Research Article
Received: 9 March 2011;
Revised: 9 August 2011;
Accepted: 13 August 2011
Published online in Wiley Online Library: 26 September 2011
(wileyonlinelibrary.com) DOI 10.1002/pca.1360
Screening and Determination for Potential
a-Glucosidase Inhibitors from Leaves of
Acanthopanax senticosus Harms by Using
UF-LC/MS and ESI-MSn
Hui Zhou,a,c Junpeng Xing,a Shu Liu,a,c Fengrui Song,a Zongwei Cai,b
Zifeng Pi,a Zhiqiang Liua* and Shuying Liua
ABSTRACT:
Introduction – Acanthopanax senticosus Harms is a typical Chinese herb with flavonoids existing in all parts of the plant but
with the largest content in leaves. However, leaves have been neglected in past research. To investigate the potential use of
leaves of A. senticosus Harms for discovering lead compounds to treat type 2 diabetes, the herb leaves were selected for
screening the potential of a-glucosidase inhibitors.
Objective – To screen for candidates of a-glucosidase inhibitors from leaves of A. senticosus Harms and evaluate the structure–
activity relationship of the a-glucosidase inhibitors.
Methodology – Ultrafiltration liquid chromatography/mass spectrometry (UF-LC/MS) assay was developed for screening candidates of a-glucosidase inhibitors from leaves of A. senticosus Harms. The interesting compounds were identified by using
reversed-phase high performance liquid chromatography with diode array detector and electrospray ionisation multiplestage tandem mass spectrometry (RP-HPLC-DAD-ESI-MSn), and confirmed by using electrospray ionisation Fourier transform
ion cyclotron resonance multiple-stage tandem mass spectrometry (ESI-FT-ICR-MSn). Furthermore, the a-glucosidase inhibitory activity of the compounds detected was estimated using in vitro assays.
Results – Eight compounds that might bind to a-glucosidase were screened and seven of them were identified successfully.
The a-glucosidase inhibitory activity of the related compounds in leaves of A. senticosus Harms was determined.
Conclusion – The results obtained provided new information for the discovery of potential a-glucosidase inhibitors and the
potential anti-diabetic application of the leaves of A. senticosus Harms. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: Ultrafiltration LC-MS; a-glucosidase inhibitors; leaves of Acanthopanax senticosus Harms
Introduction
Phytochem. Anal. 2012, 24, 315–323
* Correspondence to: Z. Liu, Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences, Changchun Center of Mass Spectrometry,
Changchun, 130022, P.R. China. E-mail: [email protected]
a
Changchun Center of Mass Spectrometry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun, P. R. China
b
Department of Chemistry, Hong Kong Baptist University, Hong Kong, SAR
China
c
Shenzhen Institute for Drug Control, Shenzhen, P. R. China
Copyright © 2011 John Wiley & Sons, Ltd.
315
Diabetes mellitus (DM), a chronic metabolic disorder, has become
a worldwide health problem. According to the World Health Organisation (WHO), approximately 171 million people worldwide suffered from diabetes mellitus in 2000, with an estimated 366 million
predicted in 2030 (Wild et al., 2004). Unfortunately, the current
treatment against DM is limited to a weak control of exacerbation.
The demand for effective therapeutic approaches to treat type 2
diabetes is increasing. a-Glucosidase inhibitors that have been
widely used in the treatment of patients with type 2 diabetes
could delay the absorption of carbohydrates from the small intestine and thus have a lowering effect on postprandial blood glucose and insulin levels (van de Laar et al., 2005). Although synthetic
a-glucosidase inhibitors exemplified by acarbose have been
approved for clinical use in the control of type 2 diabetes, adverse
side effects such as abdominal discomfort, flatulence, and diarrhoea were observed (Kihara et al., 1997). Hence, naturally existing
a-glucosidase inhibitors have attracted considerable interest for
treating type 2 diabetes mellitus.
As an important class of natural products, flavonoids have been
used in the treatment of type 2 diabetes mellitus because some of
them contain a considerable portion of a-glucosidase inhibitors
(Shibano et al., 2008). Acanthopanax senticosus Harms is a typical
Chinese herb with flavonoids existing in all parts of the plant,
but with the largest content in leaves. This herb has been
commonly used as an ingredient in folk medicine for the treatment of a variety of human diseases such as ischemic heart
diseases, hypertension, rheumatic arthritis, tumours, etc. (Chen
et al., 2002). Moreover, the extract of A. senticosus Harms leaves
was found to have the highest a-glucosidase inhibitory activity.
