Elsevier

Industrial Crops and Products

Volume 126, 15 December 2018, Pages 69-75
Industrial Crops and Products

An efficient method for the preparative separation and isolation of ginkgolic acids from the sarcotesta of Ginkgo biloba L by pH-zone-refining counter-current chromatography coupled with inner-recycling mode

https://doi.org/10.1016/j.indcrop.2018.09.034Get rights and content

Highlights

  • Ginkgolic acids (GAs) are abundant in the sarcotesta of ginkgo semen.

  • PZRCCC was used for the gram-scale preliminary separation of GAs.

  • Inner-recycling HSCCC mode was used to separate GAs with similar KD-values.

  • Five main ginkgolic acids were successfully separated with purities over 98%.

Abstract

An efficient method for the preparative separation and isolation of ginkgolic acids (GAs) from the sarcotesta of Ginkgo biloba L was developed by combining pH-zone-refining counter-current chromatography (PZRCCC) with inner-recycling mode. Two grams of GAs extract were separated by PZRCCC using a two-phase solvent system of n-heptane–ethyl acetate–methanol–water (2:1:3:1, v/v) with 10 mM HCl added to the upper phase as the retention acid and 10 mM triethylamine (TEA) in the lower phase as the elution base. In a one-step PZRCCC separation, two GA compounds, C15:1 and C17:2, were obtained with purities over 98% along with two mixtures. The mixtures were further separated by inner-recycling CCC mode with n-heptane–ethyl acetate–methanol–acetic acid (5:4:1:1, v/v) as the solvent system. Five main GAs were successfully separated with purities over 98%. The structures of the separated GAs were determined based on standard samples coupled with Electrospray Ionization Mass Spectrometry (ESI-MS) results. This separation method of coupling PZRCCC with inner-recycling CCC is effective for the preparation of GAs from the sarcotesta of G. biloba and could be further applied to separate organic acids of low polarity in other natural products.

Introduction

Ginkgo biloba L., described as a living fossil, is the last remaining plant of the Ginkgoceae family and is widely spread all over the world (Boonkaew and Camper, 2005). As a well-known medicine and dietary supplement, extracts obtained from the leaves and seeds of G. biloba play an important role in the treatment of Alzheimer’s dementia, protecting hippocampal neurons and improving cognitive performance and social functioning (Oken et al.,1998; Mazza et al., 2006; Ahlemeyer and Krieglstein, 2003; Bastianetto et al., 2000). In previous studies, the main medicinal components of Ginkgo biloba were found to be terpene trilactones (ginkgolides A, B, C, and bilobalide) and flavonoids (quercetin, kaempferol, and isorhamnetin) (Strømgaard and Nakanishi, 2004). Ginkgolic acids (GAs), on the other hand, are considered to be toxic, mutagenic, and allergenic with contents of less than 10 μg/g in the related dry G. biloba extract (Hecker et al., 2002; Benezra, 1990; Al-Yahya et al., 2006; Ahlemeyer et al., 2001). Recently, there has been more focus on pharmacological research concerning the beneficial effects of GAs. GAs possess anti-cancer, anti-parasitism, anti-bacteria, and molluscicidal activities (Yang et al., 2014; Baek et al., 2017; Wu et al., 2011; Wang et al., 2009; Kubo et al., 1993; Lee et al., 2014; Zhang et al., 2011; Li et al., 2012), in particular by inhibiting enzymes such as HIV protease, fatty acid synthase, tyrosinase and glycerol-3-phosphate dehydrogenase, as well as protein sumoylation (Lü et al., 2012; Oh et al., 2013; Fu et al., 2013; Irie et al., 1996; Fukuda et al., 2009). GAs are abundant in G. biloba, especially in the sarcotesta of ginkgo semen (over 4%, w/w) (Sun et al., 2012; Yang et al., 2002). Due to their broad pharmacological effects, abundance as resources, and the mandatory quality control of G. biloba products, an efficient method for the isolation and purification of GAs needs to be established.

GAs are natural 6-alkylsalicylic acids with a long-chain hydrophobic base of 13–17 carbons and 0–2 double bonds in the alkyl chain (Fig. 1) (Beek, 2002; Beek and Montoro, 2009). Because of their long alkyl chains, GAs are of low polarity and are strongly hydrophobic. Several studies have reported separations of GAs using silica gel, silicone oil chromatography, C18-Ag(I)-loaded cation exchange chromatography, reverse C8 and C18 HPLC and so forth (Tan et al., 2001; Itokawa et al., 1987; Nagabhushana and Ravindranath, 1995; Zhang et al., 2004; Beek and Wintermans, 2001; Li et al., 2014; Ni et al., 2001). The traditional silica gel separation method is tedious with a long separation time that uses large amounts of solvents. Hence, the cost of separation is high and it entails rigorous sample preparation. Moreover, the long-chain hydrophobic base means these compounds are strongly adsorbed on a reverse phase column. Therefore, an efficient isolation and purification method is urgently needed to develop a better way of separating GAs.

