Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2016, Article ID 4631843, 9 pages http://dx.doi.org/10.1155/2016/4631843 Research Article Components and Anti-HepG2 Activity Comparison of Lycopodium Alkaloids from Four Geographic Origins Yong-Qiang Tian,1,2 Guang-Wan Hu,1,3 and Ming-Quan Guo1,3 1 Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China 2Graduate University of Chinese Academy of Sciences, Beijing 100049, China 3Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, China Correspondence should be addressed to Ming-Quan Guo; [email protected] Received 6 November 2015; Revised 2 February 2016; Accepted 3 February 2016 Academic Editor: Raffaele Capasso Copyright © 2016 Yong-Qiang Tian et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lycopodium japonicum Thunb. has attracted great interests due to its rich alkaloids with significant anticancer activity. However, significant chemical differences often exist in a plant species from different geographic origins and affect its quality and bioactivities. Thus, it is urgent to reveal their chemical and biological distinctions at the molecular level. In this context, a comparative chemical analysis of LAs using HPLC-UV-ESI-MS/MS was firstly conducted and resulted in the detection of 46 LAs, 28 of which were identified, and a series of unique LAs markers, such as peaks 2, 9, 10,and11, were further found to be characteristic LAs and selected as markers from four different origins for their quality control. In parallel, the comparative bioactivity assay revealed that the total LAs from Hubei province exhibited much higher inhibitory rate at 65.95% against HepG2 cells than those at 26.72%, 20.26%, and 33.62% for Kenya, Guangxi province, and Zhejiang province in China, respectively. To this end, significant chemical fingerprinting differences and discrepancies in bioactivity of LAs were explored firstly, which could provide valuable information for quality control and further activity studies on LAs from different sources and promote their better pharmaceutical applications in the future as well. 1. Introduction them, geoorigin of a plant is an important factor affecting the types and chemical structures of LAs since they are produced Lycopodium japonicum Thunb is a traditional medicinal herb through plant metabolism and its complex interactions with in China, which has been used for the treatment of a variety of the growing environments. Therefore, comparative study diseases for thousands of years, such as contusion, analgesia, on the LAs of Lycopodium genus plants from different and rheumatoid arthritis [1, 2]. It has been reported that areas can provide valuable information on the evaluation of lycopodium alkaloids (LAs) were the major bioactive com- their chemical similarities and differences and further their ponents widely found in the plants of Lycopodium genus, for pharmaceutical activity discrepancies. example, L. obscurum [3], L. annotinum [4], and L. chinense To investigate LAs from a plant species, the traditional [5]. Since these LAs have been proved to possess a wide phytochemical approaches usually involved multistep iso- spectrum of bioactivities, for example, anti-inflammation, lation and structural elucidation of pure compounds from antitumor, and acetylcholineasterase inhibitory activity [6– a plant of interest, which are often tedious, complex, and 9], many efforts focusing on the isolation, synthesis, iden- time-consuming [3]. Because of the excellent performance tification, and biogenetic synthesis of LAs have been made on simultaneous separation and identification of multicom- to explore and expand this valuable medicinal resource [10]. ponent mixtures with complex background, chromatography To date, up to 300 LAs were reported mainly from various based separation techniques (i.e., GC and LC) coupled with Lycopodium and Huperzia genus plants or from one plant various detectors, such as mass spectrometry (MS), were with different growing stages and environments [11]. Among developed as useful tools in most cases [12]. Since most LAs 2 Evidence-Based Complementary and Alternative Medicine fromplantsarenonvolatile,LCtechniques,forexample,high- methanol, ethanol, chloroform, hydrochloric acid (HCl), performance LC (HPLC) and ultraperformance LC (UPLC), and ammonia, were purchased from Sino-pharm chemical were preferred over GC in obtaining the fingerprint profiles Reagent Co., Ltd. (Shanghai, China). Deionized water for ofLAs[13].However,theUVdetectorwidelyusedwithLC HPLC and LC-MS was prepared from EPED (Nanjing Yeap often failed to provide chemical structure information when