Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE Advance Publication DOI:10.1248/cpb.c21-00180 May 21, 2021
1 Chem. Pharm. Bull.
2
3 Regular Article
4
5 UHPLC/MS and NMR-Based Metabolomic Analysis of Dried Water Extract of
6 Citrus-Type Crude Drugs
7
8 Takashi Tsujimotoa,b, Ryoko Araia, Taichi Yoshitomia,c, Yutaka Yamamotod,
9 Yoshihiro Ozekib, Takashi Hakamatsukaa and Nahoko Uchiyamaa*1
10
11 aNational Institute of Health Sciences; 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki,
12 Kanagawa 210-9501, Japan:
13 bTokyo University of Agriculture and Technology; 2–24–16 Naka-cho, Koganei, Tokyo
14 184–8588, Japan:
15 cKanagawa Prefectural Institute of Public Health, 1-3-1 Shimomachiya, Chigasaki,
16 Kanagawa 253-0087, Japan: and
17 dTochimoto Tenkaido Co., Ltd.; Oniya Kaibara-cho, Tamba , Hyogo 669-3315, Japan
18
* To whom correspondence should be addressed;. e-mail: [email protected]
Ⓒ 2021 The Pharmaceutical Society of Japan 19 Summary
20 Citrus-type crude drugs (CCDs) are commonly used to formulate decoctions in Kampo
21 formula (traditional Japanese medicine). Our previous study reported metabolomic
22 analyses for differentiation of the methanol extracts of Citrus-type crude drugs (CCDs)
23 using UHPLC/MS, and 13C- and 1H-NMR. The present study expanded the scope of its
24 application by analyzing four CCD water extracts (Kijitsu, Tohi, Chimpi, and Kippi);
25 these CCDs are usually used as decoction ingredients in the Kampo formula. A principal
26 component analysis score plot of processed UPLC/MS and NMR analysis data indicated
27 that the CCD water extracts could be classified into three groups. The loading plots
28 showed that naringin and neohesperidin were the distinguishing components. Three
29 primary metabolites, α-glucose, β-glucose, and sucrose were identified as distinguishing
30 compounds by NMR spectroscopy. During the preparation of CCD dry extracts, some
31 compounds volatilized or decomposed. Consequently, fewer compounds were detected
32 than in our previous studies using methanol extract. However, these results suggested
33 that the combined NMR- and LC/MS-based metabolomics can discriminate crude drugs
34 in dried water extracts of CCDs.
35 Keywords: metabolomics; nuclear magnetic resonance; crude drug; citrus; liquid
36 chromatography/mass spectrometry; dried water extract.
37
Chemical and Pharmaceutical Bulletin Advance Publication 38 Introduction
39 In Japan, crude drugs are obtained from medicinal parts of plants or animals, cells,
40 secretions, extracts, and minerals.1,2) Numerous factors can affect the quality of these
41 crude drugs including their botanical origin, geographical divergence, growth conditions,
42 and processing method. The stable provision of crude drugs with reproducible medicinal
43 value is the first step towards establishing the global acceptance and use of traditional
44 Japanese medicines. The methods used to assure the quality of crude drugs include 1)
45 morphological methods, 2) chemical methods, and 3) genotype confirmation. The
46 chemical method involves qualitative and quantitative analyses of index components.
47 Metabolomics has gained importance in recent years as a strategy for quality evaluation
48 and control of crude drugs and the predictable reliability, quality, and efficacy of herbal
49 medicines. Metabolomics is also useful for the classification and identification of
50 natural products containing various organic compounds.3) Metabolomic studies employ
51 various analytical methods such as LC/MS,4) GC/MS,5) and NMR.6) Although LC/MS
52 and GC/MS-based metabolomics are considered superior due to sensitivity and
53 resolution, identification of marker compounds is difficult because of the structural
54 diversity of secondary metabolites of crude drugs. In contrast, NMR-based
55 metabolomics enables the determination or estimation of the structure of marker
56 compounds directly from their chemical shifts and coupling patterns. Thus, it is
57 recommended that all organic compounds with hydrogen or carbon atoms that are
58 difficult to analyze using LC/MS and GC/MS should be characterized by NMR-based
Chemical and Pharmaceutical Bulletin Advance Publication 59 metabolomic studies.
