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Xianmei Deng1 ∗∗ Jiangyong Yu2 ∗∗ Ming Zhao3 Bin Zhao4 Xingyang Xue5 ChunTao Che3 Jiang Meng1,3 Shumei Wang1 ∗ 1 Department
of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, Guangdong, China 2 Division of Traditional Chinese Medicines Standard, Chinese Pharmacopoeia Commission, Beijing, China 3 Department of Medicinal Chemistry and Pharmacognosy, and WHO Collaborating Center for Traditional Medicine, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USA 4 Zhongshan Torch Polytechnic, Zhongshan, Guangdong, China 5 Guangzhou Medical University Cancer Hospital and Institute, Guangzhou, Guangdong, China Received March 15, 2015 Revised May 8, 2015 Accepted June 2, 2015
Research Article
Quality assessment of crude and processed ginger by high-performance liquid chromatography with diode array detection and mass spectrometry combined with chemometrics A sensitive, simple, and validated high-performance liquid chromatography with diode array detection and mass spectrometry detection method was developed for three ginger-based traditional Chinese herbal drugs, Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata. Chemometrics methods, such as principal component analysis, hierarchical cluster analysis, and analysis of variance, were also employed in the data analysis. The results clearly revealed significant differences among Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata, indicating variations in their chemical compositions during the processing, which may elucidate the relationship of the thermal treatment with the change of the constituents and interpret their different clinical uses. Furthermore, the sample consistency of Zingiberis Rhizoma, Zingiberis Rhizome Preparatum, and Zingiberis Rhizome Carbonisata can also be visualized by high-performance liquid chromatography with diode array detection and mass spectrometry analysis followed by principal component analysis/hierarchical cluster analysis. The comprehensive strategy of liquid chromatography with mass spectrometry analysis coupled with chemometrics should be useful in quality assurance for ginger-based herbal drugs and other herbal medicines. Keywords: Chemometrics / Ginger / High-performance liquid chromatography / Mass spectrometry / Quality assessment DOI 10.1002/jssc.201500294
1 Introduction In Chinese medicine, there are three ginger-based herbal drugs, Zingiberis Rhizoma (Ganjiang, ZR), Zingiberis Rhizome Preparatum (Paojiang, ZRP), and Zingiberis Rhizome Carbonisata (Jiangtan, ZRC). ZR is the dried rhizome of Zingiber officinale Rosc., which is a well-known herbal medicine and edible plant widely used in China, Indian and other South-Eastern Asian countries for thousands of years [1, 2], and the latter two are the processed materials of ZR that are used for different clinical purposes in traditional Chinese medicine (TCM) [3]. The ZR is prepared by collecting the Correspondence: Prof. Jiang Meng, Department of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China E-mail:
[email protected] Fax: +86-020-2039352169
Abbreviations: ANOVA, analysis of variance; DAD, diode array detector; HCA, hierarchical cluster analysis; PCA, principal component analysis; TCM, traditional Chinese medicine; VIP, variable importance in the projection; ZR, zingiberis rhizoma; ZRP, zingiberis rhizome preparatum; ZRC, zingiberis rhizome carbonisata C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
root bark of Zingiberis Rhizoma, which is washed, cleaned, and cut into sheets or pieces and sun-dried. ZRP is usually prepared by a sand frying method, i.e. stir-frying the clear river sand in a frying vessel to the lubricating state (about 310⬚C) and then immersing ZR in the river sand with continual stir-frying until its surface turns in light brown with a brown-colored inside. It is removed, sieved, and cooled to obtain the ZRP sample. ZRC is produced by stir-frying, i.e. stir-frying the ZR in a utensil heated to a high temperature until the bark surface turn into black-brown and burnt-browncolored inside [3, 4]. According to TCM theory, proper processing of raw herbal drugs may reduce the toxicity or enhance the efficacy, and may also change the properties of the drug for different clinical practice [5, 6]. In TCM theory, ZR has the effect of warming and dispelling cold, venation restoration, warming lung to reduce watery phlegm, which can be used to cure cold, vomiting, diarrhea, and cough. ZRP can warm meridian, relieve pain, be used to cure spleen and stomach cold, vomiting, and diarrhea. While ZRC has the function of warming meridian and hemostasis and is used for ∗ Additional corresponding author: Prof. Shumei E-mail:
[email protected] ∗∗ These authors contributed equally to this work.
