with trifluoroacetic acid at 100EC for 4 hours and 20 μl of the sample wasinjected. The mobile phase and flow rate were18 mM sodium hydroxide and 1.0 ml/min.,respectively. Preparation of pectin hydroxamic acidsPectin hydroxamic acid derivatives wereprepared by the method of Hou et al. (9).Alkaline hydroxylamine (2 M, pH 12.0, 100 ml)was added to the pectin solution (1%, 100 ml)and stirred at room temperature for 4, 18, 24 or48 hours. The pH (using an Orion 3-star,Thermo Electron Corporation, Beverly, MA,USA) was adjusted to 6.5 with hydrochloricacid, and the resulting derivatives wereprecipitated with three volumes of 2-propanol,desolvated using an evaporator, and thenfreeze-dried. The pectin hydroxamic acidderivatives were designated as T4, T18, T24,and T48, depending on their reaction times (4,18, 24 and 48 hours, respectively).Characterization of pectin derivativesThe hydroxamic acid contents of the pectinderivatives were determined, with slightmodifications, by the method of Soloway et al.(21). First, the derivative solution (0.2 ml) wasmixed with hydrochloric acid (0.3 ml, 4 mM),and then 0.5 ml hydrochloric acid (0.1 mM)containing 10% ferric chloride was added. Afterstanding for 10 minutes at room temperature,the absorbance of the resulting solution wasmeasured at 540 nm with acetohydroxamic acidas a standard.This Journal is © IPEC-Americas Inc J. Excipients and Food Chem. 2 (3) 2011 -  90 Technical NoteFor the confirmation of the incorporation ofhydroxamic acid into the pectin polymer, thederivatives were ground and mixed withpotassium bromide (KBr) at a ratio of 1:20 anda thin pellet was then prepared for FT-IRanalysis (Nicolet FT-IR spectrometer,MAGNA-IR 760 E.S.P, Nicolet InstrumentCorp., Madison, WI, USA).The solubilities of native pectin and derivativesin water were determined by the method ofChang et al. (22). The derivative dispersion (10mg/ml) was agitated at 25EC for 24 hours andcentrifuged at 3,500 g for 15 minutes (VS-21SMTi, Vision Scientific Co., Kyunggi-do,Korea). The supernatant was dried at 60EC for24 hours in a vacuum oven (J-DV01, Jisico Co.,Ltd., Seoul, Korea). The solubility wascalculated from the weight of the initialdispersed solid and the weight of the residueafter drying.Statistical analysis  All experiments were performed in triplicate.The results were analyzed using StatisticalPackage for Social Science (SPSS, Version 12.0,2004, SPSS Inc., Chicago, IL, USA). Analysis ofvariance (ANOVA) was carried out todetermine any significant difference betweenthe  samples. Duncan’s multiple range test wasthen used for comparison of means at the levelof 0.05.RESULTS AND DISCUSSIONExtraction of pectin from unshiu orange peelsThe yield and chemical composition of thepectin extracted from unshiu orange peels arepresented in Table 1. The yield of the pectin inthis study was 9%, which was similar to that ofcitrus pectin extracted with citric acid (23). Ingeneral, the yields of the pectin extracted fromcitrus peels varied from 9% to 30%, dependingon the extraction methods (18, 24-25). On theother hand, the galacturonic acid content of theTable 1 Characterization of the pectin extracted fromunshiu orange peelsYIELD (%) 9Degree of esterification (%) 66.8Galacturonic acid (mg/g) 787.5Total neutral sugars (mg/g) 91.5Individual neutral sugar (mg/g) Arabinose 48.7Fructose 1.5Galactose 31.9Glucose 5Rhamnose 4.5 pectin extract was 788 mg/g which was higherthan the 343 mg/g of citrus pectin extractedwith citric acid (23).The degree of esterification (DE) is one of thekey factors affecting the practical application ofpectin in food products such as gelation,thickening, or consistency adjustment (26).Pectins are generally classified as high methoxyl(DE > 50%) or low methoxyl (DE < 50%),based on the percentage of the esterifiedcarboxyl groups. The degree of esterification(DE) of the pectin extracted in this study was67%, confirming that the unshiu orange pectinsextracted in this study are high methoxypectins. Kurita et al. (23) and Tamaki et al. (27)also reported 65% and 63% of DE of citruspectins, respectively, which compares well withthe results obtained here.Moreover, the extracted pectin was shown tohave 92 mg/g of total neutral sugars whichwere composed of arabinose (53%), galactose(35%), glucose (5%), rhamnose (5%), andfructose (2%). The pattern of the compositionof neutral sugars was similar to that of citric-extracted pectin (23), even though the contentof total neutral sugars was lower. Therefore, theyield and chemical composition of the extractedpectin appeared to be affected by the source ofpectin and the extraction method.This Journal is © IPEC-Americas Inc J. Excipients and Food Chem. 2 (3) 2011 -  91 Technical NoteFigure 3 Effect of the degree of hydroxamation onsolubilityFigure 2 FT-IR spectra of pectin and its hydroxamicacid derivativesFigure 1 Hydroxamic acid contents of pectin derivatives asa function of reaction time with alkaline hydroxylamineCharacterization of pectin hydroxamic acidderivativesHydroxamation is the generation of aminegroups in a polymer structure by introducinghydroxylamine into the carboxyl groups of the polymer chains. In this study, pectinhydroxamates with varying hydroxamic acidcontents were prepared by controlling thereaction time. Figure 1 shows that as thereaction time increased from 4 hours to 48hours, the hydroxamic acid content of thederivative increased from 4.68% to 10.43%,respectively. Notably, the hydroxamic acidpercentage gradually increased as thehydroxamation process continued (p< 0.05). Inaddition, the structure of the pectin derivativeswas investigated using FT-IR. Figure 2 showstwo new bands at 1,646 cm-1 (C=O) and 1,568cm-1 (N-H), respectively (28). Therefore, theFT-IR spectra and hydroxamic acid analysisconfirmed that the pectin hydroxamic acidderivatives were successfully produced fromunshiu orange pectins.The effect of hydroxamation on the watersolubility of pectin was investigated (Figure 3).Chemical modification by hydroxamationcaused a distinct increase in the water solubilityof pectin. In addition, water solubilitysignificantly improved in response to thecontent of hydroxamic acid (p< 0.05). Thewater solubility of the native pectin increasedfrom 47% to about 88% when 9.6% ofhydroxamic acid was incorporated into thecarboxyl groups in the pectins. It appears thatan increased incorporation of ionic groups intothe pectin structure would be responsible forthe enhanced water solubility. The positiverelationship between water solubility and theintroduction of an anionic group into thepolymer chains has been reported for severalchemical modifications of polymers includingcarboxymethylation (29), oxidation (30), andsulfation (11). It is generally recognized thatimproved water solubility is desirable forpharmaceutical formulations since it providesfor ease of administration, as well as, enhancedphysiological activity such as anti-tumor (29),cholesterol-lowering (30), and anti-oxidativeproperties (17). The increased water solubility This Journal is © IPEC-Americas Inc J. Excipients and Food Chem. 2 (3) 2011 -  92 Technical Noteof the hydroxamated pectin may partlycontribute to the potential physiologicalactivities which should be investigated further. CONCLUSIONDomestic unshiu orange peels were utilized toproduce pectin (67%  DE, 790 mg/g galacturonicacid, 92 mg/g total neutral sugars), and itsderivatives with different contents ofhydroxami hydroxamic acid (4.68% - 9.58%)were successfully prepared. The introduction ofhydroxamic acid groups into the pectinstructure was confirmed by FT-IR analysis andresulted in improved water solubility of thepectins. Further research should be carried outto investigate the effects of the hydroxamic acidcontent of different biological properties of thepectins.ACKNOWLEDGEMENTSThis work was carried out with the support ofthe "Cooperative Research Program for Agri-culture Science & Technology Development(Project No. PJ907142)" Rural DevelopmentAdministration, Republic of Korea.REFERENCES1 Kim YD, Ko WJ, Koh KS, Jeon YJ, Kim SH.Composition of flavonoids and antioxidative activityfrom juice of Jeju native citrus fruits duringmaturation. 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Preparation and Characterization of Pectin Hydroxamates from Citrus Unshiu Peels

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Preparation and Characterization of Pectin Hydroxamates from Citrus Unshiu Peels

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