- Email: [email protected]
Online screening of nitric oxide scavengers in natural products using high performance liquid chromatography coupled with tandem diode array and fluorescence detection
Accepted Manuscript Title: A novel method for online screening of nitric oxide scavengers in natural products using high performance liquid chromatography coupled with tandem diode array and fluorescence detection Author: Dapeng Li Ting Wang Yujie Guo Yuanjia Hu Boyang Yu Jin Qi PII: DOI: Reference:
S0021-9673(15)01585-X http://dx.doi.org/doi:10.1016/j.chroma.2015.10.095 CHROMA 357010
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
17-8-2015 27-10-2015 27-10-2015
Please cite this article as: D. Li, T. Wang, Y. Guo, Y. Hu, B. Yu, J. Qi, A novel method for online screening of nitric oxide scavengers in natural products using high performance liquid chromatography coupled with tandem diode array and fluorescence detection, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.10.095 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel method for online screening of nitric oxide scavengers in natural
2
products using high performance liquid chromatography coupled with
3
tandem diode array and fluorescence detection
ip t
1
4 5
Dapeng Li b, Ting Wang b, Yujie Guo b, Yuanjia Hu c, Boyang Yu a, b, *, Jin Qi a, b, *
cr
6
a State Key Laboratory of Natural Medicines, China Pharmaceutical University,
8
Nanjing 210009, China.
9
b Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China
us
7
Pharmaceutical University, Nanjing 210009, China
11
c State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
12
Medical Sciences, University of Macau, Macao 999078, China
M
an
10
13
d
14 15
te
16
18 19 20 21 22 23
Ac ce p
17
24 25 26
*Corresponding authors at: Jiangsu Key Laboratory of TCM Evaluation and
27
Translational Research, China Pharmaceutical University, Tongjia Lane 24, Nanjing
28
210009, China. Tel.: +86 25 86185157; Fax: +86 25 86185157.
29
E-mail addresses: [email protected] (Boyang Yu), [email protected] (Jin Qi). 1
Page 1 of 25
30 31
Abstract: Nitric oxide (NO) is an important cellular signaling molecule with extensive
33
physiological and pathophysiological effects. NO scavengers have the potential to
34
treat inflammation, septic shock and other related diseases, and numerous examples
35
have been chemically synthesized or isolated from natural products. The chemical
36
diversity of natural products, however, means that a huge effort is necessary to
37
efficiently screen and identify bioactive compounds, especially NO scavengers. In this
38
article, we propose an effective analytical method to screen for NO scavengers in
39
three natural products using an online system that couples high performance liquid
40
chromatography
41
(HPLC-DAD-FLD). Eighteen compounds from radix of Scutellaria baicalensis
42
Georgi and green tea displayed significant NO scavenging activity whereas
43
components of Pueraria lobata (Willd.) Ohwi had no discernable activity. The
44
structures of the active compounds were elucidated using Agilent Accurate-Mass
45
Q-TOF LC/MS system. Preliminary analysis of structure-activity relationships
46
indicated that, in flavonoids, a 2,3-double bond and a 3-H atom or a 3-OH group are
47
essential for activity. In tannins, poly-hydroxyl groups are important for NO
system, screening
50 51 52
and
fluorescence
detection
d
Ac ce p
54
49
array
te
53
48
diode
an
tandem
M
with
us
cr
ip t
32
scavenging activity. Method validation indicated that the newly developed method is both reliable and repeatable. The online method that we present provides a simple, rapid and effective way to identify and characterize NO scavengers present in natural products.
Key words: natural products, nitric oxide, scavenger, online HPLC-DAD-FLD
55
2
Page 2 of 25
55 56
1. Introduction Nitric oxide (NO) is endogenously released by several different cell types and
58
tissues in vivo during the nitric oxide synthase (NOS)-catalyzed conversion of
59
L-arginine to L-citrulline. This free radical acts as a signaling molecule with dual
60
effects. At low concentrations, NO is essential for regulating vasodilation, blood
61
pressure and neurotransmission, as well as cardiovascular and renal function[1,2].
62
Overproduction of NO, however, can promote inflammatory diseases[2,3] such as
63
arthritis, myocarditis[4], septic shock[5,6] and autoimmune disorders[7]. Correcting
64
the imbalance in physiological and pathological processes by scavenging excess NO
65
may thus provide a strategy for the treatment of inflammatory diseases.
an
us
cr
ip t
57
Numerous NO scavengers have been synthesized or isolated from natural products.
67
For instance, arylnitroalkenes[8], dithiocarbamate[9,10] and carboxy-PTIO[11] are
68
effective in animal models of inflammation and acute septic shock, and
69
edaravone[12,13], has been used in clinical practice. Some natural products, such as
70
Ginkgo biloba extract EGb 761[14], curcumin[15], green tea[16] and Pu-erh tea[17],
71
which were believed to be antioxidants, have now been shown to have the ability to
72
scavenge NO. Although there is ample evidence that constituents from natural
74 75 76 77
d
te
Ac ce p
73
M
66
products have the potential to treat NO-related diseases, it is difficult to screen effectively for NO scavengers. Separation and purification of individual compounds is difficult because of the complex nature of natural products, and analytical methods for determination of NO are challenging because of its reactivity, rapid diffusion and short half-life.
78
Various techniques[18] have, however, been developed to detect NO, including UV
79
spectroscopy, fluorescence spectroscopy, chemiluminescence, electron paramagnetic
80
resonance (EPR) and electrochemistry. In previous studies[16,19], screening for NO
81
scavengers was commonly carried out by mixing the natural product with a NO donor
82
such as sodium nitroprusside, followed by detection of the changes in NO levels. For
83
example, Yoshida et al. evaluated the NO scavenging properties of shikonin using 3
Page 3 of 25
ESR[20]. This method is rapid and accurate, but it is expensive to operate. Kang and
85
co-workers investigated the NO scavenging properties of ginseng using UV
86
spectroscopy[21]. This method, which has been widely used for analysis of a variety
87
of samples, is convenient to operate but other nitrogen oxide species, such as NO3−,
88
will interfere with the determination of NO[18]. Most of the methods currently used
89
for identifying NO scavengers from natural products are time consuming, labor
90
intensive or high cost, and can give rise to ambiguous results. Methods based on
91
hyphenated technologies, combining optimally coordinated separation of natural
92
products and measurement of NO, are likely to provide a solution to these problems.
us
cr
ip t
84
In the present report, we describe a strategy based on high performance liquid
94
chromatography (HPLC) coupled with online tandem diode array detection and
95
fluorescence detection (HPLC-DAD-FLD) (Fig. 1) to systematically identify NO
96
scavengers from natural products. Fluorescence detection is able to specifically,
97
accurately and sensitively detect NO and a large number of fluorescence probes have
98
been developed. One of the most widely used fluorescence methods, because of its
99
low cost and high sensitivity, is based on the reaction of NO with
100
2,3-diaminonaphthalene (DAN)[22]. Our online system comprises an HPLC system
101
together with a detection system. The HPLC system separates the constituents from a
107
Ac ce p
te
d
M
an
93
108
new system is an efficient and high-throughput method for identifying NO
109
scavengers.
102 103 104 105 106
complex natural product mixture and the online detection system determines activity using the fluorescence probe (DAN). NO scavengers can be identified by comparison of the two chromatograms. This novel hyphenated HPLC-DAD-FLD method has been validated and successfully applied to three natural products. The method was used to analyze and characterize the substances and structures as well as to determine the structure-activity relationships of NO scavengers from these natural products. Our
110 111
2. Experimental
112
2.1 Chemicals and reagents 4
Page 4 of 25
113
Nitric oxide gas (99%) was purchased from Nanjing Shangyuan Industrial Gas Co.,
114
Ltd. (Jiangsu, China). DAN was purchased from Aladdin Industrial Inc. (Shanghai,
115
China).
116
N-(1-naphthyl)ethylenediamine dihydrochloride (NEDD) and phosphoric acid were
117
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure
118
water (18.2 MΩ) was obtained using a Milli-Q water purification system (Millipore,
119
Bedford, MA, USA). Solvents used for extractions were analytical grade and
120
purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China).
121
Chromatographic grade acetonitrile and methanol were obtained from Tedia (Fairfield,
122
OH, USA) and Yonghua Chemical Technology Co., Ltd. (Jiangsu, China),
123
respectively.
grade
4-aminobenzenesulfonic
acid
(SULF),
an
us
cr
ip t
Analytical
Samples of rutin and quercetin for use as standards were purchased from Aladdin
125
Industrial Inc. (Shanghai, China). Dried radix of Scutellaria baicalensis Georgi and
126
Pueraria lobata (Willd.) Ohwi are traditional Chinese medicines and were obtained
127
from the local pharmacy, Nanjing ShangyuanTang Pharmacy (Zhushan Road 109,
128
Nanjing). Green tea was purchased from Yipin Xuan Organic Tea Industry Co., Ltd.
129
(Anhui, China). All three crude drugs were authenticated by one of the authors, Prof.
130
Boyang Yu. The voucher specimens (specimens number: S. baicalensis 20150215, P.
136
Ac ce p
te
d
M
124
137
absolute methanol and stored at 4°C to prevent degradation. A mixed standard
138
solution (0.5 mg/mL) was prepared by mixing the two stock solutions in a 1:1 ratio.
131 132 133 134 135
lobata 20150215, Green tea 20150215) were deposited at the Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, China. The dried crude drugs were ground to homogeneous powders.
2.2. Standard solutions
Standard stock solutions (1 mg/mL) of rutin and quercetin were prepared in
139 140
2.3. Extraction process
141
Powdered radix of Scutellariae (2.0 g) was extracted twice with 75% (v/v) aqueous
142
methanol (100 mL) for 30 min with ultrasonication at 100 Hz. The extracts were 5
Page 5 of 25
143
filtered and combined and then evaporated under vacuum at 55°C. The residue was
144
dissolved in dimethylsulfoxide (5 mL) to provide the stock solution. Powdered green tea (1.0 g) was extracted with 70% (v/v) aqueous methanol (30 mL)
145
for 45 min with ultrasonication at 100 Hz. The extract was filtered and used in the
147
following experiments.
Powdered radix of puerariae (1.0 g) was refluxed in 30% aqueous ethanol (v/v) for
148
All solutions were stored at 4°C and filtered through a 0.45 μm membrane before
150 151
cr
30 min. After cooling, the extract was filtered and used in the following experiments.
analysis.
152
2.4. Preparation of solutions for online detection
A solution of DAN (0.01 mg/mL) was prepared in 0.4 M hydrochloric acid and
154
stored in the dark at 4 °C.
M
155
an
153
us
149
ip t
146
A solution of NO was prepared as previously described[23,24], with slight
157
modifications. Briefly, absolute methanol (200 mL) was placed in a 250 mL glass
158
bottle that was to be used as the NO collection vessel. The bottle was carefully sealed
159
to minimize O2 entry and then purged of O2 by saturation with ultrapure N2 gas for 30
160
min before being linked to the rest of the apparatus (Fig. 2). The NO solution was
162 163 164 165 166
te
Ac ce p
161
d
156
obtained by saturation with NO for 1 min with stirring under O2-free conditions. The stability of the NO solution was evaluated over 150 min using the neutral Griess reagent. The NO solution was found to be stable for at least 120 min and so met the experimental requirements.
All solutions for post-chromatographic derivatization were prepared freshly before
the experiment.
167 168
2.5. Instrumentation for online HPLC-DAD-FLD
169
2.5.1 Components of the online system
170
HPLC was performed using a Shimadzu LC-2030C 3D HPLC system (Shimadzu
171
Inc.), equipped with an online vacuum degasser, a quaternary pump, a diode array
172
detection (DAD) system, an automatic sampler, a thermostatically controlled column 6
Page 6 of 25
173
compartment and a Shimadzu LabSolutions workstation.
174
Fluorescence detection was performed using a Shimadzu RF-20A fluorescence
175
detector (Shimadzu Inc.). DAN and NO solutions were delivered by two additional
176
pumps (Shimadzu LC-10AT vp and LC-10AD vp, respectively).
178
ip t
All of the instruments were interconnected with 0.5 mm I.D. polyether ether ketone
177
(PEEK) tubes and T-pieces.
