Phytosterols in sea buckthorn (Hippophae rhamnoides L.) berries: Identification and effects of different origins and harvesting times

Article abstract of DOI:10.1021/jf010813m

Sterols in seeds, pulp/peel fractions, and whole berries of sea buckthorn (Hippophae rhamnoides L.) samples belonging to two major subspecies (sinensis and rhamnoides) from Finland and China were analyzed as TMS derivatives by gas chromatography-mass spectrometry after saponification of the oils. The total sterol contents in the seeds, the fresh pulp/peel, and the whole berries were 1200-1800, 240-400, and 340-520 mg/kg, respectively. The corresponding values in the extracted oils were 12-23, 10-29, and 13-33 g/kg. Sitosterol constituted 57-76 and 61-83%, respectively, of the seed and pulp/peel sterols. The sterol content and composition showed little variation between subspecies and collection sites. Different harvesting dates showed significant effects on the levels of some sterols both in the seeds and in the pulp/peel. The sterol profiles obtained are useful for characterizing sea buckthorn and detecting adulterations of the valuable oils. The information provided by the present investigation is also important for further chemical investigation of sea buckthorn sterols and industrial utilization of the berries as a raw material of functional foods.

Full text of DOI:10.1021/jf010813m

5620
J. Agric. Food Chem. 2001, 49, 5620−5629
P h ytoster ols in Sea Bu ck th or n (Hippoph a e1 r h a m n oid es L.) Ber r ies:
Id en tifica tion a n d Effects of Differ en t Or igin s a n d Ha r vestin g Tim es
Baoru Yang,*,† Riina M. Karlsson,† Pentti H. Oksman,‡ and Heikki P. Kallio†
Department of Biochemistry and Food Chemistry and Department of Chemistry, University of Turku,
FIN-20014 Turku, Finland
Sterols in seeds, pulp/peel fractions, and whole berries of sea buckthorn (Hippophae¨ rhamnoides
L.) samples belonging to two major subspecies (sinensis and rhamnoides) from Finland and China
were analyzed as TMS derivatives by gas chromatography-mass spectrometry after saponification
of the oils. The total sterol contents in the seeds, the fresh pulp/peel, and the whole berries were
1200-1800, 240-400, and 340-520 mg/kg, respectively. The corresponding values in the extracted
oils were 12-23, 10-29, and 13-33 g/kg. Sitosterol constituted 57-76 and 61-83%, respectively,
of the seed and pulp/peel sterols. The sterol content and composition showed little variation between
subspecies and collection sites. Different harvesting dates showed significant effects on the levels
of some sterols both in the seeds and in the pulp/peel. The sterol profiles obtained are useful for
characterizing sea buckthorn and detecting adulterations of the valuable oils. The information
provided by the present investigation is also important for further chemical investigation of sea
buckthorn sterols and industrial utilization of the berries as a raw material of functional foods.
Keyw or d s: Berries; sea; buckthorn; harvesting time; seeds; sterols; Hippophae¨ rhamnoides
INTRODUCTION
buckthorn have been extensively investigated, very little
is known about their sterols. In the few existing
publications, only single samples were analyzed, and
completely different sterol profiles have been reported
(13-19). A lack of original mass spectrometric data in
most of these publications makes an objective evaluation
of their identifications impossible. Thus, there is an
urgent need for further investigation of sterols in sea
buckthorn.
In the present study, we analyzed the seeds, soft
parts, and whole berries of two major subspecies of sea
buckthorn, sp. rhamnoides in Finland and sp. sinensis
in China, to obtain a general profile of sterols in sea
buckthorn berries on the basis of a larger number of
samples. This profile will serve as a basis for further
detailed chemical investigation and nutritional evalu-
ation of the berries. The aim of the present study was
also to compare the difference between the two subspe-
cies and to follow changes in the contents and composi-
tions of sterols in the seeds, soft parts, and whole berries
during the harvesting period.
Sea buckthorn (Hippophae¨ rhamnoides L.) is a hardy
bush with nutritious berries, naturally distributed
mainly in Asia and Europe and also cultivated in North
and South America (1, 2). During the past decade, the
species has attracted intense global attention not only
because of its role in soil and water conservation and
reforestation of eroded areas but also because of the
nutritional and medicinal values of its berries. The
yellow-red, pearl-shaped berries are important sources
of vitamins for the local people at the natural growth
sites of the plant. The berries have a long history of
application in Tibetan and Mongolian medicines and are
also an ingredient for traditional Chinese medicines
found in the Chinese Pharmacopoeia. A wide spectrum
of physiological effects of the berries and berry products
has been shown by many animal experiments and the
long history of clinical and nutritional practice, espe-
cially in China and Russia (3, 4). These positive effects
include lowering plasma cholesterol level, inhibiting
platelet aggregation, regulating immune function, pro-
moting the repair of injuries on skin and mucosa, and
anticancer effects (5-12).
MATERIALS AND METHODS
Ber r ies. Wild berries of H. rhamnoides ssp. sinensis were
picked in six different natural growth sites in China around
mid-October 1997. The growth areas ranged between longi-
tudes of 104° 40′ E and 114° 41′ E, latitudes of 35° 06′ N and
40° 03′ N, and altitudes of 1370 and 2800 m. Four samples of
wild ssp. rhamnoides berries were picked on the shores of the
Gulf of Bothnia in southwestern Finland in September 1999.
The areas ranged between longitudes of 21° 04′ E and 24° 24
′E, latitudes of 60° 45′ N and 64° 47′ N, and altitudes of 0 and
50 m. To follow the changes during the harvesting period, wild
berries of subsp. sinensis were collected at their natural growth
sites (Wenshui and Xixian, Shanxi Province, People’s Republic
of China) from the end of August to the end of November 1998.
All samples were loosely frozen in sealed plastic bags im-
mediately after collection to avoid desiccation and extra
moisture condensation and stored at -20 °C until analyzed.
Recently, interest in sea buckthorn has also developed
in Europe, J apan, Canada, and the United States as
more information on the chemical composition and
physiological effects of the berries has become available.
In association with research on sea buckthorn, the
industrial cultivation and utilization of its berries as a
raw material for functional food and natural medicines
have started in these regions.
