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Research Article
Structural and Quantitative Analysis of Three
C-Glycosylflavones by Variable Temperature Proton
Quantitative Nuclear Magnetic Resonance
Jing Liu, Yang Liu, Zhong Dai, Lan He, and Shuangcheng Ma
National Institutes for Food and Drug Control, Beijing 100050, China
Correspondence should be addressed to Shuangcheng Ma; masc@nifdc.org.cn
Received October ; Revised December ; Accepted December ; Published January
Academic Editor: Hassan Y. Aboul Enein
Copyright © Jing Liu et al. is is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Quantitative nuclear magnetic resonance is a powerful tool in drug analysis because of its speed, precision, and eciency. In present
study, the application of variable temperature proton quantitative nuclear magnetic resonance (VT-1H-qNMR) for the calibration
of three C-glycosylavones including orientin, isoorientin, and schaoside as reference substances was reported. Since there was
conformational equilibrium due to the restricted rotation around the C(sp3)-C(sp2) bond in C-glycosylavones, the conformational
behaviors were investigated by VT-NMR and veried by molecular mechanics (MM) calculation. e VT-1H-qNMR method was
validated including the linearity, limit of quantication, precision, and stability. e results were consistent with those obtained
from mass balance approach. VT-1H-qNMR can be deployed as an eective tool in analyzing C-glycosylavones.
1. Introduction
Quantitative nuclear magnetic resonance (qNMR) spectrom-
etry was rst reported in the s. is method has been
widely applied to various elds such as drug analysis, ref-
erence substances quality control, and natural products due
to its high speed and precision [–]. qNMR technique has
been adopted in all major national pharmacopoeias including
US pharmacopeia, European pharmacopeia, Japanese phar-
macopeia, and Chinese pharmacopeia [–]. For qNMR,
the resonance signal is directly proportional to the number
of resonant nuclei. erefore, this approach has various
advantages such as no need for reference substances or large
amount of organic solvents.
C-glycosylavone is a unique type of natural product
with various pharmacological eects including scavenging
free radicals and protecting myocardial ischemia [, ].
Although qNMR technique has been widely used in char-
acterization of reference substances of dierent structure
types,thereisnoreportontheC-glycosylavonesdueto
poor response signal from proton NMR. Herein, orientin
(), isoorientin (), and schaoside (), three common
avone C-glycosides with sugar moieties at C6and/or C8
(Figure ), were selected for 1HqNMRstudy.Forthistype
of compounds, the restricted rotation around the C(sp3)-
C(sp2) bond results in the coexistence of rotational isomers
which might complicate the NMR spectrum. Since increasing
temperature will eliminate the carbon-carbon bond rota-
tion barrier, the conformational equilibrium of three C-
glycosylavones was directly characterized by variable tem-
perature NMR (VT-NMR). Meanwhile, the conformational
behaviors of the three C-glycosylavones were investigated
by using molecular mechanics (MM) calculation. Variable
temperature proton quantitative nuclear magnetic resonance
(VT-1H-qNMR) was also applied to directly determine the
content of orientin, isoorientin, and schaoside for the
rst time. e results are consistent with the data from
mass balance method. VT-1H-qNMR method is an eective
approach to achieve satisfactory result for C-glycosylavones.
2. Materials and Methods
2.1. Materials and Analyte Preparations. Orientin (.%),
isoorientin (.%), and schaoside (.%) (determined by
mass balance method) were from National Institutes for Food
Hindawi
Journal of Analytical Methods in Chemistry
Volume 2017, Article ID 4934309, 5 pages
https://doi.org/10.1155/2017/4934309
Journal of Analytical Methods in Chemistry
O
HO
OH O
OH
OH
O
HOHO OH
HO
1
O
HO
OH O
OH
OH
O
OH
OH
HO
OH
2
OHO
OH O
OH
O
OH
HO
HO
HO
O
HO
OH OH
3
2
3
45
6
7
89
10
F : Structures of orientin (), isoorientin (), and schaoside ().
and Drug Control, Beijing, China; ,-dinitrobenzene was
purchased from TCI chemicals (.%, Lot. EUXH-JB).
DMSO-6was from Sigma (.%, St. Louis, USA).
Test samples and internal standard ,-dinitrobenzene
were dissolved in DMSO-6to produce a concentration of
about . mol/mL and . mol/mL, respectively. For linear-
ity, dierent concentration of schaoside ranging from . to
. mg was dissolved in .mL DMSO-6.
