Comprehensive Chemical Study on Different Organs of Cultivated And
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Available online at www.sciencedirect.com Chinese Journal of Natural Medicines 2021, 19(5): 391-400 doi: 10.1016/S1875-5364(21)60038-9 •Research article• Comprehensive chemical study on different organs of cultivated and wild Sarcandra glabra using ultra-high performance liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS) WANG Cai-Yun1Δ, LU Jing-Guang1Δ, CHEN Da-Xin2, WANG Jing-Rong1, CHE Kai-Si1, ZHONG Ming3, ZHANG Wei1*, JIANG Zhi-Hong1* 1 State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China; 2 Fujian Key Laboratory of Integrative Medicine on Geriatric, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China; 3 Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Chinese Medicine and Phar- maceutical Science, Nanning 530022, China Available online 20 May, 2021 [ABSTRACT] To illuminate the similarities and differences between wild and cultivated Sarcandra glabra (S. glabra), we performed a comprehensively study on 26 batches of cultivated S. glabra and 2 batches of wild S. glabra. Chemical constituents and distribution characteristics of roots, stems and leaves in both wild and cultivated S. glabra were investigated through UHPLC-TOF-MS method. The result revealed that there were significant differences between roots, stems and leaves in S. glabra. And the chemical contents in the root part were less or even absence than those in leaf and stem, which suggested the root organ could be excluded as medicine. Meanwhile, the chemical contents of stems and leaves in cultivated S. glabra was sightly higher than that of wild samples. Therefore, cultivated S. glabra may have a high potential for substitution of wild S. glabra without affecting its pharmaceutical properties. In sum- mary, our study could provide important information to the molecular basis for quality control of S. glabra. [KEY WORDS] S. glabra; UHPLC-TOF-MS; Root; Stem; Leaf [CLC Number] R917 [Document code] A [Article ID] 2095-6975(2021)05-0391-10 ism, bone fractures and even bruises since ancient history [1, 2]. Introduction S. glabra mainly contains caffeoyl derivatives, flavonoids, [3-6] Sarcandra glabra (S. glabra) is an evergreen shrub nor- coumarins and sesquiterpenoids etc. These types of com- mally found in south China, Japan and southeastern Asia, ponents have been reported as the main active constituents which belongs to the Chloranthaceae family. The whole plant which are associated with S. glabra’ biological effects such as anti-oxidant, anti-tumor, anti-infectious and anti-inflam- of S. glabra has been used in the traditional Chinese medicin- matory [7-12]. al preparations for treating various diseases including cancer, The natural resources of S. glabra have greatly de- pneumonia, appendicitis, gastritis enteritis, diarrhea, rheumat- creased in these recent years. For a better use of this valuable resource, a large number of S. glabra have been cultivated. In [Received on] 17-Apr.-2020 order to improve rational use of S. glabra in clinical studies, [Research funding] This work was supported by the Science and Technology Development Fund, Macau SAR (No. 0023/2019/ it is critical to understand the chemical difference between AKP) and Guangxi Science and Technology Department Fund (No. cultivated and wild S. glabra. At present, the quality control AD17195002). of S. glabra is mainly determined according to the Chinese [*Corresponding author] E-mail: [email protected] (ZHANG Pharmacopoeia, in which only isofraxidin and rosmarinic Wei); [email protected] (JIANG Zhi-Hong) ∆These authors contributed equally to this work. acid are determined by HPLC-UV detection, and this analyt- These authors have no conflict of interest to declare. ical method is insufficient for a comprehensive quality con- – 391 – WANG Cai-Yun, et al. / Chin J Nat Med, 2021, 19(5): 391-400 trol of S. glabra and its preparations [1]. Several high-perform- Quality control (QC) sample was prepared by mixing every ance liquid chromatography and capillary electrophoresis batch of samples. methods have been developed for the analysis of S. glabra Preparation of standard and sample solutions and its medicinal preparations [13]. For example, LI et al. [6] Stock solutions of 5-CQA (4), 3-CQA (5), 4-CQA (6), developed a rapid analytical method based on a UHPLC sys- neoastilbin (27), astilbin (30), 3, 4-diCQA (32), isoastilbin tem coupled with a linear ion trap high-resolution mass spec- (33), 3, 5-diCQA (34), neoisoastilbin (38), 4, 5-diCQA (43) trometer for qualitative identification of the constituents of S. were prepared in 50% (V/V) methanol at the concentration of glabra and its related preparations. And then, a high-perform- 100 μg·mL−1. The stock solution was stored in a refrigerator ance liquid chromatography-electrospray