Structural characterization and discrimination of Ophiopogon japonicas (Liliaceae) from different geographical origins based on metabolite profiling analysis
Graphical abstract
Introduction
The root tuber of Ophiopogon japonicus (Thunb.) Ker-Gawl (Maidong in Chinese) is a widely used nourishes yin Chinese herb to treat inflammatory and cardiovascular diseases in clinical [1,2]. This herb is also used in many famous Chinese patent medicines such as “Shenmai injection”, “Maiwei pill”, and “Xuanmai granule” [3]. O. japonicas is widely distributed in China and herbal textural researches showed that Sichuan and Zhejiang Province have become the two major producing area of Maidong not later than Qing dynasty [1,4]. However, according to our investigation, the cultivation of O. japonicas in Zhejiang (called Zhemaidong or Hangmaidong, OJ-Z) has drastically decreased, only very small production of OJ-Z was retained in Cixi (OJ-Z-CX) and Sanmen (OJ-Z-SM) City today, which is only intended for the use of individual Chinese patent medicine business. Meanwhile, the cultivation area and yield of O. japonicas in Sichuan province (called Chuanmaidong, OJ-C) have been significantly increased and becoming the primary producing area of Maidong in about last 20 years and accounting for more than 95 % of total Maidong output at present. OJ-Z and OJ-C are different in terms of cultivation habits, environmental conditions, and genetic difference [3,5]. And the most obviously difference between the production of them is that OJ-Z is always cultivated for three years, while OJ-C is harvested yearly. This causes the production of OJ-Z much more costly than OJ-C and is thought to be the main reason for the decrease in OJ-Z cultivation.
Previous studies showed that OJ-Z and OJ-C exhibited differences in pharmacological tests in terms of antioxidation, anti-inflammation and cytotoxicity [3,5]. And some studies have been carried out for the comparison of the differences between OJ-Z and OJ-C from the point of their chemical composition. Phytochemistry studies indicate that steroidal saponins and homoisoflavonoids are the two main kinds of bioactive pharmacological compounds in Maidong medical materials [1,6,7]. More than one hundred kinds of steroidal saponins and fifty homoisoflavonoids have been isolated and identified from O. japonicas (Fig1) and some kinds of organic acids, polysaccharides, phenylpropanoids besides [1,[8], [9], [10], [11], [12], [13], [14]]. Studies based on the content determination of single components showed that OJ-Z contained more methylophiopogonanone A, methylophiopogonanone B and ophiopogonin B, ophiopogonin D′ than OJ-C, while OJ-C had a higher amount of ophiopogonin D and total saponins than OJ-Z [5,15]. Meanwhile, LC–MS method has been used for the compounds identification in Maidong and differences analyze between OJ-Z and OJ-C metabolic profiles, which showed that higher amounts of homoisoflavonoids were observed in OJ-Z than OJ-C [3,5,8,16,17]. What’s more, these papers were mainly focus on the study of homoisoflavonoids, there were also few studies on the comprehensive analysis of steroidal saponins in O. japonicas.
In this study, a metabolomics-based UPLC/Q-TOF MS method was developed to quickly differentiate OJ-Z and OJ-C by comparing steroidal saponin and homoisoflavonoid profiles. Overall, 135 steroidal saponins and 47 homoisoflavonoid were characterized, including 56 steroidal saponins were tentatively identified and reported for the first time in O. japonicas as far as we know. The metabolic profiles obtained from the MS data were then subjected to multivariate analysis techniques to screen out the discriminated chemical markers between OJ-Z and OJ-C.
Section snippets
Reagents and herbal medicine samples
Methanol and acetonitrile (HPLC grade) were purchased from Merck Co. (Darmstadt, GER). Formic acid (HPLC grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionized water (18.2 MΩ/cm) was produced by using a Milli-Q system (Millipore, MA, USA). The other reagents were obtained in analytical grade.
Standard compounds of Methyl-ophiopogonanone B (Lot number: Y02M9L60394), ophiopogonanone E (Y14M10L88268), ophiopogon Ra (P29J9S64682), ophiopogonin D (P26F9F54695) and ophiopogonin D′
Optimal UPLC/Q-TOF MS analysis conditions for O. Japonicas
To obtain satisfactory extraction efficiency and integrity of steroidal saponins and homoisoflavonoids, the extract solvents (80 % ethanol, 80 % methanol, 50 % methanol) and extract time (30 min and 60 min) were evaluated (Fig. S1). And the extract condition was finally selected as 80 % methanol and 60 min extraction time based on their higher mass response intensity.
Both the ESI (‒) and ESI (+) ion data were acquired using the optimal UPLC-MS conditions. The high CE was also optimized,
Conclusion
In this study, a metabolomics-based UPLC/Q-TOF MS method was developed to effectively differentiate between OJ-Z and OJ-C. The OJ-Z and OJ-C groups were clearly separated in PCA and OPLS-DA models and many makers were identified. When the model was used to discriminate samples from market, the results indicated that there might be OJ-C samples sold as OJ-Z. Besides, the contents of some components were quantitatively analyzed. The results indicated that there were significant differences in the
CRediT authorship contribution statement
Chao-geng Lyu: Conceptualization, Methodology, Software, Writing - original draft, Writing - review & editing. Chuan-zhi Kang: Methodology, Writing - original draft. Li-ping Kang: Conceptualization, Writing - review & editing. Jian Yang: Formal analysis, Software. Sheng Wang: Resources, Validation. Ya-li He: Writing - review & editing. Ai-ping Deng: Investigation. Hong-yang Wang: Investigation. Lu-qi Huang: Supervision, Funding acquisition. Lan-ping Guo: Supervision, Project administration,
Declaration of Competing Interest
The authors have declared no conflict of interest.
Acknowledgments
Thank Mr Tao Wang and Mr Zhi-an Wang for their support to the investigation of producing areas and sample collection. This work was supported by the National Key R&D Program of China (No. 2018YFF0214202 and No. 2017YFC1700701), National Natural Science Foundation of China (No.81891014), Ministry of Finance Central Level of the Special (No. 2060302) and China Academy of Chinese Medical Sciences Key Areas (ZZ10-027).
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These authors contributed equally to this work.