Characterization of Glyceridic and Unsaponifiable Compounds of

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Characterization of glyceridic and unsaponifiable compounds of Sacha inchi (Plukenetia huayllabambana L.) oils Nancy A. Chasquibol, Chellah del Aguila, Juan C. Yacono, Angeles Guinda, Wenceslao Moreda, RAQUEL BEATRIZ GÓMEZ-COCA, and María del Carmen Pérez-Camino J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 17 Sep 2014 Downloaded from http://pubs.acs.org on September 30, 2014

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Journal of Agricultural and Food Chemistry

Characterization of glyceridic and unsaponifiable compounds of Sacha inchi (Plukenetia huayllabambana L.) oils Nancy A. Chasquibol1, Chellah del Aguila2, Juan C. Yácono1, Ángeles Guinda2, Wenceslao Moreda2, Raquel B. Gómez-Coca2 and M. Carmen Pérez-Camino2* 1

Universidad de Lima, Av. Javier Prado Este, cuadra 46 s/n, Monterrico, Lima 33, Perú.

2

Department of Characterization and Quality of Lipids. Instituto de la Grasa -CSIC- Avda.

Padre García Tejero 4, 41012 Sevilla, Spain *

Tel: +34-9546115550 (ext 299); Fax: +34954611550. E-mail: [email protected]

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ABSTRACT: The paper deals with the characterization of the main glyceridic and

2

unsaponifiable components of oils obtained from Sacha inchi (Plukenetia huayllabambana

3

L.) seed ecotypes collected during two harvests in the Department of Amazonas in Peru. The

4

seed-oil yield was 30.3 - 41.2%, standing out the high percentage of the ω3- and ω6- fatty

5

acids series whose ranges lie within those of the present Regulation for Sacha inchi oils.

6

Triacylglycerols with even ECN (36-42) were the main components. Minor glyceridic polar

7

compounds like oxTGM, DAG, MG and FFA were determined by HPSEC. The low

8

campesterol/stigmasterol ratio (1:6), unusual in the majority of vegetable oils, stands out.

9

Regarding aliphatic hydrocarbons these oils showed a particular profile for the saturated

10

series of odd and even carbon atom number. According to our results Sacha inchi Plukenetia

11

huayllabambana oils can be offered as a good alternative to Plukenetia volubilis, the specie

12

mainly commercialized for this vegetable oil.

13

KEYWORDS: oil characterization, polar glyceridic compounds, Sacha inchi P.

14

huayllabambana oil, triglycerides, unsaponifiable components

15

16

17

18

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INTRODUCTION

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Sacha inchi plants from the Peruvian Amazon produce a kind of oilseed with high protein

25

and lipid content1. This oil can be consumed virgin (cold-pressing extraction) and is highly

26

valued for its sensory qualities. It can be considered as a gourmet oil as are olive, avocado,

27

germ wheat, rice bran, and argan oils, and it is appreciated for its beneficial health properties

28

and its unique taste and flavour2,3.

29

Nowadays, commercialized Sacha inchi oils come from Plukenetia volubilis L. (P.

30

volubilis) specie, which grows mainly in San Martin region, the most productive one in

31

Peru. In September 2004 it was declared to be of "National Genetic Heritage and alternative

32

product in the fight against poverty" by the Congress of the Republic of Peru. Extensive and

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thorough studies have been undertaken to determine the chemical composition of Sacha

34

inchi (P. volubilis) seeds and oils, leading to an exhaustive knowledge of the chemical

35

composition of this specie4-9. In addition, from 2009 on there is a Regulation (NTP) that

36

specifies the requirements of the analytical parameters that must meet Sacha inchi seed oil

37

from the Plukenetia gender10.

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There are five species of Sacha inchi growing in Peru: P. brachybotrya, P. loretensis, P.

39

volubilis, P. polyandenia, and P. huayllabambana. Bussmann and coworkers11 have

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described them morphologically. P. huayllabambana is a novel species which has larger and

41

rougher seeds than P. volubilis, the most popular specie12 and that grows wild in the

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province of Rodríguez de Mendoza, Amazonas Department. Currently, there are 220

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hectares of this specie in Rodriguez de Mendoza. This is harvested at the beginning of

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January, ending in August, yielding approximately 1.500 kg·ha-1 of seeds. An increasing

45

interest in this specie has been raised lately and thus, Muñoz Jauregui and coworkers13 have

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published a broad nutritional study, while Ruiz and coworkers14 have provided some data

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about its composition. This new specie seems a promising source of high quality vegetable

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oil, which gives to these crops a high added value since they can be considered as an

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alternative cultivar for the local inhabitants of the region.

