Accepted Manuscript Investigating chemical features of Panax notoginseng based on integrating HPLC fingerprinting and determination of multi-constituents by single reference standard Zhenzhong Yang, Jieqiang Zhu, Han Zhang, Xiaohui Fan PII:
S1226-8453(17)30029-5
DOI:
10.1016/j.jgr.2017.04.005
Reference:
JGR 276
To appear in:
Journal of Ginseng Research
Received Date: 6 February 2017 Accepted Date: 17 April 2017
Please cite this article as: Yang Z, Zhu J, Zhang H, Fan X, Investigating chemical features of Panax notoginseng based on integrating HPLC fingerprinting and determination of multi-constituents by single reference standard, Journal of Ginseng Research (2017), doi: 10.1016/j.jgr.2017.04.005. 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.
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Investigating chemical features of Panax notoginseng based on
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integrating HPLC fingerprinting and determination of
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multi-constituents by single reference standard
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Zhenzhong Yang1, , Jieqiang Zhu1, , Han Zhang2, Xiaohui Fan1,*
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University, Hangzhou 310058, China.
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Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China.
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Contributed equally to this work.
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Correspondence should be addressed to Dr. Xiaohui Fan (
[email protected])
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Running title: Investigating chemical features of Panax notoginseng
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Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang
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ACCEPTED MANUSCRIPT Abstract
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Background: Panax notoginseng is a highly valued medicine and functional food,
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whose quality is considered to be influenced by the sizes, botanical parts and growth
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environments.
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Methods: In this study, a high-performance liquid chromatography method integrating
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fingerprinting and determination of multi-constituents by single reference standard
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(DMS) was established and adopted to investigate the chemical profiles and active
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constituent contents of 215 notoginseng samples with different sizes, from different
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botanical parts and geographical regions.
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Results: Chemical differences among main root, branch root and rotten root weren’t
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distinct, while rhizome and fibrous root could be discriminated from other parts. The
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notoginseng from Wenshan Autonomous Prefecture and cities nearby was similar,
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whereas samples from cities far away wasn’t. The contents of major active
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constituents in main root didn’t correlate with the market price.
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Conclusion: This study provided comprehensive chemical evidence for the rational
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usage of different parts, sizes and growth regions of notoginseng in practice.
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Keywords:
Chemical
profile;
Geographical
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Multivariate analysis; Panax notoginseng
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region;
HPLC
fingerprinting;
ACCEPTED MANUSCRIPT 1. Introduction
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Panax notoginseng (Burk.) F. H. Chen, commonly called notoginseng, “Sanqi” or
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“Sanchi” in Chinese, is primarily produced in southwest of China, which is highly
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valued for its haemostatic and cardiovascular protective effects [1, 2]. In east Asia,
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especially China, notoginseng has been historically consumed as a medicine and
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functional food [3, 4]. Additionally, various notoginseng products are available in the
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health food market in the United States as dietary supplements [5, 6].
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Underground parts of P. notoginseng are the main portions for usage, which include
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rhizome, main root, branch root, and fibrous root (Fig. 1A) [7]. Both rhizome and root
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(Notoginseng Radix et Rhizoma) are officinal in the Pharmacopoeia of the People’s
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Republic of China. Branch root and fibrous root are often used as the raw materials of
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Chinese tonic soup, or ground to yield powder as health food. Because of the specific
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growing conditions, notoginseng is vulnerable to root rot during planting [8, 9]. The
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rotten root is called “Chouqi” in Chinese, which is considered to be of inferior quality
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and sometimes adulterated into the normal raw materials. The quality of rotten root
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was rarely studied, not to mention the comparison with the normal underground parts
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of notoginseng.
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Notoginseng primarily grows in Wenshan Autonomous Prefecture of Yunnan Province,
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China [10]. In Wenshan Autonomous Prefecture, the southwestern counties, such as
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Wenshan County, Yanshan County, Maguan County and Qiubei County, are the most
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important notoginseng-producing areas. As the demand continues to increase, the
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notoginseng production in Wenshan Autonomous Prefecture is insufficient, and
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ACCEPTED MANUSCRIPT several areas near Wenshan Autonomous Prefecture, such as Honghe Autonomous
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Prefecture, Kunming City and Yuxi City, also cultivate this herb. The geographical
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distribution of P. notoginseng is illustrated in Fig. 1B. Notoginseng is not only the
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product of the specific genome [11], and the chemical composition is strongly
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influenced by the growing conditions such as terrain, soil and climate [12]. The
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qualities of notoginseng derived from different geographical origins should be
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investigated comprehensively.