Wang
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hemorrhage of deficiency cold, hematochezia, metrorrhagia, and metrostaxis [3, 7, 8]. Modern pharmacological investigations also showed their difference in vivo experiments, ZR had the best effect in warming and dispelling cold than the other two processed drug, on the other hand ZRP had the best warming, relieve pain, and antidiarrheal. While ZRC had the best function in hemostasis [9–12]. Studies have also revealed that the stimulation effects of ZR on RAW 264.7 Cells were significantly higher than those of ZRP and ZRC, while the processed product could enhance the anticancer effects of ginger on human Hela cancer cells [13, 14]. Thus, it was necessary to discriminate ZR, ZRP, and ZRC in clinical practice. To date, a variety of methods have been developed for studying the chemical constituents of ginger [1,14–24]. However, most researchers focused on fresh or dried ginger or steamed ginger, but paid little attention to traditional processed ginger, i.e. ZRP, ZRC, and their ingredients. Here, an analytical method of HPLC coupled with diode array detector (DAD) and MS analysis combined with chemometrics was used to compare three kinds of traditional ginger drugs. By which the multivariate data from the complexity of herbal drugs can be analyzed by professional software to yield more objective, and reliable to elucidate the relationship of the thermal treatment with the constituents change. And similar attempts have also been applied successfully in some cases of other herbal medicines [24–26]. In the present study, samples of ZR, ZRP, and ZRC were analyzed by HPLC–DAD–MS, followed by chemometrics analysis, including similarity analysis, principal component analysis (PCA), hierarchical cluster analysis (HCA), and analysis of variance (ANOVA). Significant difference among ZR, ZRP, and ZRC caused by heating process was revealed.
2 Materials and methods 2.1 Materials and reagents Eighteen batches of ZR samples (No. 1–18) were collected from different geographical regions of China, and authenticated by Professor Jizhu Liu (School of Traditional Chinese Materia Medica, Guangdong Pharmaceutical University). Voucher specimens were deposited at the Herbarium Centre, Guangdong Pharmaceutical University. Part of each batch were processed to ZRP (No. 19–36) and ZRC (No. 37–54) according to Chinese Pharmacopoeia (2010 edition), respectively. The details were summarized in Table 1. The reference standards zingiberone and 6-shogaol were purchased from Chengdu Herb purify, and 6-gingerol, 8-gingerol, and 10-gingerol were obtained from the Nantong Feiyu Biological Technology. Diacetoxy-6-gingerdiol was prepared in our lab, and its structure was elucidated by comparing the spectroscopic data (ESI-MS, 1 H NMR, and 13 C NMR) in the literature [22]. The purity of all the standards C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
was all above 98% by HPLC analysis. Their structures are listed in Fig. 1. The HPLC grade acetonitrile and methanol was purchased from Merck (Darmstadt, Germany), and acetic acid was from Guangzhou East Giant Experimental Instrument (Guangzhou, China). Watson’s pure water was used throughout the study. Other chemicals were of analytical grade obtained from Guangzhou East Giant Experimental Instrument (Guangzhou, China). 2.2 Apparatus and chromatographic conditions An Agilent 1200 series HPLC system equipped with DAD (Agilent Technologies, USA), a mass spectrometer with ESI detector (Applied Biosystems, Foster City, CA, USA) and an Ultimate TM XB-C18 analytical column (250 mm × 4.6 mm, 5 m, Alltech Associates, USA) were used for all analyses. The mobile phase consisted of a mixture of acetonitrile and 0.1% v/v acetic acid in water. A gradient program was set as follows: the percentage of acetonitrile was increasing from 10 to 25% in the first 10 min, then to 35% in the second 10 min, and to 75% in the next 25 min, finally increased to 100% in another 35 min, and concluded by keeping on 100% for 10 min. The flow rate, detection wavelength, injection volume, and column temperature were set at 0.6 mL/min, 280 nm, 10 L, and 30⬚C, respectively. The data were obtained and processed with software of Chemstation (Agilent, USA). For MS part, Nitrogen was used as dry gas (40 mL/min, 150⬚C). Both positive and negative ESI modes were selected. The values for sprayer voltage, orifice voltage, and focusing ring voltage were set at 5000, 101, and 380 V, respectively. The scan range was m/z 100–1000. All data acquired were processed by MaccChrom 1.1 software (Applied Biosystems, CA, USA). 2.3 Preparation of standard solutions Primary stock solution of the six standards were prepared in methanol, to get a concentration of 0.706 mg/mL (zingiberone), 0.528 mg/mL (6-shogaol), 0.580 mg/mL (6-gingerol), 0.974 mg/mL (8-gingerol), 0.468 mg/mL (10gingerol), and 0.410 mg/mL (diacetoxy-6-gingerdiol), respectively. Working standard solutions were prepared daily by mixing and diluting the stock solutions with methanol. The standard stock and working solutions were stored at 4⬚C. 2.4 Sample preparation Each ZR, ZRP, and ZRC sample was ground and passed through a 180 mesh (80 mm) sieve. The powder (0.3 g) were accurately weighed and extracted with 10 mL of methanol for 40 min by sonication in room temperature. Additional methanol was added to make up the lost. An aliquot of 10 L solution was injected for HPLC analysis after filtration with a 0.22 m membrane filter. www.jss-journal.com
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Table 1. Sample list of ZR, ZRP, and ZRC, and the results of similarity tests
Sample No.