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Our aim was to study the P. huayllabambana oils obtained by cold extraction process

51

from seeds collected in the main plantations of the province of Rodriguez de Mendoza,

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analysing its major and minor components -glyceridic and unsaponifiable matter- with the

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purpose of serving for knowledge and supporting the potential of this specie for

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commercialization.

55 56

MATERIAL AND METHODS

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Materials. Twenty-eight batches of Sacha inchi seeds (P. huayllabambana) were

58

harvested between April 2012 and 2013 in the province of Rodriguez de Mendoza, Department

59

of Amazonas. Sampling was conducted at the proper maturation stage, considering growing

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altitude (1500-2100 m), temperature range (18.6-23.1 °C), pH of the soil (6.2), and percentage

61

of organic matter (2.3%).

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Oil extraction and composition of Sacha inchi seeds. After shelling and weighing, the

63

oils were extracted by hydraulic pressing, and then centrifuged. The process was carried out

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with a prototype designed in the Lima University laboratories, which consists of a cylinder with

65

holes of 1/16" in which the seeds were loaded and then pressed by a piston actuated by a

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hydraulic system assisted by a motor of 1 HP. An average amount of 2 kg shelled seeds was

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used for each sample in order to obtain enough oil for complete characterization. After the

68

extraction the oil was filtered with a centrifugal filter (30 min, 3600 rpm). Crude oil samples

69

were stored in dark bottles at 4 °C until analysis.

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Reagents and solutions. Acetone, diethyl ether, hexane, propionitrile, and THF were

71

supplied by VWR International, (West Chester, Pennsylvania, USA). The Si-SPE cartridges 4

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were from Varian (EA Middelburg, The Netherlands). Standards of 5-α-cholestan-3β-ol and

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trilinolenin (LnLnLn) were from Sigma-Adrich Co. (St. Louis, Missouri, USA). Hexamethyl

74

disilazane, pyridine, trimethyl chloroxilane, and standards of eicosane and tocopherols were

75

from Merck-España (Merck Group, Darmstadt, Germany). All chemical reagents were

76

analytical grade.

77 78

Physical-chemical determinations. The physical-chemical parameters included in the

79

current legislation for Sacha inchi oils and other Regulations for other edible oils were

80

determined in the crude oils10.

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Free fatty acids and peroxide values. AOCS Official Methods were followed to determine

82

FFA and peroxide indexes15, 16.

83

UV evaluation. Specific extinction at 270 nm (K270) was evaluated according to the

84

European Union Standard Methods17.

85

Oxidative stability. The induction time was evaluated using a Rancimat equipment (743

86

Rancimat Metrohm Co., Basel, Switzerland), following the standard Official Method Cd 12-

87

b-9218, 19, with a flow air of 20 L·h-1 air and 100±1 ºC.

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Other physical parameters. Parameters like viscosity and density were measured using a

89

Stabinger SVM 3000 viscometer (Anton Paar GmbH Graz, Austria). The refractive index

90

was determined with a temperature-controlled Abbe refractometer (Hilger & Watts Ltd.,

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London, U.K.)3, 20.

92 93

Major components. Triacylglycerol composition. The TG composition was obtained as

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follows: oil samples at a concentration of 50 mg·mL-1 in acetone were analysed by RP-

95

HPLC. The separations were done on a Merck Li-Chrospher 100 RP-18 column (250 mm x

96

4 mm ID, 4 µm particle size) thermostated at 20 ºC. The liquid chromatograph (Beckman5

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Coulter, Fullerton, CA, USA) was equipped with a pumping unit (118 solvent module) and

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used propionitrile as mobile phase at a flow-rate of 0.6 mL·min-1. Oil solutions in acetone

99

(10 µL) were injected using an auto-sampler (508 system). Detection was done with a

100

Perkin Elmer 200 RI detector21.

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Polar glyceridic compounds. PC were assessed with small modifications of the already

102

published method, by separating the oils in two fractions by Si-SPE using 1g cartridges22, 23.