Although many components, such as vitamin C,
sugars, acids, and fatty acids, of the berrries of sea
* Corresponding author (telephone 358-2-3336843; fax 358-
2-3336860; e-mail baoyan@utu.fi).
† Department of Biochemistry and Food Chemistry.
‡ Department of Chemistry.
10.1021/jf010813m CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/02/2001

Sea Buckthorn Sterols
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5621
Ta ble 1. Sa m p les of Sea Bu ck th or n Ber r ies fr om
Differ en t Su bsp ecies a n d Collectin g Tim es
Id en tifica tion of Ster ols. The sterol TMS derivatives were
analyzed by gas chromatography-mass spectrometry (GC-
MS). A VG Analytical 7070E double-focusing mass spectrom-
eter (VG Analytical, Altrincham, U.K.) equipped with an Opus
data system (Fisons Instruments, Manchester, U.K.) was con-
nected to a Dani 3800HR 2Ch gas chromatograph (Dani S.p.A,
Milan, Italy) without a restrictor capillary. A 30 m × 0.25 mm
i.d., 0.25 µm film DB-1701 capillary column (J &W Scientific,
Folsom, CA) was inserted directly into the ion source. The
column temperature was held at 230 °C for 1 min, increased
to 275 °C at 1 °C/min, and finally held at 275 °C for 30 min.
Helium (1.5 mL/min) was used as a carrier gas. The injector
temperature was 275 °C. The positive ion electron impact (EI)
mass spectra were recorded at an ionization energy of 70 eV
and a trap current of 100 µA with a source temperature of
180 °C. Perfluorokerosene (PFK) (Merck Art. 10145) was used
as the reference compound. The magnetic sector scans were
performed within the range of m/z 35-600 at a rate of 1 scan/s
with an intermediate time of 0.5 s between the scans, an
acceleration voltage of 6 kV, and a resolution of 1000.
collection
date
seed content
subspecies (% of fresh wt)
sample
location
F in la n d
r1
r2
r3
r4
Siikajoki
Vaasa
Pyha¨maa
Pyha¨maa
Sept 2, 1999 rhamnoides
Sept 3, 1999 rhamnoides
Sept 8., 1999 rhamnoides
Sept 8, 1999 rhamnoides
5.7
4.3
5.6
5.1
Ch in a
s1
s2
s3
s4
s5
Xunyi, Shaanxi Oct 12, 1997 sinensis
11.5
7.7
7.6
9.4
5.9
6.0
5.0
3.6
6.1
6.7
8.4
5.9
5.8
4.7
4.7
4.4
3.9
3.9
Youyu, Shanxi
Datong, Qinghai Oct 13, 1997 sinensis
Dingxi, Gansu Oct 17, 1997 sinensis
Wenshui, Shanxi Aug 30, 1998 sinensis
Sept 15, 1998
Oct 15, 1997 sinensis
Sept 30, 1998
Oct 15, 19 98
Oct 30, 1998
Nov 15, 1998
Nov 30, 1998
s6
Xixian, Shanxi
Aug 30, 1998 sinensis
Sept 15, 1998
Sept 30, 1998
Oct 15, 1998
Reference compounds, campesterol (campest-5-en-3â-ol),
sitosterol (stigmast-5-en-3â-ol), stigmastanol (5R-stigmastan-
3â-ol), R-amyrin (5R-urs-12-en-3â-ol), and â-amyrin (5R-olean-
12-en-3â-ol) were purchased from Sigma Chemical Co. (St.
Louis, MO). Sterols were identified by comparing the mass
spectra and retention times with those of reference compounds,
or the mass spectra published in the literature. For those
compounds for which neither standard compounds nor refer-
ence spectra were available, the chemical structures were
postulated according to the general patterns of mass spectro-
metric fragmentation of different sterols.
Oct 15, 1998
Nov 15, 1998
Nov 30, 1998
Detailed information of the samples is presented in Table
1.
Lip id Extr a ction . Berries (400 g) of each sample were
taken from a 5 kg lot using a sample partitioning procedure.
Seeds were isolated from frozen berries by pressing the juice
and rinsing the residue with distilled water. The press residue
was air-dried at room temperature, and seeds were separated
mechanically. For the whole berry analysis frozen berries were
freeze-dried (Dura-Top bulk tray dryer, FTS System Inc., Stone
Ridge, NY) to 15-30% of the original weight, depending on
the berry composition.
Samples (5 g) of seeds, freeze-dried whole berries, and pulp/
peel (seedless parts of the berries) were crushed in a mortar
in liquid nitrogen and the lipids isolated using a methanol/
chloroform extraction procedure (20, 21). The sample was
homogenized in methanol (50 mL) for 1 min in a blender,
chloroform (100 mL) was added, and homogenization was
continued for a further 2 min. The mixture was filtered and
the solid residue resuspended in chloroform/methanol (2:1, v/v,
150 mL) and homogenized for 3 min. The mixture was filtered
again and washed with fresh solvent (chloroform/methanol,
2:1, v/v, 150 mL). The combined filtrates were cleaned with a
repeat procedure using a 0.88% potassium chloride water
solution followed by a methanol/water (1:1, v/v) solution. The
purified lipids were filtered before the solvent was removed
on a rotary evaporator.
Qu a n ta titive An a lysis. The sterol TMS derivatives were
analyzed with a Varian 3300 gas chromatograph equipped
with a flame ionization detector (FID). The same column and
GC parameters were used as in the GC-MS analysis. The FID
temperature was 300 °C, and the split ratio of the injector was
1:40. The quantification of sterol compounds was carried out
with a cholesterol internal standard and calculated by applying
the detector reponse of sitosterol. The contents of sterols in
seeds, pulp/peel, and whole berries were calculated according
to the oil contents in these fractions and the contents of sterols
in the oils. The repeatability of the analytical procedure was
tested, and the relative standard deviation (SD) of five
repeated analyses of a single sample was <5%.
Sta tistica l An a lysis. Statistical analyses were carried out
with the statistical program SPSS 7.5. Comparison of the
absolute contents and the proportions of different sterols in
ssp. sinensis and rhamnoides were carried out with an
independent sample t test and a Mann-Whitney U test.
