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Research ArticleReceived: 17 May 2013 Revised: 17 July 2013 Accepted article published: 8 August 2013 Published online in Wiley Online Library: 4 September 2013

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6337

Fatty acid composition, physicochemicalproperties, antioxidant and cytotoxic activityof apple seed oil obtained from apple pomaceMayanka Walia,a,b Kiran Rawat,a,c Shashi Bhushan,a,c∗Yogendra S Padwada,c and Bikram Singhb

Abstract

BACKGROUND: Apple pomace is generated in huge quantities in juice-processing industries the world over and continuousefforts are being made for its inclusive utilization. In this study, apple seeds separated from industrial pomace were usedfor extraction of oil. The fatty acid composition, physicochemical and antioxidant as well as in vitro anticancer properties ofextracted oil were studied to assess its suitability in food and therapeutic applications.

RESULTS: The fatty acid composition of seed oil revealed the dominance of oleic (46.50%) and linoleic acid (43.81%). It hadhigh iodine (121.8 g I 100 g−1) and saponification value (184.91 mg KOH g−1 oil). The acid value, refractive index and relativedensity were 4.28 mg KOH g−1, 1.47 and 0.97 mg mL−1, respectively. The antioxidant potential (IC50) of apple seed oil was40.06 µg mL−1. Cytotoxicity of apple seed oil against CHOK1, SiHa and A549 cancer cell lines ranged between 0.5 ± 0.06% and88.6 ± 0.3%.

CONCLUSION: The physicochemical properties of apple seed oil were comparable with edible food oil, indicating its betterstability and broad application in the food and pharmaceutical industries. Apple seed oil could be a good source of naturalantioxidants. Also, the in vitro cytotoxic activity against specific cell lines exhibited its potential as an anticancer agent.c© 2013 Society of Chemical Industry

Keywords: apple seed oil; cytotoxicity; linoleic acid; oleic acid; palmitic acid; physiochemical properties

INTRODUCTIONApple (Malus domestica) is a delicious fruit enriched with an array ofnutrients. Fresh as well as processed apple products are consumedworldwide. Apple pomace, a left-over biomass generated in hugequantities in fruit-processing industries, contains peel, seeds, core,calyx and stem tissues.1 A significantly high moisture content andthe presence of fermentable carbohydrate make it vulnerable tomicrobial decomposition.2 Also, its disposal in open areas leads toserious environmental problems. It is mostly used for extraction ofpectin and animal feed production. It is reported that pomace canbe utilized either in a waste reduction strategy or for developing ahigh-value-added product, or preferably both.3 Apple pomace hasbeen considered a valuable biomass for the extraction/productionof products such as pectin,4 organic acids,5 protein-enrichedfeeds,6 aroma compounds,7 enzymes,8 natural antioxidants9 andedible fibers.10 It contain 4–5% seeds, a significant part afterthe peel, and can be used for oil extraction.11 Apple pomaceincluding seeds is rich in polyphenols. Phloridzin was found to bea major phenolic compound present in apple seeds. Phloretin-2′-xyloglucoside, 5-caffeoylquinic acid (chlorogenic acid), p-coumaroylquinic acid, (−)epicatechin, among other compounds,have also been reported from apple seed extracts.12 Thenutritional wealth of apple pomace has yet to find a feasible appli-cation, thus necessitating the development of a comprehensivestrategy for its management and utilization at an industrial scale.Moreover, scanty information is available on the evaluation of

apple seed oil as a dietary food ingredient.13–16 Also, in vitro cyto-toxicity studies of apple seed oil have not yet been demonstrated.

The present work is in continuation to the ongoing effortsfor utilization of the health-promoting nutritional componentspresent in apple pomace and subsequent development of an eco-nomically viable process for its value addition. Industrial pomaceis a mixture of different varieties used for juice extraction andexposed to an array of processing conditions. Thus it is pertinentto study the characteristics of oil extracted from apple seeds,separated from industrial pomace. The fatty acid composition,physicochemical properties and biological activities (antioxidantand in vitro cytotoxicity) of apple seed oil were studied in thiswork to demonstrate its suitability and stability as an ediblefood oil.