Collection location
ZR
ZRP
ZRC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mean
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
Deyang, Sichuan province, China Chengdu, Sichuan province, China Nanchong, Sichuan province, China Meishan, Sichuan province, China Qianwei, Sichuan province, China Maoming, Guangdong province, China Zhanjiang, Guangdong province, China Zhanjiang, Guangdong province, China Yangjiang, Guangdong province, China Qujing, Yunnan province, China Wenshan, Yunnan province, China Baoshan, Yunnan, China Nanning, Guangxi province, China Qinzhou, Guangxi province, China Guigang, Guangxi province, China Zunyi, Guizhou province, China Anshun, Guizhou province, China Bozhou, Anhui province, China
Collection date
2013-12-12 2013-12-25 2014-6-23 2013-11-28 2013-12-05 2013-10-13 2014-01-25 2013-02-15 2014-2-21 2014-1-13 2014-1-16 2013-2-19 2014-1-18 2014-1-17 2014-2-27 2013-10-21 2013-12-09 2013-12-26
2.5 Data analysis Similarity analysis was performed using a professional software, the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine Version 2004A (Chinese Pharmacopoeia Commission, China), which was recommended by the State Food and Drug Administration of China. The software employs correlative coefficients in the process of evaluating similarities of different chromatograms. PCA was performed with SIMCA-P + 12.0 software. Both HCA and ANOVA were calculated with SPSS16.0 software. Data are presented as mean ± SD. 2.6 Method validation The linearity of the HPLC method was evaluated by the calibration curves (Table 2). A range of concentrations of all analytes were analyzed in triplicates. The LOD and LOQ for each analyte were determined as S/N of 3 and 10, respectively. Precision of the method (Sample No. 26) was determined by intraday and interday measurements. The standard solution was analyzed in six replicates on the same day to obtain intraday precision, and they were analyzed daily (six replicates) for three successive days to obtain the interday results. The stability was assessed by analyzing the same sample solution of ZR, ZRP, and ZRC (Sample No. 8, 26, 44) at 0, 3, 6, 9, 12, 24 h, respectively. Meanwhile, to investigate the accuracy of the developed method, recovery tests (Sample No. 26) were performed according to Chinese pharmacopoeia [3].
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Similarity ZR
ZRP
ZRC
ZR– ZRP
ZR– ZRC
ZRP– ZRC
0.953 0.985 0.981 0.954 0.961 0.948 0.931 0.953 0.985 0.825 0.971 0.975 0.983 0.981 0.986 0.981 0.988 0.935 0.960 ± 0.038
0.931 0.985 0.962 0.962 0.943 0.963 0.877 0.950 0.982 0.951 0.983 0.940 0.992 0.951 0.978 0.958 0.965 0.941 0.956 ± 0.026
0.938 0.960 0.929 0.873 0.900 0.928 0.818 0.954 0.927 0.800 0.915 0.923 0.905 0.938 0.945 0.972 0.959 0.960 0.919 ± 0.047
0.904 0.880 0.867 0.885 0.886 0.798 0.796 0.830 0.884 0.713 0.871 0.93 0.873 0.924 0.912 0.892 0.914 0.827 0.866 ± 0.055
0.565 0.551 0.488 0.503 0.415 0.426 0.368 0.471 0.509 0.222 0.316 0.368 0.446 0.538 0.388 0.398 0.46 0.609 0.447 ± 0.095
0.634 0.73 0.618 0.626 0.635 0.537 0.652 0.639 0.656 0.346 0.482 0.492 0.624 0.616 0.555 0.582 0.609 0.694 0.596 ± 0.088
Different concentration levels (50, 100, 150%) of mixed standard solution were added into the known real samples of 0.15 g, and each concentration was done three copies in parallel according to the established method. The results were expressed as % RSD of the measurements (Table 3).