180
cr
179
2.5.2 HPLC and online detection conditions
Samples were separated using a Grace Alltima ODS C18 column (250mm×4.6 nm,
182
5 μm). The mobile phase consisted of (A) 0.1% (v/v) aqueous formic solution and (B)
183
acetonitrile. To achieve better results with the online system, isocratic elution
184
conditions were used as shown in Table 1.
an
us
181
Fluorescence detection was carried out with an excitation wavelength of 375 nm
186
and an emission wavelength of 415 nm. The flow rate was 0.2 mL/min for the NO
187
solution and 1.0 mL/min for the DAN solution.
d
M
185
188
2.6. HPLC-Q-TOF MS/MS analysis conditions
te
189
An Agilent 6520 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies,
196
Ac ce p
190
197
with data acquisition and analysis were controlled by Agilent MassHunter
198
Workstation Software version B.04.00. Mass spectra were recorded across the range
199
m/z 100–1000, with accurate mass measurements of all mass peaks. The samples
200
were analyzed in positive mode, according to the properties of the compounds.
191 192 193 194 195
Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) interface was coupled in parallel by splitting the mobile phase using an adjustable high-pressure stream splitter. The operating parameters were as follows: drying gas (N2) flow rate, 8.0 L/min at 325°C; sheath gas flow rate, 12 L/min at 400°C; pressure of nebulizer gas, 40 psi; capillary voltage, 4000 V; skimmer voltage, 65 V; OCT RF V, 750 V; fragmentor voltage, 100 V; sample collision energy, 5–30 V. All operations together
201 202
2.7. Statistical analysis 7
Page 7 of 25
203
Data are expressed as mean ± standard deviation (SD). Statistical analyses were
204
performed with one-way ANOVA (analysis of variance) followed by Tukey’s method
205
(GraphPad Prism 5.0). A p value < 0.05 was considered to be statistically significant
206
and p < 0.01 was considered to be highly significant.
3. Results and discussion
209
3.1. Design of online HPLC-DAD-FLD system
cr
208
ip t
207
A schematic diagram of the online HPLC-DAD-FLD system is provided in Fig. 1.
211
This apparatus integrates separation and the activity assay to screen for NO
212
scavengers continuously and efficiently. Firstly, NO and DAN solutions are pumped
213
at a constant flow rate and then react in the reactor coil. The fluorescence intensity
214
(baseline) is detected by FLD. The complex mixtures are then separated using HPLC
215
and the individual components are detected by DAD. Each peak of the chromatogram
216
is mixed with NO solution and then rapidly processed by post-column derivatization
217
with the fluorescence probe. Once an active compound has reacted with NO, the
218
corresponding baseline fluorescence will be reduced. A negative peak is, therefore,
219
recorded by FLD, allowing identification of NO scavengers.
an
M
d
te
226
Ac ce p
220
us
210
227
scavenging ratio was calculated using the formula (1):
221 222 223 224 225
3.2. Optimization of online HPLC-DAD-FLD system conditions Many factors could influence acquisition of meaningful fluorescence intensities and
hence favorable screening results. These include the stability of NO, concentrations of NO and DAN solutions, pH, flow rate and type of PEEK tubes. These factors were evaluated by calculating the scavenging ratio of standard solutions, rutin and quercetin (0.5 mg/mL), which are believed to act as NO scavengers[19]. The
Scavenging ratio = PA (FLD) / PA (UV)
228
(1)
229
where PA (UV) is the peak area of DAD and PA (FLD) is the negative peak area of
230
fluorescence when inhibited by a NO scavenger in the sample.
231
3.2.1. The stability of NO solution 8
Page 8 of 25
The stability of the NO stock solution was examined using the Griess method.
233
Briefly, NO solution was mixed with neutral Griess reagents[24] (17 mM SULF and
234
0.4 mM NEDD in pH 7.4 PBS) and the absorbance measured at 496 nm
235
(SHIMADZU UV-VIS 2550 spectrophotometer) after reaction for 30 min. Stability
236
was evaluated by monitoring the UV absorbance. The initial UV absorbance of NO
237
decreased slightly in the first 15 min and then remained stable for 120 min under an
238
NO atmosphere (Fig. 3(A)). The NO solution was thus stable for at least 120 min after
239
an initial equilibrium period of 15 min and the apparatus for preparation of the NO
240
solution was shown to meet the experimental requirements. The concentration of the
241
NO stock solution was calculated by the formula (2)
an
us
cr
ip t
232
242
(2)
Where εNO = 12,500 moL-1 L cm-1[25]. And the mean concentration was found to be
244
0.200 mM.
M
243
The effects of pH and organic solvents, which could affect stability, were also
246
investigated using the same method. Because the sensitivity of the Griess assay is
247
highly dependent on solution composition (i.e., buffer and matrix)[26], we evaluated
248
these factors using a wider fluctuation range of absorbance. Acidic conditions (pH
250 251 252 253 254
te
Ac ce p
249
d
245
<5.9) and methanol had no significant effect compared with water (control group) (Fig. 3(B)). Slightly basic conditions (pH 8.0) and acetonitrile did produce a difference, but the absorbances were only slightly higher than that of the control group. Taken together, these results suggest that pH and organic solvents have only a minor influence on NO stability. 3.2.2. Concentration of DAN and NO solution
255
Firstly, concentrations of NO solutions were prepared by different collection time
256
(5, 10, 60, 180 s). Shorter collection time would obtain less concentration of NO
257
solution. Solutions of DAN (0.005–0.04 mg/mL) in 0.4 M hydrochloric acid were
258
prepared. These concentrations were examined by assuming that the flow rates of NO
259
and DAN solutions are 0.2 mL/min and 0.7 mL/min, respectively. As shown in Fig.
260
3(C), a better scavenging ratio was obtained at 10 s for NO collection, but this time 9
Page 9 of 25
was too short to prepare a stable solution. Having analyzed all of the data, a collection
262
time of 60 s was chosen for further experiments since this gave a good scavenging
263
ratio and repeatability. The optimized concentration of DAN that gives the highest
264
scavenging ratio is 0.01 mg/mL (Fig. 3(D)). These results suggest that the
265
fluorescence intensity was genuine and the screening signal was favorable with NO
266
and DAN concentrations of 0.200 mM (60 s) and 0.01 mg/mL, respectively.
267
3.2.3 Factor of pH.
cr
ip t
261
Different pH values could affect the fluorescence intensity of the reaction
269
product[27,28]. A series of solutions with varying pH (1.5, 2.6, 3.8, 5.0, 7.1, 8.0, 10.1
270
and 13.1) were prepared using acetate, phosphate and carbonic acid buffer solutions.
271
Only acidic conditions (pH <7.1) affected the scavenging ratios (Fig. 3(E)). A pH
272
value of 1.5 (0.4 M HCl) gave the highest scavenging ratio and fluorescence intensity
273
and was chosen for subsequent experiments. DAN is a weakly basic hydrophobic
274
molecule and is most soluble under strongly acidic conditions[29]. The poor solubility
275
of DAN in neutral or alkaline solution could potentially affect NO scavenging ratios
276
at pH ≥7.1.
277
3.2.4 Factor of flow rates.
te
d
M
an
us
268
Different flow rates of NO (0.1, 0.2, 0.3, 0.5 and 0.8 mL/min) and DAN (0.1, 0.3,
284
Ac ce p
278
285
3.2.5 Type of tubes.
279 280 281 282 283
0.5, 0.7 and 1.0 mL/min) solutions were also investigated. The scavenging ratio decreased with increased flow rate of the NO solution (Fig. 3(F)). In contrast, there was a directly proportional relationship between the scavenging ratio and the flow rate of the DAN solution (Fig. 3(G)). Flow rates of 0.2 mL/min for the NO solution and 1.0 mL/min for the DAN solution were selected for further experiments to obtain reliable fluorescence intensity and a stable baseline.
286
The effects of instrumentation and the materials used to interconnect the different
287
components on baseline drift, noise and peak broadening were also investigated.
288
Different lengths (50, 100, 150 cm) and diameters (0.25, 0.5 mm) of PEEK tubing
289
were evaluated, and 50 cm tubes with 0.5 mm I.D. were found to provide a good
290
baseline, low noise levels and favorable peak shapes. 10
Page 10 of 25
291 292
3.3 Method validation Our newly developed method was validated using the known NO scavengers, rutin
293 294
and quercetin, as positive references. The precision of the method was assessed by analyzing the same sample five times.
296
The relative standard deviation (RSD) of the DAD peak area for rutin was 0.70% and,
297
for quercetin, the value was 0.90%. The RSDs of the negative peak areas recorded by
298
FLD were 2.00% and 2.30%. Repeatability was evaluated by analyzing five parallel
299
samples. The RSDs of the scavenging ratios were 2.30% and 0.96%, respectively, thus
300
meeting the requirements of validation.
us
cr
ip t
295
The linear relationships for rutin and quercetin were constructed by plotting the
302
positive (Y1, rutin; Y2, quercetin) and negative peak areas (Y1’, rutin; Y2’, quercetin)
303
versus the corresponding concentration (X). The regression equations were as follows:
304
Y1 = 3E+06X + 317067, R² = 0.9879; Y1’ = -8E+06X + 1E+06, R² = 0.9912; Y2 =
305
6E+06X + 284927, R² = 0.9975; Y2’ = -2E+07X + 467021, R² = 0.9958, indicating a
306
good linear relationship and good dose-effect relationships (Fig. 4(A–D)).
d
M
an
301
3.4. Application of method for screening NO scavengers
314
Ac ce p
308
te
307
315
tea is one of the most widely consumed beverages in the world and contains many
316
compounds believed to be beneficial to health. Green tea has been shown to contain
317
NO scavengers but, to the best of our knowledge, little effort has been made to
318
identify NO scavengers in Radix scutellariae or Radix puerariae.
309 310 311 312 313
In Asia, the natural products Radix scutellariae and Radix puerariae are widely
used and have been revealed wide and strong therapeutic functions and potentially beneficial effects. Radix scutellariae is a well-known herb medicine, which has been been used for treatment of various diseases including ulcer, hepatitis, bronchitis, fever, tumor, inflammatory[30]. Radix Puerariae is rich in isoflavonoids, and has been used to treat angina pectoris, hypertension, influenza, and neck stiffness, et al[31]. Green
319
In the present study, we used our newly developed method to test these natural
320
products for NO scavengers. To allow for baseline noise, we selected a scavenging 11
Page 11 of 25
ratio >0.1 as indicative of active peaks. We found significant differences in the NO
322
scavenging capacity of the three samples. The chromatograms of Radix scutellariae
323
(Fig. 5(A)) and green tea (Fig. 5(B)) showed significant NO scavenging activity, with
324
compounds 1, 2, 3, 4, 5, 6, 11, 15, 16, 18, and 19 in Radix scutellariae and peaks 1, 2,
325
4, 6, 7, 8, 9 and 10 in green tea developing negative peaks. By contrast, there were
326
few negative peaks in the chromatogram of Radix puerariae (Fig. 5(C)), suggesting
327
that Radix puerariae has little capacity for scavenging NO. The NO scavenging
328
compounds in these natural products displayed a diversity of chemical properties. As
329
well as having a wide range of applications in the study of natural products, our new
330
method also has the potential to analyze other complex matrices such as serum
331
samples containing drug substances.
an
us
cr
ip t
321
332
3.5. Characterization and identification of NO scavengers in Radix Scutellariae and
334
green tea
M
333
Many compounds have been isolated and purified from all three natural products
336
and most have been characterized using magnetic resonance spectroscopy and mass
337
spectrometry. There is thus abundant reference data to assist in identification of active
338
components. In the present study, HPLC-DAD-ESI-Q-TOF MS/MS was used to
344
Ac ce p
te
d
335
345
data[30,32–41].
339 340 341 342 343
provide characteristic fragmentations for rapid structural identification of potential NO scavengers from Radix Scutellariae and green tea. Diode array detection not only recorded the signal of individual components, but also provided an UV spectrum of each compound that could be used as corroborative evidence to identify the structure. Twenty nine compounds from Radix Scutellariae and green tea (Tables 2 and 3) were characterized by MS data and UV spectra by comparison with published
346 347
3.6. Scavenging activity and structure-activity relationships of NO scavengers To evaluate the activity of each component, rutin was chosen as positive compound
348 349
and these scavenging activities were calculated using the following equation: 12
Page 12 of 25
(3)
350 351
where Scavenging ratio (Peaks) is the value of every single peak. Scavenging ratio
352
(Positive control) represents the positive scavenging ratio of rutin. The scavenger activity values of the components in Radix Scutellariae are shown in
354
Fig. 6(A). The NO scavenging activities of these compounds are in the order rutin
355
(positive)
356
chrysin-6-C-arabinosyl-8-C-glucoside
357
A-7-O-glucuronide (18) > chrysin-6-C-glucosyl-8-C-arabonoside (3), scutellarin (4),
358
5,7,3,2',6'-pentahydroxy
359
5,7,8-trihydroxy-6-methoxyflavone-7-O-glucuronide
360
norwogonin-7-O-glucuronide (15).