For the investigation on the potential use of leaves of A. senticosus
Harms for lead compound discovery to treat type 2 diabetes, the
herb was selected to investigate their potential for a-glucosidase
inhibitors.
H. Zhou et al.
In vitro methods applied to the fractionated extracts of medicinal herbs have been commonly used to screen for a-glucosidase
inhibitors. However, the assays based on fractionation required
multiple-step isolations of active compounds and needed accompanying conventional structure elucidation analyses, which are
time-consuming and labour intensive (Mbaze et al., 2007). In addition, most of the in vitro screening techniques are based on optical
or radioactive detection, which may be affected by matrix interference, especially when complex samples are analysed.
To overcome the limitations of the in vitro screening
assays and enhance the throughput of drug discovery, mass
spectrometry (MS)-based techniques have been widely applied
in recent years, including size-exclusion chromatography
LC/MS (Wabnitz and Loo, 2002), ultrafiltration (UF)-LC/MS (Liu
et al., 2007), frontal affinity chromatography LC/MS (Ng et al.,
2005), capillary MS (Hodgson et al., 2005), surface plasmon
resonance (SPR)-MS (Marchesini et al., 2008), matrix-assisted
laser desorption/ionisation (MALDI) time of flight (TOF) MS
(Hannewald et al., 2006), and quantitative MALDI-Fourier transform (FT) MS (Xu et al., 2008). UF-LC/MS has been proven to be
a powerful tool for screening biologically active compounds
from botanical extracts because the ultrafiltration step facilitates the separation of ligand-receptor complexes from unbound compounds, followed by LC/MS identification of the
ligands. LC-coupled multiple-stage MS has been successfully
applied to the identification of constituents in herbal extract,
particularly discrimination of the isomers (Lin et al., 2007). Low
sample consumption, no need for immobolisation and reuse of
enzymes can be considered as the most important advantages
of UF-LC/MS for high throughput screening and identificaton
of acive compounds.
In our previous work, a UF-LC/MS assay was developed for
screening and characterising a-glucosidase inhibitors from hawthorn leaf flavonoids extract (Li et al., 2009). This powerful tool
was applied successfully to the rapid screening of a-glucosidase
inhibitors from leaves of A. senticosus Harms in this study. To
identify the potential a-glucosidase inhibitors, the chromatographic retention time, UV spectra, ESI-MSn and high resolution
MS data from Fourier transform ion cyclotron resonance mass
spectrometry (FT-ICR-MS) analysis were utilised and compared
with those of authentic standards as well as those from the literatures reported. Not only the chemical structures but also
the inhibitory activities were determined and associated with
the functional groups of the constituents.
Experimental
Materials
316
Leaves of A. senticosus Harms were collected from Baishan (Jilin, China)
in July and were identified by Professor Shumin Wang, Chang Chun
University of Chinese Medicine, Jilin Province, where a voucher specimen
is deposited. a-Glucosidase (E.C. 3.2.1.20) from yeast was obtained
from Fluka (Bueke, Switzerland). Acetonitrile and acetic acid were of
HPLC grade from Fisher Chemicals (Fair Lawn, USA). Milli-Q water
(Millipore, MA, USA) was used in all the experiments. All other chemicals
were of analytical grade from Beijing Chemical Engineering Company
(Beijing, China). Standards of quercetin, quercitrin, rutin, hyperin,
3-caffeoylquinic acid, 1,3-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid,
4,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic
acid were purchased from the Chinese Authenticating Institute of Material
Medical and Biological Products (Beijing, China).
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Preparation of extract of Acanthopanax senticosus Harms
leaves
The dried leaves of A. senticosus Harms (250 g) were crushed and
extracted three times by reflux with 2500 mL volume of 60% ethanol
for 2 h. After filtration, the combined extracts were concentrated under
reduced pressure and redissolved in water. The crude extract was then
eluted on D130 macroporous resin by eluting stepwise with water and
50% ethanol. Then, the elution with 50% ethanol was concentrated to
dryness by rotary vaporisation at 50 C under reduced pressure, and
the residue was obtained for the analysis.