3 Results and discussion 3.1 Optimization of extraction conditions To obtain the optimal extraction conditions, the use of different extraction methods (reflux or sonication), different extraction solvents (various concentration of aqueous methanol, ethanol, and ethyl acetate), and extraction time (20, 30, 40, or 50 min) were tried (Sample No. 26, Table 4). The highest yields of the selected components were obtained after 40 min of sonication in methanol. 3.2 Optimization of HPLC system After several trials on different mobile phase combination, such as methanol or acetonitrile and water or acid-modified water, a mixture of 0.1% v/v acetic acid in water and acetonitrile was selected as mobile phase. The flow rate, wavelength, and column temperature were set at 0.6 mL/min, 280 nm, and 30⬚C, respectively. Under the optimized conditions, good baseline, good resolution for target peaks, and reasonable analytical time were warranted (Fig. 2).
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Figure 1. Structures of identified compounds. (2) 1,2,4,5-tetrahydroxyphenol; (14) zingiberone; (23) 6-gingerol; (24) 8-gingerol; (25) 6-shogaol; (27) diacetoxy6-gingerdiol; (29) 1-dehydro-6-gingerdione; (30) 10-gingerol; (31) 4-[2-(5-butyl-2furanyl)ethyl]-2-methoxyphenol; (33) methyl diacetoxy-6-gingerdiol; (34) 1-dehydro-8gingerdione; (35) 10-gingerdione. Table 2. Standard curves, LOD, and LOQ values of six components
Compound
Regression equation
R2
Range (g/mL)
LOD (×10−3 g)
LOQ (×10−2 g)
Zingiberone 6-Gingerol 8-Gingerol 6-Shogaol Diacetoxy-6-gingerdiol 10-Gingerol
y = 1400.8x − 6.4591 y = 669.74x − 6.5215 y = 837.36x + 6.4058 y = 951.22x + 0.4535 y = 595.14x + 7.7187 y = 750.94x + 1.1775
0.9997 1.0000 0.9998 1.0000 0.9998 0.9999
2.65 105.90 10.15 406.00 4.87 194.80 5.28 211.20 6.14 245.70 7.02 280.80
2.239 4.105 4.312 4.987 5.109 5.397
0.746 1.368 1.437 1.662 1.703 1.799
3.3 Method validation The calibration curves of each analyte displayed linearity (R2 > 0.9997) over a range of concentrations (Table 2). LOD and LOQ were within the range 2.24–5.40 and 7.46–17.99 ng (Table 2), respectively. The RSD values of C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
precision test were 0.29–1.28% for intraday assays and 1.35–1.98% for interday assays (Table 3). The RSD values of stability test were 0.27–1.93%. The recovery of the method was above 95% (Table 3). The system was considered suitable for the chemical analysis of ZR, ZRP, and ZRC.
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Table 3. Results of precision, stability, and recovery tests
Compound
Recovery (%)
Zingiberone
6-Gingerol
8-Gingerol
6-Shogaol
Diacetoxy-6-gingerdiol
10-Gingerol
Precision RSD (%)
Low (n = 3)
Medium (n = 3)
High (n = 3)
98.91 103.54 101.58 101.52 104.96 102.87 103.82 104.83 100.52 96.92 101.82 97.85 102.45 100.79 99.31 99.7 104.25 97.23
99.78 102.31 104.32 105.28 102.48 103.47 98.48 99.29 97.89 102.76 103.03 104.73 100.05 99.43 98.79 97.48 96.95 98.64
98.64 102.31 100.37 97.64 98.40 96.26 96.78 98.64 98.41 98.78 95.71 98.57 98.76 95.89 99.73 105.63 102.12 103.78
Stability RSD (%)
Intraday (n = 6)
Interday (n = 6)
ZR
ZRP
ZRC
1.28
1.92
0.27
0.30
0.45
0.29
1.66
0.30
0.52
0.72
0.62
1.98
0.64
1.03
1.47
0.32
1.55
0.35
0.36
0.86
1.23
1.35
0.41
1.76
1.93
0.38
1.96
0.31
0.76
0.86
a) Recovery (%) = 100 × (amount found – original amount)/amount spiked. b) RSD (%) = (SD/mean) × 100. Table 4. Optimization of extraction conditions
No.