60 In our previous studies, we reported the metabolomic analyses of the methanol extracts
61 of five Citrus-type crude drugs (CCDs) using 13C- and 1H- NMR.7) We found that
62 13C-NMR-based metabolomics could be an effective method for the differentiation of
63 the five CCDs. We also compared the results from the metabolomic analysis done using
64 LC/MS and 13C-NMR techniques, illustrating differences between these analytical
65 methods;8) the study suggested that 13C-NMR metabolomics could be used for quality
66 control of crude drugs by cross-discrimination.7) Since there have been no reports on
67 13C-NMR metabolomics using processed crude drugs, we considered expanding the
68 application of this method to the discrimination studies of processed crude drugs. In the
69 present study, a metabolomic study on the differentiation of the dried boiling water
70 extracts of citrus-type crude drugs (CCDs water extracts) was conducted to evaluate
71 their quality. We performed UHPLC/MS and 13C- and 1H- NMR analyses of CCD water
72 extracts because these are usually used as the ingredient of decoction in the Kampo
73 formula. In Japan, six types of CCDs derived from the dried peels or fruits of Citrus
74 plants are regulated by the Japanese Pharmacopoeia1) and the Japanese Standards for
75 Non-pharmacopoeial Crude Drugs (non-JP crude drug standards).2) Their attributes are
76 summarized in Supporting Information (Table S1). Herein, we investigated the
77 discrimination of CCD water extracts using four of the six CCDs that are widely used in
78 various applications.
79
Chemical and Pharmaceutical Bulletin Advance Publication 80 Results and discussions
81 Overview of UHPLC/MS
82 A total of 33 CCD water extracts were analyzed in the present study (Table S2,
83 Supporting Information). Initially, 13 compounds were detected using UHPLC/MS
84 analysis and identified by direct comparison with standards or assignments of their
85 high-resolution MS and tandem MS (MS/MS) spectra (Fig. 1). The (+)-UHPLC/MS
86 chromatograms of four types of CCD water extracts are presented in Fig. 2a-d. In the
87 chromatogram of Kijitsu (Fig. 2a), characteristic flavanone neohesperidosides such as
88 naringin (1),9) neohesperidin (2),10) and melitizine (3);11) coumarin derivatives meranzin
89 hydrate (4),12) meranzin (5),13) polymethoxyflavones nobiletin (6),14) and tangeretin
90 (7)15) were observed. In the chromatogram of Tohi (Fig. 2b), two additional compounds
91 to those in Kijitsu were detected, namely neoeriocirin (8)16) and meranzin glucoside
92 (9).17) Flavanone rutinosides, narirutin (10)18) and hesperidin (11),18) and one
93 polymethoxyflavone - 3,5,6,7,8,3',4'-heptamethoxyflavone (12)19) were detected in the
94 chromatograms of Chimpi (Fig. 2c) and Kippi (type 2, Fig. 2d).
95 (-)-UHPLC/MS chromatograms of four types of CCD water extracts are presented in
96 Fig. S1a-d. In the (-)-UHPLC/MS chromatograms of Kijitsu and Tohi, 1, 2, 3, and 8
97 were detected (Fig. S1a-b). An acylated flavanone glycoside brutieridin (13)11) was
98 detected in (-)-UHPLC/MS chromatograms of Kijitsu and Tohi (Fig. S1a-b).
99
100 Overview of 13C- and 1H- NMR
Chemical and Pharmaceutical Bulletin Advance Publication 101 The evaluation of the constituents of each CCD water extract using the 13C- and
102 1H-NMR spectra was carried out and the signals of their characteristic constituents were
103 assigned. The structures of the five compounds identified in the NMR analysis are
104 presented in Fig. 1.