(1)
≈
flavanone
(6)
>
wogonoside
(19)
cr
flavone
baicalin
(11)
us
5,7,3,2',6'-pentahydroxy
(5)
an
>
ip t
353
(16)
>
>
oroxylin
> >
The scavenger activity values of the components in green tea are shown in Fig.
362
6(B). The NO scavenging activities of these compounds are in the order gallocatechin
363
(4) > (-)-epigallocatechin (6) > (-)-epicatechin (9) > rutin (positive) >
364
(-)-epigallocatechin-3-O-gallate (10) > 5-galloylquinic acid (1), gallic acid (2) >
365
caffeine (8). According to results, the compounds scavenging activity in green tea are
366
higher than components in Radix Scutellariae. And the NO scavengers that we have
372
Ac ce p
te
d
M
361
373
5,6-dihydroxybaicalein-7-O-glucuronide, suggesting that the 2,3‐double bond is an
374
essential moiety that forms a π bond conjugate system in the C ring and contributes to
375
the NO scavenging activity. The most active of all the compounds was
376
5,7,3,2',6'-pentahydroxy flavone, suggesting that a large number of phenolic hydroxyl
377
groups may be important for activity. Comparison of 5,7,3,2',6'-pentahydroxy flavone
378
with other active compounds such as wogonoside and baicalin suggests that phenolic
367 368 369 370 371
identified may play an important role in the pharmacological activities of these two natural products and may also have potential as NO scavengers for the treatment of diseases.
We also analyzed the structure-activity relationships of these NO scavengers.
Structures of compounds identified in Radix Scutellariae are shown in Fig. 7(A). The flavone,
baicalin
is
more
active
than
the
flavanone,
13
Page 13 of 25
hydroxyl groups in the B ring are not necessary for NO scavenging activity. Of the
380
compounds from Radix puerariae, isoflavones have less NO scavenging activity than
381
flavones (Fig. 5(C)), suggesting that a 3-H atom or 3-OH group in the C ring may be
382
important for activity. The presence of a 2,3-double bond with a 4-oxo group and a
383
3-H atom or 3-OH hydroxyl group were determined to be crucial for activity whereas
384
phenolic hydroxyl groups in the B ring enhanced scavenging activity but were not
385
essential.
cr
ip t
379
The graph of Fig. 7(B) displayed the structure of compounds in green tea. Tannin is
387
the main NO scavenger in green tea such as gallocatechin, (-)-epigallocatechin,
388
(-)-epicatechin, gallic acid whereas other components such as theobromine, caffeine
389
are less active. Our results are consistent with those of a previous study[16], which
390
suggested that poly-hydroxyl groups were essential for NO scavenging activity.
an
us
386
The mechanism of antioxidant activity of phenols and flavones involves the
392
formation of phenoxyl radicals[42]. The production of phenoxyl radicals from the
393
reaction of NO with a phenol moiety could be explained by two possible mechanisms:
394
H-atom abstraction to produce HNO and phenoxyl radicals or single electron transfer
395
to produce the phenol radical cation that reduces NO and immediately forms phenoxyl
396
radicals by loss of a proton[43,44]. Our results are in good agreement with previous
402
Ac ce p
te
d
M
391
403
analysis and identification of target compounds from crude extracts of natural
404
products. This approach was successfully applied to three natural products; and 18
405
active compounds were identified and characterized by HPLC-ESI-Q-TOF-MS. As
406
well as determining scavenging activity, structure-activity relationships were also
407
analyzed. Our results are important for further understanding the mechanism of NO
408
scavenging. This method is, therefore, expected to serve as a universal and practical
397 398 399 400 401
reports.
4. Conclusions
In the present study, we have successfully developed an online HPLC-DAD-FLD
system to screen for NO scavengers in natural products. This online system conveniently and efficiently solved a complex problem via continuous separation,
14
Page 14 of 25
409
approach for the discovery of potential drug leads from natural products.
410 411
Acknowledgments This project was financially supported by National Natural Science Foundation of
413
China (No. 81274004, 81473317), 2011’ Program for Excellent Scientific and
414
Technological Innovation Team of Jiangsu Higher Education and the Priority
415
Academic Program Development of Jiangsu Higher Education Institutions.
Ac ce p
te
d
M
an
us
cr
ip t
412
15
Page 15 of 25
References:
418 419
[1]
C. Nathan, Inducible nitric oxide synthase: what difference does it make? J. Clin. Invest. 100 (1997) 2417–2423.
420 421 422
[2]
D.D. Thomas, L.A. Ridnour, J.S. Isenberg, W. Flores-Santana, C.H. Switzer, S. Donzelli, et al. The chemical biology of nitric oxide: implications in cellular signaling, Free Radic. Biol. Med. 45 (2008) 18–31.
423 424 425
[3]
M.B. Grisham, D. Jourd’Heuil, D.A. Wink, Nitric oxide. I. Physiological chemistry of nitric oxide and its metabolites:implications in inflammation, Am. J. Physiol. 276 (1999) 315–321.
426 427
[4]
K.D. Kroncke, K. Fehsel, V. Kolb-Bachofen, Inducible nitric oxide synthase in human diseases, Clin. Exp. Immunol. 113 (1998) 147–156.
428
[5]
A. Cauwels, Nitric oxide in shock, Kidney Int. 72 (2007) 557–565.
429 430 431 432
[6]
K.E. Broderick, J. Feala, A. McCulloch, G. Paternostro, V.S. Sharma, R.B. Pilz, G. R. Boss, The nitric oxide scavenger cobinamide profoundly improves survival in a Drosophila melanogaster model of bacterial sepsis, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 20 (2006) 1865–1873.
433 434
[7]
C. Bogdan, Nitric oxide and the immune response, Nat. Immunol. 2 (2001) 907–916.
435 436 437
te
d
M
an
us
cr
ip t
417
Ac ce p
416
[8]
L. Celano, C. Carabio, R. Frache, N. Cataldo, H. Cerecetto, M. Gonzalez, L. Thomson, Arylnitroalkenes as scavengers of macrophage-generated oxidants, Eur. J. Med. Chem. 74 (2014) 31–40.
[9]
J. Menezes, C. Hierholzer, S.C. Watkins, V. Lyons, A.B. Peitzman, T.R. Billiar, D. J. Tweardy, B. G. Harbrecht, A novel nitric oxide scavenger decreases liver injury and improves survival after hemorrhagic shock, Am. J. Physiol. 277 (1999) 144–151.
442 443 444
[10]
E. Dickinson, R. Tuncer, E. Nadler, P. Boyle, S. Alber, S. Watkins, H. Ford, NOX, a novel nitric oxide scavenger, reduces bacterial translocation in rats after endotoxin challenge, Am. J. Physiol. 277 (1999) 1281–1287.
445 446 447
[11]
C.G.M. Millar, C. Thiemermann, Carboxy-PTIO, a scavenger of nitric oxide, selectively inhibits the increase in medullary perfusion and improves renal function in endotoxemia, Shock. 18 (2002) 64–68.
438 439 440 441
16
Page 16 of 25
[12]
K. Satoh, Y. Ikeda, S. Shioda, T. Tobe, T. Yoshikawa, Edarabone scavenges nitric oxide, Redox Rep. 7 (2002) 219–222.
450 451 452
[13]
Y. Higashi, Edaravone for the treatment of acute cerebral infarction: role of endothelium-derived nitric oxide and oxidative stress, Expert Opin. Pharmacother. 10 (2009) 323–331.
453 454 455
[14]
L. Marcocci, J.J. Maguire, M.T. Droy-Lefaix, L. Packer, The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761, Biochem. Biophys. Res. Commun. 201 (1994) 748–755.
456 457 458
[15]
I. Karmakar, N. Dolai, P. Saha, N. Sarkar, A. Bala, P. Haldar, Scavenging activity of Curcuma caesia rhizome against reactive oxygen and nitrogen species, Orient. Pharm. Exp. Med. 11 (2011) 221–228.
459 460
[16]
T. Nakagawa, T. Yokozawa, Direct scavenging of nitric oxide and superoxide by green tea, Food Chem. Toxicol. 40 (2002) 1745–1750.
461 462 463
[17]
P.D. Duh, G.C. Yen, W.J. Yen, B.S. Wang, L.W. Chang, Effects of pu-erh tea on oxidative damage and nitric oxide scavenging, J. Agric. Food Chem. 52 (2004) 8169–8176.
464 465
[18]
E.M. Hetrick, M.H. Schoenfisch, Analytical chemistry of nitric oxide, Annu. Rev. Anal. Chem. (Palo Alto. Calif). 2 (2009) 409–433.
466 467 468
[19]
S.A. van Acker, M.N. Tromp, G.R. Haenen, W.J. van der Vijgh, A. Bast, Flavonoids as scavengers of nitric oxide radical, Biochem. Biophys. Res. Commun. 214 (1995) 755–759.
Ac ce p
te
d
M
an
us
cr
ip t
448 449
[20]
L.S. Yoshida, S. Kohri, S. Tsunawaki, T. Kakegawa, T. Taniguchi, H. Takano-Ohmuro, H. Fujii, Evaluation of radical scavenging properties of shikonin, J. Clin. Biochem. Nutr. 55 (2014) 90–96.
[21]
K.S. Kang, T. Yokozawa, H.Y. Kim, J.H. Park, Study on the nitric oxide scavenging effects of ginseng and its compounds, J. Agric. Food Chem. 54 (2006) 2558–2562.
475 476
[22]
T. Nagano, Practical methods for detection of nitric oxide, Luminescence. 14 (1999) 283–290.
477 478 479 480
[23]
L.A. Ridnour, J.E. Sim, M.A. Hayward, D.A. Wink, S.M. Martin, G.R. Buettner, D.R. Spitz, A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media, Anal. Biochem. 281 (2000) 223–229.
469 470 471 472 473 474
17
Page 17 of 25
[24]
F.O. Brown, N.J. Finnerty, F.B. Bolger, J. Millar, J.P. Lowry, Calibration of NO sensors for in-vivo voltammetry: laboratory synthesis of NO and the use of UV-visible spectroscopy for determining stock concentrations, Anal. Bioanal. Chem. 381 (2005) 964–971.
485 486 487 488
[25]
R.W. Nims, J.F. Darbyshire, J.E. Saavedra, D. Christodoulou, I. Hanbauer, G.W. Cox, M.B. Grisham, F. Laval, J.A. Cook, M.C. Krishna, D.A.Wink, Colorimetric Methods for the Determination of Nitric Oxide Concentration in Neutral Aqueous Solutions, Methods. 7 (1995) 48–54.
489 490
[26]
P.N. Coneski, M.H. Schoenfisch, Nitric oxide release: part III. Measurement and reporting, Chem. Soc. Rev. 41 (2012) 3753–3758.
491 492
[27]
A.M. Miles, Y. Chen, M.W. Owens, M.B. Grisham, Fluorometric Determination of Nitric Oxide, Methods. 7 (1995) 40–47.
493 494 495
[28]
A.K. Nussler, M. Glanemann, A. Schirmeier, L. Liu, N.C. Nussler, Fluorometric measurement of nitrite/nitrate by 2,3-diaminonaphthalene, Nat. Protoc. 1 (2006) 2223–2226.
496 497 498 499
[29]
M. Akyuz, S. Ata, Determination of low level nitrite and nitrate in biological, food and environmental samples by gas chromatography-mass spectrometry and liquid chromatography with fluorescence detection, Talanta. 79 (2009) 900–904.
500 501 502 503 504
[30]
G. Liu, J. Ma, Y. Chen, Q. Tian, Y. Shen, X. Wang, B. Chen, S. Yao, Investigation of flavonoid profile of Scutellaria bacalensis Georgi by high performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry, J. Chromatogr. A. 1216 (2009) 4809–4814.
508 509 510 511 512 513 514 515 516
cr
us
an
M
d
te
Ac ce p
505 506 507
ip t
481 482 483 484
[31]
G. Wang, C. Yang, K. Zhang, J. Hu, W. Pang, Molecular clusters size of Puerariae thomsonii radix aqueous decoction and relevance to oral absorption, Molecules. 20 (2015) 12376–12388.
[32]
J. Han, M. Ye, M. Xu, J. Sun, B. Wang, D. Guo, Characterization of flavonoids in the traditional Chinese herbal medicine-Huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 848 (2007) 355–362.