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The size of main root is usually considered as an indication of its quality, which also
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greatly affect its price at the market. The size of main root is called Tou which means
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the number of individual dry main root in 500 g. At the market, the price of 20 Tou
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main root is about 2 fold higher than that of 60 Tou on December 12nd, 2016 (Fig. S1).
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Whether the main root of large size deserves the high price should be assessed.
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The chemical constituents in notoginseng are closely related to its quality, thus it is
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rational to assess the quality of notoginseng by investigating chemical composition of
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raw materials. Due to the multi-component, multi-target synergistic action of
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notoginseng [13, 14], quantification of the bioactive constituents is a key way to
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assess the quality of raw materials used. In notoginseng, saponins are thought to play
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the major role in its health function and therapeutic effect [15-17]. Several methods
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have been established to determine the contents of saponins in P. notoginseng. Jia et al
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[7] established a method for determination of 11 saponins in 1-, 2- and 3-year-old
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main root, rhizome and fibrous root of notoginseng by high-performance liquid
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chromatography-diode array detection (HPLC-DAD). Wan et al [18] developed a high
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liquid
chromatography-evaporative
light
scattering
detection
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(HPLC-ELSD) method for determining 8 major saponins in root, fibre root, rhizome,
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stem, leaf, flower and seed of notoginseng. Wang et al [19] established a method to
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determine 8 saponins in different parts of notoginseng using ultra-performance liquid
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chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS).
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Peng et al [20] developed a high performance liquid chromatography-charged aerosol
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detector (HPLC-CAD) method for the analysis of saponin contents in notoginseng.
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Among these saponins determined, notoginsenoside R1 (NG-R1), ginsenoside Rg1
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(G-Rg1), ginsenoside Re (G-Re), ginsenoside Rb1 (G-Rb1) and ginsenoside Rd (G-Rd)
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were regarded as the major active constituents [21]. In these studies, multiple
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reference standards are needed for quantitative determination of these constituents
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with routine methods, i.e., external standard method. However, shortage, cost and
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instability of chemical reference standards are issues that should be considered [22,
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23]. With this limitation in mind, determination of multi-constituents by single
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reference standard (DMS), which is also mentioned as single standard to determine
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multi-components (SSDMC) or quantitative analysis of multi-component with single
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marker (QAMS), is an outstanding solution [24-27]. Wang et al [28] established the
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DMS method for quantitative determination of saponins in notoginseng, however, few
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attention was paid to the minor constituents in notoginseng. To comprehensively
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reflect the quality of notoginseng, an ideal quality assessment should include not only
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quantitative information of contents of major active constituents from DMS, but also
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the information about minor constituents. Fingerprinting could identify as many
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constituents. In our previous study, chromatographic and spectroscopic fingerprints of
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notoginseng were developed [29]. It is urgently needed to integrate these two methods
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to holistically investigate the quality of notoginseng.
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In this study, an integrated method combining HPLC fingerprinting and DMS was
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developed to determine the contents of bioactive constituents and the chemical
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profiles meanwhile, and notoginseng samples with different sizes, from different
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botanical parts and different geographical regions were analyzed. The contents of
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major active constituents and HPLC fingerprinting coupled with multivariate analysis
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were used to investigate the quality of different notoginseng samples in term of
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chemical constituents. To the best of our knowledge, this is the most comprehensive
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report on chemical features of underground parts of P. notoginseng, and accomplished
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with the largest sample size. This study would provide comprehensive chemical
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evidence for the rational usage of different raw materials of notoginseng in practice.
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2. Materials and methods
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2.1 Chemicals and reagents
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Notoginsenoside R1 (NG-R1) and ginsenoside Rg1 (G-Rg1), ginsenoside Re (G-Re),
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ginsenoside Rb1 (G-Rb1), ginsenoside Rd (G-Rd) (purity >99%) were purchased from
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Ronghe Pharmaceutical Technology Development Co. Ltd. (Shanghai, China). HPLC
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grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany).
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Distilled water was purified by a Milli-Q system (Millipore).
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2.2 Sample preparation
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A total of 215 P. notoginseng samples were collected from different regions of Yunnan
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Province in China (Fig. 1). More specifically, these samples included rhizome (n=78),
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main root (n=119), branch root (n=6), fibrous root (n=7) and rotten root (n=5). The
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materials were authenticated by Associate Professor Liurong Chen, College of
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Pharmaceutical Sciences, Zhejiang University.
