Bioactive compounds in unsaponifiable fraction of oils from unconventional sources

$Page 1: Bioactive compounds in unsaponifiable fraction of oils from unconventional sources$
Research Article

Bioactive compounds in unsaponifiable fraction of oils fromunconventional sources

Sylwester Czaplicki1, Dorota Ogrodowska1, Dorota Derewiaka2, Małgorzata Tanska1 and

Ryszard Zadernowski1

1 Chair of Food Plant Chemistry and Processing, Faculty of Food Science, University of Warmia and Mazury,

Olsztyn, Poland2 Division of Food Quality Evaluation, Department of Biotechnology, Microbiology and Food Evaluation,

Faculty of Food Sciences, Warsaw University of Life Sciences, Warsaw, Poland

The aim of the research was to characterize bioactive components of unsaponifiable fraction of selected

unconventional oils. Nine oils were analyzed as far as the content of tocopherols, squalene, phenolic

compounds, and sterols were concerned. Tocopherols and squalene were analyzed by HPLC coupled

with diode array detector and fluorescent detector (HPLC-DAD-FLD). The content of sterols in oils was

determined by GC coupled with MS (GC-MS). The total amount of phenolic compounds in oils was

determined by the colorimetric methods using Folin–Ciocalteau phenol reagent. The examined oils were

characterized by differentiated amount of particular forms of tocopherols. The oil obtained from the

seeds of amaranth was the richest source of squalene (over 52 mg/g oil). The presence of 22 different

compounds of sterols were identified, whereas b-sitosterol was found in the largest amount. Total

amount of sterols in the oils ranged from 90 (walnut) to 850 mg/100 g (evening primrose). Significant

differentiation of total amount of phenolic compounds was observed in the examined oils. Evening

primrose oil showed the highest amount of phenolic compounds (679 mg/kg). The presented results

prove that plant oils obtained from nonconventional sources are a potential source of bioactive

compounds.

Keywords: Phytosterols / Squalene / Tocopherols / Unconventional oils / Unsaponifiable fraction

Received: July 14, 2010 / Revised: June 14, 2011 / Accepted: September 28, 2011

DOI: 10.1002/ejlt.201000410

1 Introduction

Constantly growing risk of so-called civilization diseases (car-

diovascular diseases, cancers, allergies) of general population

caused development pharmaceuticals and food supplements

global market development. There is currently an increased

demand for bioactive compounds of plant origin which are

able to affect the human health condition positively. Bioactive

components naturally occurring in vegetable unconventional

oils can reduce the risk of several diseases.

Vegetable unconventional oils contain various bioactive

components. In the composition of the unsaponifiable frac-

tion of oils several substances like phenolic acids, squalene,

tocopherols, sterols were found [1, 2]. Several preparations

composed of vegetable unconventional oils enriched with

additional substances like vitamins or compounds showing

an antioxidant activity (used to extend its shelf life) are

commonly available on the retail market.

Cold pressed virgin olive oil is considered as a high quality

product and health beneficial product. The reputation of

olive oil, undoubtedly contributes in other cold-pressed or

virgin oils growth of interest [3]. Vegetable oils are obtained

from various parts of plants, e.g., seeds, fruits, stones, or

sprouts [4]. They are produced not only from plants

traditionally regarded as oily plants, such as rapeseed, sun-

flowers, or olives, but also from many other plants, e.g., sea

buckthorn, evening primrose, walnut, or amaranth.

The vast majority of produced oils are subjected to rafi-

nation process. The production of refined oil includes the

following stages: preparation of raw material, obtaining the

crude oil (where mucus is removed), deacidification, deodor-

ization, and bleaching. These processes cause loss of several

oil components, such as phospholipids, MAGs, DAGs, free

fatty acids, dyes, aromatic compounds or metal, and sulfur

Correspondence: Dr. Sylwester Czaplicki, Chair of Food Plant Chemistry

and Processing, Faculty of Food Science, University of Warmia and

Mazury, Plac Cieszynski 1, 10-718 Olsztyn, Poland

E-mail: [email protected]

Fax: þ48-89-523-34-66

1456 Eur. J. Lipid Sci. Technol. 2011, 113, 1456–1464

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

compounds. Nutrients and antioxidants such as carotenoids

and tocopherols are also lost [5].

