Chem. Pharm. Bull. Regular Article UHPLC/MS and NMR

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.

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