Establishment of a quantitative and qualitative analysis and isolation method for tetracyclic iridoids from Morinda lucida Bentham leaves

https://doi.org/10.1016/j.jpba.2018.10.044Get rights and content

Highlights

  • Tetracyclic iridoids in Morinda lucida have anti-trypanosomal activity.

  • An analytical method and an isolation method for the compounds were developed.

  • Factors affecting the compound contents were evaluated by the analytical method.

  • New isolation method dramatically improved the yield of the compound.

  • These findings will facilitate investigation of tetracyclic iridoids as anti-trypanosomal agents.

Abstract

A new high performance liquid chromatography (HPLC) method has been established for quantitative and qualitative analysis of three tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3), which are responsible for anti-trypanosomal and anti-leishmanial activities of Morinda lucida Bentham leaves. Separation of 1–3 from dried 80% aqueous (aq.) ethanol extract was achieved on a reversed-phase cholester column packed with cholesteryl-bonded silica using an acetonitrile-0.1% aq. formic acid mobile phase system. Ultraviolet-visible (UV-VIS) spectroscopy was employed for detection of compounds, and their contents were determined by measuring absorbance at 254 nm. Depending on the above system, several factors potentially affecting the concentration of tetracyclic iridoids were evaluated resulting in several variation on plant organs, seasonality, variation between individual trees, and branch positions within the trees. Moreover, we developed a simple, quick, and effective method for tetracyclic iridoid isolation from M. lucida leaves that consisted of extraction by sonication into 80% aq. ethanol, basic hydrolysis, acid neutralization, liquid-liquid extraction into an organic solvent, and reverse phase open column chromatography. Employing this method, we have succeeded to obtain 1 as a colorless crystal yielding of 0.23%, which was 28 times higher than that of previous isolation method. Setting up methodology in this paper may be important for future in vitro and in vivo studies of tetracyclic iridoids and moreover for their applications in new drug design and development.

Introduction

Morinda lucida Bentham (Rubiaceae), a medium-sized evergreen tree with dark green, and shiny leaves, is widely grown and cultivated in West and Central Africa [1]. It has been used as a traditional remedy for fever, dysentery, abdominal colic, and intestinal worm infestation [2,3]. M. lucida extracts have been investigated frequently resulting in several activities like anti-oxidant, anti-diabetic, anti-cancer, anti-bacterial, anti-trypanosomal, and anti-malarial activities [[4], [5], [6], [7], [8], [9]]. Phytochemical studies of M. lucida have shown that it contains alkaloids, anthraquinones, triterpenes, and iridoids [1,[10], [11], [12]]. Previously, we found that the extract of M. lucida leaves had strong activity of anti-tripanosomal parasites [13]. In addition, we isolated and elucidated chemical structures of three novel tetracyclic iridoids: ML-2-3 (1), molucidin (2), and ML-F52 (3) (Fig. 1) from M. lucida leaves [[14], [15], [16]]. The absolute configuration of 2 was determined by using X-ray analysis and confirmed as an enantiomer of oruwacin [14]. Moreover, we confirmed that 1–3 exhibited strong anti-trypanosomal, anti-leishmanial, and anti-malarial activities in vitro and in vivo. [16,17]. In trypanosomes, 1 and 3 induced significant apoptosis-like cell death [15,16]. It is suggested that tetracyclic iridoids hence might be the potential lead or seed compounds of anti-trypanosomal agents. However, the yields of 1–3 from dried leaves were extremely low in our previous study like 0.0045%, 0.0032%, and 0.0005%, respectively [15,16]. Herein, we established a new method for the quantitative and qualitative analysis of 1–3 to find superior plants containing higher concentration of tetracyclic iridoids. We then examined the quantities of 1–3 in M. lucida leaves, stems, and roots, as well as potential influences of seasonality, variations between individual trees, and branch positions on the contents of the compounds. Furthermore, we report an efficient, rapid, and facile isolation and purification of tetracyclic iridoids.

Section snippets

General procedures

Specific rotations were measured with a DIP-360 digital polarimeter (JASCO, Easton, USA). Nuclear magnetic resonance (NMR) spectra were recorded on a JEOL ECX 400 FT-NMR spectrometer (JEOL, Tokyo, Japan) at 20 ◦C using JEOL’s standard pulse program, with tetramethylsilane as the internal standard and chemical shift values were expressed in δ (ppm). Electrospray ionization time-of-flight mass spectrometer (ESI-TOF-MS) experiments employed a Waters Xevo G2-XS Q-TOF mass spectrometer (Waters,

Optimal solvent system for preparation of sample solution

Frist, the extraction conditions were examined to optimize the extraction efficiency for the concentration of the target compounds (13). The contents were compared using five solvent systems (MeOH, EtOH, 80% aq. EtOH, 50% aq. EtOH, or MeCN) under sonication for 15 min. As shown in Fig. 2, 80% aq. EtOH (0.77%, contents of total tetracyclic iridoids) showed most efficacy than the other solvent systems resulted that 80% aq. EtOH was employed in this study.

Optimization of HPLC conditions

Following the sample preparation method,

Discussion

We developed an HPLC method for quantitative and qualitative analysis of three tetracyclic iridoids 13 from M. lucida. To our knowledge, this is the first report describing the quantitative and qualitative analysis of these compounds. Using this method, we examined the quantities of 13 in different plant organs, as well as other factors that could affect tetracyclic iridoid contents in M. lucida. The seasonal variation of secondary metabolite and differences depending on the place of plant

Conflict of interest

None.

Acknowledgements

This research is supported by Science and Technology Research Partnership for Sustainable Development (SATREPS) grant from the Japan Science and Agency (JST) and the Japan International Cooperation Agency (JICA) (2010 to 2015) and the Japan Initiative for Global Research Network on Infection Diseases (J-GRID) from Ministry of Education, Culture, Sports, Science & Technology (MEXT) in Japan (2015 to present).

References (20)

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  • A systematic review of antimalarial activities of Morinda species

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    For instance, Cimaga et al. reported that the activity of M. morindoides related to terpenes, steroids, and flavonoids whereas the active ingredients of M. lucida might be anthraquinones (Adewole et al., 2021; Cimanga et al., 2008; Koumaglo et al., 1992). Although there were many reports of antimalarial therapeutics of these plants (Afolabi, Abejide, 2020; Hashim et al., 2021; Ohta et al., 2019), their traditional uses were supposed to relate to their symptomatic relief rather than parasite suppression ability. Additionally, there was no comparison between the Morinda species; the active ingredients and their probable mechanisms of action had not been elucidated.

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