[33]
C.R. Horvath, P.A. Martos, P.K. Saxena, Identification and quantification of eight flavones in root and shoot tissues of the medicinal plant Huang-qin (Scutellaria baicalensis Georgi) using high-performance liquid chromatography with diode array and mass spectrometric detection, J. Chromatogr. A. 1062 (2005) 199–207. 18
Page 18 of 25
[34]
N.Z. Mamadalieva, F. Herrmann, M.Z. El-Readi, A. Tahrani, R. Hamoud, D.R. Egamberdieva, S.S. Azimova, M. Wink, Flavonoids in Scutellaria immaculata and S. ramosissima (Lamiaceae) and their biological activity, J. Pharm. Pharmacol. 63 (2011) 1346–1357.
521 522 523
[35]
J. Han, M. Ye, H. Guo, M. Yang, B. Wang, D. Guo, Analysis of multiple constituents in a Chinese herbal preparation Shuang-Huang-Lian oral liquid by HPLC-DAD-ESI-MSn, J. Pharm. Biomed. Anal. 44 (2007) 430–438.
524 525 526 527
[36] W.J. Miao, Q. Wang, T. Bo, M. Ye, X. Qiao, W.Z. Yang, C. Xiang, X.Y. Guan, D.A. Guo, Rapid characterization of chemical constituents and rats metabolites of the traditional Chinese patent medicine Gegen-Qinlian-Wan by UHPLC/DAD/qTOF-MS, J. Pharm. Biomed. Anal. 72 (2013) 99–108.
528 529 530
[37]
G.Z. Liu, N. Rajesh, X.S. Wang, M.S. Zhang, Q. Wu, S.J. Li, B. Chen, S.Z. Yao, Identification of flavonoids in the stems and leaves of Scutellaria baicalensis Georgi, J. Chromatogr. B. 879 (2011) 1023–1028.
531 532 533
[38]
D. Del Rio, A.J. Stewart, W. Mullen, J. Burns, M.E.J. Lean, F. Brighenti, A. Crozier, HPLC-MSn analysis of phenolic compounds and purine alkaloids in green and black tea, J. Agric. Food Chem. 52 (2004) 2807–2815.
534 535 536
[39]
D. Wang, J. Lu, A. Miao, Z. Xie, D. Yang, HPLC-DAD-ESI-MS/MS analysis of polyphenols and purine alkaloids in leaves of 22 tea cultivars in China, J. Food Compos. Anal. 21 (2008) 361–369.
537 538 539 540
[40]
Y. Zhao, P. Chen, L. Lin, J.M. Harnly, L. (Lucy) Yu, Z. Li, Tentative identification, quantitation, and principal component analysis of green pu-erh, green, and white teas using UPLC/DAD/MS, Food Chem. 126 (2011) 1269–1277.
Ac ce p
te
d
M
an
us
cr
ip t
517 518 519 520
[41]
L.Z. Lin, P. Chen, J.M. Harnly, New phenolic components and chromatographic profiles of green and fermented teas, J. Agric. Food Chem. 56 (2008) 8130–8140.
[42]
D. Prochazkova, I. Bousova, N. Wilhelmova, Antioxidant and prooxidant properties of flavonoids, Fitoterapia. 82 (2011) 513–523.
546 547 548
[43]
E.G. Janzen, A.L. Wilcox, V. Manoharan, Reactions of nitric oxide with phenolic antioxidants and phenoxyl radicals, J. Org. Chem. 58 (1993) 3597–3599.
549 550
[44]
A.L. Wilcox, E.G. Janzen, Nitric oxide reactions with antioxidants in model systems: sterically hindered phenols and [small alpha]-tocopherol in sodium
541 542 543 544 545
19
Page 19 of 25
dodecyl sulfate (SDS) micelles, J. Chem. Soc. Chem. Commun. (1993) 1377–1379.
551 552 553
Ac ce p
te
d
M
an
us
cr
ip t
554
20
Page 20 of 25
554
Fig captions:
556
Fig. 1. Schemes of the HPLC-DAD-FLD online system for screening nitric oxide
557
scavengers in natural products. The chromatogram of each peak was recorded by
558
DAD detector when the natural products were separated by the column. Then, the
559
eluents were mixed with nitric oxide and then rapidly for post-column derivatization
560
with fluorescence probe (DAN). Subsequently, the active profiles were obtained by
561
fluorescence detector.
cr
ip t
555
us
562
Fig. 2. Schemes of the apparatus for preparation NO methanol solution. It was
564
assembled with 99.9% NO in container connected with 4 M NaOH to remove any
565
higher oxides and control the flow rate, following 200 mL deoxygen methanol in
566
glass bottle as NO collection vessel. 30% hydrogen peroxide was used to absorb the
567
residual NO. All the connections of this apparatus should be tightness and keep it
568
oxygen-free.
d
569
M
an
563
Fig. 3. Optimization of online HPLC-DAD-FLD system conditions. (A) The stability
571
of NO methanol solution during 150 min experimented with Griess method; (B) the
572
stability influenced by the factors of pH and organic reagents (vs. water group); (C)
574 575 576 577 578
Ac ce p
573
te
570
and (D) were different concentrations of NO and DAN solutions; (E) various pH of DAN solution; (F) and (G) were different flow rate of NO and DAN solutions.
Fig. 4. The linear relationship of rutin and quercetin were constructed by plotting the positive [(A) rutin, (B) quercetin] and negative peak area [(C) rutin; (D) quercetin] versus the corresponding the concentration.
579 580
Fig. 5. The application of online HPLC-DAD-FLD system for screening NO
581
scavengers from (A) dry radix of Scutellaria baicalensis Georgi, [peaks identify: 1
582
Chrysin-6-C-arabinosyl-8-C-glucoside, 2 Unidentified, 3 Chrysin-6-C-glucosyl-8-C-arabonoside,
583
4 Scutellarin, 5 5,7,3,2',6'-Pentahydroxy flavanone, 6 5,7,3,2',6'-Pentahydroxy flavone, 7 21
Page 21 of 25
584
Unidentified, 8 5,6,7-Trihydroxy-8-methoxy flavone-7-O-glucuronide, 9 Unidentified, 10 Luteolin,
585
11
586
Apigenin-7-O-glucuronide,
587
5,7,8-Trihydroxy-6-methoxyflavone-7-O-glucuronide, 17 Baicalein-6-O-glucuronide, 18 Oroxylin
588
A-7-O-glucuronide, 19 Wogonoside]; and (B) Green tea, [peaks identify: 1 5-Galloylquinic
589
acid, 2 Gallic acid, 3 Theobromine, 4 Gallocatechin, 5 Unidentified, 6 (-)-Epigallocatechin, 7
590
Kaempferol pentose conjugate, 8 Caffeine, 9 (-)-Epicatechin, 10 (-)-Epigallocatechin-3-O-gallate];
591
(C) dry radix of Pueraria lobata (Willd.) Ohwi;
12
Unidentified,
13
5,6-Dihydroxybaicalein-7-O-glucuronide,
15
Norwogonin-7-O-glucuronide,
cr
Fig. 6. The scavenging activity of each peak investigated by online HPLC-DAD-FLD
594
system in (A) S. baicalensis Georgi and (B) Green tea. Group of rutin is a positive
595
control. [ND: Not detected.]
an
593
M
596
Fig. 7. Chemical structures of the identified compounds in (A) dry radix of S.
598
baicalensis and (B) Green tea.
600
602
603 604 605
Tables:
Ac ce p
601
te
d
597
599
7.0
16
us
592
RT(min)
14
ip t
Baicalin,
Table 1 The HPLC isocratic elution conditions for the samples Samples A:B Flow rate Injection Column (mL/min) volume (µL) temperature (°C) 79:21 1.0 5.0 35 Radix scutellariae 90:10 1.0 1.0 30 Radix puerariae Green tea 90:10 1.0 2.0 30
Detector Wavelength (nm) 280 254 275
Table 2 Retention times, UV and MS data for identification of compounds in S. baicalensis by HPLC-ESI-Q-TOF-MS. [M+H]+ (m/z)
UV λmax (nm)
272, 549.1605 315
HPLC-ESI-Q-TOF-MSn
Formula
Delta Mass (ppm)
Identification
531.1503, 489.9203, 459 1066 429 0958
C26H28O13
1.73
Chrysin-6-C-arabinosyl-8-C-glucoside
22
Page 22 of 25
315
459.1066, 429.0958
329
647.1948
501.1369, 467.1523
C31H34O15
3.47
Unidentified
8.7
273, 549.1613 315
531.1491, 459.1069, 363.0863 333.0755
C26H28O13
1.94
Chrysin-6-C-glucosyl-8-C-arabonoside
9.5
277, 463.0872 329
287.0552, 269.0498, 256.7146, 241.0462
C21H18O12
0.31
Scutellarein-7-O-glucuronide (Scutellarin)
9.9
288
305.0653
C15H12O7
1.91
5,7,3,2',6'-Pentahydroxy flavanone
13.5
303.0503
C15H10O7
1.03
312.1049, 216.0873, 166.0496 301.0709, 286.0472
-
-
C22H20O12
0.02
21.6
253, 337 280, 327 270, 332 285
287.0556, 269.0451, 153.0177 257.0438, 167.0345
23.9
258
287.0551
27.3
276, 316 281, 332 287, 350 (sh) 268, 313 279, 354 283, 322 (sh) 273, 312 271, 311 273, 340
447.0953
34.4
38.1 41.2 44.8
50.1 53.3 68.4
606 607 608 Peak
675.2244 449.1077
cr us
C15H10O6
0.25
Luteolin
271.0615, 253.0495, 529.1640, 481.1625
C21H18O11
4.80
Baicalin
-
-
Unidentified
273.0755, 173.0584, 169.0125, 168.9879
C21H20O11
1.82
5,6-Dihydroxybaicalein-7-O-glucuronide
C22H22O10
0.17
Apigenin-7-O- glucuronide
C21H18O11
3.42
Norwogonin-7-O- glucuronide
2.94
an -
284.2998, 239.0825, 136.9361 269.0354, 241.0476
-
M
325.0680
Unidentified
5,6,7-Trihydroxy-8-methoxy flavone-7-O-glucuronide Unidentified
d
31.8
477.1027
te
19.9
416.1703
5,7,3,2',6'-Pentahydroxy flavone
Ac ce p
18.6
ip t
7.7
C22H20O12
447.0965
271.0611,
C21H18O11
9.72
Baicalein-6-O-glucuronide
461.1083
285.0750, 270.0511, 168.0044 285.0751, 270.0516, 183.0296
C22H20O11
1.06
Oroxylin A-7-O-glucuronide
C22H20O11
0.03
Wogonoside
447.1284
447.0906 477.1041
461.1079
285.0769, 271.0601, 227.0666 271.0595, 225.0586, 169.0153 301.0713, 286.0491, 183.9970
5,7,8-Trihydroxy-6-methoxyflavone-7-O-glucuro
Table 3 Retention times, UV and MS data for identification of compounds in Green Tea by HPLC-ESI-Q-TOF-MS.
RT(min)
UV λmax
[M+H]+
HPLC-ESI-Q-TOF-MSn
Formula
Delta
Identification
23
Page 23 of 25
5.9
4 5
6.8 10.7
6
11.5
7
13.0
8
16.0
9
28.6
10
34.0 609 610 611
615 616 617 618
5-Galloylquinic acid
C7H6O5
0.41
Gallic acid
-
C7H8N4O2
0.51
Theobromine
177.0546, 139.0392 319.0861, 202.0619, 151.0394
C15H14O7 -
3.4 -
271.0548, 163.0376, 139.0380 339.0846, 250.1071, 177.0536 138.0645
C15H14O7
1.17
(-)-Epigallocatechin
-
-
Kaempferol pentose conjugate
207.0639, 139.0388 153.0167, 139.0375
Gallocatechin Unidentified
C8H10N4O2 11.96 C15H14O6
C22H18O11
Caffeine
3.02
(-)-Epicatechin
0.99
(-)-Epigallocatechin-3-O-gallate
Ac ce p
612 613 614
0.56
ip t
3
C14H16O10
an
4.7
189.0647, 171.0288, 153.0177 -
M
2
210, 345.0818 274 213, 171.0287 271 202, 181.0719 273 270 307.08061 206, 365,1214 259, 275(sh) 199, 307.0816 269 204, 419.1810 309 200, 195.0900 272 201, 291.0872 279 200, 459.0915 274
d
4.1
Mass (ppm)
te
1
(m/z)
cr
(nm)
us
NO.
Highlights:
We developed the online HPLC-DAD-FLD system for screening NO scavengers in natural products.
Bioactive compounds were screened out using the new online system and
619
identified by HPLC-ESI-Q-TOF-MS, then structure-activity relationships were
620
analyzed.