Due to importance of vegetable unconventional oils as an

importance source of bioactive compounds used in prophy-

lactics of present day diseases, an attempt was made to

characterize the unsaponifiable fractions of oils obtained from

uncommon oily raw plant material.

2 Materials and methods

2.1 Materials

Various cold pressed vegetable oils were investigated in this

study. Seeds of pumpkin, amaranth, sesame, linseed, camel-

ina, evening primrose, borage, poppy, and walnut came from

‘‘Szarłat’’ company (Łomza, Poland). Oils were obtained by

pressing of the raw material on a IBG Monforts & Reiners,

KometCA59G (Germany) laboratory expeller equipped with

a 4 mm diameter nozzle. Obtained oils were purified by

centrifugation on a Janetzki MLW K24D (Germany) centri-

fuge operated at 8000g.

2.2 Analysis of unsaponifiable fraction

Study included determination of the contents of unsaponifi-

able fraction, tocopherols, squalene, phenolic compounds,

and sterols. The content of unsaponifiable substances was

determined according to PN-EN ISO 3596:2002 [6].

2.3 Analysis of tocopherols

The analysis of tocopherols was carried out by HPLC,

according to the method described by Peterson and

Qureshi [7]. Briefly, 0.1 g of oil (�0.001 g) was diluted in

n-hexane in a 10 mL measuring flask. After subsequent

centrifugation (10 min at 25 000g) sample was transferred

to a chromatographic vial and 20 mL was injected into the

chromatographic system. The analysis was carried out using a

1200 series liquid chromatograph manufactured by Agilent

Technologies (Palo Alto, CA, USA), equipped with a fluor-

escent detector from the same manufacturer. Separations

were performed on a Merck LiChrospher Si60 column,

250 mm � 4 mm, 5 mm. As the mobile phase an 0.7%

iso-propanol solution in n-hexane at 0.7 mL/min flow rate

was used. Fluorescence detector was set at 296 nm of exci-

tation and 330 nm of emission. Peaks were identified on the

basis of retention times determined for the of a-, b-, g-, and

d-tocopherol standards (Calbiochem, UK) separately, and

their content was calculated using external calibration curves.

2.4 Analysis of squalene

The squalene content was determined withHPLC, according

to method used for determination of TAGs described by

Czaplicki et al. [8] with modifications. Briefly, 10 mg of

oil (�0.0001 g) was transferred into a 10 mL volumetric

flask, and filled up with n-hexane. Twenty microliter of

diluted oil sample was injected into column.

Analysis were conducted using the same chromatographic

system with DAD. Chromatographic separations were

conducted on a Merck LiChrospher RP-18 column,

250 mm � 4.6 mm, 5 mm, at 308C. A gradient system

was used for elution: A, acetonitrile; B, isopropyl alcohol;

C, hexane. The elution profile was 0–12 min, 20–22% B and

10–12% C in A (linear gradient), 12–15 min 22–25% B

and 12–25% C in A (linear gradient), 15–20 min, 25% B

and 25% C in A (isocratic), 20–25 min, 25–20% B and 25–

10%C in A (linear gradient). The mobile phase flow rate was

1 mL/min. A analytical wavelength was set at 218 nm.

External calibration curve was used for quantitative analysis.

The squalene standard was supplied by Sigma–Aldrich.

2.5 Analysis of phenolic compounds

The content of total phenolics was determined spectrophoto-

metrically, with Folin–Ciocalteau phenol reagent. Method

involved extraction of phenolic compounds with 80%

methanol v/v. The extract was concentrated with rotary evap-

orator type R210 (Buchi Labortechnik AG, Postfach,

Switzerland) to 10 mL volume. Then 0.25 mL of methanolic

extract was transferred into reaction tube and evaporated to

dryness under a gentle stream of nitrogen to remove remain-

ing methanol. 0.25 mL Folin–Ciocalteau reagent solution

was added to the flask, together with 0.5 mL of 14% (m/v)

sodium carbonate water solution. Flask was filled up with

distilled water to final volume of 5 mL. After 30 min,

absorbance at 720 nm was measured using SP6-500UV

PYE UNICAM spectrophotometer (United Kingdom).

Measurements were carried out against a blanc sample.

