Laguncularia racemosa Phenolics Profiling by Three-Phase Solvent System Step-Gradient Using High-Performance Countercurrent Chromatography with Off-Line Electrospray Mass-Spectrometry Detection

Abstract

The detailed metabolite profiling of Laguncularia racemosa was accomplished by high-performance countercurrent chromatography (HPCCC) using the three-phase system n-hexane–tert-butyl methyl ether–acetonitrile–water 2:3:3:2 (v/v/v/v) in step-gradient elution mode. The gradient elution was adjusted to the chemical complexity of the L. racemosa ethyl acetate partition and strongly improved the polarity range of chromatography. The three-phase solvent system was chosen for the gradient to avoid equilibrium problems when changing mobile phase compositions encountered between the gradient steps. The tentative recognition of metabolites including the identification of novel ones was possible due to the off-line injection of fractions to electrospray ionization mass spectrometry (ESI-MS/MS) in the sequence of recovery. The off-line hyphenation profiling experiment of HPCCC and ESI-MS projected the preparative elution by selected single ion traces in the negative ionization mode. Co-elution effects were monitored and MS/MS fragmentation data of more than 100 substances were used for structural characterization and identification. The metabolite profile in the L. racemosa extract comprised flavonoids, hydrolysable tannins, condensed tannins and low molecular weight polyphenols.

1. Introduction

Countercurrent chromatography (CCC) is an all-liquid method, with no solid support, in which the stationary liquid phase is retained in the apparatus using centrifugal force only [1]. The principle behind this technique underlies the partitioning of a sample in a biphasic liquid solvent system [2]. Among many advantages, the technique is highly versatile; has high loading capacity; is easy to scale up; and eliminates sample loss by chemical degradation and irreversible adsorption [1,3,4]. CCC is a powerful tool in the phytochemical working field as it enables plant extract fractionation with existing major compounds and also isolation or fortification of minor compounds [5,6]. This characteristic is even more noticeable when semi-preparative and preparative scales are employed [7,8]. Standard CCC separation experiments do not provide the full flexibility, solely operating with a two-phase solvent system, and the isocratic elution–extrusion mode [9,10].

To improve the polarity range in the CCC operation field, three-phase solvent systems were developed/applied, due to the great differences in polarities between the upper, middle and lower phases. Recently, some applications have been reported but only half of them actually were used as three phases in the separation process [11,12,13,14,15]. Tri-phasic systems are built from a two-phase system normally composed of n-hexane, acetonitrile and water in combination with a fourth solvent such as methyl acetate, ethyl acetate, methyl tert-butyl-methyl-ether or dichloromethane to create the third phase. Very few tri-phasic systems are described due to limited solvent combinations that form these three stable phases in a convenient volume percentage [11].

In analogy to solid phase chromatography, gradient elution in CCC intends to shorten the duration of a separation process and may also improve resolution. A common way to perform gradient elution is to change the mobile phase polarity over time [16], although gradient elution mode in CCC is less frequently used as the biphasic liquid system is in the equilibrium state and the change of composition in one phase corresponds directly to a change in the respective other liquid phase [17]. However, if a three-phase solvent system is used for gradient elution purposes, all phases involved for the experiment were previously in contact. Disturbance of the equilibrium and collapse of phase layers are omitted during the separation process while maintaining the broad polarity range for the recovery process.

In this work, a three-phase solvent system in step-gradient elution mode high-performance countercurrent chromatography (HPCCC) with off-line ESI-MS/MS detection was used for metabolite profiling of the ethyl acetate partition of the leaves from the mangrove plant Laguncularia racemosa. A similar approach was used on Anogeissus leiocarpus for compound identification but using centrifugal partition extraction (CPE), and off-line NMR detection [15]. L. racemosa (Combretaceae), popularly known as white mangrove, is the only occurring specie in the genus [18], and is considered as a strict mangrove [19], characteristic for growing in brackish coastal environments [20], and with excellent function for stabilizing shorelines against erosion [21]. From aspects of ethno-medicinal use, the plant is applied as astringent and tonic for dysentery and fever [22]. To date, there are only a few studies on the production of secondary metabolites [23,24,25,26,27], probably due to its high complexity of polar natural products.