Determination of a-glucosidase inhibitory activity
The a-glucosidase inhibition assay was performed according to the modified method of Li et al. (Li et al., 2009). A total 100 mL reaction mixture
containing 40 mL of test compounds in phosphate buffer and 40 mL of
10 mM phosphate buffer (pH 6.80) containing 0.04 U/mL a-glucosidase
were added to each well incubated at 37 C for 5 min, followed by
20 mL of 0.5 mM p-nitrophenyl a-D-glucopyranoside (PNP-G) (Biochem)
to the mixture of treatment terminated wells. The plate was incubated
at 37 C for 30 min, and then 100 mL of 0.1 M sodium carbonate solution
were added to stop the reaction. Immediately following that, the absorbance was recorded at 405 nm with a Tecan GENios multifunctional
microplate reader (Männedorf, Switzerland). Controls contained the
same reaction mixture except the same volume of phosphate buffer
was added instead of a solution of test compounds. Acarbose (Bayer)
was used as the positive control. The inhibition (%) was calculated as:
(A1 – A2)/A1 100%, where A1 is the absorbance of the control, and A2
is the absorbance of the sample tested.
Screening procedures of UF-LC/MS
The principle of the UF-LC/MS screening based on MS is as follows. A
mixture of compounds is injected into the ultrafiltration cell containing
a solution of macromolecular receptor, which in this experiment is
a-glucosidase. Components with an affinity to the receptor are bound.
The solution is subjected to ultrafiltration, facilitating the removal of
the unbound compounds with low-molecular weight from the system.
Subsequently, destabilising conditions facilitate the release of the bound
ligands from the receptor. Receptor-ligand binding is then disrupted
through a pH change or addition of an organic solvent, and the released
ligands are further analysed via LC/MS.
Leaves of A. senticosus extract (1 mg/mL, 50 mL) were incubated for
0.5 h at 37 C with 50 mL of 40 mM a-glucosidase (E.C. 3. 2. 1. 20) in
10 mM ammonium acetate buffer (pH 6.86). After incubation, the binding
mixture was filtered through the ultramembrane filter (Microcon YM-10,
Millipore, Bedford, MA) according to the modified method of Sun et al.
(2005), and centrifuged at 12000 rpm for 10 min at room temperature.
The filter was washed three times by centrifugation with 100 mL aliquots
of ammonium acetate buffer (pH 6.86) to remove the unbound compounds. The bound ligands were released by adding 100 mL of methanol:
water (50:50, v/v) (pH 3.30) followed by centrifugation at 12000 rpm for
15 min, which was repeated three times. The control experiments were
carried out in a similar manner with denatured enzyme. All the binding
assays were performed in duplicate and analysed three times.
HPLC-DAD-ESI-MSn analysis
The released ligands were redissolved in 50 mL of methanol:water (50:50,
v/v). Aliquots (10 mL) of this reconstituted ligand solution were analysed
by LC/MS, which consisted of a Waters (Milford, MA) 2690 HPLC system
coupled to a LCQTM ion trap mass spectrometer (Finnigan, San Jose,
CA, USA). HPLC separation was carried out using a C18-column (150 mm
4.6 mm, 5 mm, Agilent). The column temperature was controlled at 28 C.
The flow rate was set to 0.5 mL/min and the eluting gradient was as
follows (acetonitrile (A) and 0.5% acetic acid (B)): t = 0–14 min, 16% A;
Copyright © 2011 John Wiley & Sons, Ltd.
Phytochem. Anal. 2012, 24, 315–323
Screening and Determination of a-Glucosidase Inhibitors by MS
t = 14–30 min, 16–40% A. The mass spectrometer was operated in the
negative ion mode with a spray voltage of 5.0 kV. The metal capillary
voltage was set to 5.0 V and temperature set to 230 C, the sheath gas
(N2) flow rate was 50 arb, the scan range was m/z 100–1000 Da. The HPLC
was connected to the mass spectrometer via the UV cell outlet.
ESI-FT-ICR-MSn analysis
Mass spectrometry experiments at high mass resolution were performed
on an Ion-Spec Ultima 7.0 T FT-ICR-MS (Ion-Spec, USA) with a Waters
Z-spray source. The capillary voltage was set at 3.0 kV. The source
heater was set at 100 C and the probe heater at 80 C. The sustained
off resonance irradiation collision-induced dissociation (SORI-CID) was
performed at +2000 Hz offset frequency with SORI at 3 V. The operating
software was IonSpec99 version 7.5.10.64. All acquisitions were performed on a 1024 K data set and one scan.