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The sample was ground using a disintegrator and then passed through a sieve (hole
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diameter, 280 µm). A total of 0.5 g powdered sample was extracted ultrasonically in
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40 ml of 70% methanol (v/v) for 60 min. The mixture was then filtered and the filtrate
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was evaporated to dryness in vacuum. The residue was redissolved, transferred to a 5
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ml volumetric flask, and then diluted to this volume with 70% methanol. The solution
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was filtered through a 0.22 µm filter membrane before HPLC analysis.
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2.3 HPLC fingerprinting
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The HPLC fingerprinting was performed on an Agilent 1100 HPLC system (Agilent,
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USA) consisting of a quaternary solvent delivery system, an on-line degasser, an
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autosampler, a column temperature controller and an ultraviolet detector. The
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chromatographic separation was carried out with reference to our previous study [29].
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Briefly, The separation was performed on an Agilent Zorbax C18 column (4.6 mm×
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50 mm, 1.8 µm) at a solvent flow rate of 0.8 mL/min at 35 ◦C. Water and acetonitrile
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were used as mobile phases A and B, respectively. The solvent gradient adopted was
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ACCEPTED MANUSCRIPT as follows: 0-22 min, 17-19% B; 22-30 min, 19-27% B; 30-35 min, 27% B; 35-47
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min, 27-46% B; 47-70 min, 46-90% B. The detection wavelength was 203 nm and the
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injection volume was 3 µL.
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All peaks in the chromatograms were integrated and analyzed by the professional
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software named Similarity Evaluation System for Chromatographic Fingerprint of
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Traditional Chinese Medicine (Version 2004A, Chinese Pharmacopoeia Commission).
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The SIMCA-P 12.0 (Umetrics, Sweden) was used to perform the multivariate
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analysis.
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2.4 Determination of multi-constituents by single reference standard
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The chromatographic conditions for determination of the five major bioactive
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saponins in notoginseng by single reference standard were the same as that of HPLC
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fingerprinting in Section 2.3. G-Rb1, one of the major saponins in notoginseng, was
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selected as the reference standard. G-Rb1 served as the external standard to determine
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the content of G-Rb1, and as the internal standard to simultaneously determine the rest
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four major bioactive saponins in notoginseng according to the conversion factors (Fx).
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The Fx of the rest four major bioactive saponins could be obtained with solutions of
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reference standards and calculated via Eq. (1). With the value of Fx, the concentration
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of the saponin (cx) could be obtained via Eq. (2).
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= = 164
/ (1) / × × (2)
in which cx and Ax represent the concentration and peak area of the analyte, cr and Ar 8
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represent that of the reference standard.
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Standard method difference (SMD) [30] was applied to evaluate the DMS method and
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the external standard method, which was calculated via Eq. (3). (
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× 100% (3)
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where cES and cDMS represent the concentrations of an analyte determined by external
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standard method and DMS method, respectively.
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3. Results and discussions
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3.1 Method validation
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The linearity of the quantitative analysis method was determined by spiking a series
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of mixed standard solutions with different concentrations. The calibration curves were
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constructed by plotting the peak areas (y) vs. the concentrations (x, mg/mL) of the 5
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saponins. The results of calibration were summarized in Table 1. Within relatively
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wide ranges, all the compounds showed good linearity (r ≥ 0.999). For intra-day
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precision test, a sample solution was analyzed for 6 replicates within the same day,
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and for inter-day precision test, a sample was analyzed for 6 consecutive days. The
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intra-and inter-day precisions were 0.35-0.54% and 0.83-4.94%, respectively, which
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indicated an acceptable precision of the developed method. Repeatability was
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evaluated by analyzing 6 independent replicate preparations of a sample, and the RSD
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values of the peak area of the five saponins were 1.49-1.72%, demonstrating the
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developed method was of satisfactory repeatability. The stability test was performed
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by analyzing the same sample solution at different time intervals (0, 4, 8, 12, 16, 20
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ACCEPTED MANUSCRIPT and 24 h), and the RSD values of the peak area of the five saponins were 1.00-1.78%,
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which indicated it was feasible to analyze samples within 24 h. The accuracy was
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validated by recovery rate through the standard addition method. The samples were
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spiked with a known amount (high, mediate and low levels) of standard solution, and
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then analyzed with the established HPLC method. The results showed that the mean
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recovery rates of the 5 saponins were in the range of 95.77-104.85% with RSDs less
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than 4.80%, which demonstrated the developed method had acceptable accuracy.