2. Results and Discussion

The composition of L. racemosa ethyl acetate solvent partition (EtOAcPart) was initially investigated by TLC and LC-ESI/TOFMS analysis (Supplementary Figure S1). The liquid mass-spectrometry profile showed a high chemical complexity containing metabolites in a larger polarity range, making this mixture an ideal case study for the application of three-phase gradient elution. Some multiple-solvent biphasic systems have been proposed to great extend the polarity in CCC [28]. However, as the phases are in steady mixing contact during the complete separation process, a change of the mobile phase composition during gradient elution directly influences the stationary phase composition as well, and as consequence, disturbs the equilibrium and could lower or even lead to low chromatographic resolution [29,30]. To circumvent the equilibrium obstacle during the gradient elution procedure, a three-phase solvent system was used instead of two (or more) different biphasic systems. In this approach all phases involved in the separation were previously saturated with each other. The tri-phasic system n-hexane–tert-butyl-methyl ether–acetonitrile–water 2:3:3:2 (v/v/v/v) was used in the semi-preparative purification of L. racemosa EtOAcPart.

2.1. HPCCC of L. racemosa Metabolites by Off-Line ESI-MS/MS Profile Detection in the Sequential Order of Recovery

The L. racemosa EtOAcPart was separated by semi-preparative HPCCC chromatography, and the off-line injection profiling by injections of recovered fractions to ESI-MS/MS distinguished 17 principal phenolic constituents (Figure 1). However, as a result of the highly concentrated injections of respective HPCCC fractions, the selected single ion-based projection of the HPCCC experiment revealed more than 100 different metabolites (1–109) (Partially shown in Figure 1 and Supplementary Figure S2). Preliminary LC-ESI/TOF MS analysis was not capable of detecting all minor compounds due to concentration levels below the detection limits.

Figure 1.

Selected electrospray ionization mass spectrometry electrospray ionization mass spectrometry ions traces (negative mode) of phenolics of L. racemosa EtOAcPart detected in the off-line injected high-performance countercurrent chromatography (HPCCC) fractions. HPCCC separation using n-hexane- tert-butyl-methyl ether–acetonitrile-water 2:3:3:2 (v/v/v/v) as triphasic solvent system.

Large advantage of the off-line injection profiling methodology of preparative HPCCC-fractions by an ESI-MS detector in the sequence of recovery is the ‘on-the-fly’ delivery of the respective molecular weight- and MS/MS-fragmentation data of all ionizeable compounds in one step. This is a very fast process to get the required data for immediate compound identifications in the respective HPCCC-fractions. A full mass-spectrometry guided metabolite profile with more than ten automatically selected precuresur ions for MS/MS on a larger lab-scale preparative HPCCC fractionation can be achieved in a 60 to 100 min experimental mass-spectrometry time frame. This mass-spectromtry approach is roughly by a factor of hundred faster than the single investigation of resepctive HPCCCC fractions by LC-ESI-MS/MS analysis. The results displayed in a single data file are ready to use and not mutiple analysis sets need to be compared for guiding the decisions in fractionation process. This powerful approach was previously applied by Costa et al. [8] on the complex metabolite mixture extracted from the Brazilian plant Salicornia gaudichiana.

In case of the investigated L. racemosa ethylacetate solvent partition, the elution ranges of a large selection of higher and lower concentrated target molecules (Table 1) were visualized in the recovered HPCCC-fractions by selected single ion traces for performing the accurate fractionation, recovery and preventing unintentional mixing of already separated compounds. Additionally, the existing compound co-elution effects, and the sequential elution orders of separated isobars/ isomers were clearly detected and visualized (Figure 1).

Table 1.

Detected compounds in the HPCCC off-line ESI-MS/MS phenolic profile of Laguncularia racemosa EtOAcPart.

