Chinese Journal of Natural Chinese Journal of Natural Medicines 2018, 16(12): 09510960 Medicines Flavonoids rather than alkaloids as the diagnostic constituents to distinguish Sophorae Flavescentis Radix from Sophorae Tonkinensis Radix et Rhizoma: an HPLC fingerprint study DING Pei-Lan, HE Chang-Ming, CHENG Zhi-Hong*, CHEN Dao-Feng* Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China Available online 20 Dec., 2018 [ABSTRACT] Sophorae Flavescentis Radix (Sophora flavescens Ait., SFR) and Sophorae Tonkinensis Radix et Rhizoma (S. tonkinensis Gapnep., STR) are two commonly used traditional Chinese medicines from Sophora (Leguminosae) plants, which are believed to possess similar bioactive components with entirely different clinical applications. In order to find out the characteristic chemical constituents potentially leading to the unique medicinal properties claimed for each of the two closely related TCMs, an HPLC fingerprint method was developed for analyses of the alkaloid and flavonoid constituents of SFR and STR, respectively, which were further evaluated and compared through similarity calculation and hierarchical clustering analysis (HCA). The results from the present study showed that the alkaloid fingerprints of the two herbs were similar, with many components co-existing in both drugs and various batches of samples from different species being mixed together in the HCA dendrogram. However, their flavonoid constituents were totally different with specific fingerprints being yielded for each herb, and further HCA analysis showed that the tested samples could almost be clearly divided into two groups based on their origins of species. The results from the present study indicated that the flavonoid constituents could serve as the differentially diagnostic constituents of SFR and STR and might potentially attributed to their distinct therapeutic effects. [KEY WORDS] Sophora flavescens; Sophora tonkinensis; Alkaloids; Flavonoids; HPLC fingerprint; Hierarchical clustering analysis [CLC Number] R95, R917 [Document code] A [Article ID] 2095-6975(2018)12-0951-10 Introduction used internally to treat acute pharyngolaryngeal infections and sore throat [1-2]. Therefore, it is of great interest to find out the Sophorae Flavescentis Radix (SFR, Sophora flavescens characteristic chemical constituents potentially leading to Ait.) and Sophorae Tonkinensis Radix et Rhizoma (STR, S. their unique clinical applications claimed for the two closely tonkinensis Gapnep.) are two commonly used traditional Chinese related herbs. medicines (TCMs) from Sophora (Leguminosae) plants [1]. Although the alkaloids in these two herbs have received Both the herbs were found to contain similar chemical consti- much attention, the holistic and component-based relation- tuents such as quinolizidine alkaloids, prenylated flavonoids, ships between their constituents and different clinical applica- and oleanane triterpenoids, while their clinical applications tions are still unknown. In the Chinese Pharmacopoeia [1], are entirely different [2-3]. SFR is mostly used externally for three alkaloids (matrine, oxymatrine, and sophoridine) widely the treatments of skin diseases and gynaecological diseases, present in Sophora plants [4], are chosen as qualitative mark- such as eczema, dermatitis, and colpitis, while STR is frequently ers for identification of these two herbs in the thin layer chromatography tests, which is known to lack specificity in distinguishing SFR and STR. A similar situation exists in the [Received on] 29- Sep.-2018 quantitative tests for the two herbs recorded in the Chinese [Research funding] This work was supported by the Natural Science Pharmacopoeia [1]. These problems may arise from the vari- Foundation of China (Nos. 30672597 and 81330089). ous pharmacological findings of alkaloids that are tentatively [*Corresponding author] Tel: 86-21-51980157; E-mail: chengzhh@ fudan.edu.cn (CHENG Zhi-Hong); Tel: 86-21-51980135; E-mail: assigned as the unique active constituents of the two herbs. It [email protected] (CHEN Dao-Feng) is well known that the therapeutic efficacy of TCMs is often These authors have no conflict of interest to declare. attributed to the synergic effect of their multiple bioactive Published by Elsevier B.V. All rights reserved components and multi-targets, and thus analysis of merely – 951 – DING Pei-Lan, et al. / Chin J Nat Med, 2018, 16(12): 951960 one or a few markers is not adequately representative for unique clinical applications claimed for SFR and STR, com- quality control of herbs. In the past two decades, more and prehensive HPLC fingerprint methods of flavonoids and al- more flavonoids were isolated and identified from the two kaloids in SFR and STR are needed with more samples col- herbs [3], some of which have been gradually revealed to pos- lected from different regions of China. The objectives of the sess significant bio-activities such as anticancer [5-6], anti-in- present study were as follows: 1) to establish HPLC finger- [7] [8] [9] flammatory , anti-diabetes and