105 The C-NMR spectra of the CCD water extracts are presented in Fig. 3a–3d. In the
106 range of 110–210 ppm (aromatic region), flavanone compounds were found in Kijitsu
107 and Tohi (Fig. 3a and 3b). Specifically, aromatic carbon signals from the flavanone
108 skeleton corresponding to naringenin (an aglycon of 1 (118 and 130 ppm)) and
109 hesperetin (an aglycon of 2 (115, 120, and 150 ppm)) were observed in the spectra of
110 Kijitsu (around 165 ppm and 200 ppm, Fig. 3a).
111 In the range of 55–110 ppm (heteroatom-connected region), carbohydrate compounds
112 were found in Tohi, Chimpi, and Kippi (Fig. 3b–3d). In contrast, the carbon signals of a
113 glycoside neohesperidose (101 and 106 ppm) (a disaccharide included in 1 and 2) were
114 observed in the spectra of Kijitsu (Fig. 3a) and Tohi (Fig. 3b). For Tohi, Chimpi, and
115 Kippi (2) prepared from matured peels, the carbon signals of sucrose (14) (107 ppm)
116 and glucose (15) (95 and 100 ppm) were confirmed (Fig. 3b–3d). Compound 14 was
117 abundant in Tohi (Fig. 3b) while 15 (95 and 100 ppm) was abundant in the spectra of
118 Chimpi (Fig. 3c) and Kippi (type 2, Fig. 3d).
119 In the aliphatic region, a flavanone skeleton (corresponding to the naringenin skeleton,
120 45 ppm, C-3) was observed. The methyl group at the 6th position of rhamnose (20 ppm)
Chemical and Pharmaceutical Bulletin Advance Publication 121 included in 1 and 2 was detected in the spectra of Kijitsu (Fig. 3a). In the spectra of Tohi
122 (Fig. 3b) and Kippi (type 2, Fig. 3d), the aliphatic carbon signals of proline (16) were
123 detected (26 and 32 ppm).
124 The full range 1H-NMR spectra of the four types of CCD water extracts are presented in
125 Fig. S2 a - d; compounds 1, 2, 14, and 15 were identified (Fig. 1). There were fewer
126 compounds detected using water extract compared with our previous studies using
127 MeOH extract.7,8) This could be attributed to the volatilization or decomposition of
128 some of the compounds during the preparation of the CCDs dry extracts.
129
130 Principal component analysis (PCA) of UHPLC/MS
131 Differences in the component content of CCD extracts were also reflected in the PCA
132 plots using (+)-UHPLC/MS data (Fig. 4a, the corresponding sample number of Kijitsu
133 and Tohi were presented in Supporting information Fig. S3a). When the sample clusters
134 were in the two-dimensional space of two vectors, principal component 1 (PC1, 50%)
135 and PC2 (21%), the CCDs were classified into three groups with high statistical values
136 of Rx2 (0.973) and Q2 (0.945, Fig. 4): (A) Kijitsu, (B) Tohi, and (C) Chimpi and Kippi
137 (type 2). The PCA plot positioned group (A) Kijitsu and (B) Tohi in the positive
138 direction (in the first PC [PC1]) to the X-axis, and (C) Chimpi and Kippi (type 2) in the
139 negative direction. (A) Kijitsu and (B) Tohi (CCDs in the positive direction) are
140 commonly prepared from sources such as Citrus aurantium and C. natsudaidai, while
141 the two negatively-positioned crude drugs ((C) Chimpi and Kippi (type 2)) are derived
Chemical and Pharmaceutical Bulletin Advance Publication 142 from C. unshiu or C. reticulata. This suggested that it was possible to evaluate the
143 species of the botanical source of CCDs in the PC1 even if the crude drug dry extracts
144 were used as analytes, although our previous study suggested the possibility of CCD
145 differentiation based on their botanical origin.8) The Kijitsu group (A) was segregated
146 into two subclusters, possibly due to the differentiation between the two-botanical origin
147 of Kijitsu, C. aurantium and C. natsudaidai. In the loading plot (Supporting information
148 Fig. S4a), the flavanone neohesperidosides 1 and 2 and coumarin derivatives 4 and 9
149 contributed negatively to PC1, corresponding to (A) Kijitsu and (B) Tohi (Fig. 4a). In
150 contrast, the flavanone rutinosides 10 and 11 showed a negative contribution to the PC1,
151 corresponding to (C) Chimpi and Kippi (type 2). Furthermore, 1, 2, and 10, 11, and 12
152 showed a positive contribution to the PC2 that corresponded to (A) Kijitsu (Fig. 4a).