RESULTS AND DISCUSSION
Sa p on ifica tion of Lip id s. After the addition of cholesterol
palmitate (1.6 mg) as an internal standard, lipids (400 mg)
were saponified by refluxing in 20 mL of a 1 M KOH ethanol/
water (8:2, v/v) solution for 1 h. The refluxed mixture was then
transferred into a separatory funnel, and the reflux bottle was
washed with 40 mL of Milli-Q water. The unsaponifiables in
the combined solution were then extracted three times with
40 mL of diethyl ether. The ether phase was combined, washed
three times with 40 mL of water, and dried with sodium sulfate
overnight.
Isola tion of Ster ols a n d P r ep a r a tion of TMS Der iva -
tives. The unsaponifiables dissolved in petroleum ether (bp
) 60-80 °C) were applied to a Silica Sep-Pak Cartridge
(Waters Corp., Milford, MA) preconditioned with 5 mL of
petroleum ether. After elution of hydrocarbons with 10 mL of
petroleum ether and carotenoids with 10 mL of petroleum
ether/ether (96:4, v/v), the tocopherols and tocotrienols were
eluted with 10 mL of petroleum ether/ether (91:9, v/v) and
sterols with 20 mL of petroleum ether/ether (1:1, v/v). The
sterols were derivatized by incubation in a mixture of N,O-
bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethyl-
chlorosilane (TMCS) (2:1, v/v), at 60 °C for 2 h.
Ster ols in Seed s a n d P u lp /P eel of Ber r ies. Figure
1 presents GC-FID chromatograms of sterols in seeds
and pulp/peel, respectively. Identification of the com-
pounds in the chromatograms is summarized in Table
2. The same major sterols were found in the soft parts
and in the seeds even though differences were found in
minor compounds. In addition to sterols, a group of
triterpenoids was found, especially in the pulp/peel
(peaks 16 and 19-25), of which two compounds (peaks
24 and 25) had spectra identical to that reported and
tentatively identified as friedelan-3-ol in grape seed oil
by Kornfeldt (22). A detailed identification of these
compounds was beyond the scope of the present study.
Only sterols were included in the calculation of the
percentages of different compounds.
Compounds 1, 3, 4, and 8 were identified as TMS
ether derivatives of campesterol, sitosterol, stigmas-
tanol, and R-amyrin, respectively, by comparing the
retention times and mass spectra with those of the TMS

5622 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Yang et al.
identified as cycloartenol. Cycloartenol is commonly
known as the precursor of other sterols in plants. A third
compound was also found in the same peak in seeds but
was not identified because of its strong overlapping with
the other compounds.
Peak 10 had a mass spectrum similar to that of
cycloartenol. The ions at m/z 353 (339 + 14) and 300
(286 + 14) were characteristic of 9â,19-cyclopropane
sterols with a 24-methylene side chain (23). The com-
pound in peak 10 was suggested to be 4R,14R-dimethyl-
9â,19-cycloergost-24(241)-en-3â-ol. A minor component
with a molecular weight of 512 was also found in peak
10 and was not identified because of its small amount
and strong overlapping with the major component.
The compound in peak 11 was identified as stigmasta-
7,24(241)-dien-3â-ol on the basis of the close match of
its mass spectrum with that of avenasterol [(24Z)-5R-
stigmasta-7,24(241)-dien-3â-ol] TMS ether published by
Knights (26).
Compound 12 was found only in the pulp/peel of the
berries. Its mass spectrum was similar to those of
compound 10 and cycloartenol, suggesting a cyclopro-
pane structure in the fused ring system. However, the
absence of diagnostic ions made further indentification
of the compound impossible.
The spectrum of compound 13 fitted with those of 14R-
methyl-∆8-sterols and 14R-methyl-∆9(11)-sterols. The ion
at m/z 309 (M - 105 - 98) suggested the existence of a
24-ethylidene or 24-ethyl-∆24 side chain. Ge (17) re-
ported 4,14,241-trimethylergosta-8,24-dien-3â-ol in sea
buckthorn pulp oil. Compound 13 was possibly 4,14,-
241-trimethylergosta-8,24-dien-3â-ol, but further inves-
tigation is needed to confirm this tentative identifica-
tion.
Compound 14 was identified as 24-methylenecycloar-
tanol according to its mass spectrum matching well that
of the TMS ether of 24-methylenecycloartanol [24-
methyl-5R-cycloart-24(241)-en-3â-ol] published by Soul-
ier et al. (27).
F igu r e 1. GC-FID chromatograms of sterols of seeds (A) and
pulp/peel (B) of sea buckthorn berries (H. rhamnoides L. ssp.
rhamnoides, r3 from Pyha¨maa, Finland) analyzed as TMS
deravatives.
ethers of reference compounds. These mass spectra also
matched those published in the literature (23).
Peak 5 in seeds contained two compounds (5a and 5c),
and the major component 5a was identifed as isofucos-
terol [24(Z)-stigmasta-5,24(241)-dien-3â-ol] according to
its mass spectrum and retention time (23, 24). The mass
1
spectrum of 5c (∼ /10 of the abundancy of 5a ) matched
well that of the TMS ether of obtusifoliol [4R,14R-
dimethyl-5R-ergosta-8,24(241)-dien-3â-ol] (22), a key
intermediate compound in the most common sterol
biosynthesis pathways in plants (25). Thus, this com-
pound was identified as obtusifoliol.
In the pulp/peel, a third compound, 5b, was found at
about the same level as 5a . The strong molecular ion
(m/z 486, base peak) and the weak ions at m/z 129 and
357 (M - 129) (Table 2) suggested that the compound
was probably stigmast-8-en-3â-ol (23).
Peak 7 represents two compounds (7a and 7b) in the
seeds and is different from the corresponding one (7c)
in the pulp/peel. Compound 7a was possibly a 14R-
methyl-∆8-sterol or a 14R-methyl-∆9(11)-sterol (23). Com-
Compound 17 in pulp/peel was identified as citrosta-
dienol [(24Z)-4R-methyl-5R-stigmasta-7,24(241)-dien-3â-
ol] according to its mass spectrum, which was identical
to that of the TMS ether of citrostadienol published by
Farines et al. (24).