∗ Correspondence to: Shashi Bhushan, Division of Biotechnology, CSIR – Instituteof Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176061India. E-mail: sbhushan@ihbt.res.in

a Academy of Scientific and Innovative Research, CSIR – Institute of HimalayanBioresource Technology, Palampur, Himachal Pradesh, 176061, India

b Natural Plant Products Division, CSIR – Institute of Himalayan BioresourceTechnology, Palampur, Himachal Pradesh, 176061, India

c Division of Biotechnology, CSIR – Institute of Himalayan BioresourceTechnology, Palampur, Himachal Pradesh, 176061, India

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EXPERIMENTALMaterialApple pomace was collected from the Himachal Pradesh Horticul-tural Produce Marketing and Processing Corporation Ltd (HPMC)juice processing unit, located at Parwanoo, Himachal Pradesh,India, and brought to CSIR-IHBT, Palampur, after onsite preser-vation. The pomace was generated after extraction of juice fromdifferent apple varieties simultaneously (Red Delicious, GoldenDelicious, Royal Delicious and Red Chief). Apple seeds were sepa-rated from pomace in an indigenously developed machine. Theseseeds were sun dried and converted into powder by grindingin a blender for further processing. 1,1-Diphenyl-2-picrylhydrazyl(DPPH) and butylated hydroxyanisole (BHA) were purchased fromSigma-Aldrich Chemie (Steinheim, Germany) and Spectrochem PvtLtd (Mumbai, India). All other solvents and chemicals were of ana-lytical grade and obtained from s.d. fine-chem. Ltd (Mumbai, India).

Extraction procedureApple seed powder (100 g) was extracted with n-hexane at60 ◦C for 5 h in a Soxhlet apparatus and extraction was repeatedfive times. Hexane extracts were collected, concentrated andevaporated to dryness in a rotary evaporator. The seed oil wasstored at 4 ◦C until use.

Chemical composition analysisThe hexane extract was derivatized with methanol and sul-furic acid under a nitrogenous atmosphere by a previouslyreported method17 and analysed by gas chromatography–massspectrometry (GC-MS).

GC-MS analysisThe GC-MS analysis was performed on a Shimadzu QP 2010 seriesgas chromatograph–mass spectrometer (Tokyo, Japan), coupledto an AOC-20i auto-sampler and a DB-5 capillary column (30 m ×0.25 mm i.d., 0.25 µm). Oven temperature was programmed from40 to 220 ◦C at 4 ◦C min−1 with a 4 min hold at 40 ◦C and 15 minhold at 220 ◦C. Injector and interface temperatures were 250 ◦C. Ionsource temperature was 200 ◦C. The 20 µL sample was dissolvedin 2 mL GC-grade dichloromethane; sample injection volume was2 µL. Retention indices (RI) of the compounds relative to a mixtureof n-alkanes (C8 –C24) were calculated. The components wereidentified by comparison of their RI and mass spectral data within-house built library, Wiley, NIST, NBS and literature data.18,19

Physical and chemical analysis of apple seed oilThe procedures used for physicochemical analysis were thestandard AOAC methods.20 Density was determined using thespecific gravity bottle method. Refractive index was recordedusing an Abbe refractometer at 589 nm according to AOAC method921.08. Iodine value of oil was quantified by Wij’s method (AOACmethod 920.159). The saponification and acid value of oil weredetermined by AOAC methods 920.160 and 940.28, respectively.

Determination of antioxidant activity using the DPPHradical-scavenging methodThe antioxidant activity of apple seed oil was measured interms of hydrogen-donating or radical-scavenging ability usingthe DPPH method.21,22 Butylated hydroxyanisole (BHA) was usedas the standard lipophilic antioxidant and toluene as solvent.Different concentrations of standard (5, 10, 15, 20, 25 µg mL−1)

and apple seed oil (25, 50, 75 and 100 µg mL−1) were prepared intoluene. 0.1 mL of each sample was added to 2.9 mL fresh toluenicDPPH solution (0.1 mmol L−1). The mixture was shaken vigorouslyand left in the dark for 60 min. Absorbance was measuredagainst pure toluene (blank) at 515 nm by using a UV–visiblespectrophotometer (UV 2450 Shimadzu) and compared to control.Total antioxidant capacity was expressed as IC50, denoting theconcentration of sample required to scavenge 50% of DPPH freeradicals in the specified time period. Inhibition of free radical DPPH(I%) was calculated as follows:

% inhibition of DPPH activity = (Ac − As/Ac) × 100

where Ac is the absorbance of the control reaction and As is theabsorbance of the sample. The sample concentration providing50% inhibition (IC50) was calculated by plotting inhibitionpercentages against concentrations of the sample.

