Chemical and Genetic Discrimination of Cistanches Herba Based on UPLC-QTOF/MS and DNA Barcoding

Huang L (2014) Chemical and Genetic Discrimination of Cistanches Herba Based on UPLC-QTOF/MS and DNA Barcoding. PLoS ONE 9(5): e98061. doi:10.1371/journal.pone.0098061 Chemical and Genetic Discrimination of Cistanches Herba Based on UPLC-QTOF/MS and DNA Barcoding Sihao Zheng 0 Xue Jiang 0 Labin Wu 0 Zenghui Wang 0 Linfang Huang 0 Massimo Labra, University of Milano Bicocca, Italy 0 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College , Beijing , People's Republic of China Cistanches Herba (Rou Cong Rong), known as ''Ginseng of the desert'', has a striking curative effect on strength and nourishment, especially in kidney reinforcement to strengthen yang. However, the two plant origins of Cistanches Herba, Cistanche deserticola and Cistanche tubulosa, vary in terms of pharmacological action and chemical components. To discriminate the plant origin of Cistanches Herba, a combined method system of chemical and genetic -UPLC-QTOF/MS technology and DNA barcoding-were firstly employed in this study. The results indicated that three potential marker compounds (isomer of campneoside II, cistanoside C, and cistanoside A) were obtained to discriminate the two origins by PCA and OPLS-DA analyses. DNA barcoding enabled to differentiate two origins accurately. NJ tree showed that two origins clustered into two clades. Our findings demonstrate that the two origins of Cistanches Herba possess different chemical compositions and genetic variation. This is the first reported evaluation of two origins of Cistanches Herba, and the finding will facilitate quality control and its clinical application. - Cistanches Herba (Rou Cong Rong), known as Ginseng of the desert, originates from dried succulent stems of Cistanche deserticola Y.C. Ma and Cistanche tubulosa (Schrenk) Wig according to the Chinese Pharmacopoeia (2010 edition), and is popular for its tonifying the kidney-yin, benefiting life essence and relaxing bowel. Currently, Cistanches Herba is mainly distributed in arid and warm deserts in northwest China, particularly in Xinjiang and Inner Mongolia provinces. However, the two origins of Cistanches Herba differ in terms of their pharmacological activity and chemical components. Tu et al. investigated the decoction of three Cistanche species (C. deserticola, C. tubulosa, Cistanche salsa) and found that C. tubulosa showed the lowest effect in the Yang-deficiency mouse model [1]. Zhang et al. compared pharmacological activity between C. deserticola, C. tubulosa and C. salsa, and found that these species had medicinal functions such as anti-fatigue and hypoxia tolerance, but not on the same extent [2]. Previous research reported the chemical component, and indicated the difference of chemical component and content for plant origins of Cistanches Herba [3]. As for the clinical application and market circulation,as a tonic,C. tubulosa has been traditionally used as a blood circulation-promoting agent and in the treatment of impotence, sterility, lumbago, body weakness in Japan [48]. Consequently, it is of great significance to discriminate two origins of Cistanches Herba for the quality control and clinical application. However, there is no research focus on discrimination of two origins of Cistanches Herba. Many researched methods, including microscopy, ultraviolet and infrared detection, intersimple sequence repeats method have been used to identify the genus of Cistanches, but not only for two origins specially [920]. Here, we conjunctively utilized chemical and molecular techniques to distinguish two origins of Cistanches Herba, UPLCQTOF/MS (ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry) and DNA barcoding. UPLC-QTOF/MS provides information more rapidly and efficiently compared with other techniques. The high selectivity and sensitivity of UPLC-QTOF/MS have resulted in its application for both quantitative and qualitative analyses, as well as in metabolite analysis and identification of complex compounds in Traditional Chinese Medicine [2122]. Principal component analysis (PCA) and orthogonal projection to latent structure-discriminant analysis (OPLSDA) are also developed to identify potential marker compounds. DNA barcoding, an easier and more universal molecular marker technology, uses a DNA fragment to identify species or genera. It is objective, more accurate, and easier to perform than traditional identification methods and other molecular marker technologies. Moreover, DNA barcoding has successfully been applied to identify animal and plant, including medicinal plants [2326]. The purpose of this research is to establish a scientific method system, combined UPLC-QTOF/MS and DNA barcoding, for discrimination of two plant origins of Cistanches Herba. Materials and Methods Ethics statement We confirm that the field studies did not involve endangered or protected species. GPS coordinates have included in the sample information, please see table 1." GPS coordinates GenBank accession number WLMQ meant Wu Lu Mu Qi city; GJH meant Gan Jia Hu; HBKSEMG meant Hoboksar Mongol Autonomous County; KLMY meant Ke La Ma Yi city; BDJLSM meant Badain Jaran Desert; ALSZQ meant Alxa Left Banner; DSX meant Dong San county; CL meant Ce Le county; MF meant Min Feng county; HT meant He Tian county. doi:10.1371/journal.pone.0098061.t001 Plant materials and reagents Succulent stems of Cistanches Herba were collected from wild desert region in Inner Mongolia, Qinghai Provinces, Xinjiang Uygur Autonomous Region, Peoples Republic of China (Table 1) in May 2012. The samples of the research were all collected in wild desert region, not in private land, where no specific permissions were required. The botanical identities of the stems were confirmed by Dr. Linfang Huang. Voucher specimens were deposited at The Institute of Medicinal Plant Development. Highperformance liquid chromatography (HPLC)-grade acetonitrile (Merck KGaA, Darmstadt, Germany) and formic acid (Tedia, USA) were utilized for UPLC analysis. Deionized water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals were of analytical grade. Sample preparation Cistanches Herba samples (1.0 g, 65-mesh) were transferred into a 50-mL conical flask, and 50 mL of 70% methanol was added. After soaking for 30 min, ultrasonication (35 kHz) was performed at room temperature for 30 min. After centrifugation at 10,000 rev/min for 10 min, the supernatant was stored at 4uC and filtered through a 0.22-mm membrane before injection into the UPLC-QTOF/MS system for analysis. UPLC-QTOF/MS For UPLC analysis, the following systems/parameters were used: Waters Acquity system (Waters) equipped with a binary solvent delivery pump, auto-sampler and PDA detector connected to a Waters Empower 2 data station; ultrasonication (250 W, 50 kHz, Kunshan Ultrasonic Instrument Co., Zhejiang, China); and an electronic analytical balanc (...truncated)