The structure of compound 18 could not be defined,
although its spectrum suggested a 9,19-cyclopropane
structure in the fused ring system.
pound 7b gave a spectrum similar to that of 5a ,
5,24(241)
characteristic of ∆
-sterols and 24-ethyl-∆5,24(25)
-
sterols. According to Goad and Akihisa (23), the latter
group tends to have longer retention times, and thus
compound 7b was possibly stigmasta-5,24(25)-dien-3â-
ol. Compound 7c was possibly a stigmasta-8,24-dien-
3â-ol (23, 26). However, the final identification of this
compound has to be carried out with reference com-
pounds because of the lack of diagnostic ions in the
spectra of ∆8-sterols (23).
The structures of minor components 2, 6, and 15
cannot be defined according to their spectra alone.
Salenko et al. (13) analyzed the unsaponifiable frac-
tion of a pentane extract of the fruit pulp of sea
buckthorn, and sitosterol, 24-methylenecycloartenol,
citrostadienol, 24-ethylcholest-7-en-3â-ol, R- and â-amyr-
ins (pentacyclic triterpenes), uvaol, and erythrodiol were
identified with MS, GC-MS, and NMR. Sitosterol, 24-
methylenecycloartenol, citrostadienol, and R-amyrin
were also found in the present study. The “24-ethyl-
cholest-7-en-3â-ol” identified by Salenko et al. (13) was
probably the same compound as the stigmast-7-en-3â-
ol in our study. We did not find â-amyrin in the analyzed
samples.
Ge (17) reported 19 sterol compounds in pulp oil of
sea buckthorn (H. rhamnoides ssp. sinensis) with GC-
MS, among which sitosterol, R-amyrin, and stigmast-
7-en-3â-ol were also found in our study. Other com-
pounds reported were â-amyrin, ergosta-5,8,22-trien-3â-
ol, ergosta-5-en-3â-ol, ergosta-7-en-3â-ol, 4,14-dimethyl-
The mass spectrum of compound 9a matched well the
spectrum of TMS ether of schottenol (5R-stigmast-7-en-
3â-ol) published in the literature (23) and was identified
as stigmast-7-en-3â-ol.
Compound 9b had a molecular ion at m/z 498 in its
mass spectrum. The ions at m/z 483 and the strong ions
at m/z 408 (M - 90), m/z 393 (M - 90 - 15), m/z 286
(M - 90 - A-ring - C-6), m/z 339 (due to the loss of
TMSiOH, C-2, C-3, C-4, C-28, and C-29), and m/z 365
(M - 90 - 43) suggested that the compound had a 9â,-
19-cyclopropane structure in the ring system, a ∆24-
unsubstituted side chain, and a C-14 methyl group. The
mass spectrum matched well the published spectrum
of the TMS ether of cycloartenol (9,19-cyclo-5R,9â-
lanost-24-en-3â-ol) (23, 27). Therefore, compound 9b was

Sea Buckthorn Sterols
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5623
Ta ble 2. Ster ols a n d Tr iter p en es in Seed s a n d P u lp /P eel of Sea Bu ck th or n Ber r ies (Ma ss Sp ectr a Sh ow n Ar e of Ster ol
TMS Eth er s)
major ions in mass spectra (m/z,
intensity relative to base peak
ID
compd RRTa
identification
occurrence
methodb
refs
1
1.16 472 (62), 457 (13), 382 (85), 367 (33),
343 (96), 261 (11), 255 (16), 227 (4),
213 (11), 129 (100)
1.28 484 (100), 469 (34), 386 (30), 379 (85),
355 (11), 296 (13), 253 (38), 213 (15),
211 (22), 129 (48)
1.30 486 (65), 471 (13), 396 (91), 381 (30),
357 (93), 255 (14), 228 (2), 213 (9),
129 (100)
1.31 488 (93), 473 (53), 431 (9), 398 (24),
383 (24), 357 (11), 305 (22), 290 (8),
230 (19), 215 (60), 75 (100)
1.33 484 (15), 469 (7), 394 (5), 386 (100), 371 (12),
355 (9), 296 (57), 281 (22), 257 (22), 255 (7),
227 (6), 213 (8), 211 (8), 129 (50)
486 (100), 471 (19), 396 (6), 393 (18), 381 (12),
345 (6), 255 (17), 229 (16), 213 (20), 201 (11)
498 (65), 483 (100), 455 (2), 393 (32), 317 (5),
295 (11)
campest-5-en-3â-ol
seeds, pulp/peel
S, R
14, 17-19
(campesterol)
2
stigmastadienolc
seeds, pulp/peel
seeds, pulp/peel
seeds, pulp/peel
seeds, pulp/peel
3
stigmast-5-en-3â-ol
(sitosterol)
S, R
S, R
D, R
13-15, 17-19
4
5R-stigmastan-3â-ol
(stigmastanol)
5a
24(Z)-stigmasta-5,24(241)-
dien-3â-ol
14, 18, 19
15-18
(isofucosterol)
5b
5c
stigmast-8-en-3â-olc
pulp/peel
D, R
D, R
4R,14R-dimethyl-5R-ergosta-
8,24(241)-dien-3â-ol
(obtusifoliol)
seeds, pulp/peel
6
1.35 500 (5), 486 (7), 471 (6), 388 (100), 373 (13),
305 (10), 215 (16), 75 (37)
1.36 500 (52), 485 (51), 483 (32), 469 (26), 410 (50),
395 (100), 371 (65), 355 (8), 343 (5), 303 (5),
269 (6), 229 (17), 213 (23), 187 (33)
not identified
seeds, pulp/peel
seeds
7a
not identified
7b
484 (37), 469 (14), 394 (9), 386 (100), 371 (4),
355 (24), 296 (58), 281 (22), 257 (23), 255 (9),
227 (8), 213 (12), 211 (9), 129 (68)
stigmasta-5,24(25)-dien-3â-olc seeds
D
7c
8
484 (100), 469 (32), 394 (6), 386 (15), 379 (12),
343 (9), 281 (5), 255 (7), 229 (16), 213 (20)
1.38 498 (13), 484 (8), 408 (5), 393 (4), 388 (6),
279 (6), 269 (4), 218 (100), 207 (6), 203 (10)
