Preparative separation of three terpenoids from edible brown algae Sargassum fusiforme by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography

Highlights

• Three terpenoids were isolated from edible brown algae Sargassum fusiforme.

• High-speed countercurrent chromatography was used for separation of crude extracts.

• Preparative high performance liquid chromatography was employed to separate isomers.

• The purity of the three isolated terpenoids was all above 95%.

Abstract

Edible brown algae Sargassum fusiforme is an economic seaweed with huge yield in Asia. Terpenoids are an important class of active substances in Sargassum fusiforme, which have replaced some synthetic drugs in clinical treatment. In this work, the preparative separation protocol of three terpenoids, loliolide (1), isololiolide (2), and (3R)-4-[(2R,4S)-4-acetoxy-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-one (3), from Sargassum fusiforme by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography was presented. n-Hexane-ethyl acetate-methanol-water (1:2:1:2, v/v) was selected as biphasic solvent system through optimization. Under the optimal high-speed countercurrent chromatography separation conditions, 35.87 mg mixture of isomers 1 and 2, and 8.87 mg compound 3 were obtained from 300 mg of the crude extract of Sargassum fusiforme. The isomer mixture was further separated by preparative high-performance liquid chromatography to obtain 11.56 mg compound 1 and 12.82 mg compound 2. Their purities were all above 95%. The chemical structures of three isolated terpenoids were identified by ¹H and ¹³C nuclear magnetic resonance spectroscopy.

Introduction

In recent years, with the scarcity of land resources, the development of marine resources has been increasing. The compounds in marine organisms have properties similar to drugs and have become resources for exploring new chemical components and developing new drugs. Brown algae is the second largest species among marine algae, it includes approximately 250 genera and 1500 species [1,2]. Sargassum fusiforme, an edible brown algae, is belonging to the order Fucales, and family Sargassaceae. It is an endemic species found in the temperate region of the Northwest Pacific, and distributed mainly along the coast of China, Japan, and Korea [3,4]. It is an important economic seaweed, and has been used as an essential food and therapeutic drug for thousands of years [5]. S. fusiforme contains complex nutrients, such as polysaccharides, proteins, polyphenols, pigments, terpenoids, sterols and other small molecular compounds [3,6]. Terpenoids are a general term for hydrocarbons and their oxygen-containing derivatives that are polymerized from two or more isoprene units. They can be classified into monoterpenes, sesquiterpenes, diterpenes and polyterpenes according to their structural units [7]. Terpenoids have been confirmed to have important biological activities related to human health, such as antimicrobial, anti-inflammatory, anticancer, antidiabetic, etc. [[8], [9], [10], [11]]. Therefore, terpenoids have great potential to replace synthetic drugs in the treatment of certain diseases. In addition, terpenoids are also used in the production of advanced fragrances, cosmetics, food additives, etc. [[12], [13], [14]].

The extraction and enrichment of small molecular compounds from natural food is helpful to understand the composition of micronutrients in food and to explore the mechanism of food bioactivity. Terpenoids are widely found in plants, microorganisms and insects. However, one of the main problems inhibiting a wider commercial use of terpenoids is the low ratio among efficiency and cost associated with the extraction and purification processes used nowadays. Generally, there are more than one terpenoids in algae, and their content is extremely small, which makes the separation and purification of terpenoids very difficult [15,16]. At present, thin layer chromatography and silica gel column chromatography are widely used in the purification of terpenoids. Although their separation effect is good, they have some shortcomings, such as inconvenient operation, large solvent consumption, long separation time, and less sample loading [17,18]. High-speed countercurrent chromatography (HSCCC) is an efficient preparative separation technique, which uses the principle of liquid-liquid distribution to separate the mixture [[19], [20]]. That is, without using solid-phase carrier, the target compound is continuously distributed between the immiscible liquid-liquid phases to achieve the purpose of separation. HSCCC has the advantages of flexible elution method, large loading capacity, high product purity, simple operation, etc. [19]. Preparative high-performance liquid chromatography (Pre-HPLC) is a liquid chromatography technique that achieves high-purity separation through a preparative column with high loading and high resolution. It has the advantages of high separation efficiency and accurate collection, thus it is suitable for industrialized preparation [21,22]. At present, there have been studies combining HSCCC and Pre-HPLC to separate target mixtures from complex mixtures. For example, Fábryová et al. successfully isolated five astaxanthin monoesters from the microalgae Haematococcus pluvialis by high performance countercurrent chromatography (HPCCC) combined with semi-preparative HPLC [23]. Kim et al. successfully separated three norisoprenoids from Sargassum horneri by high-performance centrifugal partition chromatography (HPCPC) combined with semi-preparative HPLC [24]. CPC is a variant of countercurrent chromatography and the difference mainly lies in their retention mechanisms of stationary phase: hydrostatic equilibrium system for CPC and hydrodynamic equilibrium system for CCC [20]. In addition, S. fusiforme and S. horneri both belong to Sargassaceae family, however, S. fusiforme has been widely used as food and therapeutic drug for thousands of years [[3], [4], [5]]. It is a kind of more important economic brown algae and widely cultivated in Zhejiang Province of China.

There are three main ways to produce terpenoids: extraction from natural products, biosynthesis, chemical synthesis [7,25,26]. The extraction of terpenoids from natural products has the advantages of low cost, short cycle and low toxicity. Biosynthesis has the disadvantages of time-consuming and cumbersome operation. Chemically synthesized terpenoids may have toxicity and side effects. At present, there are few reports on the study of terpenoids in edible brown algae S. fusiforme. In this work, we tried to combine the advantages of HSCCC and Pre-HPLC to establish a method for preparative separation of three terpenoids, loliolide (1), isololiolide (2), and (3R)-4-[(2R,4S)-4-acetoxy-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-one (3) (Fig. 1), from S. fusiforme. The crude extracts were obtained by microwave-assisted extraction. The crude extracts were separated by HSCCC using elution-extrusion separation mode firstly, and then the isomers loliolide and isololiolide were further separated by Pre-HPLC, so as to realize the preparative separation of high-purity terpenoids from S. fusiforme.