anti-virus activities . prints of the alkaloid and flavonoid constituents for the two These findings of the flavonoids prompted us to re-examine herbs, and 2) to compare and analyze the fingerprints by comprehensively the active components of the two herbs in similarity calculation and hierarchical clustering analysis terms of alkaloids and flavonoids. (HCA). HPLC fingerprinting has been used over the last decade for authentication and quality control of herbs and their Materials and Methods preparations. This method emphasizes the whole profile of Plant materials components in a complex system, and it is a strategy recom- The reference SFR and STR herbs were obtained from mended to assess the quality of botanical products by the US the National Institute for the Control of Pharmaceutical and Food and Drug Administration (FDA), the European Medi- Biological Products of China (Beijing, China). All the other cines Evaluation Agency (EMEA), and the State Food and botanical samples studied were obtained as commercial drugs [10] Drug Administration of China (SFDA) . To the best of our from 26 different districts in 19 provinces of China. All the knowledge, few reports have been published on systemic samples were authenticated by one of the authors (Dr. CHEN comparison of the alkaloid or flavonoid constituents between Dao-Feng). Among them, 24 samples were identified as the these two herbs using fingerprinting techniques, though many roots of S. flavescens, and 19 samples were the roots and rhi- quantitative studies on the major alkaloids in SFR and STR zomes of S. tonkinensis. The specimens were deposited in the [11-14] by various chromatographic techniques have been reported . Department of Pharmacognosy, School of Pharmacy, Fudan In 2013, Ma and co-workers analyzed flavonoids in 12 batches University, Shanghai, China. The identity, sampling part, of SFR and 4 batches of STR samples by HPLC fingerprinting collection source and collection time of the 43 tested samples and found preliminarily that these two herbs had different are summarized in Table 1. All the HPLC fingerprint data [15] flavonoid profiles . To investigate systematically the char- were obtained in 2004, and the phytochemical isolation and acteristic chemical constituents that mayt contribute to their statistical analysis were accomplished recently. Table 1 Sample information of Sophorae Flavescentis Radix and Sophorae Tonkinensis Radix et Rhizoma used in the present study and their calculated similarity values for alkaloids and flavonoids Similarity No. Sample code Source Sampling part Collection time A B 1 SF1 NICPBP Roots Oct 2002 NT 0.90 2 SF2 Nanning, Guangxi Roots Oct 2001 0.95 0.80 3 SF3 Kunming, Yunnan Roots Oct 2001 0.93 0.88 4 SF4 Kunming, Yunnan Roots Jun 2003 NT 0.92 5 SF5 Guiyang, Guizhou Roots Aug 2001 0.94 0.89 6 SF6 Luoding, Guangdong Roots Oct 2001 0.97 0.96 7 SF7 Dujiangyan, Sichuan Roots Aug 2001 0.94 0.96 8 SF8 Nanchuan, Chongqing Roots Sep 2001 0.96 0.96 9 SF9 Chongqing Roots Oct 2001 0.95 0.94 10 SF10 Beibei, Chongqing Roots Jun 2003 NT 0.87 11 SF11 Fuzhou, Fujian Roots Oct 2001 0.96 0.85 12 SF12 Shanghai Roots Oct 2001 0.85 0.84 13 SF13 Tianshui, Gansu Roots Aug 2001 0.96 0.95 14 SF14 Lanzhou, Gansu Roots Jun 2003 NT 0.94 15 SF15 Yinchuan, Ningxia Roots Nov 2001 0.89 0.95 16 SF16 Hanzhong, Shaanxi Roots Jul 2001 0.94 0.38 17 SF17 Langao, Shaanxi Roots Aug 2001 0.98 0.94 18 SF18 Ziyang, Shaanxi Roots Jun 2003 NT 0.96 19 SF19 Wulumuqi, Xinjiang Roots Jun 2001 0.97 0.94 20 SF20 Nanchang, Jiangxi Roots Oct 2001 0.97 0.91 – 952 – DING Pei-Lan, et al. / Chin J Nat Med, 2018, 16(12): 951960 Continued Similarity No. Sample code Source Sampling part Collection time A A 21 SF21 Tianjin Roots Mar 2002 0.94 0.92 22 SF22 Huaiyin, Jiangsu Roots Oct 2001 0.80 0.83 23 SF23 Bozhou, Anhui Roots Sep 2001 0.84 0.86 24 SF24 Shanghai Roots Feb 2001 NT 0.86 25 ST1 NICPBP Roots and rhizomes Oct 2002 NT 0.83 26 ST2 Nanning, Guangxi Roots and rhizomes Oct 2002 NT 0.96 27 ST3 Nanning, Guangxi Roots and rhizomes Oct 2001 0.47 0.96 28 ST4 Kunming, Yunnan Roots and rhizomes Oct 2001 0.87 0.68 29 ST5 Guiyang, Guizhou Roots and rhizomes Aug 2001 0.96 0.95 30 ST6 Luoding, Guangdong Roots and rhizomes Oct 2001 0.95 0.95 31 ST7 Zhaoqing, Guangdong Roots and rhizomes Dec 2001 0.94 0.83 32 ST8 Dujiangyan, Sichuan Roots and rhizomes Aug 2001 0.83 0.94 33 ST9 Chongqing Roots and rhizomes Oct 2001 0.95 0.94 34 ST10 Fuzhou, Fujian Roots and rhizomes Oct 2001 0.97 0.97 35 ST11 Shanghai Roots and rhizomes Oct 2001 0.88 0.94 36 ST12 Lanzhou, Gansu Roots and rhizomes Oct 2001 0.91 0.94 37 ST13 Lanzhou, Gansu Roots and rhizomes Jun 2003 NT 0.91 38 ST14 Yinchuan, Ningxia Roots and rhizomes Nov 2001 0.89 0.95 39 ST15 Ankang, Shaanxi Roots and rhizomes Aug 2001 0.94 0.84 40 ST16 Shijiazhuang, Hebei Roots and rhizomes Oct 2001 0.87 0.97 41 ST17 Quzhou, Zhejiang Roots and rhizomes Nov 2001 0.94 0.93 42 ST18 Shimen, Hunan Roots and rhizomes Jul 2001 0.92 0.27 43 ST19 Shanghai Roots and rhizomes Mar 2000 NT 0.88 NT: not tested; SF: Sophora flavescens; ST: S.