Cpd	CCC-Fraction	MS [M – H]−(m/z) MS/MS [M – H]− (m/z)	LC-RT(min)	ESI/TOF MS Formula (Error in ppm)	Identification
Flavonoids and derivatives
1	11–15	255 237, 226, 209, 156	n.d.	-	Dihydrocrysin
2	21	269 151	13.7	269.04593 C15H9O5 (1.4)	Apigenin
3	19–21	271 177, 151	1.9	271.04885 C15H11O5 (45.6) *	Naringenin
4	23	273 167	29.1	273.08108 C15H13O5 (15.5)	Afzelechin
5	19	285 257, 151	40.8	285.04413 C15H9O6 (12.6)	Kaempferol
6	31–41	287 259, 151	12.1	287.05883 C15H11O6 (9.5)	Dihydrokaempferol
7	97–115	289 245, 205	6.2	289.07438 C15H13O6 (9.0)	(Epi)-catechin
8	29–33	301 179, 151	35.4	301.03937 C15H9O7 (13.3)	Quercetin
9	17–19	305 287, 249	2.4	305.0706 C15H13O7 (12.8)	(Epi)-gallocatechin
10	21–23	315 300	42.8	315.0466 C16H11O7 (14.1)	Isorhamnetin
11	25–27	317 179, 151	28.9	317.03536 C15H9O8 (16.0)	Myricetin
12	81–93	319 193	9.1	319.04883 C15H11O8 (9.0)	Dihydromyricetin
13	33–41	329 314, 299	21.7	329.05816 C17H13O7 (25.9) *	Tricin
14	133–149	393 317, 241, 169	-	-	Myricetin derivative
15	29–33	415 301	-	-	Quercetin alkyl derivative
16	127–131	419 305	-	-	(Epi)-gallocatechin alkyl derivative
17	55–57	431 317	-	-	Myricetin alkyl derivative
18	97–105	433 301, 179, 151	27.5	433.08215 C20H17O11 (10.4)	Quercetin pentoside
19	89–91	433 319, 193	-	-	Dihydromyricetin alkyl derivative
20	91–95	441 289	19.3	441.08208 C22H18O10 (1.5)	(Epi)-catechin gallate
21	105–119	447 301	29.3	447.09851 C21H19O11 (11.7)	Quercetin desoxyhexoside
22	115–133	449 317, 316	22.4	449.07395 C20H17O12 (3.1)	Myricetin pentoside
23	113–127	457 331, 305, 169	12.1	457.07859 C22H18O11 (2.1)	(Epi)-gallocatechin gallate
24	21–23	461 443, 381, 301, 193	-	-	Quercetin derivative
25	123–131	463 317, 316	24.6	463.09083 C21H19O12 (5.7)	Myricetin desoxyhexoside
26	141–145	463 301	25.9	463.09187 C21H19O12 (7.9)	Quercetin hexoside
27	153–165	467 458, 391, 301, 169	-	-	Quercetin derivative
28	67–79	469 317	n.d.	-	Myricetin galatte
29	85–91	471 319, 301, 193	-	-	Dihydromyricetin alkyl derivative
30	115–131	477 301, 179	15.2	477.06812 C21H17O13 (1.4)	Quercetin glucuronide
31	133–163	479 317, 316	22.1	479.08428 C21H19O14 (2.4)	Myricetin hexoside
32	89–97	585 433, 301	32.7	585.09204 C27H21O15 (5.9)	Quercetin pentoside gallate
33	85–95	599 447, 301	26.8	599.10714 C28H23O15 (4.8)	Quercetin desoxyhexoside gallate
34	95–113	601 449, 317	28.6	601.08787 C27H21O16 (7.3)	Myricetin pentoside gallate
35	29–33	603 301	n.d.	-	Quercetin [2M − H]−
36	125–131	611 305	-	-	(Epi)-gallocatechin [2M − H]−
37	91–115	615 463, 317, 179	23.4	615.1014 C28H23O16 (3.6)	Myricetin desoxyhexoside gallate