2.2. Instrument Conditions. e 1HNMRspectrawereac-
uired at K or K using a Bruker Ascend spec-
trometer with a BBO probe at . MHz. For qNMR, the
following parameters were applied: ∘pulse angle, spectral
width equal to ppm, acquisition time equal to . s,
receiver gain equal to , OP equal to . ppm, K data
points, scans, and relaxation time 1 equal to s. ∘
pulse calibration was conducted daily to make sure of the
performance of NMR spectrometer.
2.3. Processing Parameters. Data was processed on MestReN-
ova .. with . Hz exponential apodization applied to FID.
Manual phase correction and signal integrations were per-
formed corresponding to the IS signals and sample signals.
1H NMR shi was referenced to the solvent signal of DMSO-
d6.
2.4. Content Calculation Formula. e content of sample was
calculated by the following formula:
𝑠(%)=𝑠/𝑠×
𝑠×
𝑟
𝑟/𝑟×
𝑟×
𝑠×
𝑟× 100%,()
where 𝑠and 𝑟are the signal response of the samples and
IS, 𝑠and 𝑟arethenumbersofspinatomsintheanalyteand
IS, 𝑠is the molecular weight of samples (. g/mol for
orientin and isoorientin, . g/mol for schaoside), 𝑟is
the molecular weight of IS (. g/mol), 𝑠and 𝑟are the
masses of the analytes and IS, and 𝑟is the purity of the IS.
3. Result and Discussion
3.1. Experiments Parameters. Forpulseipangle,mostofthe
qualitative proton NMR and some of qNMR experiments
are performed with ∘pulse. Our group use ∘in our
routine 1H-qNMR experiments and get reasonable results.
2.53.54.5
5.56.5
7.58.5
9.5
11.0
1
2
3
f1 (ppm)
F : 1HNMRspectraoforientin(), isoorientin (), and
schaoside ()(K).
Although ∘pulsewillgivebetter/ than ∘,
∘in VT-
1H-qNMR can partly represent the real circumstance in using
1H-qNMR.
As a critical parameter in VT-1H-qNMR experiments,
relaxation time (1)shouldbemorethantimesthatof
longitudinal relaxation (1)toallowtheactivatedprotonto
return to equilibrium status. e 1 values were determined
by an inversion recovery method. 1 of the internal standard
and analyte signal was found to be . s and .s, respectively.
So 1 wassetassinthisstudy.
3.2. Conformational Analysis. Atropisomers occur when ro-
tation around a C-C single bond is hindered by the rotational
energy barrier. For most C(sp2)-C(sp3)singlebond,therota-
tional energy barrier is low, and the isomerism could not be
observed at room temperature. For some C-glycosylavones,
NMR spectra acquired under room temperature showed
signals corresponding to atropisomers at dierent ratio due
to the high rotational energy barrier. e phenomenon was
veried by means of variable temperature NMR experiments
and theoretical MM calculations [–].
During our study, the 1H NMR spectra acquired at K
(Figure)showedsomeimpuritysignalsaroundthearomatic
and anomeric protons for orientin and isoorientin, respec-
tively. And the spectrum for schaoside presented some
signals not splitting well. Considering the structure similarity
Journal of Analytical Methods in Chemistry
T : Linearity, range, and precision of schaoside calculated by VT-1H-qNMR ( K).
Linearity and range Precision Repeatability
Sample 𝑟(mg/mL) 𝑠(mg/mL) 𝑠/𝑟Sample 𝑟(mg/mL) 𝑠(mg/mL) 𝑠(%) No. 𝑠(%)
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . .
2. — —
RSD (%) — . .
2.53.54.55.56.57.58.59.511.0
1
2
3
f1 (ppm)
F : 1H NMR spectra of orientin (), isoorientin (), and
schaoside ()(K).
with those reported [–], the above phenomenon was
inferred from restricted rotation. erefore, VT-1H-qNMR
experiments were carried out in order to verify the deduction.
Increasing the temperature from K to K altered the
1H NMR spectra. e aromatic and anomeric proton signals
appear to undergo coalescence at K for orientin and
isoorientin as shown in Figure . Also the spectr um presented
signals splitting well, especially those around the aromatic
region for schaoside (Figure ).