Cell lines and cell cultureCHOK1 (Chinese hamster), A549 (human lung carcinoma) andSiHa (human cervical cancer cell) cells were procured from thenational animal cell culture repository at the National Centre forCell Sciences, Pune, India. Cells were grown in F-12 HAMS and MEM(Gibco, Invitrogen, Carlsbad, CA, USA) media supplemented with10% heat-inactivated fetal bovine serum (FBS; Gibco, Invitrogen)and 1% antibiotic antimycotic (Gibco, Invitrogen). The cells weremaintained at 37 ◦C with 5% CO2 and 95% humidified atmospherein a CO2 incubator (Thermo Scientific, Waltham, MA, USA).

Determination of cytotoxic activity using sulforodamine B(SRB) assayFor the experiments, the required number of wells in a 96-wellmicrotitre plate was calculated such that every concentration ofcrude drug was tested in triplicate. Accordingly 20 000 cells wereseeded per well in 100 µL medium. Seeding was done in threesuch plates, for each cell line, to obtain timely data for 24, 48 and72 h. After seeding, all the plates were incubated at 37 ◦C with 5%CO2 for 12–15 h, until cells adhered and attained morphology. Theapple seed oil samples ranged from 0.05 to 2 mg. Original stock ofdrug was prepared in DMSO at 20 mg mL−1 concentration, whilesamples were prepared in respective culture media. Samples werealso incubated in a CO2 incubator along with microtitre plates.

Incubated plates were treated and all plates were reincubatedfor 24, 48 and 72 h. After the incubation period, the cell monolayerwas fixed with 10% trichloroacetic acid (w/v) and kept at 4 ◦C for1 h. Later, these were washed with distilled water and allowed toair dry. 0.4% (w/v) SRB solution in 1% (v/v) acetic acid was used toperform SRB assay on air-dried plates.23 Wells were subjected toSRB solution for 30 min at room temperature. After staining, excessdye was removed by repeated washing with 1% (v/v) acetic acidand dried in the dark. Sequentially, the protein-bound dye wasdissolved in Tris base [tris(hydroymethyl)aminomethane] solution(10 mmol L−1) for optical density determination at 540 nm using amicroplate reader (BioTek Synergy H1 hybrid reader, Winooski, VT,USA). Percentage inhibition as well as standard deviation (SD) werecalculated with respect to control (untreated wells). Vinblastinewas taken as positive control at a concentration of 1 µg.

Statistical analysisIn general, experiments were repeated three times with fivereplicates, unless otherwise stated. SD was calculated using

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Figure 1. GC-MS chromatogram of methyl esters of fatty acids in apple seed oil.

Table 1. Fatty acid composition of apple seed oil

Compound RI Percentage Identification

Palmitic acid 1927 7.25 RI, MS

Linoleic acid 2099 43.81 RI, MS

Oleic acid 2107 46.50 RI, MS

Stearic acid 2126 1.72 RI, MS

Arachidic acid 2325 0.72 RI, MS

No. of total identified fatty acids = 5.

Microsoft Excel where required. Data on cytotoxicity studieswere analysed using a paired t-test employing SAS-JMP software,version 10, at P = 0.05.

RESULTS AND DISCUSSIONThe economical utilization of apple pomace is still a major concernto juice/cider-processing industries throughout the apple-growingregion. The fruit skin/flesh part is a good source of dietaryfibre/polyphenols and has been studied extensively. After fruitskin/flesh, apple seed is the second major ingredient of pomace.In this work, efforts were made to extract oil from separated seedspresent in industrial pomace. The extracted oil was evaluated forphysical, chemical and cytotoxic properties. The results showedgood potential of apple seed oil as a dietary ingredient in foodand pharmaceutical applications.

Fatty acids composition of seed oilThe oil was extracted from apple seeds by the Soxhlet extractionmethod using n-hexane as a solvent. The yield of oil was22.33 ± 1.5% (SD). The extracted oil was derivatized to methylesters and analysed by GC-MS (Fig. 1). Five fatty acids wereidentified as their methyl esters (Table 1). The content ofunsaturated fatty acids (90.31%) was higher than that of thesaturated ones (9.72%). Oleic acid (46.50%) and linoleic acid(43.81%) were the major unsaturated fatty acids present in appleseed oil. The saturated fatty acids present in seed oil were palmitic(7.25%), stearic (1.72%) and arachidic (0.72%) acids. Previousreports on the composition of apple seed oil showed linoleic acidas the most dominant fatty acid, followed by palmitic, linolenic,stearic and oleic acids.13 The high percentage of unsaturated fattyacid (about 90%) in apple seed oil clearly supports its suitabilityas edible oil and application as a dietary food ingredient.