621
Our method is specific, simple, reliable and efficient.
622 623 624 625
24
Page 24 of 25
te
d
M
an
us
cr
ip t
Ac ce p
626
25
Page 25 of 25
S0021-9673(15)01585-X http://dx.doi.org/doi:10.1016/j.chroma.2015.10.095 CHROMA 357010
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
17-8-2015 27-10-2015 27-10-2015
Please cite this article as: D. Li, T. Wang, Y. Guo, Y. Hu, B. Yu, J. Qi, A novel method for online screening of nitric oxide scavengers in natural products using high performance liquid chromatography coupled with tandem diode array and fluorescence detection, Journal of Chromatography A (2015), http://dx.doi.org/10.1016/j.chroma.2015.10.095 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel method for online screening of nitric oxide scavengers in natural
2
products using high performance liquid chromatography coupled with
3
tandem diode array and fluorescence detection
ip t
1
4 5
Dapeng Li b, Ting Wang b, Yujie Guo b, Yuanjia Hu c, Boyang Yu a, b, *, Jin Qi a, b, *
cr
6
a State Key Laboratory of Natural Medicines, China Pharmaceutical University,
8
Nanjing 210009, China.
9
b Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China
us
7
Pharmaceutical University, Nanjing 210009, China
11
c State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
12
Medical Sciences, University of Macau, Macao 999078, China
M
an
10
13
d
14 15
te
16
18 19 20 21 22 23
Ac ce p
17
24 25 26
*Corresponding authors at: Jiangsu Key Laboratory of TCM Evaluation and
27
Translational Research, China Pharmaceutical University, Tongjia Lane 24, Nanjing
28
210009, China. Tel.: +86 25 86185157; Fax: +86 25 86185157.
29
E-mail addresses: [email protected] (Boyang Yu), [email protected] (Jin Qi). 1
Page 1 of 25
30 31
Abstract: Nitric oxide (NO) is an important cellular signaling molecule with extensive
33
physiological and pathophysiological effects. NO scavengers have the potential to
34
treat inflammation, septic shock and other related diseases, and numerous examples
35
have been chemically synthesized or isolated from natural products. The chemical
36
diversity of natural products, however, means that a huge effort is necessary to
37
efficiently screen and identify bioactive compounds, especially NO scavengers. In this
38
article, we propose an effective analytical method to screen for NO scavengers in
39
three natural products using an online system that couples high performance liquid
40
chromatography
41
(HPLC-DAD-FLD). Eighteen compounds from radix of Scutellaria baicalensis
42
Georgi and green tea displayed significant NO scavenging activity whereas
43
components of Pueraria lobata (Willd.) Ohwi had no discernable activity. The
44
structures of the active compounds were elucidated using Agilent Accurate-Mass
45
Q-TOF LC/MS system. Preliminary analysis of structure-activity relationships
46
indicated that, in flavonoids, a 2,3-double bond and a 3-H atom or a 3-OH group are
47
essential for activity. In tannins, poly-hydroxyl groups are important for NO
system, screening
50 51 52
and
fluorescence
detection
d
Ac ce p
54
49
array
te
53
48
diode
an
tandem
M
with
us
cr
ip t
32
scavenging activity. Method validation indicated that the newly developed method is both reliable and repeatable. The online method that we present provides a simple, rapid and effective way to identify and characterize NO scavengers present in natural products.
Key words: natural products, nitric oxide, scavenger, online HPLC-DAD-FLD
55
2
Page 2 of 25
55 56
1. Introduction Nitric oxide (NO) is endogenously released by several different cell types and
58
tissues in vivo during the nitric oxide synthase (NOS)-catalyzed conversion of
59
L-arginine to L-citrulline. This free radical acts as a signaling molecule with dual
60
effects. At low concentrations, NO is essential for regulating vasodilation, blood
61
pressure and neurotransmission, as well as cardiovascular and renal function[1,2].
62
Overproduction of NO, however, can promote inflammatory diseases[2,3] such as
63
arthritis, myocarditis[4], septic shock[5,6] and autoimmune disorders[7]. Correcting
64
the imbalance in physiological and pathological processes by scavenging excess NO
65
may thus provide a strategy for the treatment of inflammatory diseases.
an
us
cr
ip t
57
Numerous NO scavengers have been synthesized or isolated from natural products.
67
For instance, arylnitroalkenes[8], dithiocarbamate[9,10] and carboxy-PTIO[11] are
68
effective in animal models of inflammation and acute septic shock, and
69
edaravone[12,13], has been used in clinical practice. Some natural products, such as
70
Ginkgo biloba extract EGb 761[14], curcumin[15], green tea[16] and Pu-erh tea[17],
71
which were believed to be antioxidants, have now been shown to have the ability to
72
scavenge NO. Although there is ample evidence that constituents from natural
74 75 76 77
d
te
Ac ce p
73
M
66
products have the potential to treat NO-related diseases, it is difficult to screen effectively for NO scavengers. Separation and purification of individual compounds is difficult because of the complex nature of natural products, and analytical methods for determination of NO are challenging because of its reactivity, rapid diffusion and short half-life.
78
Various techniques[18] have, however, been developed to detect NO, including UV
79
spectroscopy, fluorescence spectroscopy, chemiluminescence, electron paramagnetic
80
resonance (EPR) and electrochemistry. In previous studies[16,19], screening for NO
81
scavengers was commonly carried out by mixing the natural product with a NO donor
82
such as sodium nitroprusside, followed by detection of the changes in NO levels. For
83
example, Yoshida et al. evaluated the NO scavenging properties of shikonin using 3
Page 3 of 25
ESR[20]. This method is rapid and accurate, but it is expensive to operate. Kang and
85
co-workers investigated the NO scavenging properties of ginseng using UV
86
spectroscopy[21]. This method, which has been widely used for analysis of a variety
87
of samples, is convenient to operate but other nitrogen oxide species, such as NO3−,
88
will interfere with the determination of NO[18]. Most of the methods currently used
89
for identifying NO scavengers from natural products are time consuming, labor
90
intensive or high cost, and can give rise to ambiguous results. Methods based on
91
hyphenated technologies, combining optimally coordinated separation of natural
92
products and measurement of NO, are likely to provide a solution to these problems.
us
cr
ip t
84
In the present report, we describe a strategy based on high performance liquid
94
chromatography (HPLC) coupled with online tandem diode array detection and
95
fluorescence detection (HPLC-DAD-FLD) (Fig. 1) to systematically identify NO
96
scavengers from natural products. Fluorescence detection is able to specifically,
97
accurately and sensitively detect NO and a large number of fluorescence probes have
98
been developed. One of the most widely used fluorescence methods, because of its
99
low cost and high sensitivity, is based on the reaction of NO with
100
2,3-diaminonaphthalene (DAN)[22]. Our online system comprises an HPLC system
101
together with a detection system. The HPLC system separates the constituents from a
107
Ac ce p
te
d
M
an
93
108
new system is an efficient and high-throughput method for identifying NO
109
scavengers.
102 103 104 105 106
complex natural product mixture and the online detection system determines activity using the fluorescence probe (DAN). NO scavengers can be identified by comparison of the two chromatograms. This novel hyphenated HPLC-DAD-FLD method has been validated and successfully applied to three natural products. The method was used to analyze and characterize the substances and structures as well as to determine the structure-activity relationships of NO scavengers from these natural products. Our
110 111
2. Experimental
112
2.1 Chemicals and reagents 4
Page 4 of 25
113
Nitric oxide gas (99%) was purchased from Nanjing Shangyuan Industrial Gas Co.,
114
Ltd. (Jiangsu, China). DAN was purchased from Aladdin Industrial Inc. (Shanghai,
115
China).
116
N-(1-naphthyl)ethylenediamine dihydrochloride (NEDD) and phosphoric acid were
117
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure
118
water (18.2 MΩ) was obtained using a Milli-Q water purification system (Millipore,
119
Bedford, MA, USA). Solvents used for extractions were analytical grade and
120
purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China).
121
Chromatographic grade acetonitrile and methanol were obtained from Tedia (Fairfield,
122
OH, USA) and Yonghua Chemical Technology Co., Ltd. (Jiangsu, China),
123
respectively.
grade
4-aminobenzenesulfonic
acid
(SULF),
an
us
cr
ip t
Analytical
Samples of rutin and quercetin for use as standards were purchased from Aladdin
125
Industrial Inc. (Shanghai, China). Dried radix of Scutellaria baicalensis Georgi and
126
Pueraria lobata (Willd.) Ohwi are traditional Chinese medicines and were obtained
127
from the local pharmacy, Nanjing ShangyuanTang Pharmacy (Zhushan Road 109,
128
Nanjing). Green tea was purchased from Yipin Xuan Organic Tea Industry Co., Ltd.
129
(Anhui, China). All three crude drugs were authenticated by one of the authors, Prof.
130
Boyang Yu. The voucher specimens (specimens number: S. baicalensis 20150215, P.
136
Ac ce p
te
d
M
124
137
absolute methanol and stored at 4°C to prevent degradation. A mixed standard
138
solution (0.5 mg/mL) was prepared by mixing the two stock solutions in a 1:1 ratio.
131 132 133 134 135
lobata 20150215, Green tea 20150215) were deposited at the Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, China. The dried crude drugs were ground to homogeneous powders.
2.2. Standard solutions
Standard stock solutions (1 mg/mL) of rutin and quercetin were prepared in
139 140
2.3. Extraction process
141
Powdered radix of Scutellariae (2.0 g) was extracted twice with 75% (v/v) aqueous
142
methanol (100 mL) for 30 min with ultrasonication at 100 Hz. The extracts were 5
Page 5 of 25
143
filtered and combined and then evaporated under vacuum at 55°C. The residue was
144
dissolved in dimethylsulfoxide (5 mL) to provide the stock solution. Powdered green tea (1.0 g) was extracted with 70% (v/v) aqueous methanol (30 mL)
145
for 45 min with ultrasonication at 100 Hz. The extract was filtered and used in the
147
following experiments.
Powdered radix of puerariae (1.0 g) was refluxed in 30% aqueous ethanol (v/v) for
148
All solutions were stored at 4°C and filtered through a 0.45 μm membrane before
150 151
cr
30 min. After cooling, the extract was filtered and used in the following experiments.
analysis.
152
2.4. Preparation of solutions for online detection
A solution of DAN (0.01 mg/mL) was prepared in 0.4 M hydrochloric acid and
154
stored in the dark at 4 °C.
M
155
an
153
us
149
ip t
146
A solution of NO was prepared as previously described[23,24], with slight
157
modifications. Briefly, absolute methanol (200 mL) was placed in a 250 mL glass
158
bottle that was to be used as the NO collection vessel. The bottle was carefully sealed
159
to minimize O2 entry and then purged of O2 by saturation with ultrapure N2 gas for 30
160
min before being linked to the rest of the apparatus (Fig. 2). The NO solution was
162 163 164 165 166
te
Ac ce p
161
d
156
obtained by saturation with NO for 1 min with stirring under O2-free conditions. The stability of the NO solution was evaluated over 150 min using the neutral Griess reagent. The NO solution was found to be stable for at least 120 min and so met the experimental requirements.
All solutions for post-chromatographic derivatization were prepared freshly before
the experiment.
167 168
2.5. Instrumentation for online HPLC-DAD-FLD
169
2.5.1 Components of the online system
170
HPLC was performed using a Shimadzu LC-2030C 3D HPLC system (Shimadzu
171
Inc.), equipped with an online vacuum degasser, a quaternary pump, a diode array
172
detection (DAD) system, an automatic sampler, a thermostatically controlled column 6
Page 6 of 25
173
compartment and a Shimadzu LabSolutions workstation.
174
Fluorescence detection was performed using a Shimadzu RF-20A fluorescence
175
detector (Shimadzu Inc.). DAN and NO solutions were delivered by two additional
176
pumps (Shimadzu LC-10AT vp and LC-10AD vp, respectively).
178
ip t
All of the instruments were interconnected with 0.5 mm I.D. polyether ether ketone
177
(PEEK) tubes and T-pieces.
180
cr
179
2.5.2 HPLC and online detection conditions
Samples were separated using a Grace Alltima ODS C18 column (250mm×4.6 nm,
182
5 μm). The mobile phase consisted of (A) 0.1% (v/v) aqueous formic solution and (B)
183
acetonitrile. To achieve better results with the online system, isocratic elution
184
conditions were used as shown in Table 1.
an
us
181
Fluorescence detection was carried out with an excitation wavelength of 375 nm
186
and an emission wavelength of 415 nm. The flow rate was 0.2 mL/min for the NO
187
solution and 1.0 mL/min for the DAN solution.
d
M
185
188
2.6. HPLC-Q-TOF MS/MS analysis conditions
te
189
An Agilent 6520 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies,
196
Ac ce p
190
197
with data acquisition and analysis were controlled by Agilent MassHunter
198
Workstation Software version B.04.00. Mass spectra were recorded across the range
199
m/z 100–1000, with accurate mass measurements of all mass peaks. The samples
200
were analyzed in positive mode, according to the properties of the compounds.