ionization-tandem at 4 °C for analysis. mass spectrometry (HPLC-ESI-MS/MS) method was further The powdered sample of S. glabra (0.1g) was accurately established to quantify 17 main compounds isolated from S. weighed and placed into a 50 mL centrifugal tube. Then glabra and its preparations [14]. However, there is no compre- 25.00 mL of 50% methanol was accurately added. The total hensive research that evaluate the similarity and identify the weight of the tube with the solution was weighted. The mix- differences between cultivated and wild S. glabra, although a ture was sonicated for 30 min with occasional shakings at few reports of the analysis of chemical constituents of S. room temperature and was centrifuged at 1800 g for 5 min glabra have already been published. This becomes the major subsequently. The weight of the tube with the sample solu- obstacle in clinical usage of S. glabra. tion was weighted again to ensure the lossless of the solvent. In this study, a comprehensive and systematically analys- The supernatant was filtered through a 0.22 μm PTFE filter to is of the chemical constituents was studied in both wild and afford sample solution for analysis. Quality control (QC) cultivated S. glabra samples using UHPLC-TOF-MS. sample was prepared following the same procedure. Moreover, the distribution characteristics of chemical com- UHPLC-TOF-MS conditions ponents was also described for the roots, stems and leaves. The liquid chromatography was assembled by an Agi- The aim of this study was to illuminate the similarities and lent 1290 infinity system consisting of binary pumps with in- differences in complex fingerprints between wild and cultiv- tegrated vacuum degasser (G4220A), thermostat (G1330B), ated S. glabra samples. This is very important for authoriza- standard autosampler (Model G4226A) and thermo statted column compartment (Model G1316C). The mass spectro- tion and quality control of S. glabra. meter was an Agilent 6230 TOF mass spectrometer equipped Materials and Methods with Agilent Jet Stream (AJS) source. Data acquisition was Chemicals carried out by Agilent Mass hunter® workstation B.05.00 software on a DELL computer (Agilent, Singapore). HPLC grade acetonitrile and methanol were purchased The chromatographic separations were achieved on an from Merck (Darmstadt, Germany). HPLC grade formic acid Agilent Extend C RRHD column (1.8 μm, 100 mm × 2.1 was purchased from Fluka (Buchs, Switzerland). Water used 18 mm I.D., Agilent). A gradient program was used with mobile in this study was deionized and further purified by Milli-Q phase consisting of solvent A (0.1% (V/V) formic acid in wa- Plus system (Millipore, Inc., MA, USA) at 18.2 MΩ cm. Oth- ter) and solvent B (0.1% (V/V) formic acid in acetonitrile) as er reagents used were of analytical grade. Marker com- follows: 0−12 min, 10%−20% B; 12−20 min, 20%−95% B; pounds of 5-O-caffeoylquinic acid (5-CQA), 3-O-caf- 20−23 min, 95% B; 23−26 min, 10% B. The flow rate was feoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (4-CQA), 0.3 mL·min−1. The injection volume was 2 μL and the column 3, 4-O-dicaffeoylquinic acid (3,4-diCQA), 3, 5-O-dicaf- temperature was maintained at 30 °C. feoylquinic acid (3, 5-diCQA) and 4, 5-O-dicaffeoylquinic The mass spectrometer was operated in negative ion acid (4,5-diCQA) were purchased from Chengdu MUST Bio- mode with mass scanning range of m/z 100 to m/z 1700. The Technology Co., Ltd. (Chengdu, China). Neoastilbin, astilbin, optimized MS conditions were set as the followings: drying neoisoastilbin and isoastilbin were bought from Chengdu gas temperature, 325 °C; drying gas flow, 9.0 L·min−1; nebuli- Alfa Biotechnology Co., Ltd. (Chengdu, China). zer, 35 psi; sheath gas temperature, 325 °C; sheath gas flow, Plant materials 11.0 L·min−1; fragmentor, 150 V; skimmer, 65 V; and octo- Twenty-six batches of cultivated S. glabra (9 seeding pole RF, 500 V; VCap., 3500 V; nozzle, 1500 V. Agilent TOF samples and 17 sprouting samples) from different proven- reference solution was applied for calibration of mass drift by ance places of China were collected for the experiment. Two using the reference mass ions of m/z 112.9855 and m/z batches of wild S. glabra were collected at Youxi country, 966.0007. Sanming city, Fujian Province. All the plant materials collec- Results and Discussion ted were taxonomically identified and provided by Prof. JI- ANG Zhi-Hong (Table 1). Each batch of sample was firstly Optimization of sample preparation separated into different parts, i.e., leaves, stems and roots. In order to achieve improved separations and higher ex- Then the samples of each part were cut into smaller pieces, traction efficiency, the extraction conditions (solvents and ex- grounded into powder and passed through a 50-mesh sieve. traction times) for sample solutions were optimized carefully.