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Thus, 200 mg oil samples were weighted in 10 mL volumetric flasks and dissolved in

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hexane. On each case an aliquot of this solution (2 mL) was introduced into the cartridge

105

and then eluted with 15 mL of the hexane:diethyl ether 87:13 v/v admixture. The fraction

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containing the non-altered TG was rejected. A second fraction was collected eluting with 15

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mL of diethyl ether. The latter fraction was evaporated until dryness, re-dissolved in 1 mL

108

THF and injected onto the HPLC (Hewlett Packard 1050 series). The HPSEC system is

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equipped with an Agilent PL gel of 5 µm, a 100 Å column, and a Merck La Chrom RI

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Detector L-7490. THF was used as eluent at a flow rate of 0.7 mL·min-1. The injection

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volume was 20 µL. The compounds eluted from the columns according to their molecular

112

size, so those with the largest molecular sizes were eluted earlier and were quantified as

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individual percentages of the total eluted compounds and also as absolute quantities using an

114

external standard of pure TG in THF at concentrations of 5-10 mg·mL-1.

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Fatty acid composition. The FFA composition was determined as the composition of FAME

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by GC after trans-esterification of the oils with 2N KOH in methanol, according to the

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IUPAC Standard Method20,

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5890 GC system (Palo Alto, CA) equipped with a split injector (1:50 split ratio), a polar

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capillary column (poly-90% biscyanopropyl-10% cyanopropylphenyl siloxane, 60 m x 0.25

120

mm ID, 0.20 µm film thickness) and a FID. Hydrogen was used as carrier gas at a flow rate

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of 1.0 mL·min-1. The detector and injector temperatures were 225 and 250 °C, respectively.

24

. The chromatographic analysis was done using an Agilent

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The initial oven temperature was 180 °C and a temperature gradient from 180 to 220 °C at 3

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°C min-1 was applied. 1 µL Injections were performed automatically.

124 125

Minor unsaponifiable compounds. The percentage of the total unsaponifiable fraction

126

was calculated gravimetrically after subjecting the oil samples to a saponification procedure

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carried out according to the IUPAC 2401 Official Method25.

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Tocopherols. Tocopherols were determined according to the IUPAC Standard Method

129

243226. An oil solution in hexane at a concentration of 10 mg·mL-1 was prepared and

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analysed by HPLC fitted with a Si-column (250 mm x 4 mm ID, 4 µm particle size). The

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elution solvent was a hexane:2-propanol (99:1, v/v) mixture at a flow rate of 1 mL·min-1.

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Detection was done by fluorescence (RF-10AXL Shimadzu fluorescence detector), setting

133

excitation and emission at λ = 290 and λ = 330 nm, respectively. For quantitative

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determinations standards of tocopherols in hexane at concentrations of 4 - 6 µg·mL-1 were

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prepared and injected.

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Sterol composition. The sterol fraction was isolated from the unsaponifiable matter by silica

137

TLC using plates impregnated with potassium hydroxide with the purpose of retaining the

138

remaining saponified compounds. The plate was developed twice with a mixture of

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petroleum ether:diethyl ether (87:13, v/v). The isolated fraction was scratched off and

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extracted with hot chloroform and diethyl ether. The solution was evaporated until dryness,

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derivatized with 500 µL of the trimethyl chloroxilane:hexamethyl disilazane:pyridine 1:3:9

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(v/v/v) admixture and analysed by GC. The gas chromatograph (Agilent 6890N) was

143

equipped with a fused silica low polarity capillary column (poly-5% diphenyl-95% dimethyl

144

siloxane, 30 m x 0.25 mm ID x 0.25 µm film thickness), and FID. The oven program was

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isothermal at 260 ºC with a split ratio of 1:50. Hydrogen was used as carrier gas at a flow 7

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rate of 1 mL·min-1. The temperatures of the injector and detector were 300 ºC. The

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quantitative determination was done using α-cholestanol as internal standard.

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Aliphatic

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chromatography filled with 15 g silica-gel (Si-60) impregnated with AgNO327. A sample of

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0.5 g oil was introduced into the column and the fraction containing the aliphatic

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hydrocarbons was eluted with 80 mL petroleum ether. This fraction was evaporated until

152

dryness, re-dissolved in 0.5 mL hexane and analysed by GC. The gas chromatograph was

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equipped with a low polarity capillary column (poly-5% diphenyl-95% dimethyl siloxane,

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12 m x 0.32 mm ID x 0.1 µm film thickness) and an on-column injection system. The initial

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oven temperature was set at 80 ºC for 2 min, the rate was established at 12 ºC·min-1 up to

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280 ºC, then at 7 ºC·min-1, and then up to 340 ºC. The injector temperature was 80 ºC and let

157

follow the oven temperature program. The FID temperature was 350 ºC. Eicosane (C20:0) at

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a concentration of 0.05 mg·mL-1 was used as internal standard.

saturated

hydrocarbons.

