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J Sci Food Agric 1998, 78, 141È146
Effect of Storage and Extraction on Ratio of
Soyasaponin I to 2,3-Dihydro-2,5-dihydroxy-6methyl-4-pyrone-Conjugated Soyasaponin I in
Dehulled Peas (Pisum sativum L)
Ylva Dandanell Daveby,1* Per AŽ man,1 Joseph M Betz2 and Steven M Musser2
1 Department of Food Science, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden
2 US Food and Drug Administration, Center for Food Safety and Applied Nutrition, HFS-347, 200 C St
SW, Washington, DC 20204, USA
(Received 5 March 1997 ; revised version received 4 August 1997 ; accepted 20 October 1997)
Abstract : The e†ects of solvent and time on extraction of 2,3-dihydro-2,5dihydroxy-6-methyl-4-pyrone (DDMP)-conjugated soyasaponin I in dehulled
peas (Pisum sativum L) were studied. Extraction in 80% ethanol gave a higher
and more stable yield than 100% methanol over the time studied. Acetonitrile
did not extract any detectable saponins. Both soyasaponin I and the DDMPconjugated form were present in the extracts of dehulled, dried peas. The proportion of soyasaponin I to the DDMP-conjugate, calculated as the ratio of the
molecular ions, increased with increasing extraction time, from an initial 0É24
after 0É5 h to 0É55 after 24 h of extraction in 80% ethanol, showing that the
DDMP-conjugate was hydrolysed during the extraction. Storage of dried dehulled peas also increased the proportion of soyasaponin I, from 0É063 in newly
harvested peas to 0É34 after 9 months of storage. Peas harvested in 1988 and
stored for 7 years had a ratio of 0É78. These results clearly show that the DDMPconjugate was converted to soyasaponin I both during storage and extraction of
the peas. ( 1998 Society of Chemical Industry.
J Sci Food Agric 78, 141È146 (1998)
Key words : Pisum sativum, dehulled seeds, hulls, soyasaponins, DDMPconjugate, storage, extraction.
Phaseolus coccineus (Yoshiki et al 1994), L upinus angustifolius (Ruiz et al 1995), Medicago sativa and Glycine
max (Massiot et al 1992) are soyasaponins conjugated
2,3-dihydro-2,5-dihydroxy-6-methyl-4-pyrone(DDMP) (Fig 1). Traditional methods of extracting,
derivatising and analysing soyasaponins have used hot
exhaustive extractions. It has been suggested that
heating converts DDMP-conjugated soyasaponin I to
soyasaponin I (Fig 2) (Kudou et al 1992). Later studies
on soyasaponins in legumes using mild (eg room
temperature) extractions have shown DDMPconjugated soyasaponins to be the predominant soyasaponins (Kudou et al 1992 ; Massiot et al 1992). The
DDMP-conjugated soyasaponins have a strong superoxide anion (O~) scavenging activity, suggesting a pre2
ventive role against biomolecular damage due to radical
Saponins are steroid or triterpenoid glycosides present
in a wide variety of plants consumed by humans and
animals. The soyasaponins comprise a triterpenoid soyasapogenol with one or two (mono- or bidesmosidic)
carbohydrate sidechains attached. They have a bitter
taste (Price and Fenwick 1984), inhibitory e†ects against
infectivity of the AIDS virus (Okubo et al 1994) as well
as possible cholesterol-binding and growth retarding
activities (Cheeke 1996). These properties are, however,
dependant upon the individual structure of the soyasaponins (Okubo et al 1994). It has been reported that the
native saponins in Pisum sativum (Tsurumi et al 1992),
* To whom correspondence should be addressed.
( 1998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50.
Printed in Great Britain
Y D Daveby et al
Fig 1. MS/MS product ion spectrum of m/z 1069 (DDMP-conjugate soyasaponin I).
attack (Yoshiki and Okubo 1995). It has also been suggested to function as a reductant in root growth regulation (Tsurumi and Tsujino 1995).
