Structural characterization and discrimination of Ophiopogon japonicas (Liliaceae) from different geographical origins based on metabolite profiling analysis

doi:10.1016/j.jpba.2020.113212

Journal of Pharmaceutical and Biomedical Analysis

Volume 185, 5 June 2020, 113212

https://doi.org/10.1016/j.jpba.2020.113212 Get rights and content

Highlights

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A comprehensive comparison between the metabolic profile data of O. japonicas from different origins was carried out.
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191 metabolites were characterized, including 56 potential new steroidal saponins in O. japonicas.
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PCA and OPLS-DA analysis can clearly separate of O. japonicas samples from Sichuan and Zhejiang.
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OPLS-DA analysis for samples from market indicated that OJ-C might be used to substitute for or adulterate OJ-Z in some cases.

Abstract

The root tuber of Ophiopogon japonicus (Thunb.) Ker-Gawl (“Maidong” in Chinese), with steroidal saponins and homoisoflavonoids as its representative chemical compositions, is a representative medicinal herbs with multiple major producing areas. This study aimed to distinguish the O. japonicas samples from Zhejiang and Sichuan by using an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS)-based metabolome analysis. Firstly, a global chemical constituent identification of O. japonicas was carried out by using both automatic and manual methods. An integrated steroidal saponins structural identification strategy in O. japonicas based on exact mass information, fragmentation characteristics and retention time was developed. Overall, 135 steroidal saponins, 47 homoisoflavonoids and 9 other metabolites were quickly identified or tentatively identified from the MS^E continuum data. Furthermore, multivariate statistical analysis revealed that O. japonicas from Zhejiang and Sichuan can clearly be separated and some markers were screened. Moreover, some major active components including total soluble sugar, total soluble polyphenol, total flavonoid, total saponin and 10 specific compounds were analyzed quantitatively. In general, these results showed that there were many differences between the metabolic profile data of O. japonicas from different producing areas, O. japonicas from Sichuan showed higher level steroidal saponins and samples from Zhejiang had higher contents of homoisoflavonoids specifically, and indicated that metabolite profiling by UPLC/Q-TOF MS is an effective approach for the discrimination of medicinal herbs from different geographical origins.

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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Homoisoflavonoids and the antioxidant activity of Ophiopogon japonicus root

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Evaluation of anti-inflammatory activity of compounds isolated from the rhizome of Ophiopogon japonicas

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¹: These authors contributed equally to this work.

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Structural characterization and discrimination of Ophiopogon japonicas (Liliaceae) from different geographical origins based on metabolite profiling analysis

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Reagents and herbal medicine samples

Optimal UPLC/Q-TOF MS analysis conditions for O. Japonicas

Conclusion

CRediT authorship contribution statement

Declaration of Competing Interest

Acknowledgments

Ophiopogon japonicus-A phytochemical, ethnomedicinal and pharmacological review

J. Ethnopharmacol.

Antithrombotic activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents

Biol. Pharm. Bull.

Comparison of bioactive components and pharmacological activities of Ophiopogon japonicas extracts from different geographical origins

J. Pharm. Biomed. Anal.

Textual research of chinese herb maidong

China Pharmacist

Characterization of homoisoflavonoids in different cultivation regions of Ophiopogon japonicus and related antioxidant activity

J. Pharm. Biomed. Anal.

Steroidal saponins from the tuber of Ophiopogon japonicus

Steroids

Anti-inflammatory homoisoflavonoids from the tuberous roots of Ophiopogon japonicus

Fitoterapia

Rapid identification of ophiopogonins and ophiopogonones in Ophiopogon japonicus extract with a practical technique of mass defect filtering based on high resolution mass spectrometry

J. Chromatogr. A

New sesquiterpenoid glycoside and phenylpropanoid glycosides from the tuber of Ophiopogon japonicus

J. Asian Nat. Prod. Res.

Enrichment and separation of steroidal saponins from the fibrous roots of Ophiopogon japonicus using macroporous adsorption resins

RSC Adv.

Homoisoflavonoids and the antioxidant activity of Ophiopogon japonicus root

Iran. J. Pharm. Res.

Homo-aro-cholestane, furostane and spirostane saponins from the tubers of Ophiopogon japonicus

Phytochemistry

Cytotoxic homoisoflavonoids from Ophiopogon japonicus tubers

Chem Pharm Bull (Tokyo)

Evaluation of anti-inflammatory activity of compounds isolated from the rhizome of Ophiopogon japonicas

BMC Complement. Altern. Med.