The contents of phenolic compounds was calculated on

the basis of D-catechin calibration curve [9].

2.6 Analysis of sterols

The content of sterols in oils was determined by GC coupled

with MS (GC-MS) according to method described by

Vlahakis and Hazebroek [10]. The sample was saponificated

by adding 0.5 mL 2 MNaOH solution in methanol at ambi-

ent temperature for 2 h. Unsaponifiables were extracted with

diethyl ether, which was subsequently evaporated under

nitrogen conditions. The dry residues were re-dissolved in

1.5 mL of n-hexane and 0.2 mL of 5a-cholestane internal

standard solution was added (0.4 mg/g). The extract was

transferred into a vial and evaporated under a nitrogen

stream. The residues were re-dissolved in 100 mL of pyridine

and 100mL N,O-bis (trimethylsilyl) trifluoroacetamide

(BSTFA) with 1% trimethylchlorosilan (TMCS) and left

in the dark for 24 h to complete derivatization. Then

1 mL of hexane was then added and 1 mL of obtained mix-

ture was analyzed by GC-MS.

Eur. J. Lipid Sci. Technol. 2011, 113, 1456–1464 Unsaponifiable bioactive compounds in selected oils 1457


A DB-5ms capillary column was used for separations of

phytosterols with helium as a carrier gas at a flow rate of

0.9 mL/min. The injector temperature was 2308C, and the

column temperature was programmed as follows: 508Cfor 2 min, a subsequent increase to 2308C at the rate of

158C/min, to 3108C at the rate of 38C/min 10 min hold.

The interface temperature of GC-MS was 2408C. The

temperature of the ion source was 2208C and the electron

energy was 70 eV. The total ion current (TIC) mode was

used for quantification (100–600 m/z range). The quantifi-

cations were carried out using internal standard method.

2.7 Quality analysis of oil

Evaluation of freshly pressed oil quality was done on the basis

of acid value, peroxide value, and anisidine value (determined

according to PN-ISO 660:1998 [11], PN-EN ISO

3960:2005 [12], PN-EN ISO-6885:2008 [13], respectively).

3 Results and discussion

3.1 Oil quality

Undoubtedly, the content and the composition of the unsa-

ponifiable fraction have a significant effect on the quality and

the stability of the oil. While assessing the initial oils

parameters, it was found that directly after the pressing, they

were characterized by a varied quality (Table 1). Themajority

of them meet the requirements of standards specified for

cold-pressed oils. Pursuant to those requirements, the acid

value of oil should not be higher than 4 mg KOH/g and

peroxide value should not exceed 15 mEq O2/kg [14].

Acid value proves the presence of free fatty acids in oil,

which results from hydrolysis of triglycerides and/or incom-

plete synthesis of lipids in seeds [15]. The research results

showed a broad range of acid value determined for inves-

tigated oil samples, from 0.80 mg KOH/g for walnut oil to

4.82 mg KOH/g for poppy seed oil. Evening primrose oil

proved to be the most oxidized oil of the oils examined. This

oil was characterized by a high content of secondary products

of oxidation, and its anisidine value was 8.25 (Table 1). This

can be explained by the fact that polyunsaturated acids are the

dominating in the composition of fatty acids of evening

primrose oil, accounting for 80% of total fatty acid contents

[16]. These acids, although beneficial from a nutritional point

of view, could cause the reduction of this oil stability [17].

The rate of linoleic acid oxidation is 10–40 times higher than

that of oleinic acid, while the linolenic acid oxidation rate of is

2–4 times higher than that of linoleic acid [15].

3.2 Unsaponificable fraction

Oils are generally composed of triacylglycerides and about

1% of so-called unsaponifiable substance. Unsaponificable

fraction contains different substances like, vitamins (A, D),

preservatives (benzoic acid, sorbic acid), antioxidants (toco-

pherols, gallates), or pigments (carotene) used deliberately as

additives [18].

Among examined oil samples, poppy seed oil had the

lowest content of unsaponifiable fraction (0.48%).

Linseed, camelina, and pumpkin seed oils were characterized

by a similar content of unsaponifiable fraction, which

amounted to 0.78, 0.74, and 0.70%, respectively (Table 2).