1.39 486 (100), 471 (15), 396 (4), 381 (9), 345 (5),
303 (4), 255 (45), 229 (11), 213 (12)
498 (2), 483 (11), 408 (100), 393 (45), 365 (37),
339 (38), 297 (5), 286 (20), 271 (9), 69 (63)
1.41 498 (2), 483 (3), 408 (100), 393 (42), 365 (2),
353 (6), 300 (4), 283 (7), 269 (3), 217 (5),
189 (10)
1.43 484 (21), 469 (10), 394 (3), 386 (100), 371 (8),
343 (88), 296 (6), 281 (6), 255 (13), 253 (8),
229 (5), 211 (3)
1.45 498 (4), 483 (6), 408 (100), 393 (44), 311 (12),
297 (3), 269 (12), 216 (15), 201 (6)
1.46 512 (81), 497 (100), 485 (7), 458 (4), 407 (59),
368 (13), 353 (3), 329 (5), 309 (17), 278 (7),
255 (3), 215 (12), 213 (11), 69 (57)
1.49 512 (4), 497 (8), 422 (100), 407 (40), 379 (33),
353 (15), 339 (3), 323 (2), 300 (13), 297 (6),
255 (3), 241 (3)
1.50 498 (100), 483 (18), 422 (15), 408 (10), 396 (53),
391 (15), 383 (11), 356 (7), 342 (6), 269 (6),
253 (11), 243 (8), 241 (9), 227 (15)
1.53 571 (2), 496 (100), 481 (3), 391 (2), 216 (83),
203 (34), 189 (15), 133 (7), 73 (41)
stigmasta-(8,24)-dien-3â-olc
pulp/peel
D
5R-urs-12-en-3â-ol
(R-amyrin)
stigmast-7-en-3â-ol
seeds, pulp/peel
seeqds, pulp/peel
seeds, pulp/peel
seeds, pulp/peel
S, R
D, R
D, R
D
13, 15-18
13-15, 17-19
18
9a
9b
10
9,19-cylco-5R,9â-lanost-24-
en-3â-ol (cycloartenol)
4R,14R-dimethyl-9â,19-cyclo-
ergost-24(241)-en-3â-olc
11
stigmasta-7,24(241)-dien-3â-ol seeds, pulp/peel
D, R
14, 19
12
13
not identified
pulp/peel
4R,14R,241-trimethylergosta-
8,24(241)-dien-3â-olc
seeds, pulp/peel
D
17
14
15
24-methyl-5R-cycloart-24(241)- seeds, pulp/peel
en-3â-ol (24-
methylenecycloartanol)
not identified
D, R
13, 18
seeds, pulp/peel
16
17
unidentified triterpene,
isomer of 19
seeds, pulp/peel
1.57 498 (10), 483 (9), 400 (100), 393 (5), 385 (5),
357 (71), 310 (7), 295 (6), 267 (9), 241 (8),
227 (11)
24(Z)-4R-methyl-5R-stigmasta- seeds, pulp/peel
D, R
13
7,24(241)-dien-3â-ol
(citrostadienol)
18
19
1.58 512 (2), 422 (100), 407 (45), 379 (1), 367 (7),
325 (11), 312 (3), 309 (2), 283 (7), 69 (29)
1.60 571 (3), 496 (100), 481 (2), 393 (2), 391 (2),
216 (37), 203 (36), 188 (13), 133 (11),
not identified
pulp/peel
unidentified triterpene,
isomer of 16
seeds, pulp/peel
95 (12), 73 (37)
20
21
22
23
24
25
a
1.65 586 (7), 571 (4), 496 (85), 483 (57), 393 (29),
374 (10), 279 (13), 203 (56), 189 (56), 129 (28)
1.73 586 (12), 482 (31), 320 (50), 307 (5), 279 (8),
219 (5), 203 (100), 189 (30), 133 (8), 73 (64)
1.78 587 (5), 496 (2), 481 (2), 279 (6), 216 (100),
201 (11), 189 (11), 145 (10), 120 (46), 73 (25)
1.83 586 (11), 512 (2), 482 (13), 320 (100), 307 (10),
279 (12), 203 (91), 189 (29), 133 (32), 73 (74)
1.88 512 (5), 497 (2), 407 (1), 422 (2), 279 (15),
232 (69), 203 (100), 190 (57), 175 (13), 131 (18)
2.00 512 (5), 497 (2), 407 (1), 422 (2), 279 (17),
232 (22), 203 (100), 190 (47), 175 (9), 133 (19)
b
unidentified
pulp/peel
pulp/peel
pulp/peel
pulp/peel
pulp/peel
pulp/peel
unidentified triterpene,
isomer of 23
unidentified triterpene
unidentified triterpene,
isomer of 21
friedelan-3-olc
R
R
friedelan-3-olc
Retention time relative to cholesterol. S, identified with standard compound; R, identified with published reference spectrum; D,
deduced according to the mass spectrum in the present study. c Tentative identification.
ergosta-8,24(28)-dien-3â-ol [4,14-dimethylergosta-8,24-
(241)-dien-3â-ol], stigmasta-5-en-3-one, lanosta-8,24-
dien-3â-ol, 9,19-cyclolanosta-25-en-3â-ol series (three
separated compounds), stigmasta-7,16-dien-3â-ol, 24-
methylenelanosta-8-en-3â-ol, 4,14,28-trimethylergosta-
8,24(28)-dien-3â-ol [4,14,28-trimethylergosta-8,24(241)-
dien-3â-ol], 4-methylstigmasta-7,16-dien-3â-ol, 14-methyl-
R-sitosterol, and 14,15-dimethyl-R-sitosterol. According
to the structure presented in the publication, the last
two compounds were 4,14-dimethylstigmasta-7,24(241)-

5624 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Yang et al.
F igu r e 2. Major sterols found in sea buckthorn seeds and berries and possible precursor-product relationships between these
compounds. Arrows with solid tails represent the dominating pathways proposed in plants; arrows with dotted tails represent
less common pathways.
dien-ol and 4,14,15-trimethylstigmasta-7,24(241)-dien-
ol, respectively (17).
Ge (17), the identifications of Salenko et al. (13), Schiller
(14), and Bat (19) were more consistent with that in the
present study, although we did not find detectable
amount of cholesterol or stigmasterol. The discrepancy
among the identifications in the investigations sug-
gested cautions should be excercized when sterol com-
positions are adopted as a criterion for assessing
adulteration of sea buckthorn oils.