38	137–153	615 463, 301	31.6	615.10412 C28H23O16 (8.1)	Quercetin hexoside gallate
39	113–127	629 477, 317, 316, 289	21.5	629.07893 C28H21O17 (0.8)	Quercetin glucuronide gallate
40	133–139	631 479, 317	28.9	631.09859 C28H23O17 (7.2)	Myricetin hexoside gallate
41	45–57	635 317	n.d.	-	Myricetin [2M − H]−
42	87–91	639 319, 301	11.2	639.05562 C29H19O17 (11.2)	HHDP Dihydromyricetin
43	25	657 317	-	-	Myricetin derivative
44	89	697 599	-	-	Quercetin desoxyhexoside gallate derivative
45	89–93	737 585, 301	n.d.	-	Quercetin pentoside digalloyl
46	93–103	753 601, 449, 317	32.5	753.09740 C34H25O20 (3.9)	Myricetin pentoside digalloyl
47	85–89	773 471, 301	-	-	Quercetin derivative
48	97–103	867 433, 301	n.d.	-	Quercetin pentoside [2M − H]−
49	117–127	883 449, 317	-	-	Myricetin pentoside derivative
50	115–123	892 457, 433	-	-	(Epi)-gallocatechin gallate derivative
51	117–127	899 463, 449, 317	-	-	Myricetin pentoside derivative
52	87–93	901 599, 301	-	-	Quercetin desoxyhexoside gallate derivative
53	111–121	905 469, 457, 447, 425, 301	-	-	Quercetin desoxyhexoside derivative
54	113–125	907 449, 317	-	-	Myricetin pentoside derivative
55	113–123	915 457	-	-	(Epi)-gallocatechin gallate [2M − H]−
56	129–131	927 463, 317	n.d.	-	Myricetin desoxyhexoside [2M − H]−
Hydrolisable tannins and deivatives
57	93–115	169 125	12.1	169.01664 C7H5O5 (14.2)	Gallic acid
58	61–81	183 124	5.9	183.01418 C8H7O5 (12.2)	Methyl gallate
59	43–57	197 169, 125	14.3	197.04741 C9H9O5 (9.5)	Ethyl gallate
60	81–93	301 283, 257, 229, 163	18.5	300.99939 C14H5O8 (1.3)	Ellagic acid
61	19–21	315 300	6.1	315.01809 C15H7O8 (10.9)	Ellagic acid methyl ether
62	97–103	321 169	3.9	321.03300 C14H9O9 (24.3) *	Galloyl gallate
63	133–151	325 169	3.3	325.06016 C14H13O9 (11.3)	Galloyl shikimate
64	33–39	329 314	44.2	329.02154 C16H9O8 (8.9)	Ellagic acid dimethyl ether
65	163	331 271, 169, 125	11.5	331.06888 C13H15O10 (5.5)	Galloyl hexoside
66	81–87	335 183	9.2	335.02817 C15H11O9 (37.9) *	Galloyl methyl gallate
67	21–23	343 328	44.1	343.04787 C17H11O8 (5.6)	Ellagic acid trimethyl ether
68	59–79	349 197	13.9	349.0416 C16H13O9 (42.7) *	Galloyl ethyl gallate
69	105–119	425 301	15.8	425.01469 C20H9O11 (0.8)	Ellagic acid pyrogallol ether
70	103–119	469 425	15.7	469.0039 C21H9O13 (2.1)	Valoneic acid dilactone
71	161–165	481 439, 331, 301, 169	1.2	481.06556 C20H17O14 (6.6)	HHDP hexoside
72	155–165	483 439, 331, 313, 169	11.5	483.07806 C20H19O14 (0.1)	Digalloyl hexoside
73	85–89	497 301	23.4	497.03631 C23H13O13 (0.3)	Valoneic acid dilactone ethyl ether
74	83–91	625 471, 301	28.6	625.07458 C26H25O18 (28.1) *	Ellagic acid dihexoside
