全 文 :广 西 植 物 Guihaia 32(1):129-133 2012 年 1 月
DOI:10.3969/j.issn.1000-3142.2012.01.025
Application of ESI-MS combined with 1H NMR in
analyzingseed phospolipids of six species of Michelia
TANG An-Jun1,Naomichi BABA2
(1.College of Life Sciences,Chongqing Normal University,Chongqing 400047,China;2.Graduate School
of Natural Science and Technology,Okayama University,Tsushimanaka Okayama 700-8530,Japan)
Abstract:To date,there was no example to authenticate seed phospholipids based on their fingerprinting.By means
of electrospray ionization mass spectrometry(ESI MS)and proton nuclear magnetic resonance(1 H NMR)spectrosco-
py,phospholipids fraction extracted from seeds of six species from the genus Michelia were detected.It was firstly
found that distinct difference in spectral fingerprinting region between m/z895-910in ESI MS and 5.30-5.40mg/
L in 1 H NMR among these six species.Thus,it suggested that the spectral differences shown by ESI MS and 1 H
NMR among seeds of these six species could be applied to identify them.And in a wider sense,for analyzing seed
phospholipids of other plant species,a combination of ESI MS AND NMR was an effective tool.
Key words:Seed phospholipid;ESI MS;NMR;Fingerprinting;Michelia
CLC Number:Q945 Document Code:A Article ID:1000-3142(2012)01-0129-05
*
联合应用ESI MS和1H NMR分析
含笑属植物种子磷脂
唐安军1,Naomichi BAbA2
(1.重庆市重庆师范大学生命科学学院,重庆401331,中国;2.日本冈山大学
自然科学与技术研究生院,冈山县津岛北700-8530,日本 )
摘 要:首次尝试利用电喷雾电离质谱(ESI MS)和氢原子核磁共振(1 H NMR)技术分析了含笑属六种植物
种子的磷脂特性,发现在两个指纹图谱区发现明显的差异,即在质荷比(m/z)895-910(ESI MS)和5.30~5.40
mg/L(1 H NMR)两个特异的区域存在显著差异。这些源于种子磷脂ESI/MS和1 H NMR的谱带差异可以被
用来分析不同植物的种子的磷脂特征。而且,相似地,在更广的层面上,特异性的谱带差异可用于分析其他植
物种子的磷脂组成和特性,辅助鉴定种子。
关键词:种子磷脂;质谱;核磁共振;指纹;含笑属
As for lipids,they provide both the structural ba-
sis of cel membranes and fuel for metabolism.Lipids
consist of diverse types of hydrophobic biomolecules.
One lipid classification system contrasts neutral lipids,
such as sterols and acylglycerols,with polar lipids.
Another scheme contrasts complex lipids,which were
lipids that contain one or more fatty acyl species,with
simple lipids that contain only a single building block,
such as fatty acids or sterols(Fahy et al.,2005).Polar
lipid classes,which include the major membrane lipids,
* 收稿日期:2011-05-06 修回日期:2011-09-008
基金项目:中国科学院生物保护专项(KSCX2-YW-Z-0925);重庆市教委科技项目(KJ100610)[Supported by the Particular Biological Conservation
Foundation of the Chinese Academy of Sciences(KSCX2-YW-Z-0925);Chongqing Education Foundation(KJ100610)]
作者简介:唐安军(1976-),男,湖南永州人,博士,主要从事植物生理与分子生态学研究,(E-mail)tanganjun@mail.kib.ac.cn。
were defined by a specific‘head group’or polar moiety
(Fig.1).Each polar lipid class was composed of vari-
ous molecular species whose fatty acids or other hydro-
carbon portions vary in chain length and degree of sat-
uration(Brügger et al.,1997).In plants,increasing ev-
idence has demonstrated roles for lipids in celular
processes that include photosynthesis,signal transduc-
tion,vesicle traficking,secretion and cytoskeletal rear-
rangement(Halett and Bewley,2002;Wang,2002;
Welti and Wang,2004;Meijer and Munnik,2003).
Presumedly,there was certain relationship between lip-
id profiles and classification of species.To date,al-
though various mass spectrometry(MS)and nuclear
magnetic resonance(NMR)spectroscopy were usualy
the methods of choice for the structure determination
of natural(or synthesized)product and for both identif-
ying and quantifying the metabolites in a cel or tissue
type for being extremely rapid and without bias(Krish-
nan et al.,2005;Ward et al.,2007;Colquhoun,2007).