Physicochemical properties of seed oilThe apple seed oil is yellowish in colour. The relative density (25 ◦C)was found to be 0.97 mg mL−1 (Table 2). The relative density ofoil is known to be influenced by degree of unsaturation. The acidnumber signifies the number of carboxylic acid groups presentin the fatty acids. A high acid value of oil indicates the presenceof more free fatty acid. The acid value of the apple seed oil was4.28 mg KOH g−1 oil – comparatively higher than sunflower andmustard oils (Table 2). The saponification value is a measure of theaverage molecular weight (or chain length) of all the fatty acidspresent in the oil. The saponification value of apple seed oil was

Table 2. Physicochemical characteristics of apple seed, sunflower and mustard oil

Apple variety (seed oil)15

Physicochemical parameters Apple seed oil Sunflower oil24 Mustard oil24 Fuji New Red Star

Colour Yellow Pale yellow Brownish yellow — —

Relative density (25 ◦C) (mg mL−1) 0.97 0.9182 0.9072 0.903 0.902

Acid value (mg KOH g−1) 4.28 3.89 1.21 4.036 4.323

Saponification value (mg KOH g−1) 184.91 188.0 174.0 179.01 197.25

Iodine value (g 100 g−1) 121.8 128.0 108.0 94.14 101.15

Refractive index 1.47 1.4672 1.4655 1.465 1.466

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found to be 184.91 mg KOH g−1 oil. The recorded value is higherthan mustard oil but comparable to previous reports on apple seedoil. The iodine number determines the amount of unsaturationpresent in the fatty acids. The higher the iodine number, the moreunsaturated fatty acid bonds will be present in the oil. The iodinevalue of apple seed oil was 121.8 g I 100 g−1 oil, which is quitehigh compared to mustard oil, but lower than sunflower oil. A highrefractive index of oil indicates a high degree of unsaturation andlong chain length of fatty acids. The refractive index of apple seedoil was 1.47, which is on a par with other edible oils (sunflower andmustard oils).

Apple seed oil recorded a higher acid value in comparisonto sunflower oil and mustard oil,24 signifying the presenceof unsaturated fatty acids in oil. However, it is well withinthe acceptable limits of edible vegetables oils.25 Most of thephysicochemical properties are in accordance with an earlier reportpublished on seed oil, extracted from specific apple varieties.15

Owing to the presence of high unsaturated fatty acid content(over 90%) and comparable edible oil properties, apple seed oilseems to have the edge over sunflower and mustard edible oils.It could be a good nutritional supplement or fortifying agent inpharmaceuticals.

Antioxidant activity of apple seed oilThe antioxidant activity of apple seed oil was evaluated by DPPHradical-scavenging assay. DPPH is a stable free radical, frequently

Table 3. Antioxidant activity of apple seed oil

Sample IC50 values (µg mL−1)

BHA 9.97

Apple seed oil 40.06

used to determine the radical-scavenging activity of naturalcompounds.26 DPPH, due to its stability, is considered a goodkinetic model for peroxy radicals.27 The scavenging effect of appleseed oil on DPPH radical is depicted in Fig. 2. Apple seed oil atconcentrations of 25, 50, 75 and 100 µg mL−1 scavenged 38.12%,60.34%, 71.23% and 88.74% of DPPH radicals, respectively. Theconcentration of the sample that reduced radical absorbance by50% (IC50) served as an index to compare antioxidant activity. Alower IC50 value indicates higher antioxidant activity. Apple seedoil showed concentration-dependent scavenging of the DPPHfree radical. The IC50 value of the apple seed oil was quite high,i.e. 40.06 µg mL−1, as compared to BHA standard (9.97 µg mL−1)(Table 3). However, the amount of seed oil required to scavenge50% of DPPH radical (IC50) is much lower than in earlier reportson seed oil of specific apple varieties (8.34 mg mL−1 in Fuji and7.91 mg mL−1 in New Red Star).15 This variation can be attributedto the fatty acid composition as well as the concentration ofindividual compounds present in oil and varieties used for juiceextraction. In the present study, the apple seed oil is mainly

Figure 2. Antioxidant activity of (a) BHA and (b) apple seed oil.