153 Also, 4 and 9 showed a negative contribution equivalent to (B) Tohi (Fig. 4a). The
154 contributors assigned from the loading plot are illustrated in Fig. 6. Thus, discrimination
155 of the powdered extracts of CCDs was based on their botanical origin through the
156 observation of differences in the sugar chains of the flavanone glycosides in the PC1.
157 The PCA plot of the (-)-UHPLC/MS (Rx2 (0.981) and Q2 (0.954)) showed differences in
158 the constituents of the above-mentioned botanical sources (Fig. 4b, the corresponding
159 sample number of Kijitsu and Tohi were presented in Supporting information Fig. S3b).
160 It is likely that differentiation according to the botanical origin took place as well as in
161 the case of (+)-UHPLC/MS. The loading plot (Supporting information Fig. S4b)
162 differentiated compounds based on the original plant species, similarly to
Chemical and Pharmaceutical Bulletin Advance Publication 163 (+)-UHPLC/MS. Compounds 3 and 8 were observed as contributors of (B) Tohi. The
164 UHPLC/MS conditions used in this study also provided information on secondary
165 metabolites. The flavanone glycosides in the Citrus species contained sugar chains
166 according to the plant species. Therefore, the results of differentiation using secondary
167 metabolites also reflected the differences in botanical origin.
168
169 PCA of NMR
170 The PCA score plot of the 13C-NMR spectra data derived from the four CCD water
171 extracts (statistical values: Rx2 (0.877) and Q2 (0.721)) and 1H-NMR spectra (Rx2
172 (0.941) and Q2 (0.907)) are shown in Fig. 5 (The corresponding sample number were
173 presented in Supporting information Fig. S5a and S5b). The PCA models indicated that
174 the CCD water extracts were classified into the following three groups in both the 13C-
175 and 1H- NMR-based analyses: (A) Kijitsu, (B) Tohi, (C), and Chimpi and Kippi (type 2).
176 This classification of the type of CCD water extracts indicates a high distinguishability
177 of these models. The loading plots of the PC1 and PC2 are shown in Supporting
178 information Fig. S6 (Fig. S6a: 13C-NMR and Fig. S6b: 1H-NMR). The assignment of
179 the 13C-NMRspectra revealed the characteristics of compounds 1, 2, 14, and 15 (Fig.
180 S6a); these compounds were responsible for the differentiation in the PCA score plot.
181 The multivariate analysis based on the 13C-NMR spectra (Fig. S6a) identified an area
182 corresponding to the Kijitsu score plot (Fig. 5a, A) that indicated the presence of
183 compounds 1 and 2 in greater abundance than in the other three CCD water extracts
Chemical and Pharmaceutical Bulletin Advance Publication 184 (Fig. S6a). A section corresponding to Tohi (Fig. 5a, B) showed that compound 14 was
185 present in a greater abundance than other CCDs. The area corresponding to Chimpi and
186 Kippi (type 2) in the score plot (Fig. 5a, B and C) indicated a greater abundance of
187 compound 15 than other CCDs (Fig. S6a). Similar contributors were detected in the
188 loading plot of 1H-NMR (Fig. S6b) for CCDs extracts differentiation except the
189 compound 2.
190 Conclusions
191 Four types of CCD water extracts were analyzed by UHPLC/MS and 13C- and 1H-NMR
192 and the data were used for metabolomic evaluation. PCA revealed that the
193 discrimination of CCD water extracts was successfully achieved irrespective of the
194 analytical method. An analysis of the loading plot identified the contributing
195 components for the differentiation of CCDs (Fig. 6).
196 Similar to our previous studies, UHPLC/MS and NMR-based metabolomic analysis
197 enabled the discrimination of CCD water extract, however, with differences in
198 components that contributed to group discrimination. It was confirmed that
199 metabolomic analysis using NMR and UHPLC/MS could be applied to distinguish CCD
200 water extracts. The metabolomic analysis using 13C-NMR spectra was also applicable to
201 distinguishing the CCD water extracts as well as methanol extracts. Further
202 development of the quality evaluation using metabolomics for the discriminant and
203 integrated analyses using multiple analytical methods is currently underway.