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G-Rb1 was selected as internal reference standard, due to that it was easy to obtain,
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low cost and steady. The conversion factors were calculated via Eq. (2) in Section 2.4,
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and the conversion factors of NG-R1, G-Rg1, G-Re, and G-Rd were 0.73, 0.78, 0.77
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and 0.79, respectively. Taguchi design was utilized to evaluate the stability of the
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DMS method. According to the Taguchi’s L9 (33) design, 9 experiments were
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conducted under various conditions of three factors, namely HPLC instruments
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systems (Agilent 1100, Agilent 1260 and Waters 2695), analytical columns (SB-C18,
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Eclipse Plus C18 and Extend-C18) and concentrations of analytes (high, middle and
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low), as listed in Table S1. Signal-to-noise (S/N) was introduced to evaluate the
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influence of these factors to conversion factors.
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As shown in Table 2, analysis of variance revealed that all of these three factors, i.e.,
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HPLC instruments systems, columns and concentrations of analytes, didn’t
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significantly fluctuate the conversion factors. Though the conversion factors was
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inevitably influenced under different analytical conditions, the variation was
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acceptable. Thus, in a certain range, this DMS method was robust enough to be
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Standard method difference (SMD), which represented the difference between the
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results assayed by external standard method and DMS, were used to evaluate the
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accuracy of the DMS. SMDs of DMS were below 5% for NG-R1, G-Rg1, G-Re and
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G-Rd, which implied that the DMS method had a high accuracy in comparison with
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external standard method and could be used as the substitutive method in the
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quantitative determination of multiple active constituents.
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3.2 Comparison of different parts of notoginseng
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Underground parts of P. notoginseng are the main portions for usage, and individual
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parts were used separately. Rhizome and root are both officially approved for the
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medicinal usage. However, there are still different applications of these two parts, for
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example, rhizome and main root are the raw materials of Chinese medicine
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“Xuesaitong” [31] and “Xueshuantong” [32], respectively. Branch root and fibrous
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root are mainly used for producing health food and food supplement. Due to the
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special growing environment, notoginseng is easily given rise to various diseases.
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Among the commonly seen diseases, root rot is a vital one and is thought to harm
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notoginseng mostly [33]. The rotten root with poor appearance, is available in the
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market at a low price, and even sometimes adulterated into the normal raw materials.
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To investigate the variation of the chemical profiles among different botanical parts of
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notoginseng, samples of rhizome, main root, branch root, fibrous root, as well as
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rotten root, were collected.
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root, and rotten root were displayed in Fig. 2. A total of 19 peaks were selected as the
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common peaks for further multivariate analysis. Principal component analysis (PCA)
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was adopted for the exploratory analysis. The first components of samples explained
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47.2 % of the systematic variation. The PCA score plot is displayed in Fig. 3A. The
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rhizome samples tended to cluster to the right part, while the other parts of
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notoginseng scattered on the left. Interestingly, there was no clear separation among
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main root, branch root, as well as rotten root samples. The fibrous root was mainly
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located at the higher left quarter, separated from other parts of samples to a certain
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extent. The result indicated that the chemical profiles of rhizome and fibrous root
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were different from other underground parts of notoginseng, while it was highly
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similar among that of main root, branch root and rotten root.
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The quantitative results were shown in Fig. 3B. The contents of NG-R1, G-Rg1, G-Re,
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G-Rb1 and G-Rd in rhizome samples were all higher than that in main root, branch
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root, fibrous root and rotten root samples (p main root ≥ branch root > fibrous root
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[19], which was consistent with the results here. Differentiating main root, branch root
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and fibrous root is mainly based on the diameter of the parts. Within a certain range of
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the diameter, the chemical profiles of the parts will not vary dramatically. Previous
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studies about chemical information of rotten root were quite limited. It was interesting
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to find that the chemical profiles of rotten root could not be differentiated from that of
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main root, which indicated the high similarity of the chemical profiles between
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normal and rotten root. And the quantification results also confirmed this opinion. In
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an early report, the saponins in rotten root had been contrasted with that in normal
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root collected from corresponding fields [34]. The contents of NG-R1, G-Rg1 and
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G-Rb1 in rotten root ranged from 64% to 99% of that in normal root. In view of these
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findings, rotten root should be rationally utilized. For safety reasons, the rotten root
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might not be a suitable raw material for food or medicine, however, it might be
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proposed to be collected for manufacturing active constituents.