In addition to the compounds reported earlier in sea
buckthorn, five compounds were identified (or postu-
lated) in the present study, of which the occurrence in
sea buckthorn seeds and berries is reported for the first
time (Table 2).
4,14-Dimethyl-ergosta-8,24(241)-dien-3â-ol and 4,14,-
28-trimethylergosta-8,24(241)-dien-3â-ol in Ge’s identi-
fication were possibly the same as compounds 5c
(obtusifoliol) and 13 in the present study, respectively.
The mass spectrum of “4-methylstigmasta-7,16-dien-3â-
ol” in the publication (17) resembled that of citrostadi-
enol published in the literature (28), and it is possible
that this compound was actually citrostadienol. The
“ergost-5-en-3â-ol” was probably the same compound as
campesterol [24(R)-ergost-5-en-3â-ol or campest-5- en-
3â-ol] identified in the present study.
Wang et al. (15) reported 22 sterols and triterpenes
in pulp oil and 10 in seed oil of sea buckthorn ssp. sin-
ensis. Only R- and â-amyrins, 4,14-dimethyl-ergosta-8,-
24(241)-dien-3â-ol, and 9,19-cyclolanosta-25-en-3â-ol were
claimed to be the common components found in both
seed and pulp oils. Sitosterol was reported to be absent
in the seed oil. In contrast to this, we found that the
sterol compositions in seed and pulp/peel of sea buck-
thorn berries were rather similar, except for a few minor
components, and sitosterol was always the most abun-
dant component (60-80%) in both seeds and pulp/peel.
Schiller (14) reported sitosterol (86% of fruit pulp
sterols, 73% of seeds sterols), campesterol (2% of fruit
pulp sterols and 4% of seed sterols), stigmasta-5,24-dien-
ol (6% in fruit pulp sterols and 17% in seed sterols),
stigmast-7-en-ol (2% in fruit pulp sterols and 1% in
seeds sterols), stigmasta-7,24-dien-ol (1% in fruit pulp
and seed sterols), a small amount of cholesterol (1.5-
2.0%) in seeds and fruit flesh, and a trace amount of
stigmasterol (0.1% of total sterols) in berry pulp. The
same compounds were found by Bat (19) in oils from
seeds, fruit flesh, and press cake of sea buckthorn
berries. Compared with those of Wang et al. (15) and
A dominating biosynthetic pathway of sterols has
been proposed to operate in most vascular plants based
on the sterols identified and the enzymological inves-
tigations of sterol biosynthesis in a number of plant spe-
cies (25, 29). Deviations from the proposed biosynthetic
pathway have also been suggested in some species (25,
29). The differences found in the biosynthesis of sterols
in different plant species are possibly related to the
phylogenetical regulation of the expression of genes
coding for some enzymes involved in the process (29).
The biosynthesis of sterols in sea buckthorn has not
been investigated or discussed before. On the basis of
the existing knowledge of biosynthesis of plant sterols,
the major sterols identified in the present study are
presented in a scheme as Figure 2, suggesting the pos-
sible precursor-product relationships between these
compounds. The scheme is in agreement with the dom-
inating pathway proposed by Benveniste (25) and Nes
and Venkatramesh (29). Further investigations are
needed to demonstrate the biosynthesis of sterols in sea
buckthorn.
Ster ol Con ten t in Seed s. Table 3 presents the sterol
contents in seeds of different origins of the two subspe-

Sea Buckthorn Sterols
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5625
Ta ble 3. Ster ol Con ten t (Milligr a m s p er Kilogr a m ) in Seed s of Tw o Su bsp ecies of Sea Bu ck th or n
subspecies
sample
sinensis
mean
rhamnoides
mean SD
sterol
s1
31
s2
32
805
53
292
s3
39
s4
31
r1
35
763
59
189
5
r2
37
822
56
160
3
27
25
15
14
7
r3
51
1164
70
282
8
14
34
29
14
9
r4
35
976
16
172
3
SD
p value
1
3
4
33
934
46
244
33
22
25
12
26
7
4
40
931
50
201
5
17
19
17
12
7
8
179
24
55
2
0.20
0.98
0.75
0.31
0.03
0.28
0.52
0.41
0.01
1.00
0.54
0.76
0.74
992
52
243
39
21
24
12
26
9
1041
30
166
49
17
11
9
18
5
10
3
24
896
49
276
12
23
25
10
23
7
105
11
56
19
4
5a + 5c
6
7a + 7b
9a + 9b
10
27
39
18
35
8
39
1
64
17
10
13
12
10
13
6
10
6
7
11
4
7
2
13
2
17
13
9
3
3
9
11
7
13
3
14
20
6
44
16
4
37
43
2
40
48
2
58
11
6
21
4
42
29
4
39
19
2
14
15
17
30
26
total
1519
1413
1422
1409
1162
1251
1783
1280
1441
52
1369
281
0.63
R-amyrin and unidentified triterpenes
8
7
13
36
12
8
6
9
7
8
10
15
6
9
7
11
12
9
7
11
9
2
17
23
8
10
18
3
2
13
7
12
12
4
3
7
0.47
0.31
0.46
16
19
13
total
56
33
22
33
22
32
27
42
36
14
31
9
0.55
Ta ble 4. Ster ol Con ten t (Milligr a m s p er Kilogr a m ) in F r esh P u lp /P eel of Ber r ies of Tw o Su bsp ecies of Sea Bu ck th or n
subspecies
sample
s4
sinensis
mean
rhamnoides
mean SD
sterol
s1
s2
s3
r1
r2
r3
r4
SD
1.3
p value
1
2
3
4
6
7
328
4
10
3
5
5
5
186
3
20
4
4
3
4
4.8
5.5
218.5
2.3
15.3
3.8
3.3
0.5
0.5
24.1
0.5
8.5
2.8
2.8
1.5
3.1
1.7
3.1
1.7
2.4
1.2
1.0
1.7
0.19
0.02
0.62
0.25
0.49
4
164
1
6
196
1
3
183
1
3
225
1
4
211
2
3
173
1
1.3
74.2
1.5
198.0
1.3
5a + 5b + 5c
14
17
8
5
28
22
9
17
4.3
18.8
7.0
6
7a + 7b + 7c
9a + 9b
2
9
3
3
7
6
8
18
8
6
8
19
4
10
10
10
6
5
8
7
6
5
7
3
6
4
4
7
3
3
6
3.8
8.5
1.7
1.3
6.1
2.1
1.2
2.5
6.8
5.0
0.52
0.02
0.32