204
Chemical and Pharmaceutical Bulletin Advance Publication 205 Experimental
206 Chemicals
207 Naringin, neohesperidin, methanol (HPLC grade), 0.1% formic acid, methanol-d4, and
208 acetonitrile (containing 0.1% formic acid) were purchased from Kanto Chemical Co.,
209 Ltd. (Tokyo, Japan); neoeriocitrin and nobiletin from Extrasynthase SAS (Lyon,
210 France); meranzin hydrate and meranzin from Ark Pharm Inc. (Arlington Heights, IL,
211 USA); narirutin and 3,5,6,7,8,3′,4′-heptamethoxyflavone from Sigma-Aldrich (St. Louis,
212 MO, USA); hesperidin from ACROS (Geel, Belgium); tangeretin from Tokyo Chemical
213 Industry Co., Ltd (Tokyo, Japan); and sodium 4,4-dimethyl-4-silapentane-1-sulfonate-d6
214 (DSS-d6) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
215
216 Plant Materials
217 All the crude drugs were obtained as JP17 or Non-JPS2018 grade; details are
218 summarized in Supporting Information Table S2. Commercially available products for
219 11 Kijitsu species (JP17 product), 8 Tohi species (JP17 product), 11 Chimpi species
220 (JP17 product), and 3 Kippi species (type 2, Non-JPS2018 product) were used.
221
222 Preparation of CCD Water Extracts and Sample Preparation
223 Twenty-five grams of each drug was suspended in water (500 mL). After boiling the
224 resulting suspension for 1 h, the mixture was filtered and the filtrate was lyophilized to
225 produce the powdered form of the water extract. For UHPLC/MS analysis, 100 mg of
Chemical and Pharmaceutical Bulletin Advance Publication 226 the powdered extract was suspended in 1 mL of methanol, sonicated for 10 min, and
227 centrifuged at 2500 ×g for 10 min. The supernatant was filtered through a membrane
228 filter (0.45 μm; Merck, Kenilworth, NJ, USA) to obtain a sample stock solution of 100
229 mg/mL. This stock solution was diluted 20-fold and used as the sample solution (5
230 mg/mL) for UHPLC/MS measurements. For NMR analysis, the powdered extract (100
231 mg) was suspended in solvent (1 mL methanol-d4, containing 0.5 mg/mL DSS-d6 as an
232 internal standard). The resulting suspension was subjected to ultrasonication for 10 min,
233 centrifuged at 2500 ×g for 10 min, and filtered through a membrane filter (0.45 m;
234 Merck). The methanol-d4 filtrate of each sample was transferred to a 5 mm NMR
235 sample tube for spectral measurements.
236
237 UHPLC/MS Data Analysis
238 The UHPLC system was interfaced with a Q Exactive hybrid quadrupole-orbitrap mass
239 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Two microliters of each
240 sample were introduced using full-loop injection into an UltiMate 3000 RS LC system
241 equipped with a photodiode array detector (Thermo Fisher Scientific) onto an Acquity
242 UPLC HSS T3 column (100 × 2.1 mm, particle size 1.8 μm, Waters) maintained at
243 40 °C. The mobile phase consisted of a 0.1% aqueous solution of formic acid (phase A)
244 and acetonitrile containing 0.1% formic acid (phase B), at a flow rate of 0.4 mL/min.
245 Gradient elution was performed using the following parameters: 5% to 40% B in the
246 initial 5 min, 95% B in successive 9 min increments, holding for 3 min, and then
Chemical and Pharmaceutical Bulletin Advance Publication 247 returned to the initial ratio (5%B) in 0.2 min. MS was measured in the positive- and
248 negative-ion electrospray modes. Nitrogen was used as the desolvation gas at 300 °C.
249 The capillary and cone voltages were set to 4000 and 35 V, respectively. Data were
250 collected over the range of m/z 150 to 2000 and were centroided during the acquisition.
251 All data obtained from the four assays in the two systems in both the positive and
252 negative ion modes were processed using Progenesis QI data analysis software
253 (Nonlinear Dynamics, Newcastle upon Tyne, UK). This was used for peak picking,
254 alignment, and normalization to produce peak intensities for retention time and m/z data
255 pairs. The ranges of the automatic peak picking assays were between 1.5 and 14 min.