To our knowledge,NMR spectroscopy in combination
with multivariate analysis has been applied to a number
of classification problems including species and cultivar
discrimination and quality assessment of foods and
herbal medicines(Le Gal et al.,2003;Walis and
Browse,2002;Colquhoun,2007),but few findings on
seed lipids for Michelia plants by means of both MS
and NMR have been reported.Mass spectrometry has
established itself as the method of choice,but comple-
mentary information from other techniques,particularly
NMR spectroscopy was potentialy useful in extending
Fig.1 A general structure of phospholipid with double bonds(olefinic structure)
and chemical shift(δ,mg/L)in NMR spectrophotometry
the coverage of information.
To exactly test whether the new method was reli-
able,we used six species(which were wel classified to
certain systermatic position)from the genus Michelia.
In the investigation,both tandem electrospray ioniza-
tion(ESI)MS and 1 H NMR profiling were harnessed,
which were already wel-suited to generating the neces-
sary data in this study.Seed phospholipid profiles of
M.hedyosperma,M.fulgens,M.yunnanensis,M.cha-
pensis,M.foveolataand M.floribunda were analyzed,
which can be hardly diferentiated by morphological
features(Nooteboom,2000;Liu et al.,2004)by means
of tandem ESI MS and 1 H NMR,using spectral data
on seed lipids of each species as chemical descriptors.
1 Materials and Methods
Mature seeds of M.yunnanensis,M.chapensis,M.
floribundaand M.hedyospernia were colected at the
Magnolia garden of the Kunming Institute of Botany,
Chinese Academy of Sciences(25°07′24″N,102°44′37″
E,Alt.1 982m)on October 3,2009.Seeds of M.fove-
olataand M.fulgens growing in east Yunnan prov-
ince,China were colected(23°22.28′N,103°47.71′E,
Alt.2 103m)on 17October,2009.
Seeds(0.5g)were crashed in a china mortar with
031 广 西 植 物 32卷
pestle,and then total lipids were extracted in a mixture
of CHCl3/CH3OH(2∶1,v/v)with addition of a trace
of butylated hydroxytoluene(BHT)as an anti-oxidant
to prevent further oxidation of non-conjugated olefinic
structure in polyunsaturated fatty acyl group.After e-
vaporation of the solvent under reduced pressure,the
residue was rinsed with acetone to remove neutral lipid
such as cholesterol and triglyceride to obtain polar
phospholipids such as phosphatidylcholine(PC),phos-
phatidylethanolamine(PE),phosphatidylserine(PS),
phosphatidylinsitol(PI),phosphatidylglycerol(PG)and
sphingolipids(Bligh & Dyer,1959).The polar lipid
fraction was dissolved in a mixture of methanol/aceto-
nitrile/water(194∶215∶16)with 0.1%ammonium
acetate to enhance protonation of phospholipids for ESI
MS measurement.The tests of both ESI MS and fol-
lowing 1 H NMR were repeated three times.ESI MS
was conducted using a Perkin-Elmer SCIEX(Thorn-
hil,ON,Canada)API-III tandem quadrapole mass
spectrometer.The sample solution was introduced by
direct infusion for spectral acquisition.Positive ion was
scanned fromm/z500-900for most of the polar phos-
pholipids.1 H NMR spectra were recorded on 600MHz
Varian INOVA UNITY 600spectrophotometer and
chemical shift(δ)were expressed in mg/L given relative
to CD3OD(3.30mg/L).
Fig.2 ESI MS spectra of phospholipid fractions of six species in the genus Michelia
2 Results and Discussion
An ESI MS spectrum for the extract from M.
floridunda was given in Fig.2-A.Although a
number of signals were observed,the complex
spectrum was surprisingly similar to those of phos-
pholipids from other seed species (data not
shown).When a region between m/z895-910was
expanded to a ful scale(Fig.2),distinct differences
in signal patterns were observed among seed spe-
cies investigated(spectra A-F).For example,be-
tween m/z 895-910in spectrum A,there were at
least three kinds of signals at m/z896.6(peak a),
898.6(peak b)and 900.6(peak c).The signal at
m/z896.6(peak c)overlaped a second isotopic peak
of m/z 896.6(peak a).Also,the signal at m/z
900.6(peak c)overlaped a second isotopic peak of
m/z898.6.The first important point was that the
difference in m/zbetween peak a and b,and peak b
and c was equal to 2.This result suggested that
chemical structures of three kinds of phospholipids
(peak a,b and c)were very similar,and the differ-
ences in m/z 2between them indicated that the
1311期 唐安军等:联合应用ESI MS和1 H NMR分析含笑属植物种子磷脂
difference was due to the number of double bond
(Fig.1)since the difference in mass number be-
tween-CH2-CH2-and-CH=CH-was equal to 2.A
similar pattern can be seen in Fig.2B-1Ffor the
other five species.The second important feature
was that,although three peaks at m/z 896.6(peak
a),898.6(peak b)and 900.6(peak c)were found for
al the spectra,their relative intensities were signif-
icantly different among seed species.This indica-
ted that al the seed lipids contained the same three
kinds of phospholipids,their relative amounts were
different species by species.Using this method(e.