0.17
0.88
0.29
0.48
3
4
4
2
9
5
4
5
3
6
6
7
9
6
9
5.3
10
10
5
11
4
10.3
7.6
6.5
11
7.3
5.5
12
4
2
3
4
5
5.0
4.8
13
4
5
4.8
6.5
14
7
8
9.3
6.5
15
2
3.3
17
6
6
6
5
12
9
6
6
5
6
6
7.5
6.5
2.0
1.7
5.8
0.31
0.15
18
6
4.5
total
397.3
237
319
284
243
313
305
254
309.3
67.6
278.8
35.4
0.45
R-amyrin and unidentified triterpenes
8
0
19
88.1
4
3
6
3
6
2
8
37
3
30
4
6
5
4
7
11
58
4
3
5
2.0
9.8
42.8
2.7
24.0
6.0
52.8
4.5
18.8
1.4
6.2
31.1
1.5
21.0
4.2
51.5
2.4
8.5
4.8
6.5
1.7
3.1
18.0
1.4
2.4
0.5
1.2
1.7
8.2
0.05
0.39
0.61
0.58
0.09
0.16
0.13
0.87
0.81
16
19
20
21
22
23
24
25
27
1
14
3
19
34
2
18
1
22
1
33.0
2.0
55
9
8
8
5
5
2
5.0
3
3
3
2
2.8
121
15
7
12
11
6
31
64
2
14
8
6
8
6
7.0
3
18
7
5
4
3
4.8
30
24
13
14
20.3
total
317.1
88
84
160
102
71
113
58
162.3
109.0
86.0
25.8
0.22
cies. In the seed samples analyzed, the total sterol
content varied from 1200 to 1800 mg/kg, of which 57-
76% was sitosterol and 13-21% isofucosterol. Stigmas-
tanol, citrostadienol, and campesterol constituted 1-5,
2-5, and 2-3% of total seed sterols, repectively. The
proportion of the other sterols was typically 1-2% each.
No significant difference was found in the level of most
of the compounds or the total sterols between the two
subspecies. The contents of compound 6 (33 ( 19 vs 5
( 2 mg/kg, p < 0.05) and stigmasta-7,24(241)-dien-3â-
ol (compound 11) (26 ( 7 vs 12 ( 3 mg/kg, p < 0.05)
were higher in seeds of ssp. sinensis than in those of
ssp. rhamnoides. In the extracted seed oil, the total
sterol content was 12.4-23.0 g/kg in the samples
analyzed.
of the sterols were similar in the two subspecies even
though the berries have been collected in different years
from growth sites with different geographic, soil, and
climate conditions. Only compounds 6 (2.3 vs 0.4%) and
11 [stigmasta-7,24(241)-dien-3â-ol] (1.8 vs 0.9%) repre-
sented a higher proportion of total sterols in ssp.
sinensis than in ssp. rhamnoides (p < 0.05).
From the end of August to the end of November 1998,
an increase was found in the content of campesterol
(compound 1), whereas the levels of stigmastanol (com-
pound 4), compound 6, and stigmasta-7,24(241)-dien-3â-
ol (compound 11) decreased (Figure 3A1) in seeds of ssp.
sinensis. The sum of isofucosterol and obtusifoliol
(compounds 5a + 5c) reached a maximum at mid-
October (Figure 3A2). The total sterol content and the
level of sitosterol (compound 3) remained rather con-
stant, even though the values at mid-October were
Comparison of the sterol composition in seeds of the
two subspecies also showed that the proportions of most

5626 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Yang et al.
F igu r e 3. Changes in sterol contents in seeds (A) and pulp/peel (B) of sea buckthorn (H. rhanmoides L. ssp. sinensis) growing
in Xixian County, People’s Republic of China, from the end of August to the end of November 1998.
slightly higher than those at other dates (Figure 3A3).
During the period of investigation, the proportions of
sitosterol (from 69 to 76%) and campesterol (from 1.8
to 2.4%) of total seed sterols increased, whereas those
of stigmastanol (from 3.3 to 0.8%) and compound 6 (from
1.2 to 0.3%) decreased. The proportions of other com-
pounds remained fairly constant.
The results show that, like many other components,
the levels of most sterols in the seeds of ripe berries
remained rather constant during the harvesting period.
The clear increase in campesterol and decrease in
stigmastanol suggested that a complete balance was not
yet reached in the major biosynthetic pathways at the
beginning of the harvesting period.
When the two subspecies were compared, no signifi-
cant difference was found in the absolute content or the
proportion of sitosterol and most of the other sterols in
the soft parts. The absolute levels of compound 2 (a
stigmastadienol) and the sum of stigmasta-7-en-3â-ol
and cycloartenol (compounds 9a + 9b) were higher in
ssp. sinensis (p < 0.05). In addition, the unidentified
compound 15 was not detected in this subspecies (Table
4).
The total content of the unidentified triterpenes
varied from 58 to 317 mg/kg in the fresh pulp/peel of
the berries. The average level was higher in ssp. sinensis
(162 vs 86 mg/kg). However, the difference was not
statistically significant because of the high deviation
(Table 4).
From the end of August to the end of November 1998,
the level of campesterol (compound 1) increased, whereas
the contents of stigmasta-7,24(241)-dien-3â-ol (com-
pound 11) and the sum of stigmast-7-en-3â-ol and
cycloartenol (compounds 9a + 9b) decreased signifi-
cantly (Figure 3B1). A significant decrease was also
found in the sum of isofucosterol, stigmast-8-en-ol, and
obtusifoliol (compounds 5a + 5b + 5c). The levels of
4R,14R-dimethyl-9â,19-cycloergost-24(241)-en-3â-ol (com-
pound 10), 24-methylenecycloartanol (compound 14),
and compound 18 reached a maximum at the end of
October (Figure 3B2). A slight decrease was found both
in the total sterol content (from 213 to 186 mg/kg) and
in the content of sitosterol (from 150 to 130 mg/kg) of
pulp/peel during this period (Figure 3B3).