Screening of a-glucosidase ligands in the leaves of
Acanthopanax senticosus Harms by UF-LC/MS
Figure 1A shows the LC-chromatogram of A. senticosus Harms
leaves. It is clear that 13 constituents were separated and
detected within 30 min. After incubation with a-glucosidase and
ultrafiltration affinity purification, the trapped ligands in leaves
of A. senticosus Harms were analysed by HPLC. After the specific
binding to a-glucosidase, the peaks showed higher intensities
for the compounds incubated with a-glucosidase than those of
control samples incubated with denatured enzyme. As shown in
Fig. 1B, eight trapped ligands (compounds 3, 4, 5, 6, 7, 8, 10 and
11) from leaves of A. senticosus Harms were observed. The compounds 1, 2, 9, 12 and 13 were not considered as a-glucosidase
ligands because they could not be distinguished from the control
sample in the ultrafiltration screening assay.
Identification of a-glucosidase inhibitors by LC-DAD-MSn
Results and Discussion
Evaluation of a-glucosidase inhibitory activity
The extracts of A. senticosus Harms leaves inhibited the aglucosidase activity by 73.83% at a concentration of 1 mg/mL,
which was evaluated by in vitro assays. Comparing the activity
of the acarbose (78.90% inhibition at 1 mg/mL), the data
obtained clearly demonstrated the potential application of using
A. senticosus Harms leaves for the determination of a-glucosidase
inhibitors. Therefore, it would be valuable if the active compounds in the herbal plant could be screened and identified.
In order to identify the ligands screened from leaves of A. senticosus
Harms, LC/MSn analysis was performed. Prior to the analysis, the
ESI-MS parameters including electrospray voltage, capillary voltage and capillary temperature were optimised for the ligands.
The mass spectral data of the A. senticosus Harms leaves extract
were obtained in negative ion mode, which provided more
structural information than those obtained in positive ion mode.
The data related to retention time (tR), UV spectra and ESI-MSn
data are summarised in Table 1. By comparing retention time
(tR), UV spectra and ESI-MSn fragmentation patterns with those
of the corresponding reference compounds and the literature,
Phytochem. Anal. 2012, 24, 315–323
Copyright © 2011 John Wiley & Sons, Ltd.
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317
Figure 1. LC chromatogram of extract of Acanthopanax senticosus Harms leaves (A) before and (B) after performing ultrafiltration.
H. Zhou et al.
seven ligands were identified, within that four compounds were
found to be flavonoids and three others could be determined
as phenolic acids. The detailed compound identification is
discussed below.
Compound 3 gave a [M H] ion at m/z 609 in the full scan
MS. It could yield the product ions at m/z 301 with a relative
abundance of 100% as well as m/z 300 with a relative abundance
of 35% in the ESI-MS2 experiment by using a CID energy of 23 eV.
The ion at m/z 301 with highest abundance could be related to
the elimination of rhamnosylglucoside (146 Da + 162 Da), while
the ion at m/z 300 could be considered to lose one more proton.
Ions with the loss of one sugar moiety were not observed in
the mass spectrum, which might be due to the relatively
high collision energy. Furthermore, we applied a CID energy
of 23 eV to perform the ESI-MS3 experiment for the ion at
m/z 301. The daughter ion at m/z 179 could be observed
resulting from cleavage of ring C, corresponding to the neutral
molecule of 122 Da. Then elimination of CO could produce
the ion at m/z 151 (Scheme 1). By comparing the retention time,
[M H]- ion and fragmentation pathway with those of the
Table 1. LC/MSn data in the negative ion mode of a-glucosidase ligands from leaves of Acanthopanax senticosus Harms
Peak
tR
(min)
3
12.53 254, 353
4
13.05 254, 335
5
15.17 254, 353
6
16.32 254, 353
7
19.62
218,
241, 328
8
22.34
216,
241, 328
10 23.68 254, 353
11 25.68
MSn (m/z) and relative
abundance (%)
UVlmax
(nm)
220,
242, 328
318
MS: 609(100)
MS2[609]: 301(100), 300(35)
MS3[609 ! 301]:179(100), 151(62),
273(18), 257(13), 229(8), 193(5)
MS: 609(100)
MS2[609]: 301(100), 300(35)
MS3[609 ! 301]:179(100), 151(60),
273(18), 257(13), 229(5), 193(5)
MS: 463(100)
MS2[463]: 301(100), 300(40)
MS3[463 ! 301]:179(100), 151(73),
273(15), 257(13), 229(8), 193(5)
MS: 463(100)
MS2[463]: 301(100), 300(45)