High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatographic technique that eliminates the irreversible adsorption of the sample onto the solid support. Furthermore, HSCCC is characterized by high target recovery and separation capabilities, as well potential to easily scale up the sample size, simple operation, low solvent consumption, and rapid separation (Jin et al., 2013; Gao et al., 2017; Zhang et al., 2017; Zhao et al., 2015). It has gradually become a useful tool for preparative isolation and purification of various natural products (Li et al., 2013; Chen et al., 2018; Zou et al., 2018). pH-Zone-refining counter-current chromatography (PZRCCC) is a variation of HSCCC. It can separate organic acids and alkaloids into a succession of highly concentrated rectangular elution peaks according to their pKa values and hydrophilicities. PZRCCC has many advantages including a 10-fold sample loading capacity, concentration of minor impurities, and highly concentrated fractions (Sun et al., 2016; Zhu et al., 2015; Ito, 2013; Ito and Ma, 1996). Nevertheless, as it is restricted to an insufficient theoretical plate number compared with HPLC, especially for compounds with similar KD values, it is difficult to separate similar compounds using 1D HSCCC. The traditional way to separate compounds with similar KD values is to increase the separation time, which results in increased peak broadening and wastes organic solvents. Recently, 2D/multi-D HSCCC methods were developed to enhance peak resolution. Of the various modes available to date, e.g. tandem HSCCC (Zeng et al., 2015), off-line 2D/multi-D CCC (Shi et al., 2012), on-line inner-recycling CCC (Zhang et al., 2016), online-storage recycling CCC (He et al., 2016). Inner-recycling CCC is an excellent solution to separate compounds with similar KD-values. The amount of the solvent required using this technique is greatly reduced.

Here, PZRCCC coupled with an inner-recycling mode was introduced to separate GAs. The purpose of this study is to develop an effective method for the preparative isolation and purification of GAs in the sarcotesta of G. biloba using this system. To the best of our knowledge, this is the first report using PZRCCC and inner-recycling mode for separating GAs in the sarcotesta of G. biloba. The chemical structures of the isolated GAs were shown in Fig. 1.

Section snippets

Materials, chemicals and reagents

Fresh sarcotesta of G. biloba was obtained from ginkgo trees in the city of Jinan (Shandong, China) and identified by Dr. Jia Li (College of Pharmacy, Shandong University of Traditional Chinese Medicine). A voucher specimen (2016100701) has been deposited at Shandong Analysis and Test Center.

n-Hexane, n-heptane, ethyl acetate, methanol, acetic acid, petroleum ether (60–90 °C), hydrochloric acid (HCl) and triethylamine (TEA) used for the preparation of crude extract and CCC separations were

Selection of the PZRCCC solvent systems

In order to separate the GAs by PZRCCC, a suitable two-phase solvent system was needed. Such a system requires an appropriate partition coefficient (Kacid >> 1 and Kbase << 1, organic phase as the stationary phase) as well as good sample solubility (Ito, 2005). Various solvent systems were tested including ethyl acetate–n-butanol–water (4:1:5, v/v), chloroform–methanol–water (4:3:3, v/v), n-hexane–ethyl acetate–methanol–water (5:5:2:8, v/v), and n-heptane–ethyl acetate–methanol–water (2:1:3:1,

Conclusion

The present study represents a novel and efficient method combination PZRCCC and inner-recycling mode for the preparative separation and isolation GAs from sarcotesta of G. biloba. Initially, a two-phase solvent system of n-heptane-ethyl acetate-methanol-water (2:1:3:1, v/v) with 10 mM HCl in the upper phase and 10 mM TEA in lower was chosen to separate GAs by PZRCCC. Two pure compounds of GA3 (C15:1) and GA5 (C17:2) were obtained in one-step PZRCCC separation and coupled with two fractions.

Funding

This work was supported by funding: National Natural Science Foundation of China (21506119), Shandong Province Major Scientific and Technological Innovation Project (2017CXGC1209, 2017CXGC1308) and Primary Research & Development Plan of Shandong Province (2017GSF216002).

Acknowledgment

These funders played no roles in the study design, data collection and analysis, and decision to publish. We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

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