191 192 193 194 195
Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) interface was coupled in parallel by splitting the mobile phase using an adjustable high-pressure stream splitter. The operating parameters were as follows: drying gas (N2) flow rate, 8.0 L/min at 325°C; sheath gas flow rate, 12 L/min at 400°C; pressure of nebulizer gas, 40 psi; capillary voltage, 4000 V; skimmer voltage, 65 V; OCT RF V, 750 V; fragmentor voltage, 100 V; sample collision energy, 5–30 V. All operations together
201 202
2.7. Statistical analysis 7
Page 7 of 25
203
Data are expressed as mean ± standard deviation (SD). Statistical analyses were
204
performed with one-way ANOVA (analysis of variance) followed by Tukey’s method
205
(GraphPad Prism 5.0). A p value < 0.05 was considered to be statistically significant
206
and p < 0.01 was considered to be highly significant.
3. Results and discussion
209
3.1. Design of online HPLC-DAD-FLD system
cr
208
ip t
207
A schematic diagram of the online HPLC-DAD-FLD system is provided in Fig. 1.
211
This apparatus integrates separation and the activity assay to screen for NO
212
scavengers continuously and efficiently. Firstly, NO and DAN solutions are pumped
213
at a constant flow rate and then react in the reactor coil. The fluorescence intensity
214
(baseline) is detected by FLD. The complex mixtures are then separated using HPLC
215
and the individual components are detected by DAD. Each peak of the chromatogram
216
is mixed with NO solution and then rapidly processed by post-column derivatization
217
with the fluorescence probe. Once an active compound has reacted with NO, the
218
corresponding baseline fluorescence will be reduced. A negative peak is, therefore,
219
recorded by FLD, allowing identification of NO scavengers.
an
M
d
te
226
Ac ce p
220
us
210
227
scavenging ratio was calculated using the formula (1):
221 222 223 224 225
3.2. Optimization of online HPLC-DAD-FLD system conditions Many factors could influence acquisition of meaningful fluorescence intensities and
hence favorable screening results. These include the stability of NO, concentrations of NO and DAN solutions, pH, flow rate and type of PEEK tubes. These factors were evaluated by calculating the scavenging ratio of standard solutions, rutin and quercetin (0.5 mg/mL), which are believed to act as NO scavengers[19]. The
Scavenging ratio = PA (FLD) / PA (UV)
228
(1)
229
where PA (UV) is the peak area of DAD and PA (FLD) is the negative peak area of
230
fluorescence when inhibited by a NO scavenger in the sample.
231
3.2.1. The stability of NO solution 8
Page 8 of 25
The stability of the NO stock solution was examined using the Griess method.
233
Briefly, NO solution was mixed with neutral Griess reagents[24] (17 mM SULF and
234
0.4 mM NEDD in pH 7.4 PBS) and the absorbance measured at 496 nm
235
(SHIMADZU UV-VIS 2550 spectrophotometer) after reaction for 30 min. Stability
236
was evaluated by monitoring the UV absorbance. The initial UV absorbance of NO
237
decreased slightly in the first 15 min and then remained stable for 120 min under an
238
NO atmosphere (Fig. 3(A)). The NO solution was thus stable for at least 120 min after
239
an initial equilibrium period of 15 min and the apparatus for preparation of the NO
240
solution was shown to meet the experimental requirements. The concentration of the
241
NO stock solution was calculated by the formula (2)
an
us
cr
ip t
232
242
(2)
Where εNO = 12,500 moL-1 L cm-1[25]. And the mean concentration was found to be
244
0.200 mM.
M
243
The effects of pH and organic solvents, which could affect stability, were also
246
investigated using the same method. Because the sensitivity of the Griess assay is
247
highly dependent on solution composition (i.e., buffer and matrix)[26], we evaluated
248
these factors using a wider fluctuation range of absorbance. Acidic conditions (pH
250 251 252 253 254
te
Ac ce p
249
d
245
<5.9) and methanol had no significant effect compared with water (control group) (Fig. 3(B)). Slightly basic conditions (pH 8.0) and acetonitrile did produce a difference, but the absorbances were only slightly higher than that of the control group. Taken together, these results suggest that pH and organic solvents have only a minor influence on NO stability. 3.2.2. Concentration of DAN and NO solution
255
Firstly, concentrations of NO solutions were prepared by different collection time
256
(5, 10, 60, 180 s). Shorter collection time would obtain less concentration of NO
257
solution. Solutions of DAN (0.005–0.04 mg/mL) in 0.4 M hydrochloric acid were
258
prepared. These concentrations were examined by assuming that the flow rates of NO
259
and DAN solutions are 0.2 mL/min and 0.7 mL/min, respectively. As shown in Fig.
260
3(C), a better scavenging ratio was obtained at 10 s for NO collection, but this time 9
Page 9 of 25
was too short to prepare a stable solution. Having analyzed all of the data, a collection
262
time of 60 s was chosen for further experiments since this gave a good scavenging
263
ratio and repeatability. The optimized concentration of DAN that gives the highest
264
scavenging ratio is 0.01 mg/mL (Fig. 3(D)). These results suggest that the
265
fluorescence intensity was genuine and the screening signal was favorable with NO
266
and DAN concentrations of 0.200 mM (60 s) and 0.01 mg/mL, respectively.
267
3.2.3 Factor of pH.
cr
ip t
261
Different pH values could affect the fluorescence intensity of the reaction
269
product[27,28]. A series of solutions with varying pH (1.5, 2.6, 3.8, 5.0, 7.1, 8.0, 10.1
270
and 13.1) were prepared using acetate, phosphate and carbonic acid buffer solutions.
271
Only acidic conditions (pH <7.1) affected the scavenging ratios (Fig. 3(E)). A pH
272
value of 1.5 (0.4 M HCl) gave the highest scavenging ratio and fluorescence intensity
273
and was chosen for subsequent experiments. DAN is a weakly basic hydrophobic
274
molecule and is most soluble under strongly acidic conditions[29]. The poor solubility
275
of DAN in neutral or alkaline solution could potentially affect NO scavenging ratios
276
at pH ≥7.1.
277
3.2.4 Factor of flow rates.
te
d
M
an
us
268
Different flow rates of NO (0.1, 0.2, 0.3, 0.5 and 0.8 mL/min) and DAN (0.1, 0.3,
284
Ac ce p
278
285
3.2.5 Type of tubes.
279 280 281 282 283
0.5, 0.7 and 1.0 mL/min) solutions were also investigated. The scavenging ratio decreased with increased flow rate of the NO solution (Fig. 3(F)). In contrast, there was a directly proportional relationship between the scavenging ratio and the flow rate of the DAN solution (Fig. 3(G)). Flow rates of 0.2 mL/min for the NO solution and 1.0 mL/min for the DAN solution were selected for further experiments to obtain reliable fluorescence intensity and a stable baseline.
286
The effects of instrumentation and the materials used to interconnect the different
287
components on baseline drift, noise and peak broadening were also investigated.
288
Different lengths (50, 100, 150 cm) and diameters (0.25, 0.5 mm) of PEEK tubing
289
were evaluated, and 50 cm tubes with 0.5 mm I.D. were found to provide a good
290
baseline, low noise levels and favorable peak shapes. 10
Page 10 of 25
291 292
3.3 Method validation Our newly developed method was validated using the known NO scavengers, rutin
293 294
and quercetin, as positive references. The precision of the method was assessed by analyzing the same sample five times.
296
The relative standard deviation (RSD) of the DAD peak area for rutin was 0.70% and,
297
for quercetin, the value was 0.90%. The RSDs of the negative peak areas recorded by
298
FLD were 2.00% and 2.30%. Repeatability was evaluated by analyzing five parallel
299
samples. The RSDs of the scavenging ratios were 2.30% and 0.96%, respectively, thus
300
meeting the requirements of validation.
us
cr
ip t
295
The linear relationships for rutin and quercetin were constructed by plotting the
302
positive (Y1, rutin; Y2, quercetin) and negative peak areas (Y1’, rutin; Y2’, quercetin)
303
versus the corresponding concentration (X). The regression equations were as follows:
304
Y1 = 3E+06X + 317067, R² = 0.9879; Y1’ = -8E+06X + 1E+06, R² = 0.9912; Y2 =
305
6E+06X + 284927, R² = 0.9975; Y2’ = -2E+07X + 467021, R² = 0.9958, indicating a
306
good linear relationship and good dose-effect relationships (Fig. 4(A–D)).
d
M
an
301
3.4. Application of method for screening NO scavengers
314
Ac ce p
308
te
307
315
tea is one of the most widely consumed beverages in the world and contains many
316
compounds believed to be beneficial to health. Green tea has been shown to contain
317
NO scavengers but, to the best of our knowledge, little effort has been made to
318
identify NO scavengers in Radix scutellariae or Radix puerariae.
309 310 311 312 313
In Asia, the natural products Radix scutellariae and Radix puerariae are widely
used and have been revealed wide and strong therapeutic functions and potentially beneficial effects. Radix scutellariae is a well-known herb medicine, which has been been used for treatment of various diseases including ulcer, hepatitis, bronchitis, fever, tumor, inflammatory[30]. Radix Puerariae is rich in isoflavonoids, and has been used to treat angina pectoris, hypertension, influenza, and neck stiffness, et al[31]. Green
319
In the present study, we used our newly developed method to test these natural
320
products for NO scavengers. To allow for baseline noise, we selected a scavenging 11
Page 11 of 25
ratio >0.1 as indicative of active peaks. We found significant differences in the NO
322
scavenging capacity of the three samples. The chromatograms of Radix scutellariae
323
(Fig. 5(A)) and green tea (Fig. 5(B)) showed significant NO scavenging activity, with
324
compounds 1, 2, 3, 4, 5, 6, 11, 15, 16, 18, and 19 in Radix scutellariae and peaks 1, 2,
325
4, 6, 7, 8, 9 and 10 in green tea developing negative peaks. By contrast, there were
326
few negative peaks in the chromatogram of Radix puerariae (Fig. 5(C)), suggesting
327
that Radix puerariae has little capacity for scavenging NO. The NO scavenging
328
compounds in these natural products displayed a diversity of chemical properties. As
329
well as having a wide range of applications in the study of natural products, our new
330
method also has the potential to analyze other complex matrices such as serum
331
samples containing drug substances.
an
us
cr
ip t
321
332
3.5. Characterization and identification of NO scavengers in Radix Scutellariae and
334
green tea
M
333
Many compounds have been isolated and purified from all three natural products
336
and most have been characterized using magnetic resonance spectroscopy and mass
337
spectrometry. There is thus abundant reference data to assist in identification of active
338
components. In the present study, HPLC-DAD-ESI-Q-TOF MS/MS was used to
344
Ac ce p
te
d
335
345
data[30,32–41].
339 340 341 342 343
provide characteristic fragmentations for rapid structural identification of potential NO scavengers from Radix Scutellariae and green tea. Diode array detection not only recorded the signal of individual components, but also provided an UV spectrum of each compound that could be used as corroborative evidence to identify the structure. Twenty nine compounds from Radix Scutellariae and green tea (Tables 2 and 3) were characterized by MS data and UV spectra by comparison with published
346 347
3.6. Scavenging activity and structure-activity relationships of NO scavengers To evaluate the activity of each component, rutin was chosen as positive compound
348 349
and these scavenging activities were calculated using the following equation: 12
Page 12 of 25
(3)
350 351
where Scavenging ratio (Peaks) is the value of every single peak. Scavenging ratio
352
(Positive control) represents the positive scavenging ratio of rutin. The scavenger activity values of the components in Radix Scutellariae are shown in
354
Fig. 6(A). The NO scavenging activities of these compounds are in the order rutin
355
(positive)
356
chrysin-6-C-arabinosyl-8-C-glucoside
357
A-7-O-glucuronide (18) > chrysin-6-C-glucosyl-8-C-arabonoside (3), scutellarin (4),
358
5,7,3,2',6'-pentahydroxy
359
5,7,8-trihydroxy-6-methoxyflavone-7-O-glucuronide
360
norwogonin-7-O-glucuronide (15).