Analyzing seven cold-pressed linseed oils, Choo et al. [19]

reported contents of unsaponifiable fraction ranging from

0.39 to 0.71%. Esuoso et al. [20] found 0.03% of unsapo-

nifiable fraction contribution in their research, while

El-Adawy andTaha [21] found 0.85% contribution in pump-

kin seed oil. In our results we obtain 0.70% of this fraction.

The divergence in results could be explained by, e.g., differ-

ences between varieties of raw material, cultivation areas, or

research material storage conditions. A similar content of

unsaponifiable fraction was found in evening primrose oil

(1.30%) and sesame oil (1.37%). Mohamed and Awatif

[22] found that the content of the examined fraction in

sesame oil was similar and ranged from 1.1 to 1.3%. The

Table 1. Quality of freshly pressed oils

Acid value

(mg KOH/g oil)

Peroxide value

(mEq O2/kg oil)

Anisidine

value

Linseed 1.38 � 0.01 0.90 � 0.00 2.02 � 0.24

Poppy seed 4.82 � 0.57 4.37 � 0.16 3.97 � 0.93

Camelina 2.22 � 0.02 2.64 � 0.04 0.70 � 0.06

Pumpkin 1.95 � 0.01 2.04 � 0.08 3.00 � 0.72

Amaranth 1.56 � 0.02 3.04 � 0.23 0.87 � 0.05

Evening primrose 1.56 � 0.01 3.86 � 0.14 8.25 � 0.59

Borage 3.20 � 0.25 1.50 � 0.14 4.72 � 0.85

Walnut 0.80 � 0.07 4.28 � 0.05 0.73 � 0.05

Sesame 1.22 � 0.19 2.34 � 0.04 2.60 � 0.23

Values are mean � SD (n ¼ 3).

Table 2. Unsaponifiable compounds of oils

Sample

Unsaponifiable

matter (%)

Phenolic compounds

(mg/kg oil)

Squalene

(mg/g oil)

Linseed 0.78 � 0.05 294 � 3.81 nd

Poppy seed 0.48 � 0.02 190 � 18.0 nd

Camelina 0.74 � 0.07 400 � 14.6 traces

Pumpkin 0.70 � 0.01 192 � 23.5 5.23 � 0.26

Amaranth 7.12 � 0.01 335 � 23.9 52.2 � 0.3

Evening primrose 1.30 � 0.29 679 � 16.4 traces

Borage 0.96 � 0.13 581 � 14.3 0.22 � 0.01

Walnut 3.73 � 0.40 554 � 28.1 28.3 � 0.5

Sesame 1.37 � 0.10 283 � 6.12 nd


1458 S. Czaplicki et al. Eur. J. Lipid Sci. Technol. 2011, 113, 1456–1464


highest contribution of unsaponifiable fraction among

examined oils was found in walnut oil (3.73%) and amaranth

oil (7.12%). According to Gamel et al. [23], the contribution

of unsaponifiable fraction in amaranth oil amounts to about

8.8%. It is a high value in respect to other vegetable oils, e.g.,

contribution of olive oil unsaponifiable fraction, varies

between 0.5 and 1.5% [24]. Presence of squalene can explain

high contribution of the unsaponifiable fraction in amaranth

oil. Amount of squalene in amaranth oil is several times

higher in comparison to other analyzed oil samples: walnuts

– 28.3 mg/g of oil (Fig. 1), amaranth – 52.2 mg/g of oil

determined concentration. Berganza et al. [25] reported

squalene content in amaranth oil at the level of 3.20–

5.80%, depending on the plant speciment and area of culti-

vation. This compound occurs also in high concentrations in

the sharks liver and kidneys. However, because of the concern

arising on sea animal protection, new sources of squalene

are being searched. Amaranth seed oil (6–8%) and olive oil

(0.3–0.7%), are potentially valuable sources of squalene [26].

3.3 Phenolic compounds

Phenolic compounds are very numerous and important

group of compounds showing antioxidizing properties.