HC

were

isolated

by

low-pressure

column

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Statistical analysis. Each sample was analysed in triplicate and data were presented as

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the average of the fourteen data obtained in each crop. The statistical analyses were run

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using the GraphPad Prism© version 5.01 for Windows software. The crops were compared

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by ANOVA. When significance was observed the Tukey test was applied. Significant

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differences (p > 0.05) are specified with different letters.

164 165

RESULTS AND DISCUSSION

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Oil yield. Compared to Sacha inchi P. volubilis, currently the most widely used cultivar

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for oil extraction and commercialization, P. huayllabambana specie is almost twice its size

168

(1.7 - 4.1 cm3 vs. 4.1 - 8.0 cm3,) and weight having an oil yield after pressing and filtration

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in the range of 30.3 - 41.2% referred to shelled seeds.

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Analytical indexes and oxidative stability. The values of the indexes commonly used

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to evaluate initial quality of vegetable oils, i.e., FA, PV, and K270, were found within the

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expected ranges for crude oils of good quality, with some remarkable exceptions. Regulation

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for extra virgin and virgin Sacha inchi oils establishes a maximum of 1.0 and 2.0 (expressed

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as % of oleic acid) of FA, respectively. Table 1 shows the results of the quality parameters

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and it can be observed that the average value obtained for the 28 oil samples from the two

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harvests under study is within the established ranges for virgin oils. Half of the samples can

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be classified as extra virgin, 29% as virgin, and 21% are neither extra virgin nor virgin oils.

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These results are indicating that Sacha inchi P. huayllabambana seeds are highly susceptible

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to hydrolysis, yielding oils with acidity over the established limits. The high hydrolyzation

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level showed is not an exclusive characteristic of P. huayllabambana oil samples. Actually

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some published data on P. volubilis also present acidity values over the limits established in

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the Regulations28, 29.

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Regarding the oxidation state of the oils, the mean values showed to be within the limits

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included in the Regulations, although three of the samples overpassed the 10 meq O2·kg-1

185

maximum established in the NTP.

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Concerning the absorbance at 270 nm there is not limit established in the NTP

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Regulation. Olive oil Regulation includes a limit of 0.22 for extra virgin and 0.25 for virgin

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olive oils. The results on the analysed Sacha inchi oils drove us to categorize a high number

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of samples (24) as extra virgin, and 3 as virgin, whereas only one dropped out of both

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categories.

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Most of the PV data reported for P. huayllabambana were within the limits established

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by the Regulations. It can be observed that some of the samples from the first harvest

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yielded oils with PV over the limit, but that the oils obtained from the second harvest

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showed a significantly lower value, which means lower oxidation level, undoubtedly due to 9

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a more suitable storage period in the second repetition of the study. Nevertheless their

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absorbance at 270 nm is higher, although with no meaningful differences.

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The oxidative stability determined by Rancimat gave IP of 2.0 - 3.4 h, at 100 ºC. These

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values are rather low compared to those of other edible oils as olive or sunflower oils, but

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they are similar or even higher than those of oils with similar unsaturation level, as safflower

200

or flax oils30. No significant differences are found between the two harvests under study.

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We have to consider that these oils were obtained from raw seeds. In the case of carrying out

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a previous seed roasted process, an increase of the oil stability is to be expected, as occurred

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with argan seeds2,3. This kind of processes have been recently carried out with Sacha inchi

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(P. volubilis)31.

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Density, refractive index and viscosity are physical parameters determined to

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differentiate and to characterize the oils and mainly are used for commercial purposes. The

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density values at 20 ºC were in the 0.920 - 0.930 g·cm-3 range and were within the

208

requirements of the NTP. These values are slightly higher than those reported at the same

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temperature for other vegetable oils such as sunflower or olive (0.898 - 0.906)3.

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The refractive index at 20 °C for the first harvest ranged from 1.480 - 1.482 and is

211

within the ranges set in the NTP. For the second harvest the average of the fourteen samples

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under study was over the limit established in the NTP. These results were also higher than

213

those reported for other vegetable oils (1.461 to 1.474)32.