75	147–155	631 479, 301	19.4	631.09323 C27H19O18 (26.3) *	NHDP hexoside
76	155–165	633 479, 301	7.8	633.07511 C27H21O18 (2.8)	HHDP galloyl hexoside
77	133–151	635 483, 465, 313	15.6	635.08832 C27H23O18 (1.0)	Trigalloyl hexoside
78	135–139	733 635	n.d.	-	Trigalloyl hexoside derivative
79	149	781 631, 301	3.7	781.06132 C34H21O22 (10.7)	Punicalin
80	155–167	783 481, 301	2.7	783.07063 C34H23O22 (2.5)	DiHHDP hexoside
81	133–165	785 633, 481, 301, 275	9.6	785.08378 C34H25O22 (0.7)	HHDP digalloyl hexoside
82	133–155	787 635, 617, 483, 465, 301	21.1	787.09741 C34H27O22 (3.2)	Tetragalloyl hexoside
83	145–167	935 917, 633, 571, 365, 329, 299, 275	6.0	935.07728 C41H27O26 (2.5)	Galloyl diHHDP hexoside
84	131–169	937 785, 769, 633, 617, 301	6.0	937.28345 C41H29O26 (12.6) *	HHDP trigalloyl hexoside
85	155–167	939 787, 769, 617, 465	26.2	939.11228 C41H31O26 (1.5)	Pentagalloyl hexoside
86	155–169	951 907, 783, 605	-	-	DiHHDP hexoside derivative
Condensed tannins
87	121–133	577 463, 425, 313, 289	3.9	577.13515 C30H25O12 (2.9)	(Epi)-catechin dimer
88	123–125	593 575, 467, 441, 425, 305	2.6	593.15119 C30H25O13 (4.3)	(Epi)-catechin-(epi)-gallocatechin dimer
89	107–119	609 457, 439, 321, 169	4.1	609.12858 C30H25O14 (5.9)	(Epi)-gallocatechin dimer
90	125–129	897 745, 575, 463, 449, 423	13.0	897.14880 C44H33O21 (3.6)	(Epi)-catechin gallate -(epi)-gallocatechin gallate dimer
91	123–131	913 463, 449, 317	8.0	913.14548 C44H33O22 (1.5)	(Epi)-gallocatechin gallate dimer
92	137–155	913 761, 573, 449, 423	24.6	913.16762 C45H37O21 (17.1)	(Epi)-gallocatechin trimer
Others
93	73–85	109 -	3.0	109.02893 C6H5O2 (5.3)	Catechol
94	67–81	124 -	n.d.	-	Amino catechol
95	93–115	125 -	12.1	125.02756 C6H5O3 (17.2)	Pyrrogallol
96	77–83	153 109	9.1	153.02038 C7H5O4 (6.9)	Protocatechuic acid
97	23–25	167 125	9.1	167.03454 C8H7O4 (2.6) *	Vanillic acid
98	67–79	168 124	n.d.	-	Amino protocatechuic acid
99	85–95	193 111	9.1	193.01665 C9H5O5 (12.5)	Trihydroxychromone
100	15	209 187, 165, 125	n.d.	-	Jasmonic acid
101	59–69	217 155	-	-	Unknown
102	13–15	279 277, 243, 237	73.4	279.23401 C18H31O2 (3.8)	Linoleic acid
103		281 277, 255	75.8	281.24987 C18H33O2 (4.5)	Oleic acid
104	11–15	295 277, 275, 265, 251, 249, 185	70.2	295.2304 C18H31O3 (8.6)	Hydroxy linoleic acid
105	125–131	305 221, 219, 179, 165, 125	12.1	305.06942 C12H17O7S (2.1)	5′-hydroxysulphonyloxy jasmonic acid
106	11–17	383 337	-	-	Unknown
107	157–159	707 687, 671, 533, 359	n.d.	-	Integracin D
108	97–99	875 441, 433, 289	-	-	Unknown
109	89–93	887 585, 301	-	-	Unknown