Because their common occurrence in plants, phenolic com-

pounds are important food components [27]. Evening prim-

rose, borage, and walnut were characterized by a similar

content of phenolic compounds, with concentrations at

679, 581, and 554 mg/kg of oil, respectively. The content

of phenolic compounds in camelina oil was 400.39 mg/kg of

oil. Abramovic et al. [28] specified the content of phenolic

compounds in Slovenian camelina oil. The total amount of

the examined compounds measured in that oil was much

lower (128 mg/kg). Amaranth oil (Amaranthus cruentus) was

characterized by the content of phenolic compounds at the

level of 335 mg/kg of oil. By way of example, Amaranthus

hypochondriacus and Amaranthus hybridus seeds are charac-

terized by the presence of polyphenols, in the amounts of 41.5

and 40.5 mg/100 g d.m. [29]. Linseed and sesame oils were

characterized by a similar content of phenolic compounds, at

concentrations of 294 and 283 mg/kg of oil, respectively. The

lowest content of phenolic compounds was found in pumpkin

seed oil (192 mg/kg of oil) and poppy seed oil (190 mg/kg of

oil).

3.4 Tocopherols

Tocopherols (vitamin E) are compounds, which in various

amounts occur in all vegetable oils. Vitamin E is represented

by a family of chemical compounds linked to each other in

terms of structure. Eight forms of this vitamin can be found

under natural conditions, namely: a-, b-, g-, and d-tocopher-

ols, as well as a-, b-, g-, and d-tocotrienols. Vegetable oils

prove the richest source of vitamin E for the everyday diet.

Palm oil contains 600–1000 mg/kg of vitamin E. It is charac-

terized by particularly high tocotrienol content in comparison

to common vegetable oils [30]. It was found that the content

of tocopherols in food is inversely proportional to the mortality

rate related to cardiovascular diseases. Additionally, tocopher-

ols, due to their ability to scavenge free radicals, play a

Figure 1. Chromatogram of phytosterols of walnut oil.



prophylactic role in Alzheimer’s disease and cancer preven-

tion [31]. The oils analyzed in this study were characterized

by a significant amount of tocopherols (Table 3). In this

group, only g-tocopherol was found in sesame seed oil

(837 mg/kg of oil). Namiki et al. [32] also confirmed the

highest contribution of this isomer in sesame seeds. a- and

b-tocopherol were not identified, but trace amounts of d-

tocopherol was found. For example, Mohamed and Awatif

[22] while analyzing tocopherols in Egyptian sesame seeds,

obtained values of 40.4 mg/100 g for white unroasted ses-

ame, 33.0 mg/100 g for white roasted sesame, 54.0 mg/

100 g for brown unroasted sesame, and 39.0 mg/100 g of

for brown roasted sesame. This data demonstrate that cli-

matic conditions, species, and type of the technological proc-

ess used can have a strong effect on the tocopherol content.

According to Reblova [33], significant differences in the

decrease of a- and d-tocopherols can be observed when oils

are heated oils at temperatures above 1008C. In addition, a

high content of tocopherols was reported for borage oil

(1603 mg/kg of oil). Just as in case of sesame seed oil, borage

oil did not contain a- or b-tocopherol isomers, but was also

characterized by a high content of d-tocopherol (1432 mg/kg

of oil), contributing over 89% of determined tocopherols

contents. Similar results for borage oil were presented by

Shahidi [1], who determined the content of d-tocopherols

at a level of 1350 mg/kg. Pumpkin, camelina, evening prim-

rose, walnut, amaranth, and linseed oils were characterized

by a similar total contents of tocopherols, amounting to: 848,

674, 662, 634, and 563 mg/kg, respectively. While analyzing

the Slovenian linseed oil, Abramovic et al. [28] found

approximately tenfold lower contents of tocopherols

(751 mg/kg) in comparison to this research, with g-toco-

pherol as the most abundant isomer (710 mg/kg). In the

linseed oil analyzed in this study, g-tocopherol contributed

to 89% of the total determined tocopherol contents. While b-

tocopherols was not detected, in evening primrose oil. This

results are in concordance with data reported by Shahidi [1].