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The cinematic viscosity data at 20 ºC were in the 38.9 - 44.0 mm2·s-1 range, being the

215

average 42.8 and 41.1 mm2·s-1 for the first and second harvests, respectively. These values

216

are higher than those reported at the same temperature for Plukenetia volubilis8.

217 218

Major saponifiable compounds

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Triglyceride molecular species. A detailed study of the TG was carried out by means

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of RP-HPLC-RI and it is summarized in Table 2. The eluting order of each TG molecular

221

species can be determined using the standard of LnLnLn, which elutes as the first TG peak.

222

One should take into account that under the isocratic conditions used in this work the

223

retention times for TG are directly proportional to the number of carbon atoms, and

224

inversely proportional to the total number of double bonds in the three fatty acyl chains. TG

225

molecular species can be grouped according to their ECN, defined as the total acyl-carbon

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number in the molecule minus twice the total number of double bonds of the fatty acids

227

comprising the TG21.

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As one can observe in Fig. 1A the ECN of the main TG identified are within the range

229

from 36 – 42, normally with combinations of Ln and L. Thus, the four main TG are:

230

LnLnLn, LnLnL, LnLL and LLL (ECN 36, 38, 40, and 42, respectively). Also TG with

231

ECN 44, 46, 48 and 50 are found, but in minor proportions. Fanali and coworkers6 reported

232

the TG composition of Sacha inchi P. volubilis oils from Peruvian producers for the first

233

time. Remarkable differences can be found with the P. huayllabambana species presently

234

studied. Thus, although in both species LnLnL (ECN 38) is the major TG, for P.

235

huayllabambana this TG is higher than the whole group of TG with ECN 40 (26-% vs. 21-

236

%), which is the main TG group reported for P. volubilis (28.7%)6.

237 238

Glyceridic polar compounds. The quantitative determination of glyceridic polar

239

compounds gives a direct evaluation of all the compounds having polarity higher than that

240

of the TG, the major compounds in edible fats and oils. The composition and profile of the

241

PC is the most valuable and common methodology utilized for evaluating the alteration in

242

heated oils. The fraction isolated by Si-SPE was quantified using HPSEC, separating the

243

compounds according to their molecular size: D-TG, oxTG, DG, MG, and FFA (Fig. 1B1 11

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and 1B2). Quantitative PC data (Table 2), which include values between 2.3 and 7.5% for

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Sacha inchi P. huayllabambana oils, shows percentages below 5-6 % in most of the samples

246

studied, which is indicative of good quality oils34. No comparison can be done with other

247

Sacha inchi species since no PC data have been reported. As can be observed, the PC

248

fraction is mainly composed of DG and FFA in a ratio of 2:1 approximately. It is noteworthy

249

the presence of MG in proportions around 0.6-3.3% in all analysed samples, confirming that

250

the susceptibility of the seed to hydrolization goes beyond the DG formation. Some of the

251

evaluated samples exhibited a high level of compounds related to hydrolization such as DG

252

and FA, and also important quantities of oxTG and D-TG (Fig. 1B2). The PC method,

253

designed in the beginning for oils subjected to frying processes, gives us valuable

254

information on the alteration of the Sacha inchi seeds due to hydrolysis. Moreover, all oil

255

samples contain oxTG as has been seen in other edible oils but an important number of them

256

also have high quantities of minor glyceridic components, which indicates the facility of

257

Sacha inchi oils to oxidative alteration, which is expected due to their high unsaturation

258

degree.

259 260

Fatty acid composition. A representative chromatogram of the FAME of Sacha inchi

261

P. huayllabambana oil is presented in Fig. 1C. There we can observe the high percentage of

262

Ln, as well as the presence of ω7-C18:1 and the absence of C20:1. The results (Table 2)

263

indicate no significant differences between the two studied harvests. The high contents of Ln

264

(55.5 - 60.7%) and L (25.0 - 27.3%) stand out. These values are slightly different to those

265

reported for Sacha inchi P. volubilis28, 35. The ω6:ω3 ratio determined in this work for Sacha

266

inchi P. huayllabambana is 0.45:1, which is lower than those reported for Sacha inchi P.

267

volubilis (0.75:1), since for the commercial species the Ln content is low (47-50%).

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Main unsaponifiable components

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We have studied the total quantity of the unsaponifiable matter as well as the sterol,

270

tocopherols and aliphatic hydrocarbons profiles and contents.