MS² numbers in bold indicate the most intense product ion. * indicate very minor compounds. HHTP = hexahydroxydiphenoyl ester; NHTP = nonahydroxytriphenoyl ester.

One of the special cases of isomer/isobar separation by HPCCC is the selected ion trace [M − H]⁻ at m/z 615, as the HPCCC experiment separated flavonoid-glycosides with identical molecular weights as displayed in the low resolution ESI-MS injection profile (Figure 1). A set of two partly co-eluting positional isomers of myricetin-desoxy-hexoside-gallate (37) (fraction range 91–115) were absolutely separated from the later eluting isobar quercetin-hexoside-gallate (38) (fraction range 137–153).

The selected ion trace at m/z 635 displayed two strong HPCCC elution ranges with compeletly separated compound areas with 41 (range 45–57), and 77 (133–151) (Figure 1). However, the metabolite 41 with lower elution volume in the HPCCC run was identified by the ESI-MS/MS profile data as myricetin whereby the ESI-ion-source dimer [2M − H]⁻ was generated in dominant intensity. This was confirmed by the exact identical position of m/z 317 ([M − H]⁻) in the HPCCC profile. Nevertheless, the late eluting metabolite 77 was identified as a [M − H]⁻-signal with a hexosid unit substituted by three galloyl-moieties indicated by MS/MS neutral loss cleavage (Δm/z 152) to m/z 483, and 331 of gallic acid releases. The tetra-galloyl-hexoside with [M – H]⁻ at m/z 787 (82) (Table 1) co-eluted in this HPCCC run as seen in Figure 1, as well as the penta-galloyl-hexoside (85) (Table 1). A very large elution volume for recovery in the triphasic HPCCC experiment displayed the galloyl diHHDP hexoside (83) seen by [M − H]⁻ at m/z 935 (range 145–167) (Figure 1 and Table 1). Although the constitution of certain compounds had been different, the polarity differences were not sufficient for a successful HPCCC separation as seen for the selected ion traces [M – H]⁻ m/z 585 (quercetin pentoside gallate, 32), and m/z 599 (quercetin desoxyhexoside gallate, 33) (Figure 1).

Using literature to guide the identification process of the minor, and very minor concentrated derivatives, literature was verified and the few previously isolated compounds in L. racemosa were listed with molecular weights as a comparative database. Most of the unknown compounds were characterized by ESI-MS/MS fragmentation and indicative neutral loss pattern. High accuracy molecular weights acquired by LC-ESI/TOF MS were used to ratify and/or verify the proposed molecular formulas. Phytochemical investigations describing chemical compounds on other genus of Combretaceae helped to support the results based on chemotaxonomic knowledge. From the aspect of natural product classes, the chemical composition of the EtOAcPart was distinguished in four main groups as flavonoids, hydrolysable tannins, condensed tannins and other low molecular weight polyphenols (Supplementary Figure S2). The chemical structures and substitution patterns of fractionated and identified compounds are shown in Figure 2.

Figure 2.

Laguncularia racemosa EtOAcPart general structures and tentative substitution patterns of some of the existing compounds. (a) Flavonoids, (b) hydrolysable tannins, (c) condensed tannins and (d) other low molecular weight polyphenols.

Fractions had been combined on the basis of TLC analysis and the electrospray mass-spectrometry profiling experiment. Supplementary Figure S3 displays the TLC-analysis on the combined fractions of the HPCCC experiment. Table 1 lists HPCCC chromatographic elution, and ESI-MS/MS informations; LC ESI-TOF-MS data (when present) and tentative identification. Although L. racemosa EtOAcPart showed quite complex constituents, most phenolic compounds were well separated.

2.1.1. Flavonoids and Derivatives

Flavonoid derivatives were detected and identified in L. racemosa EtOAcPart by ESI-MS/MS as principal compounds in the recovered HPCCC fractions (Table 1, compounds 1-56). Flavonoids including flavonols, flavones, flavanols and flavanones were found in free form, linked to one sugar unit, as well as in the presence of galloyl substituents. The tentative identification of the flavonoid-aglyca (compounds 1–13) was done by comparison to specific fragmentation patterns, as the spectra of this flavonoid often displayed loss of small neutral fragments contributing to structure information [31,32,33]. The free flavonoid aglyca eluted during the first step of the gradient before the glycoside linked flavonoids, in accordance to mobile phase/compound polarity in the tail-to-head mode.

The flavonoid-O-substituted characteristically exhibited the neutral loss [34] attributed to a pentose unit [M − H − 132]⁻, hexose unit [M − H − 162]⁻, desoxy-hexose unit [M − H − 146]⁻, glucuronyl unit [M − H − 176]⁻, galloyl moiety [M − H − 152]⁻ and combination of these substituents. A set of quercetin-O-pentoside, -O-desoxy-hexoside, -O-hexoside, -O-glucuronide and myricetin-O-pentoside, -O-desoxy-hexoside, -O-hexoside were detected in compounds 18, 21, 22, 25, 26, 30 and 31 [35,36,37,38,39,40,41]. The substituent gallate was found connected to (epi)-catechin (20), (epi)-gallocatechin (23) and myricetin (28) as well as in glycosylated forms of quercetin and myricetin (32–34, 37–40) [41]. The digallate derivative of quercetin and myricetin-O-pentoside were also recognized in compounds 45 and 46.