There is a little literature data concerning the tocopherol

content in amaranth, although Leon-Camacho et al. [34]

identified three forms of tocopherols in Amaranthus cruentus

oil at 248 ppm of a-tocopherol, 546 ppm of b-tocopherol,

and 8 ppm of d-tocopherol. On the other hand, those

researchers reported no presence of g-tocopherol is studied

samples. The contribution of g-tocopherol was 16% in the

sum of examined isomers in this study. Bozan and Temelli

[35] also did not find detectable amounts of this isomer in

linseed oil, while the total content of tocopherols was

79.4 mg/100 g. The same researchers analyzed the oil

derived from poppy seeds, obtaining a total amount of toco-

pherols at the level of 309 mg/kg. In the current study, poppy

seed oil was characterized by the lowest tocopherol content

(293 mg/kg) out of all analyzed samples. g-Tocopherol was

the main isomer determined in poppy seed oil (201 mg/kg),

while neither b- and d-tocopherols were found.

3.5 Sterols

Contents of sterols determined in analyzed samples are

shown in Tables 4 and 5. The lowest total sterol content

was found in poppy seed oil (139 mg/100 g), while the high-

est content of sterols was determined for amaranth oil

(1991 mg/100 g). Desmethyl, 4-monomethyl, and 4,4-

dimethyl sterol classes were determined. Analyzed oil

samples contained desmethyl sterols commonly found in

vegetable oils. Mainly b-sitosterol, campesterol, stigmasterol,

brassicasterol, as well as negligible amounts of cholesterol

were found. Additionally several substances like 4-monodi-

methyl sterols (gramisterol, citrostadienol) and 4,4-dimethyl

sterols, i.e., cycloartenol, a-amyrin, b-amyrin, erythrodiol,

uvaol, lanosterol, 28-methylobtusifoliol, 24-methylenecy-

cloartanol were determined. While applying another

classification of sterols, the oils contained D5-sterols (D5-

avenasterol, D5,24-stigmastadienol), D7-sterols (D7-avenas-

terol, D7,22,25-stigmastatrienol), and stanols (sitostanol).

The oils characterized by a low content of sterols included

camelina, evening primrose, and poppy seed oil.

Dominating sterols found in amaranth oil included:

a-spinasterol (30–34%) and D7-sterols i.e., D7-ergosterol,

Table 3. Tocopherol content in oils (mg/kg)

Sample a-Tocopherol b-Tocopherol g-Tocopherol d-Tocopherol

Linseed 64.4 � 5.21 nd 495 � 40.3 3.46 � 0.25

Poppy seed 91.7 � 8.18 nd 201 � 18.2 nd

Camelina 69.5 � 4.98 nd 599 � 39.4 5.15 � 0.39

Pumpkin 94.3 � 7.52 58.5 � 3.45 694 � 61.1 1.51 � 0.09

Amaranth 234 � 20.8 191 � 18.3 102 � 8.83 90.9 � 8.38

Evening primrose 222 � 18.3 nd 433 � 38.2 7.72 � 0.69

Borage nd nd 171 � 15.4 1432 � 119

Walnut 167 � 16.8 107 � 9.25 306 � 22.2 54.2 � 5.30

Sesame nd nd 837 � 26.0 nd




D7-stigmasterol, and D7-awenasterol; other phytosterols

were: 24-methylenecholesterol, D5-avenasterol, citrostadie-

nol, cycloartenol, camepsterol, sitostanol, and stigmasterol.

Comparing the results of previous research concerning

identification of phytosterols in amaranth oil, it is clear that

qualitative analysis of sterols can be questionable. For

instance, Leon-Camacho et al. [34] found that the main

phytosterol occurring in amaranth oil is clerosterol, while

they did not report any presence of a-spinasterol.

Chernenko et al. [36] found that amaranth oil, analyzed

by MS, contained a significant amount of a-spinasterol

and b-sitosterol, stigmasterol, campesterol, and cholesterol,

while the presence of D7-sterol was not detected [36].

However, Marcone et al. [37] reported that amaranth con-

tained only b-sitosterol, stigmasterol, and campesterol.

Camelina oil was characterized by a high content of

b-sitosterol (58%) and campesterol (23%) and significantly

lower content of D5-avenasterol, cholesterol, and brassicas-

terol (Table 4). Schwartz et al. [38] determined the content of

sterols in the Finnish camelina oil. The total sterol content

observed in the oil was higher – 500 mg/100 g, but percent-

age of individual sterols was identical to the composition of

camelina oil investigated in current study. A similar phytos-

terol content in camelina oil (360 mg/100 g of oil) was

observed by Shukla et al. [39]. Evening primrose oil was

characterized by the presence of b-sitosterol, campesterol,

and D5-avenasterol. The total content of sterols in evening

primrose oil was 856.6 mg/100 g. Primrose evening oil is

similar to literature sources, which report that the content

of b-sitosterol amounts to 787–814 mg/100 g, campesterol –

70, and D5-avenasterol – 122–123 mg/100 g [40]. The sterol

fraction of poppy seed oil contained over 70% of b-sitosterol,

15 and 13% of campesterol and D5-avenasterol, respectively.