(1)
≈
flavanone
(6)
>
wogonoside
(19)
cr
flavone
baicalin
(11)
us
5,7,3,2',6'-pentahydroxy
(5)
an
>
ip t
353
(16)
>
>
oroxylin
> >
The scavenger activity values of the components in green tea are shown in Fig.
362
6(B). The NO scavenging activities of these compounds are in the order gallocatechin
363
(4) > (-)-epigallocatechin (6) > (-)-epicatechin (9) > rutin (positive) >
364
(-)-epigallocatechin-3-O-gallate (10) > 5-galloylquinic acid (1), gallic acid (2) >
365
caffeine (8). According to results, the compounds scavenging activity in green tea are
366
higher than components in Radix Scutellariae. And the NO scavengers that we have
372
Ac ce p
te
d
M
361
373
5,6-dihydroxybaicalein-7-O-glucuronide, suggesting that the 2,3‐double bond is an
374
essential moiety that forms a π bond conjugate system in the C ring and contributes to
375
the NO scavenging activity. The most active of all the compounds was
376
5,7,3,2',6'-pentahydroxy flavone, suggesting that a large number of phenolic hydroxyl
377
groups may be important for activity. Comparison of 5,7,3,2',6'-pentahydroxy flavone
378
with other active compounds such as wogonoside and baicalin suggests that phenolic
367 368 369 370 371
identified may play an important role in the pharmacological activities of these two natural products and may also have potential as NO scavengers for the treatment of diseases.
We also analyzed the structure-activity relationships of these NO scavengers.
Structures of compounds identified in Radix Scutellariae are shown in Fig. 7(A). The flavone,
baicalin
is
more
active
than
the
flavanone,
13
Page 13 of 25
hydroxyl groups in the B ring are not necessary for NO scavenging activity. Of the
380
compounds from Radix puerariae, isoflavones have less NO scavenging activity than
381
flavones (Fig. 5(C)), suggesting that a 3-H atom or 3-OH group in the C ring may be
382
important for activity. The presence of a 2,3-double bond with a 4-oxo group and a
383
3-H atom or 3-OH hydroxyl group were determined to be crucial for activity whereas
384
phenolic hydroxyl groups in the B ring enhanced scavenging activity but were not
385
essential.
cr
ip t
379
The graph of Fig. 7(B) displayed the structure of compounds in green tea. Tannin is
387
the main NO scavenger in green tea such as gallocatechin, (-)-epigallocatechin,
388
(-)-epicatechin, gallic acid whereas other components such as theobromine, caffeine
389
are less active. Our results are consistent with those of a previous study[16], which
390
suggested that poly-hydroxyl groups were essential for NO scavenging activity.
an
us
386
The mechanism of antioxidant activity of phenols and flavones involves the
392
formation of phenoxyl radicals[42]. The production of phenoxyl radicals from the
393
reaction of NO with a phenol moiety could be explained by two possible mechanisms:
394
H-atom abstraction to produce HNO and phenoxyl radicals or single electron transfer
395
to produce the phenol radical cation that reduces NO and immediately forms phenoxyl
396
radicals by loss of a proton[43,44]. Our results are in good agreement with previous
402
Ac ce p
te
d
M
391
403
analysis and identification of target compounds from crude extracts of natural
404
products. This approach was successfully applied to three natural products; and 18
405
active compounds were identified and characterized by HPLC-ESI-Q-TOF-MS. As
406
well as determining scavenging activity, structure-activity relationships were also
407
analyzed. Our results are important for further understanding the mechanism of NO
408
scavenging. This method is, therefore, expected to serve as a universal and practical
397 398 399 400 401
reports.
4. Conclusions
In the present study, we have successfully developed an online HPLC-DAD-FLD
system to screen for NO scavengers in natural products. This online system conveniently and efficiently solved a complex problem via continuous separation,
14
Page 14 of 25
409
approach for the discovery of potential drug leads from natural products.
410 411
Acknowledgments This project was financially supported by National Natural Science Foundation of
413
China (No. 81274004, 81473317), 2011’ Program for Excellent Scientific and
414
Technological Innovation Team of Jiangsu Higher Education and the Priority
415
Academic Program Development of Jiangsu Higher Education Institutions.
Ac ce p
te
d
M
an
us
cr
ip t
412
15
Page 15 of 25
References:
418 419
[1]
C. Nathan, Inducible nitric oxide synthase: what difference does it make? J. Clin. Invest. 100 (1997) 2417–2423.
420 421 422
[2]
D.D. Thomas, L.A. Ridnour, J.S. Isenberg, W. Flores-Santana, C.H. Switzer, S. Donzelli, et al. The chemical biology of nitric oxide: implications in cellular signaling, Free Radic. Biol. Med. 45 (2008) 18–31.
423 424 425
[3]
M.B. Grisham, D. Jourd’Heuil, D.A. Wink, Nitric oxide. I. Physiological chemistry of nitric oxide and its metabolites:implications in inflammation, Am. J. Physiol. 276 (1999) 315–321.
426 427
[4]
K.D. Kroncke, K. Fehsel, V. Kolb-Bachofen, Inducible nitric oxide synthase in human diseases, Clin. Exp. Immunol. 113 (1998) 147–156.
428
[5]
A. Cauwels, Nitric oxide in shock, Kidney Int. 72 (2007) 557–565.
429 430 431 432
[6]
K.E. Broderick, J. Feala, A. McCulloch, G. Paternostro, V.S. Sharma, R.B. Pilz, G. R. Boss, The nitric oxide scavenger cobinamide profoundly improves survival in a Drosophila melanogaster model of bacterial sepsis, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 20 (2006) 1865–1873.
433 434
[7]
C. Bogdan, Nitric oxide and the immune response, Nat. Immunol. 2 (2001) 907–916.
435 436 437
te
d
M
an
us
cr
ip t
417
Ac ce p
416
[8]
L. Celano, C. Carabio, R. Frache, N. Cataldo, H. Cerecetto, M. Gonzalez, L. Thomson, Arylnitroalkenes as scavengers of macrophage-generated oxidants, Eur. J. Med. Chem. 74 (2014) 31–40.
[9]
J. Menezes, C. Hierholzer, S.C. Watkins, V. Lyons, A.B. Peitzman, T.R. Billiar, D. J. Tweardy, B. G. Harbrecht, A novel nitric oxide scavenger decreases liver injury and improves survival after hemorrhagic shock, Am. J. Physiol. 277 (1999) 144–151.
442 443 444
[10]
E. Dickinson, R. Tuncer, E. Nadler, P. Boyle, S. Alber, S. Watkins, H. Ford, NOX, a novel nitric oxide scavenger, reduces bacterial translocation in rats after endotoxin challenge, Am. J. Physiol. 277 (1999) 1281–1287.
445 446 447
[11]
C.G.M. Millar, C. Thiemermann, Carboxy-PTIO, a scavenger of nitric oxide, selectively inhibits the increase in medullary perfusion and improves renal function in endotoxemia, Shock. 18 (2002) 64–68.
438 439 440 441
16
Page 16 of 25
[12]
K. Satoh, Y. Ikeda, S. Shioda, T. Tobe, T. Yoshikawa, Edarabone scavenges nitric oxide, Redox Rep. 7 (2002) 219–222.
450 451 452
[13]
Y. Higashi, Edaravone for the treatment of acute cerebral infarction: role of endothelium-derived nitric oxide and oxidative stress, Expert Opin. Pharmacother. 10 (2009) 323–331.
453 454 455
[14]
L. Marcocci, J.J. Maguire, M.T. Droy-Lefaix, L. Packer, The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761, Biochem. Biophys. Res. Commun. 201 (1994) 748–755.
456 457 458
[15]
I. Karmakar, N. Dolai, P. Saha, N. Sarkar, A. Bala, P. Haldar, Scavenging activity of Curcuma caesia rhizome against reactive oxygen and nitrogen species, Orient. Pharm. Exp. Med. 11 (2011) 221–228.
459 460
[16]
T. Nakagawa, T. Yokozawa, Direct scavenging of nitric oxide and superoxide by green tea, Food Chem. Toxicol. 40 (2002) 1745–1750.
461 462 463
[17]
P.D. Duh, G.C. Yen, W.J. Yen, B.S. Wang, L.W. Chang, Effects of pu-erh tea on oxidative damage and nitric oxide scavenging, J. Agric. Food Chem. 52 (2004) 8169–8176.
464 465
[18]
E.M. Hetrick, M.H. Schoenfisch, Analytical chemistry of nitric oxide, Annu. Rev. Anal. Chem. (Palo Alto. Calif). 2 (2009) 409–433.
466 467 468
[19]
S.A. van Acker, M.N. Tromp, G.R. Haenen, W.J. van der Vijgh, A. Bast, Flavonoids as scavengers of nitric oxide radical, Biochem. Biophys. Res. Commun. 214 (1995) 755–759.
Ac ce p
te
d
M
an
us
cr
ip t
448 449
[20]
L.S. Yoshida, S. Kohri, S. Tsunawaki, T. Kakegawa, T. Taniguchi, H. Takano-Ohmuro, H. Fujii, Evaluation of radical scavenging properties of shikonin, J. Clin. Biochem. Nutr. 55 (2014) 90–96.
[21]
K.S. Kang, T. Yokozawa, H.Y. Kim, J.H. Park, Study on the nitric oxide scavenging effects of ginseng and its compounds, J. Agric. Food Chem. 54 (2006) 2558–2562.
475 476
[22]
T. Nagano, Practical methods for detection of nitric oxide, Luminescence. 14 (1999) 283–290.
477 478 479 480
[23]
L.A. Ridnour, J.E. Sim, M.A. Hayward, D.A. Wink, S.M. Martin, G.R. Buettner, D.R. Spitz, A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media, Anal. Biochem. 281 (2000) 223–229.
469 470 471 472 473 474
17
Page 17 of 25
[24]
F.O. Brown, N.J. Finnerty, F.B. Bolger, J. Millar, J.P. Lowry, Calibration of NO sensors for in-vivo voltammetry: laboratory synthesis of NO and the use of UV-visible spectroscopy for determining stock concentrations, Anal. Bioanal. Chem. 381 (2005) 964–971.
485 486 487 488
[25]
R.W. Nims, J.F. Darbyshire, J.E. Saavedra, D. Christodoulou, I. Hanbauer, G.W. Cox, M.B. Grisham, F. Laval, J.A. Cook, M.C. Krishna, D.A.Wink, Colorimetric Methods for the Determination of Nitric Oxide Concentration in Neutral Aqueous Solutions, Methods. 7 (1995) 48–54.
489 490
[26]
P.N. Coneski, M.H. Schoenfisch, Nitric oxide release: part III. Measurement and reporting, Chem. Soc. Rev. 41 (2012) 3753–3758.
491 492
[27]
A.M. Miles, Y. Chen, M.W. Owens, M.B. Grisham, Fluorometric Determination of Nitric Oxide, Methods. 7 (1995) 40–47.
493 494 495
[28]
A.K. Nussler, M. Glanemann, A. Schirmeier, L. Liu, N.C. Nussler, Fluorometric measurement of nitrite/nitrate by 2,3-diaminonaphthalene, Nat. Protoc. 1 (2006) 2223–2226.
496 497 498 499
[29]
M. Akyuz, S. Ata, Determination of low level nitrite and nitrate in biological, food and environmental samples by gas chromatography-mass spectrometry and liquid chromatography with fluorescence detection, Talanta. 79 (2009) 900–904.
500 501 502 503 504
[30]
G. Liu, J. Ma, Y. Chen, Q. Tian, Y. Shen, X. Wang, B. Chen, S. Yao, Investigation of flavonoid profile of Scutellaria bacalensis Georgi by high performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry, J. Chromatogr. A. 1216 (2009) 4809–4814.
508 509 510 511 512 513 514 515 516
cr
us
an
M
d
te
Ac ce p
505 506 507
ip t
481 482 483 484
[31]
G. Wang, C. Yang, K. Zhang, J. Hu, W. Pang, Molecular clusters size of Puerariae thomsonii radix aqueous decoction and relevance to oral absorption, Molecules. 20 (2015) 12376–12388.
[32]
J. Han, M. Ye, M. Xu, J. Sun, B. Wang, D. Guo, Characterization of flavonoids in the traditional Chinese herbal medicine-Huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 848 (2007) 355–362.