In peas, a domestic protein crop in Sweden, only soyasaponin I has been quantiÐed (Curl et al 1985 ; Price et
al 1986 ; Daveby et al 1997). DDMP-conjugated soyasaponin I was quantiÐed in 7-day-old aetiolated pea
seedlings (Tsurumi et al 1992). The latter study
reported production of soyasaponin I from DDMPconjugated soyasaponin I in frozen tissues or from
prolonged extraction.
We have investigated the efficiency of di†erent solvents for extraction of DDMP-conjugated soyasaponin
I as well as the e†ect of storage and extraction time on
the ratio of soyasaponin I to DDMP-conjugated soyasaponin I in dehulled peas.
Swedish light-coloured and leaÑess peas (Pisum sativum
L ; subsp hortense ; cv Capella) grown during 1988 at
Ultuna, Sweden (60¡N) and during 1995 at SvaloŽv,
Sweden (56¡N) were studied.
The peas were soaked in distilled water at 4¡C overnight, dehulled by hand (the germs were included with
the dehulled seeds) and the dehulled seeds were freezedried. The peas harvested in 1988 were stored dry as
whole seeds in room temperature until analysis. Peas
harvested in 1995 were stored dehulled in a desiccator
until analysis. Prior to analysis samples were ground in
a Tecator cyclone sample mill to pass a 0É5 mm sieve.
One batch each from the 1995 (in a desiccator) and the
1988 harvest were stored whole and ground (0É5 mm)
prior to analysis. All results are based on duplicate
analysis with \5% and \8% as highest acceptable differences between duplicate samples for HPLC analyses
and LC-MS calculated ratios, respectively.
To investigate the extraction efficiency, the dehulled
peas (0É5 g) were extracted in a shaker at room temperature for 2, 4, 6, 8 and 10 h in 80% aqueous ethanol,
100% acetonitrile or 100% methanol (5 ml). The
samples were centrifuged (875 ]g, 10 min) and Ðltered
(0É45 lm) prior to analysis by HPLC.
Ratios of soyasaponin I to DDMP-conjugated
soyasaponin I
The ratios of the area of the molecular ion of soyasa-
E†ect of storage and extraction on soyasaponins
Fig 2. MS/MS product ion spectrum of m/z 943 (soyasaponin I).
ponin I ([MH]` \ 943) to the corresponding area of
the DDMP-conjugated soyasaponin I ([MH]` \ 1069)
were analysed using LC-MS after extraction of the
dehulled peas at room temperature in 80% ethanol for
0É5, 2, 4, 6, 12, 18 and 24 h. The ratios were also
analysed after 0É5 h extraction in 80% ethanol, followed
by Ðltration and standing at room temperature for 1É5,
5É5, 11É5, 17É5 and 23É5 h prior to analysis.
Saponins were analysed by HPLC on a YMC (YMC
Inc., Wilmington, NC, USA) J-sphere ODS-L80 S-4 lm
(4É6 ] 250 mm) steel cartridge. After initial scanning
with a photodiode array detector, detection was carried
out at 205 and 292 nm. The following equipment was
used : Waters 600 E Multisolvent Delivery System
(Waters Associates, Milford, CA, USA, LDC spectromonitor 3100 (LDC Analytical, Riviera Beach, FL,
USA), Waters 991 Photodiode Array Detector, BioRad
AS-100 autosampler (BioRad Inc, Hercules, CA, USA)
and SP 4270 integrator (Spectra Physics, San Jose, CA,
USA). The column was eluted at a Ñow rate of
1 ml min~1 starting with 35% CH CN followed by a
linear gradient to 60% CH CN in 40 min ; a concentra3
tion of 40 mM acetic acid was maintained throughout
For post-column collection for MS/MS experiments
the 80% ethanol extract was eluted through a YMC
Inc. J-sphere ODS-L80 S-4 lm (10 ] 250 mm) steel cartridge with a Ñow rate of 2É4 ml min~1. All other conditions were the same as above. The fractions (26É71 and
27É37 min) were collected over dry ice and immediately
stored in a freezer ([30¡C). Prior to analysis the acetonitrile was evaporated under nitrogen, and the sample
LC-MS experimental
A Hewlett-Packard Model 1050 LC pump (Palo Alto,
CA, USA) was used to provide linear gradients and a
constant Ñow rate of 200 ll min~1. All chromatography
was performed on a YMC Inc. J-sphere ODS-L80 LC
column (2 ] 250 mm). Chromatographic elution for
positive ion electrospray analysis started with 18%
CH CN followed by a linear gradient to 81% CH CN
in 30 min ; a concentration of 40 mM formic acid was
maintained throughout elution. Under these conditions
soyasaponin I elutes at 20É0 min and DDMPconjugated soyasaponin I at 22É1 min. A Finnigan
Y D Daveby et al
Model TSQ-7000 triple-quadrupole mass spectrometer
with the standard Finnigan electrospray ion source was
used for ionisation of the analytes. Nitrogen was used as
a nebulising gas and the capillary temperature was
225¡C. The instrument was scanned over the range of
600È1400 amu at 1 s per scan. The entire 200 ll min~1
column effluent was directed into the ion source. For
MS/MS experiments argon was used as the collision gas
along with a collision energy of 35 V.