1 2 3 4 5 6 7 8 9 10 11
Content (mg/g) Zingiberone
6-gingerol
8-gingerol
6-shogaol
Diacetoxy6-gingerdiol
10-gingerol
0.39 ± 0.13 0.36 ± 0.10 0.29 ± 0.08 0.31 ± 0.23 0.36 ± 0.16 0.23 ± 0.09 0.19 ± 0.07 0.27 ± 0.03 0.29 ± 0.06 0.36 ± 0.02 0.36 ± 0.17
2.05 ± 0.08 2.12 ± 0.05 1.92 ± 0.03 2.03 ± 0.03 2.14 ± 0.05 1.77 ± 0.08 1.76 ± 0.04 1.85 ± 0.03 1.98 ± 0.02 2.14 ± 0.07 2.15 ± 0.06
0.43 ± 0.21 0.59 ± 0.14 0.40 ± 0.11 0.47 ± 0.14 0.58 ± 0.03 0.49 ± 0.04 0.48 ± 0.04 0.48 ± 0.02 0.50 ± 0.03 0.57 ± 0.03 0.57 ± 0.13
2.44 ± 0.10 2.32 ± 0.07 1.98 ± 0.08 2.14 ± 0.05 2.31 ± 0.02 1.94 ± 0.05 1.86 ± 0.01 2.32 ± 0.13 2.36 ± 0.11 2.31 ± 0.06 2.32 ± 0.06
1.19 ± 0.07 1.28 ± 0.12 1.06 ± 0.14 1.16 ± 0.16 1.23 ± 0.05 1.10 ± 0.13 1.20 ± 0.33 1.11 ± 0.22 1.17 ± 0.10 1.23 ± 0.10 1.25 ± 0.08
0.99 ± 0.12 1.15 ± 0.06 0.71 ± 0.07 0.88 ± 0.04 1.14 ± 0.03 0.85 ± 0.05 0.88 ± 0.01 0.89 ± 0.02 1.01 ± 0.03 1.15 ± 0.05 1.14 ± 0.02
a) 1–2: Extracted with methanol for 60 min by reflux and sonication methods, respectively; 3–7: Sonicated for 40 min with 50% aqueous methanol, 80% aqueous methanol, methanol, ethanol, and ethyl acetate, respectively; 8–11: Sonicated with methanol for 20, 30, 40, and 50 min, respectively.
3.4 Sample analysis ZR, ZRP, and ZRC samples (18 batches each) were analyzed under optimized HPLC–DAD–MS conditions. Twelve peaks in samples were identified by comparing their UV profiles, retention times, and MS data with those of reference standards (Table 5). Based on the individual LC chromatograms of ZR, ZRP, and ZRC samples, a reference chromatogram for them (Fig. 2B, C, and D) was generated respectively using the software of the Similarity Evaluation System for Chromato-
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graphic Fingerprint of Traditional Chinese Medicine [27]. The reference chromatograms were subsequently used in the similarity test for each sample by comparing retentions time and peak area (Table 1). In similarity test, high average indexes of 0.960, 0.956, and 0.919 were observed within ZR, ZRP, and ZRC samples, respectively, indicating the high consistency of the samples. On the other hand, low similarities between ZR, ZRP, and ZRC samples (Table 1) indicated significant difference, revealing significant changes happened during the thermal
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Figure 2. Representative chromatograms of reference substances (A) and samples (BZR, C-ZRP, D-ZRC) (UV280 nm). (2) 1,2,4,5-tetrahydroxyphenol; (14) zingiberone; (23) 6-gingerol; (24) 8-gingerol; (25) 6-shogaol; (27) diacetoxy-6-gingerdiol; (29) 1-dehydro-6-gingerdione; (30) 10-gingerol; (31) 4-[2(5-butyl-2-furanyl)ethyl]-2methoxyphenol; (33) methyl diacetoxy-6-gingerdiol; (34) 1-dehydro-8-gingerdione; (35) 10-gingerdione.