256 The assignment of individual peaks was performed by direct comparison of the
257 UHPLC/MS data of the CCDs with those of authentic compounds.
258
259 NMR Data Acquisition
260 1H-NMR and 13C-NMR were measured at 800 and 201 MHz, respectively, using a
261 JEOL JNM-ECZ800 spectrometer (JEOL, Tokyo, Japan) equipped with the Ultracool™
262 probe. For the 1H-NMR spectra, the water signal was suppressed using the presaturation
263 method. The measurement parameters were: 32768 data points; spectral width -5 to 15
264 ppm; acquisition time 1.64 s; delay time 5.36 s; and 64 scans. The parameters of the
265 13C-NMR spectra were: 65536 data points; spectral width -25 to 225 ppm; acquisition
266 time 1.04 s; delay time 1.96 s; and 3000 scans. The NMR data were processed using the
267 Alice2 metabolome program (JEOL). The NMR spectral data were reduced to the
Chemical and Pharmaceutical Bulletin Advance Publication 268 integral region of equal width (0.04 ppm) corresponding to the region of -0.1 to 10.1
269 ppm spectral buckets for 1H-NMR and 0.2 ppm corresponding to -10 to 210 ppm
270 spectral buckets for 13C-NMR. All spectra were aligned and normalized to DSS using
271 the Alice2 metabolome program. Before the multivariate analysis, the data
272 corresponding to residual water (4.70 - 4.90 ppm for 1H-NMR), methanol (3.20 - 3.40
273 ppm for 1H-NMR, 48.0 - 52.0 ppm for 13C-NMR), and DSS (-0.10 to 0.10 ppm for
274 1H-NMR, -10.0 to 5.0 ppm for 13C-NMR) were removed from the data sets. The
275 assignment of each signal was performed by a direct comparison of the NMR spectra of
276 the crude drugs with those of authentic compounds.
277
278 Data Analysis
279 The resulting data sets were imported into SIMCA version 14.0 (Umetrics, Umeå,
280 Sweden) for further multivariate statistical analysis. The resultant data matrices were
281 mean-centered and then scaled using Pareto scaling. Hotelling’s T2 region shown as an
282 ellipse in the score plots defined the 95% confidence interval of the modeled variation.
283 The quality of the model was checked using Rx2 and Q2 values.
284
285 Acknowledgments
286 This research was supported by the Japan Agency for Medical Research and
287 Development (AMED), grant number JP19mk0101102. This work was also supported
288 by the Japan Food Chemical Research Foundation.
Chemical and Pharmaceutical Bulletin Advance Publication 289 Conflict of Interest
290 The authors declare no conflict of interest.
291
292 Supplementary Materials
293 The online version of this article contains supplementary materials.
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Chemical and Pharmaceutical Bulletin Advance Publication 1
2
3 Fig. 1. Structures of the compounds detected in the UHPLC/MS analysis (1 - 13) and
4 NMR (1, 2, 14 - 16).
5
Chemical and Pharmaceutical Bulletin Advance Publication A: Kijitsu (K-6)
B: Tohi (T-4)
C: Chimpi (C-5)
D: Kippi (KP-2)
6
7 Fig. 2. (+)-UHPLC/MS chromatograms of CCD water extracts.
Chemical and Pharmaceutical Bulletin Advance Publication 8
9 Fig. 3. 13C-NMR spectra of CCD water extracts.
Chemical and Pharmaceutical Bulletin Advance Publication 10
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12 Fig. 4. PCA score plot of CCD water extracts. a): ESI (+)-UHPLC/MS; b): ESI
13 (-)-UHPLC/MS.
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Chemical and Pharmaceutical Bulletin Advance Publication 21 22 Fig. 5. PCA score plot of CCD water extracts based on a) 13C-NMR and b) 1H-NMR
23 spectra.
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Chemical and Pharmaceutical Bulletin Advance Publication (+)-ESI-MS (+/-)-ESI-MS (+/-)-ESI-MS NMR NMR
26 27
28 Fig. 6. Structures of contributors derived from UHPLC/MS and NMR metabolomics of
29 CCD water extracts.
Chemical and Pharmaceutical Bulletin Advance Publication