271

The total unsaponifiable matter (Table 3) differs for both harvests and in some samples

272

is out of the limits established in the Regulations (0.36%). At this point it is important to

273

highlight that the limit established in the Regulation is not really reasonable since just the

274

addition of the values regarding valuable unsaponifiable components such as sterols and

275

tocopherols exceeds it.

276 277

Fig. 2 represents the chromatograms of the main unsaponifiable compounds of Sacha inchi P. huayllabambaba oils studied here.

278

Tocopherols. γ- and δ-Tocopherols are the main species that can be directly separated

279

and quantified by HPLC using fluorescence detector (Fig. 2A). In a few number of samples

280

also α-tocopherol is present, but with percentages lower than 0.1. γ-Tocopherol was the

281

major tocopherol with percentages above 65% in most of the samples. High variability is

282

found in their total quantities not only between harvests but also among samples within the

283

same harvest (Table 3). This fact can be explained taking into account that these

284

compounds, specially δ –tocopherol, are powerful antioxidants and protect the highly

285

unsaturated TG of Sacha inchi oils against oxidation, resulting in their degradation. The

286

results obtained regarding the tocopherol content of the Sacha inchi P. huayabambana are

287

slightly higher than data published for volubilis species6,28. However, they are above the

288

maximum limit established in the most recent Peruvian Regulations for Sacha inchi P.

289

volubilis oils36. This recent Regulation concerning only to P. volubilis species establishes a

290

maximum limit of 0.7, 0.29, 136.7 and 85.6 mg·kg-1 for α, β, γ and δ, respectively, which is

291

at the least strange.

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Sterols. Related to the sterols GC profile (Fig. 2B) twelve peaks can be separated

294

including α-cholestanol, which is added as internal standard for quantitation purposes. The

295

major peak corresponds to β-sitosterol (peak 7), as in the majority of vegetable oils, with

296

percentages above 60%. Stigmasterol (peak 4) is the second most important sterol in Sacha

297

inchi oils, which is in agreement with recent data reported by Muñoz Jáuregui and

298

coworkers13 where sterols were determined by HPLC. In rare cases, as occur in Sacha inchi

299

oils, including the P. volubilis species, the stigmasterol content is higher than the

300

campesterol concentration5. For most edible oils, the ratio campesterol/stigmasterol is higher

301

than 137. This is the case of olive oil, where its Regulation establishes explicitly that the

302

stigmasterol content must be lower than that of campesterol17. The campesterol/stigmasterol

303

ratios calculated in this work are within the 0.14-0.21 range, being the stigmasterol

304

concentration six times that of campesterol. This high stigmasterol content can provide

305

interesting properties to Sacha inchi oils since this phytosterol plays an important role in

306

reducing the inflammatory processes.

307 308

Aliphatic saturated hydrocarbons. HC form a remarkable group of minor

309

unsaponifiable compounds present in edible fats and oils. At present, they are considered as

310

intermediate metabolites in the formation of other minor compounds, such as aliphatic

311

alcohols, with specific roles. Fig. 2C shows an on-column gas chromatogram corresponding

312

to the aliphatic saturated hydrocabons of Sacha inchi P. huayllabambana oil. The main

313

peaks correspond to: H21 Heneicosane; H22 Docosane; H23 Tricosane; H24 Tetracosane,

314

and H25 Pentacosane. Minor amount of H19 Nonadecane; H26 Hexacosane, H27

315

Heptacosane, H28 Octacosane and H29 Nonacosane are also present. Surprisingly both the

316

odd and even series were present. The procedure was applied using impregnated silver

317

nitrate silica to ensure that only saturated hydrocarbons eluted from the silica column. 14

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Results of the mean concentrations of the main aliphatic saturated hydrocarbons and also

319

their total quantities evaluated using as standard a solution of heneicosane (n-H20:0) at

320

concentration of 0.05 mg·mL-1 are included in Table 3. The high concentrations of the H23:0

321

hydrocarbon with values of 36-38%, followed by those of H24 (17-19%) and H25 (13-15%)

322

are remarkable. As can be observed, the area relation between hydrocarbons with even and

323

odd carbon-atom number is around 2, although the odd series is the most abundant one, as in

324

the main edible vegetable oils. However the even carbon number series is present in

325

important concentrations. This fact must be pointed out as distinctive of Sacha inchi oils

326

when compared with other vegetable oils37. Data on hydrocarbons and their relative

327

concentrations in edible oils have been scarcely studied albeit their usefulness. In this sense

328

it has been proposed as a good parameter for beeswaxes authentication, allowing

329

discriminate in the adulterations with paraffin33 and also to distinguish among olive oil

330

varieties38.