Attempts were made to isolate a standard of DDMPconjugated soyasaponin I. Owing to degradation during
the isolation procedure we were not able to isolate a
pure compound which could be used as a standard.
Chromatographic runs with a photodiode array detector were compared for a standard of soyasaponin I and
the ethanol extract. The soyasaponin I eluted at
26É71 min with a maximum absorbance at 205 nm. The
ethanol extract showed two distinct and well-separated
peaks at 26É71 and 27É37 min, the latter with maximum
absorption at 292 nm and the former with smaller
absorption at 205 nm. These data are in very good
agreement with earlier reports for soyasaponin I and
DDMP-conjugated soyasaponin I (Kudou et al 1992 ;
Massiot et al 1992 ; Yoshiki et al 1995).
MS/MS product ion spectrum of m/z 943 from the
26É71 min collected fraction gave a molecular ion at m/z
943 and fragment ions at 797, 635, 459, 441 and 423
(Fig 2). These are expected fragment ions for soyasaponin I and veriÐes the identify of this peak as soyasaponin
I. MS/MS product ion spectrum of the 27É37 min collected fraction gave a molecular ion at m/z 1069 and
fragment ions at 923, 759, 581, 567, 423, 144 and 126
(Fig 1). These data are in very good agreement with
earlier reports on DDMP-conjugated soyasaponin I
(Kudou et al 1993 ; Yoshiki et al 1994). Together with
the chromatographic appearance the compound was
identiÐed as DDMP-conjugated soyasaponin I.
The yield of DDMP-conjugated soyasaponin I (in
relative area units) from the extraction procedures are
shown in Fig 3. Extracting with 80% ethanol gave the
highest and most stable recovery over the time studied.
The amount of saponin extracted increased up to about
4 h. Extraction times from 4 to 32 h gave similar yields.
Extracting with 100% methanol gave a very low yield of
saponins. One hundred percent acetonitrile did not
extract any detectable amounts of DDMP-conjugated
Both soyasaponin I and DDMP-conjugated soyasaponin I were found in the extracts of mature dehulled
peas. However, the ratio of the two saponins changed
during storage and with di†erent extraction times. In
newly harvested mature dried dehulled peas the ratio of
Fig 3. Yield of DDMP-conjugated soyasaponin I with di†erent extraction solvents. =, Methanol ; K, 80% ethanol.
the areas of the molecular ions of 943 : 1069 after 0É5 h
extraction was 0É063. After storage of the dehulled peas
in a desiccator for 5 and 7 months, the ratio had
increased to 0É19 and 0É24, respectively. When stored
whole in a desiccator and ground without dehulling
after 9 months the ratio was 0É34. The same cultivar of
peas harvested in 1988, stored as whole seeds at room
temperature for 7 years and dehulled and ground prior
to analysis, had a ratio of 0É76. In peas from the 1988
harvest, stored at room temperature for 7 years and
analysed without dehulling, the ratio was 0É78.