Esselte Technology Development Co., Nanjing, China). the authentic standards of LAs were unavailable. Thus, LC- MS was used in this work for the identification of LAs of 2.3. Alkaloids Extraction. For Hubei origin Lycopodium Lycopodium japonicum Thunb from different geoorigins. It japonicum, the pulverized powder (40.0 g) was immersed in is reported that the types and contents of LAs did not only 90% ethanol for 12 h and sonically extracted for 30 min three differ in different plant species but also differ in the same plant times. After filtration, the filtrate was combined and then ∘ species originated from different places [14, 15]. It is reported concentrated at 45 C with a vacuum rotatory evaporator. The that Lycopodium japonicum Thunb is widely distributed in residue was dissolved in 0.5% HCl and then extracted with China in Flora of China, which was close but different from chloroform three times. The remaining aqueous phase was Lycopodium clavatum [16]. However, lycopodium plants with adjusted to pH 10 using NH4OH and further extracted with different growing environments may have significant differ- chloroform to afford the crude alkaloids in this study. In ences in LAs compositions, and very few efforts have been thesameway,thetotalLAsinLycopodium japonicum from made to illustrate these differences. Most of the existing study Guangxi, Zhejiang province, and Kenya were prepared. focused on the chemical composition and bioactivity of one or several components of LAs in Lycopodium plant species from one place [2], which could pose high risk in practical 2.4. Instrumentation and Chromatographic Conditions medical applications or evaluations on the Lycopodium plant 2.4.1. High Performance Liquid Chromatography. AThermo due to the inconsistent responses of LAs when a lycopodium Accela 1250 HPLC system (Thermo Fisher Scientific, San Jose, plant species from different places were utilized. Thus, a CA, USA) equipped with a vacuum degasser, an autosampler, comparative investigation on the chemical composition and and a variable-wavelength detector (VWD) was used for the the corresponding bioactivity of LAs in a Lycopodium species HPLC-UV analysis. The separation was carried out on a from different places will be of special interest in exploring Waters Xbridge Sunfire C18 (4.6 × 150 mm, 3.5 m, Waters and expanding these natural resources for human health and Technology, Ireland, UK). The column temperature was well-being. ∘ maintained at 30 C and the flow rate was set at 0.6 mL/min. In this work, the fingerprinting profiles of LAs in Lycopo- The mobile phase consisted of 10 mM ammonium acetate dium japonicum Thunb from four geoorigins, that is, Hubei, aqueous solution (A) and acetonitrile (B). The gradient Guangxi, and Zhejiang province of China and Kenya, were elution profiles were as follows: 0–3 min, 90% (B); 3–35 min, firstly determined and compared using high performance 90%–50% (B); 35–37 min, 50%–10% (B); 37–40 min, 10% (B). liquid chromatography coupled with electrospray ionization The LC chromatogram was monitored at a wavelength of mass spectrometry (HPLC/ESI-MS/MS), and the anti-HepG2 279 nm. activities of the corresponding total LAs were also tested in vitro with Cell Counting Kit-8 (CCK-8). In this way, the correlations between total LAs and their associated 2.4.2. Mass Spectrometry. For the LC-ESI-MS/MS experi- activities were firstly explored, which could provide valuable ment, a Thermo Accela 600 HPLC system with a UV detector information for quality control and further activity studies on coupled with a TSQ Quantum Access MAX mass spectrom- LAs from different natural resources and could thus promote eter (Thermo Fisher Scientific, San Jose, CA, USA) was used. their better applications in the future. LC-MSanalyseswereconductedinthepositivemode.MS conditions were as follows: mass range from 200 to 1000, ∘ spray voltage, 3.0 kV; capillary temperature, 250 C; sheath gas 2. Materials and Methods pressure, 40 psi; aux gar pressure, 10 psi. 2.1. Plant Materials. Crude Lycopodium plants of three geo- 2.5. Anti-HepG2 Activity In Vitro. The anti-HepG2 activity of origins (Hubei, Guangxi and Zhejiang) were obtained from the crude extracts was tested using human hepatic carcinoma Xinhui Pharmaceutical Factory (Anhui, China). The Kenya cell line (HepG2, from China Center For Type Culture origin Lycopodium material was collected from Mountain Collection) with Cell Counting Kit-8 (CCK-8). Cells were Kenya, Kenya. The plant materials of the four origins were cultured in a 96-well plate at a density of 5000 cells per identified as Lycopodium japonicum Thunb of