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3.3 Comparison of notoginseng from different geographical regions
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Geographical region is usually regarded as one of the most important factors which
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would affect the quality of traditional Chinese medicines [35-37]. The traditional best 13
ACCEPTED MANUSCRIPT cultivated region of herbs is called “Daodi” region in China [38]. The “Daodi” region
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of P. Notoginseng is Wenshan Autonomous Prefecture of Yunnan Province, China,
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where over 90% of the total production of this herb all over the world is produced. In
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Wenshan Autonomous Prefecture, the cultivated areas for P. notoginseng are mainly
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located in the southwestern parts [39]. Wenshan County, Yanshan County, Maguan
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County and Qiubei County were all traditional cultivated regions of P. Notoginseng in
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Wenshan Autonomous Prefecture. Main root and rhizome were collected from these
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four counties, and the PCA score plot is displayed in Fig. 5A and 5C. The main root
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from these four counties were superimposed with each other, which indicated that the
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chemical profiles of the main root from these four counties were not significantly
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different.
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There was no significant differences among the contents of NG-R1, G-Rg1, G-Re and
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G-Rd in the main root samples collected from these four counties, except the content
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of G-Rb1 (the content of this saponin in main root from Wenshan County was lower
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than that from Maguan County and Qiubei County). The quantification results agreed
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well with the results of PCA.
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The rhizome samples from these four counties exhibited a similar chemical profile
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pattern as shown in Fig. 5C, and there was also no significant differences in the
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contents of these five saponins among the four counties.
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Thus, there is no significant difference in the chemical profiles and the major active
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constituents’ contents of rhizome and main root derived from traditional cultivated
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regions of P. Notoginseng in Wenshan Autonomous Prefecture, including Wenshan
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The demand of notoginseng, both for medicine and food, continues increasing.
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However, the cultivation of P. Notoginseng needs appropriate soil, climate and
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geographical conditions, and is also affected by continuous cropping obstacle.[40, 41]
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The production of notoginseng in Wenshan Autonomous Prefecture can’t meet the
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enormous demands. Thus, several regions nearby, i.g., Honghe Autonomous
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Prefecture, Kunming City and Yuxi City, begin to cultivate P. Notoginseng [42]. A
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total of 58 batches of main root were collected from these four Prefectures/Cities. The
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PCA score plot is displayed in Fig. 5B. Samples of main root from Wenshan
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Autonomous Prefecture, Honghe Autonomous Prefecture and Kunming City were
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superimposed with each other, however, samples from Yuxi City were located on the
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lower left right away from other three Prefectures/Cities.
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The quantification results were showed in Fig. S2. The contents of G-Rb1 and the sum
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of these five saponins in Wenshan Autonomous Prefecture were higher than that in
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Kunming City and Yuxi City. The levels of G-Rg1 and G-Rd in Wenshan Autonomous
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Prefecture were higher than in Yuxi City. The contents of NG-R1 in Honghe
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Autonomous Prefecture were higher than that in other three Prefectures/Cities.
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As shown in Fig. 5D, the chemical profile pattern of rhizome samples from Wenshan
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Autonomous Prefecture, Honghe Autonomous Prefecture and Kunming City were
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similar, while samples from Yuxi City were located left lower of the PCA score plot.
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The contents of the five saponins in rhizome samples from Wenshan Autonomous
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Prefecture, Honghe Autonomous Prefecture and Kunming City were not significant
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Thus, to a certain range, there is no significant difference in the chemical profiles and
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the major active constituents’ contents of rhizome and main root among adjacent
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geographical regions. When beyond the range, the difference could not be ignored.
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3.4 Comparison of notoginseng with different sizes
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Size is an important parameter for the commercial grades of the main root, which is
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thought to be associated with its quality [43]. However, the correlation between size
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and chemical profile has not yet been investigated. The main root samples collected
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were classified to 3 parts. Samples above 40 Tou were treated as large group, below
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80 Tou as small group, and 40-80 Tou as middle group. The PCA score plot is
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displayed in Fig. 6A. The samples of large, middle and small group were
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superimposed with each other, which indicated that the chemical profiles of main root
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with different sizes were similar to a certain degree. In the quantitative assays, the
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contents of NG-R1 and G-Rd in large main root samples higher than that in small
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group, while G-Rg1, G-Re and G-Rb1 were not significantly different among different
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sizes (Fig. 6B).
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In main root of different Tou, collected from the same field and in the same harvest
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time, the contents of these five saponins were compared. Among main root of 20, 30,
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40, 60, 80, 120 and