The proportion of soyasaponin I to the conjugated
form, also increased with increasing extraction time,
from an initial ratio of 0É24 after 0É5 h in 80% ethanol
to 0É55 after 24 h of extraction (Fig 4). Exclusion of
light during extraction had no e†ect. When extracting
for 0É5 h, Ðltering and then standing at room temperature prior to analysis, the ratio of the areas of the
molecular ions increased in a similar manner but at a
lower initial rate. These results suggest continued
release of DDMP-conjugated soyasaponin I from the
pea extract up to approximately 4 h. Prolonged extraction in 80% ethanol seems to enhance the conversion of
DDMP-conjugated soyasaponin I to soyasaponin I, the
rate after about 4 h being similar with or without the
ground peas in the extract.
The half life of DDMP-conjugated soyasaponin I
(approximately 150 h) was calculated from the results
for the breakdown of DDMP-conjugated soyasaponin I
to soyasaponin I at room temperature after 0É5 h
extraction. The ratio of [MH]` \ 943 : [MH]` \ 1069
during the extraction may be determined by the rate of
extraction for the compounds, half life of DDMPconjugated soyasaponin I and the relative composition
in the starting material. The Ðtted line for the ratio of
E†ect of storage and extraction on soyasaponins
[MH]` \ 943 : [MH]` \ 1069 after storage. With the
ratio in whole peas being similar to the ratio in dehulled
peas, no e†ect of soaking, dehulling or freeze-drying was
found. Neither did exposure to light a†ect the ratio
compared to extraction in darkness.
It is thus suggested that not only heating or prolonged extraction but also storage after harvesting releases
soyasaponin I from the DDMP-conjugate form, which
could be due to natural enzymatic processes in the cotyledon. Further evaluation of the compositional changes
of soyasaponins during storage and extraction is
4. E†ect
[MH]` \ 943 : [MH]` \ 1069. =, Extracted in 80% ethanol
for 0É5 h and then standing in 80% ethanol at room temperature ; K, extraction with 80% ethanol. The Ðtted line represents the ratio obtained by simulation of the course of
[MH]` \ 943 : [MH]` \ 1069 after extraction in 80%
ethanol in Fig 4 represents the ratio obtained by simulation of the course of extraction using the previously
calculated half-life as well as the optimised rate of
extraction and starting ratio.
Earlier results from Glycine max seeds (Kudou et al
1993), Phaseolus coccineus hypocotyls (Yoshiki et al
1994) and L upinus angustifolius seeds (Ruiz et al 1995)
showed only the DDMP-conjugated soyasaponins to be
present after mild extraction. In 7-day-old aetiolated
pea seedlings only DDMP-conjugated soyasaponin I
was reported and soyasaponin I was not found in the
free form. The concentration of DDMP-conjugate was
higher in the hook and root tip than in other nongrowing tissues (Tsurumi et al 1992). This study also
reported release of soyasaponin I from the DDMPconjugated form when extracting frozen tissues or with
prolonged extraction of unfrozen tissues. Soybean hypocotyls extracted for 30 min at room temperature had
DDMP-conjugated saponins I and II as major compounds whereas after extraction for 5 h at 80¡C the
major compounds were soyasaponin I and II (Kudou et
al 1992). Heating of DDMP-conjugated soyasaponins
at 100¡C for 1 h completely converted them into the
corresponding soyasaponins (Kudou et al 1994).
DDMP-conjugated soyasaponin I was the only saponin
detected in unprocessed chickpeas and lentils. Soaking
of the seeds for 24 h at 25¡C at di†erent pH values did
not a†ect the saponin composition (Ruiz et al 1996).
The present study indicates that both soyasaponin I
and the DDMP-conjugate are present in the mature
dehulled peas, with an increasing ratio of the areas of
The authors are indebted to those at the US Food and
Drug Administration who provided the opportunity to
perform this work. Special thanks to Dr Samuel W
Page for placing the resources of his research group at
the authors disposal and to Dr James A Sphon for
allowing access to the LC-MS instrumentation used in
these studies.