Table 5. Peaks identification
Peak No.
Retention time (min)
ESI-MS (m/z) [M–H]−
[M+H]+
2 14 23 24 25 27 29 30 31
10.04 25.21 40.60 48.10 49.98 52.56 53.98 55.67 56.28
141 193 294 321 276 380 289 349 273
195 295 323 277 381 291 351 275
33
61.03
393
395
34 35
67.23 70.37
317 347
319 349
Identification
1,2,4,5-tetrahydroxyphenol zingiberone 6-gingerol 8-gingerol 6-shogaol diacetoxy-6-gingerdiol 1-dehydro-6-gingerdione 10-gingerol 4-[2-(5-butyl-2-furanyl) ethyl]-2-methoxyphenol methyl diacetoxy-6gingerdiol 1-dehydro-8-gingerdione 10-gingerdione
processing operation (Fig. 2). Based on their chromatograms, after processing from ZR to ZRP and ZRC, several new peaks were detectable, such as peaks 4–6, 8, 9, 14, 19, 31, and 36 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in both ZRP and ZRC, and peaks 3, 7, 11, 13, and 26 for ZRC, while peaks 12 and 16 disappeared. The relative content of some peaks 1, 2, 15, 23, 24, 30, and 33, became more or less. Quantitative analysis of six selected components was also carried out (Fig. 3). The contents of 6-gingerol, 8-gingerol, 10gingerol, and diacetoxy-6-gingerdiol were higher in ZR than that in ZRP and ZRC. Zingiberone was only detectable in ZRP and ZRC. Compared to the content in ZR, 6-shogoal was higher in ZRP but lower in ZRC. By one-way ANOVA analysis (Fig. 3), the contents of markers showed significant difference between ZR, ZRP, and ZRC samples with the exception of 8-gingerol and 6-shogoal between ZR and ZRP. All these changes above might be attributed to decomposing or cracking during the process on high temperature. During the heating process, gingerols transformed to shogaols or zingiberone by dehydration, so the concentrations of gingerols decreased but the levels of shogaols or zingiberone increased. However, heating treatment to a certain extent, shogaol will crack and its content lower in ZRC [14,22,28,29]. The study elucidate the relationship of the thermal treatment with the constituents change, which may also interpret the different effect of various processed drugs partially, and even demonstrate the significance of Chinese Materia Medica processing. www.jss-journal.com
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Figure 3. Contents of six components in ZR, ZRP, and ZRC samples. Compared with ZR * p < 0.05, ** p < 0.01; Compared with ZRP 䊉 p < 0.01.
Figure 4. The PCA results for ZR, ZRP, and ZRC samples. (A) Scores plot; (B) Loading plot.
3.5 PCA and HCA analyses PCA was employed to compare 54 samples of ZR, ZRP, and ZRC (Fig. 4). The peak areas of all peaks in the chromatograms and 54 samples were taken as variables and observation objects, respectively. First, the 54 samples fell into three distinct clusters corresponding to the sample properties (Fig. 4A), indicating significant differences existed among ZR, ZRP, and ZRC. Within individual clusters, samples of ZR and ZRP were tightly distributed, while those of ZRC were dispersed, revealing the C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 5. The HCA results for ZR (A), ZRP (B), and ZRC (C) samples.
lower consistency of ZRC samples. Since ZRC and ZRP were all from the same origin of ZR, the low consistency of ZRC samples may due to the unhomogenized sampling or / and processing operation. The PCA loading www.jss-journal.com
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plot (Fig. 4B) also indicated 1,2,4,5-tetrahydroxyphenol (peak at tR = 10.039), 6-gingerol (peak at tR = 40.602), 8-gingerol (peak at tR = 48.100), as well as other components at tR = 4.989, 12.071, and 13.978, which are the most discriminating variables between three classes, had great influence on the scores. For HCA, only peaks with variable importance in the projection (VIP) > 1 in partial least squares-discriminate analysis were selected, which working as potential markers can minimize the interference of noise, improve the classification efficiency. While peaks removed (VIP