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Table 4. Cytotoxicity assay of apple seed oil, showing percent inhibition

Apple seed oil (mg mL−1)

Cell line Time exposure Vinblastine (1 µg) 0.05 0.1 0.5 1 1.5 2

CHOK1 24 h 79.7 ± 0.25 3 ± 0.3 −8.5 ± 0.26 0.8 ± 0.27 −6.5 ± 0.34 19 ± 0.45 26.5 ± 0.3

48 h 65.6 ± 0.07 0.5 ± 0.06 3.8 ± 0.17 15.2 ± 0.11 31.6 ± 0.08 53.6 ± 0.08 72.8 ± 0.05

72 h 90 ± 0.2 −19 ± 0.35 37 ± 0.1 38 ± 0.32 53.8 ± 0.23 85.7 ± 0.24 88.6 ± 0.3

SiHa 24 h 64.4 ± 0.15 −42.5 ± 0.09 −26.3 ± 0.1 −9.1 ± 0.25 12.3 ± 0.03 26.1 ± 0.35 47 ± 0.07

48 h 53.5 ± 0.14 0.7 ± 0.09 1.7 ± 0.06 19.2 ± 0.09 39.8 ± 0.02 40.4 ± 0.15 41.2 ± 0.05

72 h 54.2 ± 0.13 16.6 ± 0.06 2.2 ± 0.19 17.2 ± 0.2 30.1 ± 0.18 48.1 ± 0.17 56.2 ± 0.21

A549 24 h 74.1 ± 0.21 17.9 ± 0.21 17.8 ± 0.34 24.4 ± 0.15 36.4 ± 0.28 46.7 ± 0.17 52.9 ± 0.23

48 h 50.3 ± 0.55 16 ± 0.72 20.6 ± 0.5 23.8 ± 0.7 25.8 ± 0.69 28.3 ± 0.6 32.1 ± 0.6

72 h 51.2 ± 0.54 6.4 ± 0.75 54 ± 0.5 64 ± 0.5 62.7 ± 0.9 59.1 ± 0.7 64.4 ± 0.6

constituted of unsaturated fatty acids (about 90 %), which couldbe responsible for higher antioxidant activity. Oleic and linoleicacids are reported to have good antioxidant potential.28 Therefore,apple seed oils can be considered as a good source of unsaturatedfatty acids for fortification or development of nutritionalsupplement, especially in the management of cardiometabolicdisorders.

Cytotoxic activity of apple seed oilThe effects of apple seed oil were tested in vitro against Chinesehamster (CHOK1), A549 (human lung carcinoma) and humancervical cancer cells (SiHa) using different concentrations (Table 4)by SRB assay. Apple seed oil displayed varying growth inhibitionpotentials at different concentrations on respective cell lines(Fig. 3). Higher cytotoxicity was observed with increase in oilconcentration. Vinblastine (1 µg) reached a maximum of 90%cytotoxicity on CHOK1, while the tested sample (2 mg) reached88.6% in 72 h. Interestingly, apple seed oil displayed a similarcytotoxicity trend on the SiHa cell line. It was able to inhibit56% cells at the concentration of 2 mg. The drug was alsotested on A549 and gave no proliferation at all. 24 h studydisplayed a steady rise in cytotoxicity followed by 48 and 72 h.Although the effect of vinblastine was gradually reduced, theseed oil displayed a better potential at 72 h. Although oleic acidis reported to have cytotoxic effects against cancer cell lines,29

the observed cytotoxicity against specific cell lines could beattributed to a synergistic effect of all the fatty acids presentin the apple seed oil.30 The cytotoxicity results of apple seed oilagainst CHOK1, A549 and SiHa showed good pharmacologicalpotential.

CONCLUSIONThe management of industrial residues like apple pomace wouldonly be possible through its inclusive utilization. The resultsof the present study clearly demonstrate the use of appleseeds for extraction of oil, which showed good potential as anedible food oil and nutraceutical/pharmaceutical supplement.The physicochemical properties were comparable with qualityedible oils and were even better than mustard oil. The presence ofunsaturated fatty acid in high concentration with good antioxidantpotential and in vitro cytotoxic activity further add value tothe extracted oil. These results will definitely pave the way forthe development of a comprehensive apple pomace utilizationstrategy, feasible at an industrial scale.

Figure 3. Antiproliferative activity of apple seed oil against (a) CHOK1; (b)SiHa; and (c) A549 cell line.

ACKNOWLEDGEMENTSThe authors gratefully acknowledge the Director, CSIR-IHBTPalampur (HP), India, for continuous encouragement and forproviding the necessary facilities during the course of theinvestigation. The authors would also like to thank the Ministry of

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Food Processing Industries, Government of India, for funding theproject, and Himachal Pradesh Horticultural Produce Marketingand Processing Corporation Ltd (HPMC), Parwanoo (HP), India, forproviding the apple pomace.

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