The poppy seed oil sterols were determined. The results

were compared to obtained by Ryan et al. [31] and the main

difference was absence of D5-avenasterol, but content of b-

sitosterol and campesterol was 145.8 and 24.5 mg/100 g and

it was similar to our study.

Table 4. Phytosterol content in oils

Linseed Poppy Camelina Pumpkin seed Amaranth

Desmethylsterols (mg/100 g of oil)

24-Methylcholesta- nd nd nd nd nd

5,23-Dienol

24-Methylenecholesterol nd nd nd nd 76.7 � 6.8

Cholesterol nd nd 16.2 � 2.3 nd nd

Brassicasterol traces nd 9.4 � 0.9 nd nd

Campesterol 36.3 � 2.4 21.1 � 1.2 82.1 � 4.7 nd 34.3 � 4.2

Stigmasterol 8.8 � 0.9 traces traces nd 22.2 � 3.6

b-Sitosterol 103 � 1.8 100 � 3.3 221 � 8 nd 31.8 � 2.0

D5-Avenasterol 24.8 � 0.1 17.6 � 0.2 18.6 � 0.7 nd 105 � 12.2

D7-Avenasterol nd nd nd 32.6 � 3.6 356 � 34.2

D7-Stigmasterol nd nd nd nd 458 � 33.1

a-Spinasterol nd nd nd nd 787 � 55.3

a-Spinasterol þ b-sitosterol nd nd nd 96 � 7.4 nd

D5,24-Stigmastadienol nd nd nd 42.8 � 5.4 nd

D7,22,25-Stigmastatrienol nd nd nd 7.6 � 0 nd

4-Monomethylsterols

Gramisterol nd nd nd nd nd

Citrostadienol nd nd nd nd 70.8 � 9.3

4,4-Dimethylsterols

a-Amyrin nd nd nd nd nd

b-Amyrin nd nd nd nd nd

Cycloartenol 87.5 � 1.6 nd nd nd 49.0 � 6.7

24-Methylenecycloartanol 18.6 � 0.9 nd nd nd nd

28-Methylobtusifoliol nd nd nd nd nd

Erythrodiol þ citrostadienol nd nd nd nd nd

Uvaol nd nd nd nd nd

Other compound

Sesamin nd nd nd nd nd

Oleanol aldehyde nd nd nd nd nd

S 277 W 1.1 139 W 2.4 349 W 8 179 W 16.5 2616 W 188




The total sterol content in pumpkin seed oil was 179 mg/

100 g of oil and most characteristic sterols for this oil were a-

spinasterol, D5,24-stigmastadienol, and D7,22,25-stigmasta-

trienol (Table 4). The results of analyses conducted by

Mandl et al. [41] confirm the composition of the sterol

fraction of pumpkin seed oil obtained in our experiment.

On the other hand, Nyam et al. [42] reported that the total

content of sterols in pumpkin seed oil was 274 mg/100 g, of

which over 75% was b-sitosterol, while the remainder was

made up of campesterol, stigmasterol, and ergosterol. The

discrepancies could have resulted from difficulties in qual-

itative analysis of sterols due to appliance of an FID detector

[42].

Walnut oil contained significant amounts of cycloartenol

(2%) and citrostadienol (6%). The total content of sterols in

sesame oil was 354 mg/100 g, and in walnut oil – 485 mg/

100 g. A typical profile of sesame oil was described by

Schwartz et al. [38], and Kamal-Eldin et al. [43]. Five

varieties of the Turkish walnut were analyzed to reveal the

sterol fraction composition: total sterol content ranged from

119 to 153 mg/100 g and was significantly lower than in the

walnut oil examined in the current study. The content of the

sterol fraction determined in given oils were different in

comparison to the results in the current study. Presence of

cholesterol, clerosterol, stigmasterol, D7-avenasterol, and

D7-stigmasterol were also observed [44]. Sterol fraction of

borage, beside typical desmethyl sterols also contained 24-

methylcholesta-5,23-dienol and 4-mono and 4,4-dimethyl

sterols, i.e., gramisterol, cycloarenol and citrostadienol.