331

To summarize we will conclude as follows: The concentration ranges of minor and major

332

components of cold pressing Sacha inchi P. huayllabambana oils studied can serve as a

333

valuable guide in forthcoming enlargement of the Regulations. Changes and extension in the

334

normative are necessary as it would ensure better control of these oils avoiding the presence

335

of adulteration with other seeds of lower prices. It also serves to encompass analytical

336

parameters of other Sacha inchi species since there are almost no studies concerning to this

337

respect. It should be pointed out that some objective analytical determinations can help to

338

characterize Sacha inchi oils both P. volubilis and P. huayllabambana. Such is the case of

339

their characteristic sterol profile with its particular campesterol/stigmasterol ratio.

340

Determinations of FAME and TG compositions indicate that P. huayallabambana oils are

341

more unsaturated than P. volubilis, which affects to the presence of TG molecular species

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342

with lower ECN for the first of the species. Results demonstrate that the studied species

343

have a seasonal stable composition, not influenced by the harvest year significantly.

344

Finally, their high susceptibility to oxidative and hydrolytic alterations is other common

345

characteristic of these valuable oils. At this point it is convenient to emphasize the need to

346

protect the product from the time of seed harvest, passing by the oil extraction up to their

347

marketing, in order to guarantee their unique and valuable properties.

348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 16

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ABBREVIATIONS USED

368

DG, diglycerides; ECN, equivalent carbon number; FA, free acidity; FAME, fatty acid

369

methyl esters; FFA, free fatty acids; FID, flame ionization detector; GC, gas

370

chromatography; HC, hydrocarbons; RP-HPLC, reverse phase-high performance liquid

371

chromatography; HPSEC high performance size exclusion chromatography; ID, internal

372

diameter; IP, induction periods; L, linoleic acid; Ln, linolenic acid; MG, monoglycerides;

373

oxTG, oxidized triglycerides; PC, glyceridic polar compounds; PV, peroxide value; RI,

374

refractive index; Rt, retention time; Si-SPE, silica-solid phase extraction; TG, triglycerides;

375

THF, tetrahydrofurane; TLC, thin layer chromatography; UC, unsaponifiable compounds.

376

377

ACKNOWLEDGMENT

378

The authors would like to thank Ms Diana Gómez Castillo for her assistance in the

379

laboratory.

380

381

382

383

384

385

386

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REFERENCES

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(10) Norma Tecnica Peruana 151.400. Requisitos aceite sacha inchi. In N. T. Peruana (Ed.) (Peru) 2009. (11) Bussmann, R.; Téllez, C.; Glenn, A. Plukentia huayllambambana sp.nov (Euphorbiaceae) from upper Amazon of Perú. Nord. J. Bot. 2009, 27, 313-315.

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propuesta de una nueva especie. Rev. Peru. Biol. 2010, 17, 325-330.

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grasos y de aminoácidos de semillas y tortas de 2 especies de sacha inchi (plukenetia

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volubilis y plukenetia huayllabambana). Rev. Soc. Quim. Perú. 2013, 79, 29-36.

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the characteristics of olive oil and olive-residue oil and on the relevant methods of

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analysis. 2011, pp. 1-14.

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triacylglycerols in olive oils by high performance liquid chromatography. Grasas Aceites.

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2003, 54, 175-179.

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glycerol in fats by means of gel permeation chromatography [C-VI 5b(02)]. Eur. J. Lipid

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oils and fats by HPLC. In Standard methods for the analysis of oils, fats and derivatives.

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Supercritical CO2 extraction of omega-3 rich oil from Sacha inchi (Plukenetia volubilis

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L.) seeds. J. Supercritical Fluids. 2009, 49, 323-329.

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(29) Liua, Q.; Xua, Y.; Zhanga, P.; Naa, Z.; Tanga, T.; Shia, Y. Chemical composition and

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oxidative evolution of Sacha Inchi (Pluketia volubilis L.) oil from Xishuangbanna

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(China). Grasas Aceites. 2014, 65: e012. doi: http://dx.doi.org/10.3989/gya.075713.