[33]
C.R. Horvath, P.A. Martos, P.K. Saxena, Identification and quantification of eight flavones in root and shoot tissues of the medicinal plant Huang-qin (Scutellaria baicalensis Georgi) using high-performance liquid chromatography with diode array and mass spectrometric detection, J. Chromatogr. A. 1062 (2005) 199–207. 18
Page 18 of 25
[34]
N.Z. Mamadalieva, F. Herrmann, M.Z. El-Readi, A. Tahrani, R. Hamoud, D.R. Egamberdieva, S.S. Azimova, M. Wink, Flavonoids in Scutellaria immaculata and S. ramosissima (Lamiaceae) and their biological activity, J. Pharm. Pharmacol. 63 (2011) 1346–1357.
521 522 523
[35]
J. Han, M. Ye, H. Guo, M. Yang, B. Wang, D. Guo, Analysis of multiple constituents in a Chinese herbal preparation Shuang-Huang-Lian oral liquid by HPLC-DAD-ESI-MSn, J. Pharm. Biomed. Anal. 44 (2007) 430–438.
524 525 526 527
[36] W.J. Miao, Q. Wang, T. Bo, M. Ye, X. Qiao, W.Z. Yang, C. Xiang, X.Y. Guan, D.A. Guo, Rapid characterization of chemical constituents and rats metabolites of the traditional Chinese patent medicine Gegen-Qinlian-Wan by UHPLC/DAD/qTOF-MS, J. Pharm. Biomed. Anal. 72 (2013) 99–108.
528 529 530
[37]
G.Z. Liu, N. Rajesh, X.S. Wang, M.S. Zhang, Q. Wu, S.J. Li, B. Chen, S.Z. Yao, Identification of flavonoids in the stems and leaves of Scutellaria baicalensis Georgi, J. Chromatogr. B. 879 (2011) 1023–1028.
531 532 533
[38]
D. Del Rio, A.J. Stewart, W. Mullen, J. Burns, M.E.J. Lean, F. Brighenti, A. Crozier, HPLC-MSn analysis of phenolic compounds and purine alkaloids in green and black tea, J. Agric. Food Chem. 52 (2004) 2807–2815.
534 535 536
[39]
D. Wang, J. Lu, A. Miao, Z. Xie, D. Yang, HPLC-DAD-ESI-MS/MS analysis of polyphenols and purine alkaloids in leaves of 22 tea cultivars in China, J. Food Compos. Anal. 21 (2008) 361–369.
537 538 539 540
[40]
Y. Zhao, P. Chen, L. Lin, J.M. Harnly, L. (Lucy) Yu, Z. Li, Tentative identification, quantitation, and principal component analysis of green pu-erh, green, and white teas using UPLC/DAD/MS, Food Chem. 126 (2011) 1269–1277.
Ac ce p
te
d
M
an
us
cr
ip t
517 518 519 520
[41]
L.Z. Lin, P. Chen, J.M. Harnly, New phenolic components and chromatographic profiles of green and fermented teas, J. Agric. Food Chem. 56 (2008) 8130–8140.
[42]
D. Prochazkova, I. Bousova, N. Wilhelmova, Antioxidant and prooxidant properties of flavonoids, Fitoterapia. 82 (2011) 513–523.
546 547 548
[43]
E.G. Janzen, A.L. Wilcox, V. Manoharan, Reactions of nitric oxide with phenolic antioxidants and phenoxyl radicals, J. Org. Chem. 58 (1993) 3597–3599.
549 550
[44]
A.L. Wilcox, E.G. Janzen, Nitric oxide reactions with antioxidants in model systems: sterically hindered phenols and [small alpha]-tocopherol in sodium
541 542 543 544 545
19
Page 19 of 25
dodecyl sulfate (SDS) micelles, J. Chem. Soc. Chem. Commun. (1993) 1377–1379.
551 552 553
Ac ce p
te
d
M
an
us
cr
ip t
554
20
Page 20 of 25
554
Fig captions:
556
Fig. 1. Schemes of the HPLC-DAD-FLD online system for screening nitric oxide
557
scavengers in natural products. The chromatogram of each peak was recorded by
558
DAD detector when the natural products were separated by the column. Then, the
559
eluents were mixed with nitric oxide and then rapidly for post-column derivatization
560
with fluorescence probe (DAN). Subsequently, the active profiles were obtained by
561
fluorescence detector.
cr
ip t
555
us
562
Fig. 2. Schemes of the apparatus for preparation NO methanol solution. It was
564
assembled with 99.9% NO in container connected with 4 M NaOH to remove any
565
higher oxides and control the flow rate, following 200 mL deoxygen methanol in
566
glass bottle as NO collection vessel. 30% hydrogen peroxide was used to absorb the
567
residual NO. All the connections of this apparatus should be tightness and keep it
568
oxygen-free.
d
569
M
an
563
Fig. 3. Optimization of online HPLC-DAD-FLD system conditions. (A) The stability
571
of NO methanol solution during 150 min experimented with Griess method; (B) the
572
stability influenced by the factors of pH and organic reagents (vs. water group); (C)
574 575 576 577 578
Ac ce p
573
te
570
and (D) were different concentrations of NO and DAN solutions; (E) various pH of DAN solution; (F) and (G) were different flow rate of NO and DAN solutions.
Fig. 4. The linear relationship of rutin and quercetin were constructed by plotting the positive [(A) rutin, (B) quercetin] and negative peak area [(C) rutin; (D) quercetin] versus the corresponding the concentration.
579 580
Fig. 5. The application of online HPLC-DAD-FLD system for screening NO
581
scavengers from (A) dry radix of Scutellaria baicalensis Georgi, [peaks identify: 1
582
Chrysin-6-C-arabinosyl-8-C-glucoside, 2 Unidentified, 3 Chrysin-6-C-glucosyl-8-C-arabonoside,
583
4 Scutellarin, 5 5,7,3,2',6'-Pentahydroxy flavanone, 6 5,7,3,2',6'-Pentahydroxy flavone, 7 21
Page 21 of 25
584
Unidentified, 8 5,6,7-Trihydroxy-8-methoxy flavone-7-O-glucuronide, 9 Unidentified, 10 Luteolin,
585
11
586
Apigenin-7-O-glucuronide,
587
5,7,8-Trihydroxy-6-methoxyflavone-7-O-glucuronide, 17 Baicalein-6-O-glucuronide, 18 Oroxylin
588
A-7-O-glucuronide, 19 Wogonoside]; and (B) Green tea, [peaks identify: 1 5-Galloylquinic
589
acid, 2 Gallic acid, 3 Theobromine, 4 Gallocatechin, 5 Unidentified, 6 (-)-Epigallocatechin, 7
590
Kaempferol pentose conjugate, 8 Caffeine, 9 (-)-Epicatechin, 10 (-)-Epigallocatechin-3-O-gallate];
591
(C) dry radix of Pueraria lobata (Willd.) Ohwi;
12
Unidentified,
13
5,6-Dihydroxybaicalein-7-O-glucuronide,
15
Norwogonin-7-O-glucuronide,
cr
Fig. 6. The scavenging activity of each peak investigated by online HPLC-DAD-FLD
594
system in (A) S. baicalensis Georgi and (B) Green tea. Group of rutin is a positive
595
control. [ND: Not detected.]
an
593
M
596
Fig. 7. Chemical structures of the identified compounds in (A) dry radix of S.
598
baicalensis and (B) Green tea.
600
602
603 604 605
Tables:
Ac ce p
601
te
d
597
599
7.0
16
us
592
RT(min)
14
ip t
Baicalin,
Table 1 The HPLC isocratic elution conditions for the samples Samples A:B Flow rate Injection Column (mL/min) volume (µL) temperature (°C) 79:21 1.0 5.0 35 Radix scutellariae 90:10 1.0 1.0 30 Radix puerariae Green tea 90:10 1.0 2.0 30
Detector Wavelength (nm) 280 254 275
Table 2 Retention times, UV and MS data for identification of compounds in S. baicalensis by HPLC-ESI-Q-TOF-MS. [M+H]+ (m/z)
UV λmax (nm)
272, 549.1605 315
HPLC-ESI-Q-TOF-MSn
Formula
Delta Mass (ppm)
Identification
531.1503, 489.9203, 459 1066 429 0958
C26H28O13
1.73
Chrysin-6-C-arabinosyl-8-C-glucoside
22
Page 22 of 25
315
459.1066, 429.0958
329
647.1948
501.1369, 467.1523
C31H34O15
3.47
Unidentified
8.7
273, 549.1613 315
531.1491, 459.1069, 363.0863 333.0755
C26H28O13
1.94
Chrysin-6-C-glucosyl-8-C-arabonoside
9.5
277, 463.0872 329
287.0552, 269.0498, 256.7146, 241.0462
C21H18O12
0.31
Scutellarein-7-O-glucuronide (Scutellarin)
9.9
288
305.0653
C15H12O7
1.91
5,7,3,2',6'-Pentahydroxy flavanone
13.5
303.0503
C15H10O7
1.03
312.1049, 216.0873, 166.0496 301.0709, 286.0472
-
-
C22H20O12
0.02
21.6
253, 337 280, 327 270, 332 285
287.0556, 269.0451, 153.0177 257.0438, 167.0345
23.9
258
287.0551
27.3
276, 316 281, 332 287, 350 (sh) 268, 313 279, 354 283, 322 (sh) 273, 312 271, 311 273, 340
447.0953
34.4
38.1 41.2 44.8
50.1 53.3 68.4
606 607 608 Peak
675.2244 449.1077
cr us
C15H10O6
0.25
Luteolin
271.0615, 253.0495, 529.1640, 481.1625
C21H18O11
4.80
Baicalin
-
-
Unidentified
273.0755, 173.0584, 169.0125, 168.9879
C21H20O11
1.82
5,6-Dihydroxybaicalein-7-O-glucuronide
C22H22O10
0.17
Apigenin-7-O- glucuronide
C21H18O11
3.42
Norwogonin-7-O- glucuronide
2.94
an -
284.2998, 239.0825, 136.9361 269.0354, 241.0476
-
M
325.0680
Unidentified
5,6,7-Trihydroxy-8-methoxy flavone-7-O-glucuronide Unidentified
d
31.8
477.1027
te
19.9
416.1703
5,7,3,2',6'-Pentahydroxy flavone
Ac ce p
18.6
ip t
7.7
C22H20O12
447.0965
271.0611,
C21H18O11
9.72
Baicalein-6-O-glucuronide
461.1083
285.0750, 270.0511, 168.0044 285.0751, 270.0516, 183.0296
C22H20O11
1.06
Oroxylin A-7-O-glucuronide
C22H20O11
0.03
Wogonoside
447.1284
447.0906 477.1041
461.1079
285.0769, 271.0601, 227.0666 271.0595, 225.0586, 169.0153 301.0713, 286.0491, 183.9970
5,7,8-Trihydroxy-6-methoxyflavone-7-O-glucuro
Table 3 Retention times, UV and MS data for identification of compounds in Green Tea by HPLC-ESI-Q-TOF-MS.
RT(min)
UV λmax
[M+H]+
HPLC-ESI-Q-TOF-MSn
Formula
Delta
Identification
23
Page 23 of 25
5.9
4 5
6.8 10.7
6
11.5
7
13.0
8
16.0
9
28.6
10
34.0 609 610 611
615 616 617 618
5-Galloylquinic acid
C7H6O5
0.41
Gallic acid
-
C7H8N4O2
0.51
Theobromine
177.0546, 139.0392 319.0861, 202.0619, 151.0394
C15H14O7 -
3.4 -
271.0548, 163.0376, 139.0380 339.0846, 250.1071, 177.0536 138.0645
C15H14O7
1.17
(-)-Epigallocatechin
-
-
Kaempferol pentose conjugate
207.0639, 139.0388 153.0167, 139.0375
Gallocatechin Unidentified
C8H10N4O2 11.96 C15H14O6
C22H18O11
Caffeine
3.02
(-)-Epicatechin
0.99
(-)-Epigallocatechin-3-O-gallate
Ac ce p
612 613 614
0.56
ip t
3
C14H16O10
an
4.7
189.0647, 171.0288, 153.0177 -
M
2
210, 345.0818 274 213, 171.0287 271 202, 181.0719 273 270 307.08061 206, 365,1214 259, 275(sh) 199, 307.0816 269 204, 419.1810 309 200, 195.0900 272 201, 291.0872 279 200, 459.0915 274
d
4.1
Mass (ppm)
te
1
(m/z)
cr
(nm)
us
NO.
Highlights:
We developed the online HPLC-DAD-FLD system for screening NO scavengers in natural products.
Bioactive compounds were screened out using the new online system and
619
identified by HPLC-ESI-Q-TOF-MS, then structure-activity relationships were
620
analyzed.
621
Our method is specific, simple, reliable and efficient.
622 623 624 625
24
Page 24 of 25
te
d
M
an
us
cr
ip t
Ac ce p
626
25
Page 25 of 25