g.,ESI MS),profiling of total phospholipid molec-
ular species was considered better to analyze lipid
Fig.3 1 H NMR spectra of phospholipid fractions of six species from Michelia
Solvent:deuteriomethanol;Abscissa:chemical shift(δ,mg/L)
molecular species in seeds.
As described above,the diference in ESI MS
spectrum(m/z885-910)among 6species was supposed
to be originated from the diference in structure of PC
and number of the olefinic bond(Fig.1),similar spec-
tral diference should be observed in 1 H NMR since
non-conjugated olefinic protons(Scheme 1)gave reso-
nance signals specificaly atδ5.3-5.5mg/L in gener-
al.1 H NMR was measured for the 6phospholipid
fractions extracted from each species in deuteriometha-
nol.The 1 H NMR spectrum of the each species was
also found to be highly similar to each other,and they
were those of typical glycerophospholipids.Terminal
methyl group of fatty acyl group at sn-1and 2-positi-
ton on glycerol backbone gave resonance signal at typi-
calyδ0.9mg/L for methyl protons,δ2.0-2.1mg/
L(m,alylic protons;-CH2CH2CH=CH),δ2.8mg/L
(m,bis-alylic protons;-[CH=CH-CH2-CH=CH])
andδ5.30-5.40mg/L(m,olefin protons;CH=CH)
andδ5.24-5.29pm for a methyne proton(Baba et
al.,2001;Yamomoto et al.,2007)(Fig.1).Unfortu-
nately,analyses of multiplet signals atδ5.3-5.5mg/
L for olefine protons was not possible since the multi-
plicity was due to proton-proton coupling,magnetic
non-equvalence of each olefine proton and overlap of
olefinic signals from diferent phospholipids.Although
significant diferences seen between spectra,they were
not as clear as ESI MS spectra in Fig.2.The diference
between spectrum B and F in Fig.3was not clear.An-
other feature seen from Fig.3was the relative intensity
of the olefinic proton signals betweenδ5.29-5.40
mg/L and those of multiplet signals of the hydrogen
231 广 西 植 物 32卷
(5.25mg/L)on sn-2carbon of glycerol moiety in
phospholipids.For example,the spectrum C(M.yun-
nanensis),the ratio was 9∶1and in spectrum E(M.
foveolata),the ratio was 27∶1.This indicated that
the total number of double bonds in the phospholipid
fractions of seed lipids from M.yunnanensis was less
than that in the seed lipid fractions fromM.foveolata.
This outcome was consistent with the results from
comparison between the spectra C and E in Fig.2.In
spectrum E,the height of a peak at m/z896.6was ap-
parently higher than those at m/z 898.6and 900.6
comparing from the relative intensity of the peak at m/
z896.4in the spectrum C,indicating that the number
of double bonds in phospholipid fractions from M.fo-
veolata appears to be more than those in phospholipid
fractions fromM.yunnanensis.Similarly,NMR finger-
printings were applied to discrimination of three Ephe-
dra species(Kimet al.,2005)and Strychnos species
(Frédérichet al.,2004).Based on our results and oth-
er studies published,a successful model could be estab-
lished for every species,better in combination with MS
spectral data.
As observed from the ESI MS and 1 H NMR spec-
tra of 6species,the phospholipids patterns were simi-
lar,but their fingerprint regions were significantly dif-
ferent from one species to another.Therefore,it was
possible that spectral diference may be utilized to ana-
lyze seed phospholipids species and to a certain extent,
potentialy used to classify plant species.It should be a
valuable approach to detect lipid based on spectral ana-
lytical methodology in seeds in the future.
Acknowledgements We would like to express our
warmest gratitude to Professor Kyozo Chiba,the Presi-
dent of Okayama University through the Center of Ex-
celence of Okayama University for providing financial
support to do laboratory studies.Also,we thank the
laboratory of SC-NMR and the laboratory of the API
Ⅲ Mass spectrometry in Okayama University,Japan.
This work was partly supported by the Chinese Acade-
my of Sciences(KSCX2-YW-Z-0925)and Chongqing
Normal University(09XLB016).
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3311期 唐安军等:联合应用ESI MS和1 H NMR分析含笑属植物种子磷脂