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poppy seed and seed oils. Biores. Technol. 2008, 99, 6354-6359.

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(31) Cisneros, F.H.; Paredes, D.; Arana, A.; Cisneros-Zevallos, L. Chemical composition,

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oxidative stability and antioxidant capacity of oil extrated from roasted seeds of sacha

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inchi (plukenetia volubilis L). J. Agric. Food Chem. 2014, 62, 5191-5197.

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(32) Norma del CODEX (STAN 210-1999) para aceites vegetales especificados. 1999, pp. 114).

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(33) Jiménez, J.; Bernal, M.; del Nozal, L.; Toribio, J.; Bernal, J. Detection of beeswax

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adulterations using concentration guide-values. Eur. J. Lipid Sci. Technol. 2007, 109,

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(34) Santos, M. F. G.; Marmesat, S.; Brito, E. S.; Alves, R. E.; Dobarganes, M. C.; Santos, M.

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Major components in oils obtained from Amazonian palm fruits. Grasas Aceites. 2013,

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Saldaña, L.; Cardozo Filho, I. Phase Equilibrium Measurements of Sacha Inchi Oil

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(Plukenetia volubilis) and CO2 at High Pressures. J. Am. Oil Chem. Soc. 2011, 88, 1263-

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(37) Moreda, W.; Pérez-Camino, M. C.; Cert, A. Neutral lipids: Unsaponifiable matter. In

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(38) Guinda, A.; Lanzón A.; Albi, T. Differences in Hydrocarbons of virgin olive oils obtained from several olive varieties. J. Agric. Food Chem. 1996, 44, 1723–1726.

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Notes

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The Spanish AECID funded the project AP/036672/11 and the Scientific Institute of

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Investigation of Lima University (IDIC)- Perú, funded part of this work.

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The authors declare no competing financial interest.

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Table 1. Physical-chemical Parameters Of The Sacha Inchi (P. Huayllabambana L.) Oils. Data Are The Average Of 14 Samples And Are Expressed As The Mean ± SD.

Harvest 1 M (n=14) (%) Acidity value Peroxide value (meq O2·kg-1) K270 Stability (h) Density (25 ºC) (g·cm-3) Refractive Index Viscosity (20 ºC) (mm2·s-1) a,b

1.8a ± 0.1 8.4a ± 0.2 0.14b± 0.01 2.8a ± 0.1 0.927a± 0.001 1.480a ± 0.0001 42.8a ± 0.9

Harvest 2 Range

0.5 – 4.7 1.5 – 19.1 0.10 – 0.28 2.0 - 3.4 0.926 – 0.930 1.480 – 1.481 41.2 – 44.0

M (n=14) (%) 1.6b± 0.1 3.3b± 0.2 0.19a± 0.01 3.0a ± 1.2 0.925a ± 0.002 1.482a ± 0.0004 41.1a ± 2.2

Range 0.5-3.7 2.1-5.6 0.14-0.23 1.8 ± 5.5 0.920 – 0.930 1.481 – 1.482 38.9 – 43.8

Different superscript letters represent significant differences

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Table 2. Major Compounds Of Sacha Inchi (P. Huayllabambana L.) Oils: Triglyceride, Polar Glyceridic Compounds And Fatty Acid Methyl Esters. Data Are The Average Of 14 Samples And Are Expressed As The Mean ± SD.

Samples Harvest 1 M (n=14) Range

Harvest 2 M (n=14)

Range

TRIGLYCERIDES (wt% on total) ECN 36 (LnLnLn) ECN 38 (LnLnL) ECN 40 (LnLL+LnLnP+LnLnO)

20.0±1.18 26.3 ± 0.7 20.6±0.79

17.7-22.5 25.3-27.5 19.2-21.8

20.0±1.84 26.0±1.21 20.9±0.86

18.0-23.0 22.6-27.7 19.4-23.1

ECN 42 (LLL+OOLn+LLP+POLn) ECN 44 (LLO+OOLn+LLP+POLn) ECN 46 (OOL+LnPP+PLO)

20.7±1.20 7.0±0.70 2.5±0.48

19.4-22.8 6.0-8.3 1.8-3.4

22.5±1.10 7.6±0.94 2.4±0.79

19.0-24.0 6.0-10.5 1.39-5.0

ECN 48 (OOO+POO) ECN 50 (SSO)

1.3±0.75 0.7±0.7

0.5-2.7 0.0-2.5

0.5±0.28