The total content of R-amyrin and the unidentified
triterpenes in the seeds varied from 22 to 56 mg/kg
without any significant difference between the two
subspecies (Table 3).
Ster ol Con ten t in P u lp /P eel of Ber r ies. Table 4
presents the contents of sterols in pulp/peel of the
berries. The total sterol content in the fresh soft parts
varied within the range of 240-400 mg/kg. The total
sterol content in the extracted pulp/peel oil was 10.3-
28.7 g/kg in the analyzed samples. Sitosterol constituted
61-83% of the total sterols in the soft parts of the
berries. Isofucosterol, stigmast-8-en-3â-ol, and obtusi-
foliol together (compounds 5a + 5b + 5c) comprised
3-9%, showing a significantly lower proportion of
isofucosterol than in the seeds. This was consistent with
the results (17% of seed sterols and 6% of fruit pulp
sterols were stigmasta-5,24-dien-ol) of Schiller (29). The
proportions of the rest were typically 1-3% each of the
total sterols.
Ster ol Con ten t in Wh ole Ber r ies. Table 5 presents
the sterol contents in fresh whole berries of different
origins. The total sterol contents of the samples varied

Sea Buckthorn Sterols
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5627
F igu r e 4. Changes in sterol contents in fresh whole berries of sea buckthorn growing in Wenshui (A) and Xixian (B) counties,
People’s Republic of China, from the end of August to the end of November 1998.
Ta ble 5. Ster ol Con ten t (Milligr a m s p er Kilogr a m ) in F r esh Ber r ies
subspecies
sample
s4 r1
sinensis
mean
rhamnoides
mean SD
sterol
s1
s2
s3
r2
r3
r4
SD
p value
1
3
4
10
317
7
66
1
5
215
2
43
1
2
11
15
9
9
7
257
4
42
1
6
10
16
9
7
7
247
1
31
9
6
6
8
275
4
47
1
5
12
15
12
7
8
13
4
2
48
2
7
256
2
32
9
6
9
11
5
4
8
13
4
15
6
1
30
1
0.32
0.52
0.11
0.09
309
2
243
1
299
2
233
2
5a + 5b + 5c
6
7c
37
28
42
27
13
0
7
7
8
16
9
6
6
12
1
15
7
6
10
9
6
10
11
7
5
7
15
5
5
6
10
3
13
7
3
1
2
3
1
3
2
3
1
4
4
0.69
0.25
0.05
0.07
0.27
0.56
0.89
0.82
0.90
0.37
9a + 9b
19
13
21
10
9
10
7
8
10
5
2
8
11
5
11
3
5
10
1
11
4
6
12
5
8
14
5
7
15
2
8
2
13
8
11
16
2
1
14
13
4
2
15
4
17
14
11
16
9
13
7
16
3
19
12
15
9
1
18
2
total
515
355
444
399
354
358
451
344
432
94
385
60
0.27
R-amyrin and triterpenes
8
10
32
119
5
2
11
37
1
26
4
30
14
82
3
6
14
1
14
2
19
4
7
3
8
24
2
13
3
16
5
8
7
4
17
2
9
1
11
5
22
4
4
7
8
3
5
5
14
49
2
4
12
48
2
27.8
8
73.9
5
35.8
5
5
22
2
2
2
10
0
2.6
1
2.6
2
4.0
0.74
0.19
0.32
0.80
0.13
0.28
0.22
0.86
0.65
16
19
20
21
22
23
24
25
13
2
35
2
22
2
72
18
169
3
8
6
3
31.3
6.5
2
4
2
7
2
7
9
5
58.5
7
8
5
9
5
6
18
16
25
18
28.8
20.25
total
436
205
67
79
71
57
98
62
196.8
171.3
72
18.3
0.20
within the ranges of 340-520 mg/kg and 13.0-33.2 g/kg
in fresh whole berries and the extracted berry oil,
respectively. Comparison of the two subspecies showed
that the total sterol content in ssp. sinensis berries was
typically higher than in ssp. rhamnoides (432 ( 94 vs
385 ( 60 mg/kg), but the difference was not statistically

5628 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Yang et al.
ACKNOWLEDGMENT
significant (p ) 0.27). Difference between the two
subspecies was found in the combined level of isofucos-
terol, stigmast-8-en-3â-ol, and obtusifoliol (compounds
5a + 5b + 5c, p ) 0.09) and the contents of 4,14-
dimethyl-9,19-cyclo-ergost-24(241)-en-3â-ol (compound
10, p < 0.05) and stigmasta-7,24(241)-dien-3â-ol (com-
pound 11, p ) 0.07), the levels of these compounds being
higher in ssp. sinensis berries.
We thank Aromtech Oy (Tornio, Finland) for provid-
ing the berries. We also thank Nina Rostiala and Kirsti
Wiinama¨ki for their participation in the analytical work.
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triterpenes in the whole berries showed considerable
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kg, respectively (Figure 4A3). No clear trend was found
in the other compounds.
During the same period, in Xixian berries (Figure 4B),
the levels of 4,14-dimethyl-9,19-cycloergost-24 (241)-en-
3â-ol (compound 10), 24-methylenecycloartanol (com-
pound 14), and unidentified compunds 12 and 18
reached maxima around the middle to the end of
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found in the levels of stigmastanol (compound 4);
stigmasta-5,24-dien-â-ol, stigmast-8-en-3â-ol, and ob-
tusifoliol (5a + 5b + 5c); stigmast-7-en-3â-ol and
cycloartenol (9a + 9b); and stigmasta-7,24(241)-dien-
3â-ol (compound 11) (Figure 4B2). The total sterol
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and whole berries. The information obtained in the
present investigation is very useful for characterizing
sea buckthorn oils, detecting adulterations, and further
chemical and nutritional investigations of sterols in sea
buckthorn. The results are also important for industrial
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Received for review J une 18, 2001. Revised manuscript
received August 30, 2001. Accepted September 3, 2001. We
thank Kiinteisto¨kartio Oy (Turku, Finland) for financial
support.
J F010813M