It was demonstrated that steric hindrance was the main
eect that inuences the rotational isomerism []. In this
study, the dierence between the isomers of orientin and
isoorientin is the dierent position of the glucosyl sub-
stituent. Due to the bigger substitute at C9compared to that
at C5,itwouldbesubjectedtogreaterrotationhindrancefor
orientin compared to isoorientin. As a result, it is obvious that
the 1H NMR spectrum of orientin presented impurity signals
around the aromatic region corresponding to the rotational
isomer. For the C-diglycosylavone of schaoside, it was
obviouslymorediculttoovercometherotationbarrier.e
conformational analysis for three compounds was performed
via molecularmechanicsusingtheMMforceeldinChem-
Bio D Ultra soware (Figure ). Since structures of the three
compounds were dierent, the absolute energy was useless for
comparison, and the energy dierence between conformers
of the same compound is meaningful. e calculated energy
dierence for orientin, isoorientin, and schaoside was .,
., and . kcal/mol, respectively. Bigger energy dier-
ence represents the higher rotational barrier.
3.3. Selection of Sample Signals and IS Signals. ,-Dinitrob-
enzenewasselectedastheinternalstandardduringthe
experiment due to the following reasons: high solubility
and the chemical shi of the aromatic protons provide
a well-separated signal (.) without any interference
with orientin, isoorientin, and schaoside in the integration
region. In our experiments, the singlet signal at . for
orientin and isoorientin and . for schaoside were used
for quantication, respectively (Figure ).
3.4. Method Validation
3.4.1. Linearity and Range. Schaoside was used as a model
compound to validate VT-1H-qNMR.
e solutions for linearity test were prepared by dissolv-
ing dierent amount of schaoside and IS to the required
concentrations. e calibration curve was drawn for the ratio
of sample to IS () versus the ratio of selected sample signal
to IS signal () (Table ). e correction coecient showed it
had good linearity within .∼. mg/mL concentration
ranges ( = 0.068 + 0.007,2= 0.998).
3.4.2. Limit of Quantication (LOQ). It is reported that the
signal to noise ratio (/)shouldbemorethaninquan-
titative experiments to produce good quantication results
[]. Here, / = 150 was used to calculate LOQ. LOQ for
schaoside is . mg/mL.
3.4.3. Precision, Repeatability, and Stability. Precision tests
were carried out by characterizing the same sample six
times. And repeatability was achieved by characterizing ve
independent solutions containing both the sample and IS.
Both RSD indicated the good accuracy of the method. e
stability was assessed by analyzing one sample at -, -, -, -,
and -hour interval. e results indicated that schaoside was
stable aer hours in solution.
Method validation results were summarized (Table ).
Journal of Analytical Methods in Chemistry
123
F : MM computed structures of the lowest energy conformers of orientin (), isoorientin (), and schaoside ().
2.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5
IS
f1 (ppm)
348 K
298 K
F : VT-1H-qNMR spectra of schaoside and internal stan-
dard (IS).
T : Content from VT-1H-qNMR ( K) and mass balance
method (%).
Orientin Isoorientin Schaoside
VT-1H-
qNMR
. (RSD
.%)
. (RSD
.%)
. (RSD
.%)
Mass balance . . .
3.5. Comparison Results from VT-1H-qNMR with Mass Bal-
ance Method (Table 2). e established VT-1H-qNMR meth-
od was applied for the calibration of orientin, isoorientin,
and schaoside. Also the mass balance approach was used for
calculation []. Table shows that the results of the three C-
glycoavones by VT-1H-qNMR are similar to data from mass
balance method.
4. Conclusions
Contents of some avone C-glycosides cannot be achieved
due to the existence of isomers. is study developed a
reliable VT-1H-qNMR method to determine the content of
three common avone C-glycosides: orientin, isoorientin,
and schaoside. Comparing the qNMR method with the
massbalanceapproach,thecontentsoforientin,isoorientin,
andschaosideweresimilar.VT-
1H-qNMR method could be
complementary to the mass balance approach for the value
assignment of the reference substances. is technology is a
powerful tool in drug quality control.
Competing Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Authors’ Contributions
Jing Liu and Yang Liu contributed equally to this work.
Acknowledgments
e investigation was nancially supported by national spe-
cial topic of “Major New Drug Discovery” in th Five-Year
Platform of traditional Chinese medicine quality and safety
testing and risk control, Project no. ZX-.
e authors are thankful to Institute for Reference Standards
and Standardization, NIFDC.
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