MS3[463 ! 301]:179(100), 151(75),
273(15), 257(13), 229(6), 193(6)
MS: 515(100)
MS2[515]: 353(100), 299(55), 203(45),
317(30), 335(20), 255(18), 173(6)
MS3[515 ! 353]:173(100), 179(60),
191(50)
MS: 515(100)
MS2[515]: 353(100), 191(28), 335(6)
MS3[515 ! 353]:191(100), 179(5)
MS: 447(100)
MS2[447]: 301(100), 300(45)
MS3[447 ! 301]:179(100), 151(75),
273(15), 257(13), 229(8), 193(5)
MS: 515(100)
MS2[515]: 353(100), 299(15), 203(14),
317(10), 255(7), 173(6), 179(5), 135(5)
MS3[515 ! 353]: 173(100), 179(70),
191(30), 135(10)
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reference compound, compound 3 could be identified as rutin
(quercetin-3-O-rhamnosylglucoside). With regard to compound
4, interestingly, the multistage MS spectra showed the same
result as compound 3. By taking the UV absorption value into
account, compound 3 at the retention time of 12.53 min
exhibited the highest absorption wavelengths of 254 nm and
353 nm, whereas compound 4 at the retention time of
13.05 min shifted its highest absorption at the wavelengths of
254 nm and 335 nm, suggesting that compounds 3 and 4 could
be the isomers. However, for more confirmation, compound 4
should be identified by isolation of the pure compound.
In the MS spectra, both compounds 5 and 6 exhibited their
[M H] ions at m/z 463, which indicated that they were the isomers. In order to identify these two compounds, ESI-MS2 analysis
was employed and it was demonstrated that the two ions at
m/z 463 could produce two ion peaks at m/z 301 and 300,
indicating that both possess the quercetin skeleton. The fragmentation pathway of compound 5 was shown to be similar to
hyperin. The retention time in LC/MS analysis was also confirmed by an authentic standard. Therefore, compound 5 was
identified as hyperin (quercetin-3-O-galactoside), which is one
of the major constituents in leaves of A. senticosus Harms. Interestingly, the ESI-MSn behavior of compound 6 was found to be
the same as that of compound 5. With regard to the literature
(Huang et al., 2010) and the discussion mentioned, compound
6 could be identified as isoquercitrin (quercetin-3-O-glucoside),
which is also in agreement with the result reported by Parejo
et al. (2004). Nevertheless, these compounds could also be
discriminated by the elution order in the LC chromatogram. As
a result, by interpretation of MSn spectra, the proposed fragmentation pathway of rutin and the chemical structures of hyperin
and isoquercitrin are shown in Scheme 1.
Similarly, compound 10 (tR = 23.68 min) was identified as
quercitrin (quercetin-3-O-rhamnoside) by comparing the fragmentation patterns with those of an authentic standard. The
[M H]- precursor ion at m/z 447 gave rise to ions at m/z 301
and 300. In the MS3 spectrum of the ion at m/z 301, ions at
m/z 179, 151, 273, 257, 229 and 193 were observed. All of these
data are well in agreement with those of the quercitrin standard.
Three compounds can be considered to be in a different category. Compounds 7, 8 and 11 eluted at 19.62, 22.34 and
25.68 min gave the same deprotonated ion at m/z 515 in the full
scan MS analysis, which indicated that these three compounds
were the isomers. In the MS2 spectrum, the ion at m/z 515
yielded a neutral loss of a sugar moiety (162 Da) to form the
prominent ion at m/z 353. The ion at m/z 353 could further give
rise to the daughter ions at m/z 191 and 179, which are similar to
those of caffeoylquinic acid (Clifford et al., 2003). As reported
in the literature, (Alonso-Salces et al., 2009), the MS2 ions at
m/z 353 and the MS3 ions at m/z 191 and 179 are characteristic
fragments of dicaffeoylquinic acids and could be assigned as
[M H–caffeoyl]-, [quinic acid–H]-, and [caffeic acid–H]-, respectively. Therefore, compounds 7, 8 and 11 are tentatively characterised as dicaffeoylquinic acid isomers. To the best of our
knowledge, this is the first report of dicaffeoylquinic acids in
leaves of A. senticosus Harms. To differentiate the three isomers,
multiple-stage mass spectrometry was performed.
In the MS2 spectrum, peaks 7 and 11 both gave rise to a
product ion at m/z 173. Clifford et al. (2003) have demonstrated
that the presence of the dehydrated quinic residue ion [quinic
acid–H–H2O] at m/z 173 revealed that one of the caffeoyl
moieties was bonded to quinic acid at position 4. In MS2
Copyright © 2011 John Wiley & Sons, Ltd.