Cheeke P R 1996 Biological e†ects of feed and forage saponins and their impacts on animal production. In : Saponins
used in Food and Agriculture, ed Waller G R & Yamasaki
K. Plenum Press, New York, USA, pp 377È385.
Curl C L, Price K R, Fenwick G R 1985 The quantitative
estimation of saponin in pea (Pisum sativum L.) and soya
(Glycine max). Food Chem 18 241È250.
Daveby Y D, Óman P, Betz J M, Musser S M, Obermeyer W
R 1997 The variation in content and changes during development of soyasaponin I in dehulled Swedish peas (Pisum
sativum L). J Sci Food Agric (In press).
Kudou S, Tonomura M, Tsukamoto C, Shimoyamada M,
Uchida T, Okubo K 1992 Isolation and structural elucidation of the major genuine soybean saponin. Biosci Biotechnol Biochem 56 142È143.
Kudou S, Tonomura M, Tsukamoto C, Uchida T, Sakabe T,
Tamura N, Okubo K 1993 Isolation and structural elucidation of DDMP-conjugated soyasaponins as genuine
saponins from soybean seeds. Biosci Biotechnol Biochem 57
Kudou S, Tonomura M, Tsukamoto C, Uchida T, Yoshikoshi
M, Okubo K 1994 Structural elucidation and physiological
properties of genuine soybean saponins. In : Food Phytochemicals for Cancer Prevention I. Fruit and V egetables, ed
Huang M-T et al . American Chemical Society, Washington
DC, USA, pp 340È348.
Massiot G, Lavaud C, Benkhaled M, Le Men-Olivier L 1992
Soyasaponin VI, a new maltol conjugate from alfalfa and
soybean. J Nat Prod 55 1339È1342.
Okubo K, Iudou S, Uchida T, Yoshiki Y, Yoshikoshi M, Tonomura M 1994 Soybean saponin and isoÑavonoids. In :
Food Phytochemicals for Cancer Prevention I. Fruit and
V egetables, ed Huang M-T et al. American Chemical
Society, Washington DC, USA, 330È339.
Price K R, Curl C L, Fenwick G R 1986 The saponin content
and sapogenol composition of the seed of 13 varieties of
legume. J Sci Food Agric 37 1185È1191.
Price K R, Fenwick G R 1984 Soyasaponin I, a compound
possessing undesirable taste characteristics isolated from
the dried pea (Pisum sativum L) J Sci Food Agric 35 887È
Ruiz R G, Price K R, Rose M E, Rhodes M J C, Fenwick G R
1995 Determination of saponins in lupin seed (L upinus
angustifolius) using high-performance liquid chromatography : comparison with a gas chromatographic method. J
L iquid Chromatogr 18 2843È2853.
Ruiz R G, Price K R, Arthur A E, Rose M E, Rhodes M J,
Fenwick R G 1996 E†ect of soaking and cooking on the
saponin content and composition of chickpeas (Cicer
arietinum) and lentils (L ens culinaris). J Agric Food Chem 44
Y D Daveby et al
Tsurumi S, Tsujino Y 1995 Chromosaponin I stimulates the
growth of lettuce roots. Physiologica Plantarum 93 785È789.
Tsurumi S, Takagi T, Hashimoto T 1992 A y-pyronyl-triterpenoid saponin from Pisum sativum. Phytochemistry 31
Yoshiki Y, Kim J-H, Okubo K 1994 Saponins conjugated
with 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one
from Phaseolus coccineus. Phytochemistry 36 1009È1012.
Yoshiki Y, Kim J H, Okubo K, Nagoya I, Sakabe T, Tamura
N 1995 A saponin conjugated with 2,3-dihydro-2,5dihydroxy-6-methyl-4H-pyran-4-one from Dolichos lablab.
Phytochemistry 38 229È231.
Yoshiki Y, Okubo K 1995 Active oxygen scavenging activity
of DDMP (2, 3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran4-one) saponin in soybean seed. Biosci Biotechnol Biochem
59 1556È1557.
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