Similar results were presented by Wretensjo and Karlberg

[45], who obtained a much richer profile of sterols found in

the borage oil by using thin-layer chromatography, e.g., the

presence of isofucosterol, obtusifoliol, and a-amyrin was

found. Sterol content in linseed oil amounted to 277 mg/

100 g. The presence of desmethyl sterols (b-sitosterol, cam-

pesterol, D5-avenasterol, and stigmasterol, trace amounts of

brassicasterol) and 4,4-dimethyl sterols (cycloartenol and 24-

metylenocycloartanol) was established for the sterol profile.

Schwartz et al. [38] reported that Finnish linseed oil con-

tained 689 mg sterols in 100 g. They also found the presence

Table 5. Phytosterol content in oils

Evening primrose Borage Walnut Sesame

Desmethylsterols (mg/100 g of oil)

24-Methylcholesta-5,23-dienol nd 21 � 1.1 nd nd

24-Methylenecholesterol nd nd nd nd

Cholesterol nd nd nd nd

Brassicasterol nd nd nd nd

Campesterol 55.3 � 2.9 43.8 � 2.1 83.2 � 2.7 62.6 � 1.2

Stigmasterol traces nd 196 � 6.3 15 � 0.2

b-Sitosterol 749 � 48.9 56.6 � 2.3 104 � 3.3 241 � 3.6

D5-Avenasterol 52.2 � 5.9 43.6 � 4.9 62 � 2 35.2 � 1.3

D7-Avenasterol nd nd nd Traces

D7-Stigmasterol nd nd nd nd

a-Spinasterol nd nd nd nd

a-Spinasterol þ b-sitosterol nd nd nd nd

D5,24-Stigmastadienol nd nd nd nd

D7,22,25-Stigmastatrienol nd nd nd nd

4-Monomethylsterols

Gramisterol nd 11.2 � 0.1 nd nd

Citrostadienol nd 3.2 � 0.4 29.7 � 0.9 Traces

4,4-Dimethylsterols

a-Amyrin nd nd nd nd

b-Amyrin nd nd nd nd

Cycloartenol nd 17.2 � 0.7 10 � 0.3 Traces

24-Methylenecycloartanol nd nd nd nd

28-Methylobtusifoliol nd nd nd nd

Erythrodiol þ citrostadienol nd nd nd nd

Uvaol nd nd nd nd

Other compound

Sesamin nd nd nd 162 � 2.7

Oleanol aldehyde nd nd nd nd

S 857 W 57.1 196 W 11.6 484 W 15.5 516 W 6

Values are mean � standard deviation (n ¼ 3).



of campesterol, sitostanol, stigmastadienol, gramisterol, a-

amyrin, D7-avenasterol, and citrostadienol.

4 Conclusions

In the human diet raw materials are important source of

ingredients that contribute to the proper functioning of the

organism. In search of bioactive fat-soluble components oils

obtained from unconventional oil raw materials were ana-

lyzed. Studies have shown that they are rich in the unsapo-

nifiable fraction, which was identified components such as

phenolic compounds, squalene, tocopherols, and sterols.

Particular attention was paid to oil from amaranth seeds,

evening primrose, borage oil, and the oil obtained from wal-

nut. Oil from the seeds of amaranth and walnut oil contained

the highest amounts of unsaponifiable fraction components,

more than 7 and 3.73%, respectively. These oils were also

characterized by the highest content of squalane, 52.2 mg/kg

of oil (amaranth oil) and 28.28 mg/kg (walnut oil). The

evening primrose oil was a rich source of sterols (857 mg/

100 g of oil), but the richest in the compounds of this group

proved to be amaranth oil (2616 mg/100 g oil). These oils

were also characterized by the highest content of a-toco-

pherol, but the richest in tocopherols was oil obtained from

borage seed.

The authors have declared no conflict of interest.

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Bioactive compounds in unsaponifiable fraction of oils from unconventional sources