Phytochem. Anal. 2012, 24, 315–323
Screening and Determination of a-Glucosidase Inhibitors by MS
A Rutin
OH
OH
OH
OH
HO
O
O
O
OH
-308
HO
O
O
HO
-122
O
O
OH
OH
OHOH
OH
O- O
OH
Rhamnosylglucoside
m/z 301
O
O-
O
H3C
OH
O
OO
m/z 179
m/z 609
-28
CO
-28
OH
CO
OH
HO
HO
O
O
OH
O-
O-
m/z 151
m/z 273
B Hyperin
C Isoquercitrin
OH
OH
OH
O
HO
O
O-
O
OH
O
HO
OH
OH
OH OH
O
OH
O
O
O-
m/z 609
O
OH OH
O
OH
m/z 609
Scheme 1. (A) Proposed fragmentation pathways of rutin. (B) Chemical structure of hyperin. (B) Chemical structure of isoquercitrin.
spectrum of compound 7, the relative intensities of ions at
m/z 299 and m/z 203 were higher (>40% base peak) than other
ions, and the relative intensities of ions at m/z 335, m/z 317 and
m/z 255 were almost 20% of base peak. By comparison with
the MS data reported in the literature (Clifford et al., 2005),
compound 7 could be identified as 1,4-dicaffeoylquinic acid.
In contrast to peak 7, the relative intensities of ions at
m/z 299, m/z 317, m/z 255 and m/z 203 in the MS2 spectrum of
compound 11 were much more lower (<20% base peak).
Additionally, the fragment ion at m/z 335, as expected, should
be detected if the compound was 3,4-dicaffeoylquinic
acid. The results obtained through the characterisation of
Table 2. ESI-FT-ICR-MSn data of compounds 8 and 11
Peak
8
11
Measured m/z
Phytochem. Anal. 2012, 24, 315–323
Formula element [M H]-
DBE
Error (ppm)
Neutral loss
C9H6O3
C9H8O4
C18H12O6
515.11950
353.08781
335.07724
191.05611
[C25H23O12]
[C16H17O9][C16H15O8][C7H11O6]-
14.5
8.5
9.5
2.5
0
1.4
3.3
0.4
191.05611
179.03498
515.11950
353.08781
317.06668
299.05611
255.06628
203.03498
179.03498
173.04555
135.04515
[C7H11O6][C9H7O4][C25H23O12][C16H17O9][C16H13O7][C16H11O6][C15H11O4][C11H7O4][C9H7O4][C7H9O5][C8H7O2]-
2.5
6.5
14.5
8.5
10.5
11.5
10.5
8.5
6.5
3.5
5.5
0.6
0.06
0
0.06
1.6
0.27
0.20
0.1
0.11
0.17
0.15
C9H6O3
C9H10O5
C9H12O6
C10H12O8
C14H16O8
C16H16O8
C18H14O7
C15H16O10
191.05611
179.03498
173.04555
[C7H11O6][C9H7O4][C7H9O5]-
2.5
6.5
3.5
0.31
0.22
0.29
C9H6O3
C7H10O
5
C9H8O4
Copyright © 2011 John Wiley & Sons, Ltd.
C9H6O3
C7H10O
5
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319
515.11950 >
353.08776
335.07834
191.05618
515.12001 > 353.08776 >
191.05608
179.03499
515.11950 >
353.08783
317.06617
299.05619
255.06633
203.01343
179.03500
173.04552
135.04513
515.11743 > 353.08783 >
191.05605
179.03502
173.04560
Theory m/z
H. Zhou et al.
Figure 2. FT-ICR-MSn in the negative ion mode spectra of compound 8. (A) Full-scan mass spectrum of compound 8. (B) MS/MS of [M H]- ion at
m/z 515. (C) MS3 of the daughter ion at m/z 353.
320
compound 11, however, demonstrated a markedly different
fragmentation pattern from those of 1,4-dicaffeoylquinic acid
or 3,4-dicaffeoylquinic acid. Therefore, compound 11 was
tentatively identified to be 4,5-dicaffeoylquinic acid.
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The same deprotonated molecular ion ([M H]-) at m/z 515
was also found in the full scan MS for compound 8. The
dehydrated quinic residue ion [quinic acid–H–H2O]+ at m/z 173,
however, was not found in the MS2 spectrum of compound 8,
Copyright © 2011 John Wiley & Sons, Ltd.
