Offline preparative 2-D polar-copolymerized reversed-phase chromatography × zwitterionic hydrophilic interaction chromatography for effective purification of polar compounds from Caulis Polygoni Multiflori

Highlights

• An offline 2-D RPLC/HILIC method was developed to purify polar compounds.

• A polar-copolymerized XAqua C18 and zwitterionic Click XIon column were selected and combined.

• The orthogonality was 72% in the separation of Caulis Polygoni Multiflori.

• Three polar compounds with high purity were first purified from this plant.

Abstract

Polar compounds are abundant in the water decoction of Traditional Chinese Medicines (TCMs) and possess important biological activities. However, purifying these compounds has faced great difficulties largely due to poor retention and insufficient selectivity. To solve this problem, an offline orthogonal 2-D RPLC coupled with hydrophilic interaction chromatography (HILIC) method was developed to achieve purification of polar compounds from Caulis Polygoni Multiflori. A polar-copolymerized XAqua C18 column and a zwitterionic Click XIon column exhibited satisfactory retention and separation for polar compounds. Therefore, they were adopted to construct the offline 2-D LC system. Furthermore, the method presented a high orthogonality, which was calculated to be 72%. The XAqua C18 column was used in the first dimension to fractionate the 5.1 g polar fraction with a recovery of 85% within 10 h. In the second dimension, three representative fractions were purified using the Click XIon column. Finally, three compounds with purity higher than 95% were identified for the first time from this plant. This offline 2-D RPLC/HILIC method was shown to be an effective approach to purifying polar compounds from Caulis Polygoni Multiflori.

Introduction

Polar compounds are an important component of Traditional Chinese Medicines (TCMs) because most TCMs are prepared and administered as water decoctions. Polar compounds like alkaloids, polar glycosides, organic acids, and polyphenols are responsible for a variety of biological activities in TCMs [[1], [2], [3], [4]]. Recently, natural polar compounds as hit compounds have been increasingly important for a drug discovery program [5,6]. Consequently, the isolation of natural polar compounds has gradually become a research hotspot.

Caulisol Pygoni Multiflori, the dried caulis of Polygonum multiflorum Thunb, is one of the commonly used TCMs owing to its therapeutic application for sleeping disorder [7] and hypoglycemic activities [8]. Previous chemical investigations showed that Caulis Polygoni Multiflori mainly contains stilbenes, phenolic acids, flavonoids, anthraquinones, etc. [9]. Few reports focused on polar compounds in Caulis Polygoni Multiflori. In our ongoing work, the crude extract of Caulis Polygoni Multiflori was found active on dopamine-2 (D2) receptor (Supporting information Fig. S1), and the polar fractions exhibited D2 receptor agonistic activity in further tests. Therefore, estimation of polar components is favorable for understanding their bioactivities.

Up to now, purifying polar compounds from TCMs remained challenging. The conventional preparative low- and medium-pressure columns have proved to be inefficient when handling polar compounds. Recently, many modern purification techniques have been employed, such as high-speed-countercurrent chromatography (HSCCC) [10,11] and preparative HPLC (prep-HPLC) [[12], [13], [14]]. HSCCC is a convenient technique with a high total sample recovery and a large load capacity. Nevertheless, it is inefficient to prepare several high-purity compounds simultaneously because of its insufficient peak resolution [15]. Alternatively, taking advantages of high-performance separation, excellent repeatability, online detection and automatic control [16], prep-HPLC is considered as one of the most popular techniques and it has been extensively applied in the purification of polar compounds from TCMs [17,18].

As a dominant HPLC separation mode, RPLC served as a very effective preparative technique for compounds from TCMs [16,19]. However, conventional RPLC stationary phase fails in polar compounds separation since such analytes are regularly poorly retained on a silica-based octadecyl (C18) reversed-phase packing [20,21]. To solve this problem, different polar-modified C18 stationary phases were developed. Polar-embedded and polar-endcapped stationary phases among them have gained increasing popularity. Wang et al. [22] developed a series of C10 dipeptide-embedded stationary phases, which increased the retention for polar compounds and displayed different selectivity, compared to conventional C18 columns. Liu et al. [23] prepared a reversed-phase packing containing both polar-embedded and polar-endcapped moieties to further increase the resolution for polar analytes. In our group, based on a polar-copolymerized approach, a series of polar-modified RP stationary phases were developed and applied for purifying polar compounds. Li et al. [19] established an efficient method to separate polar compounds from water-soluble extract of toad skin, based on a C18HCE column. Zeng et al. [24] used a C18HC column to purify polar compounds from Radix isatidis. RPLC with these polar-copolymerized C18 stationary phases exhibited increased retention, unique selectivity, and good resolution for polar compounds. Nevertheless, it is intractable to effectively separate the polar components with high resolution employing 1-D RPLC separation alone, due to its inadequate selectivity and peak capacity.

Presently, many studies tend to use 2-D LC to deal with complex samples [[25], [26], [27], [28]]. Li et al. used an online comprehensive 2-D NPLC × RPLC system to separate Toad Venom and isolate 19 compounds with high purity [25]. By combining two orthogonal LC separations, this system will maximize its resolving power and achieve good separation. Hydrophilic interaction chromatography (HILIC), with different separation mechanism from RPLC, serves as an effective technique for separating polar compounds [[29], [30], [31]]. Therefore, a 2-D method composed of RPLC and HILIC would offer sufficient peak capacity for isolating polar compounds from complex samples. To obtain better retention and resolution, both RPLC and HILIC columns required optimization respectively. Our previous work developed some appropriate stationary phases that might deal with polar compounds in complex samples, such as C18 HCE (in which H stands for horizontal, C for chloropropyl and E for endcapping process) [32], C18 HC (the same as C18 HCE for each symbol) [24], Click TE-Cys (in which TE refers to Thiol-Ene, and Cys represents Cysteine) [33], etc. In addition, the offline 2-D mode allowed the fraction eluted from the first dimension to be prepared in the second dimension with no time restriction, and thus fractions could be well prepared in the second dimension [28]. We selected offline 2-D LC in this work to maximize resolving power of the two dimensions. To the best of our knowledge, few reports focused on the purification of natural polar compounds using an offline 2-D LC method.

In this study, an offline preparative 2-D RPLC/HILIC method was developed to provide a solution to the purification of polar compounds from Caulis Polygoni Multiflori. This method addressed the problem of poor retention and exhibited superior separation orthogonality. A polar-copolymerized XAqua C18 column was used for RPLC separation in the first dimension, and a zwitterionic Click XIon column for HILIC separation in the second dimension. In the first dimension, 11 fractions were prepared according to their UV absorbance, and three were taken as an example for purification in the second dimension. Three compounds were first identified from this plant. The results demonstrated that this offline 2-D LC method was efficient in the purification of polar compounds from Caulis Polygoni Multiflori.