Phytochem. Anal. 2012, 24, 315–323
Screening and Determination of a-Glucosidase Inhibitors by MS
OH
OH
O
OH
OH
O
OH
PathA
O
OOC
OH
-
OOC
-162
OH
O
OH
OH
-
OOC
O
B
A
OH
-
OH
OH
Caffeoyl
m/z 191.05618
m/z 353.08776
OH
PathB
O
O
OH
OH
OH
-
OOC
OH
OH
OH
O
O
m/z 515.11950
O-
OH
HO
m/z 335.07834
m/z 179.03499
Scheme 2. Proposed fragmentation pathways of compound 8.
suggesting that there was no caffeoyl moiety bonded to quinic
acid at position 4. The presence of the product ion at m/z 335 in
the MS2 spectrum of compound 8 suggests 1,3-dicaffeoylquinic
acid or 1,5-dicaffeoylquinic acid rather than 3,5-dicaffeoylquinic
acid. In MS3 spectrum, the relative abundance of the ion at
m/z 191 was 100% (the base peak), while the relative abundance
of the ion at m/z 179 was much lower (about 5%). Using the hierarchical key developed by Schram et al. (2004), compound 8 was
identified as 1,5-dicaffeoylquinic acid.
Identification of the compounds of peaks 8 and 11 by
ESI-FT-ICR-MSn
Since compounds 8 and 11 were tentatively identified by
multiple-stage mass spectrometry, ultrahigh resolution MS
was needed for confirmation of the elemental composition.
Prior to ESI-FT-ICR-MSn experiments and for more evidence,
compounds 8 and 11 were purified by preparative HPLC. The
MS data, including the proposed product ions, measured and
theoretical masses, double bond equivalents (DBE), measurement error and proposed neutral losses are summarised in
Table 2.
The deprotonated ion at m/z 515.11950 for compound 8 was
confirmed as [C25H23O12]1 ([M H]-). In addition, this result
was supported by SORI-CID analysis (Fig. 2). The proposed fragmentation mechanism is shown in Scheme 2. In the MS2 spectrum of compound 8, product ions [M H–C9H6O3]- at m/z 353,
[M H–C9H8O4]- at m/z 335 and [M H–C18H12O6]- at m/z 191
were detected. In the MS3 spectrum of the ion at m/z 353, product ions at m/z 191 and 179 were observed by losing C9H6O3 and
C7H10O5, respectively. All the above data of compound 8 are
comparable with the literatures cited for 1,5-dicaffeoylquinic
acid (Schram et al., 2004). Similarly, the elemental composition
of compound 11 was confirmed to be C25H24O12. The ultrahigh
resolution MSn data were consistent with the results reported
by Zhang et al. (2007). Thus compound 11 was identified as
4,5-dicaffeoylquinic acid.
Table 3. a-Glucosidase inhibition activity of the related
compounds
Samples
Inhibition (%)a
Quercetin
72.51 (0.2 mM)
Quercitrin (quercetin-3-O-rhamnoside)
71.35
Hyperin (quercetin-3-O-galactoside)
52.51
Rutin (quercetin-3-O-rhamnosylglucoside)
36.62
3-Caffeoylquinic acid
30.53
1,5-Dicaffeoylquinic acid
61.41
1,3-Dicaffeoylquinic acid
35.23
3,5-Dicaffeoylquinic acid
62.51
4,5-Dicaffeoylquinic acid
66.51
3,4-Dicaffeoylquinic acid
68.81
a
Inhibition by 1 mM standards
Less than 40% inhibition at concentration of 1 mM
6
5
IC50
(mM)
0.03
0.4
0.5
NIb
NIb
0.66
NIb
0.63
0.57
0.51
OR4
OR3
HOOC
2
R1 O 1
O
4
O
3
OR2
Quinic acid
Compound
OH
HO
C = Caffeic acid residue
R1
R2
R3
R4
3-Caffeoylquinic acid
H
H
H
H
1,3-Dicaffeoylquinic acid
C
C
H
H
1,5-Dicaffeoylquinic acid
C
H
H
C
4,5-Dicaffeoylquinic acid
H
H
C
C
3,5-Dicaffeoylquinic acid
H
C
H
C
3,4-Dicaffeoylquinic acid
H
C
C
H
b
Copyright © 2011 John Wiley & Sons, Ltd.
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321
Phytochem. Anal. 2012, 24, 315–323
Scheme 3. The chemical structures of the six phenolic acids.
H. Zhou et al.
Evaluation of a-glucosidase inhibition activity of the ligands
identified in leaves of A. senticosus Harms
322
In order to evaluate the structure–activity relationship of
a-glucosidase inhibitors from leaves of A. senticosus Harms,
a-glucosidase inhibitory activity of related compounds was
estimated using in vitro assays. It can be seen from Table 3
that the inhibitory activity of flavonoids was: quercetin
quercitrin > hyperin > rutin. Based on the structure–activity
relationship of these compounds, it was found that quercetin
is the most potent inhibitor compared with its glycosides. A
possible explanation is that the hindrance of the glycosylation group of quercetin weakens the inhibitory activity of
flavones against a-glucosidase. In addition, by detailed comparison of the a-glucosidase inhibitory activity for quercitrin,
hyperin and rutin, it was found that the type of sugar moiety
substituted on ring C of the flavonol quercetin also affected
the inhibitory activity of the corresponding flavonoid glycosides. When the flavonoid aglycones were substituted with
a monosaccharide, the corresponding flavonoid glycosides
exhibited a decreased a-glucosidase inhibitory activity. If substitution of disaccharide occurred, a-glucosidase inhibitory
activity of the corresponding flavonoid glycosides would be
reduced markedly.
Although only three phenolic acids were identified as the
ligands in the UF-LC/MS analysis, we assayed six phenolic acids
for evaluation of the structure–activity relationship. First of all,
the chemical structures of the six compounds were summarised
in Scheme 3. As can be seen in Table 3, the a-glucosidase
inhibition activity of the related phenolic compounds can
be ordered as 3,4-dicaffeoylquinic acid > 4,5-dicaffeoylquinic
acid > 3,5-dicaffeoylquinic acid > 1,5-dicaffeoylquinic acid > 1,3dicaffeoylquinic acid > 3-caffeoylquinic acid, revealing that all
of the five dicaffeoylquinic acids tested exhibited more enhanced a-glucosidase inhibitory activity compared with monocaffeoylquinic acid. Therefore, it is reasonable to assume that
the a-glucosidase inhibition activity of the related phenolic compounds was related to the conjugated rings and hydroxyl
groups. Among the dicaffeoylquinic acid isomers tested,
a-glucosidase inhibition activity of 4,5-dicaffeoylquinic acid and
3,4-dicaffeoylquinic acid was more than those of 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 1,3-dicaffeoylquinic
acid, in which 1,3-dicaffeoylquinic acid showed the weakest
a-glucosidase inhibition activity. Because these five isomers of
dicaffeoylquinic acids are discriminated by the positions of
caffeoyl moieties, the difference in the a-glucosidase inhibitory
activity could be the result of the linkage positions of caffeoyl
groups on the quinic core. The results obtained indicated that
the caffeoyl moieties substitution at position 4 played a crucial
role in the enhanced inhibitory activity. Dicaffeoylquinic acids
in leaves of A. senticosus Harms contributed to the a-glucosidase
inhibition activity, giving support to the validity of dicaffeoylquinic
acids in treating type 2 diabetes mellitus. More recently, Wang
et al. (2008) reported that dicaffeoylquinic acid could inhibit
glucose uptake, demonstrating that dicaffeoylquinic acid was associated epidemiologically with a reduced risk of developing type
2 diabetes mellitus. Therefore, the observation of dicaffeoylquinic
acid binding to a-glucosidase in the present work is a further indication of which dicaffeoylquinic acid can be a potential class of
natural a-glucosidase inhibitors. In addition, comparing the
activity of the related phenolic compounds suggested that the
a-glucosidase inhibition potency was not only dependent on
wileyonlinelibrary.com/journal/pca
the number of caffeoyl groups attached to the quinic core, but
also related to the linkage positions of caffeoyl groups on the
quinic core.
Summary
The results from this study demonstrated that UF-LC/MS is a
powerful tool for rapid screening and detection of a-glucosidase
inhibitors from complex herb extracts. The data obtained on
inhibitory activity and structure–activity relationships may be
valuable for the determination of a-glucosidase inhibitors in
leaves of A. senticosus Harms and the development of efficient
anti-diabetic drugs.
Acknowledgements
The authors would like to thank the financial support from the
State Project of Innovative Approach Research (2009IM030400),
the National Natural Science Foundation of China (No.
20905067, 21005075) and the Science and Technology Foundation of Jilin Province (20080736, 20101501).
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