Aglycones apigenin, kaempferol, quercetin and tricin were previously reported in L. racemosa [8,26] in addition to the glycosylated derivatives quercetin-3-O-arabinoside and quercetin-3-O-rhamnoside [24]. Not fully identified derivatives could be distinguished by observed aglycone fragment ions in MS/MS.

2.1.2. Hydrolysable Tannins

Hydrolysable tannins, well-known in Combretaceae, were the second main class of natural compounds detected by the HPCCC and off-line injection ESI-MS/MS experiment (Table 1, compounds 57–86) [36,37,38,39,40,42,43]. It included derivatives of gallic acid, ellagic acid, gallotannins and ellagitannins. Some of the ellagic acid and its methyl-, dimethyl- and trimethyl ether derivatives were previously reported in L. racemosa [26]. Several studies describing the detection of hydrolysable tannins in species of Combretaceae can be found [44,45,46].

Common neutral loss cleavages observed in the MS/MS for simple gallic acid and its derivatives were related to the cleavage of carboxyl [M − H − 44]⁻, methyl [M − H − 15]⁻, ethyl [M − H − 29]⁻ and galloyl [M − H − 152]⁻. They were found as ester or ether arrangements. Compounds 57–59, 62, 63, 66 and 68 were identified as gallic acid, methyl gallate, ethyl gallate, galloyl gallate, galloyl shikimate, galloyl methyl gallate and galloyl ethyl gallate, respectively [37,38,42].

Ellagic acid derivatives were characterized by the fragment ion m/z 301. At this point, LC ESI-TOF-MS was essential to distinguish derivatives from quercetin and ellagic acid. The sequence of compounds comprised ellagic acid itself and the -methyl, -dimethyl, -trimethyl, -pyrogalloyl and dihexoside ether forms (60, 61, 64, 67, 69, 74) [36,39,40,43]. Additionally, valoneic acid dilactone (70) and its ethyl ether derivative (73) were detected [36,40].

By comparison to literature [47], the molecular masses of compounds 65, 72, 77, 82 and 85 showed that they consist of a gallotannin series of molecules (mono-, di-, tri-, tetra- and penta-galloyl hexosides) [37,38,42]. A similar series of monomeric ellagitannins (HHDP-, NHDP-, HHDP galloyl-, diHHDP-, HHDP digalloyl-, diHHDP galloyl- and HHDP trigalloyl-) were found in compounds 71, 75, 76, 80, 81, 83 and 84 [36,38,40]. The ellagic acid punicalin (79) was further detected at m/z 781.

2.1.3. Condensed Tannins

Condensed tannins (proanthocyanidins), formerly observed in L. racemosa wood and leaves [48,49,50], were recognized and characterized based on the detected flavanol-aglyca (4, 7, 9) and its gallate derivatives (20, 23). They were found as homo-dimers consisting of (epi)-catechin (87), (epi)-gallocatechin (89) and (epi)-gallocatechin gallate (91) [40]. Additionally, as hetero-dimers, existing as (epi)-catechin-(epi)-gallocatechin (88) and (epi)-catechin gallate-(epi)-gallocatechin gallate (90). The trimeric (epi)-gallocatechin (92) was also encountered. Compounds had fragmentation patterns related to the cleavage of flavanol units according to literature [51]: [M − H − 289]⁻ for (epi)-catechin loss, [M − H − 305]⁻ (epi)-gallocatechin loss, [M − H − 441]⁻ (epi)-gallocatechin gallate loss and [M − H − 162]⁻ for gallate loss.

Considering the elution order of compounds in respect to gradient polarity range, the flavonol-aglyca eluted before the gallate derivatives, both in the first step, while dimers and trimers stayed retained in the column until extrusion started.

2.1.4. Low Molecular Weight Polyphenols

Other compounds were recognized and characterized based on precursors/derivatives of existing identified compounds in the off-line ESI-MS/MS profile or on the L. racemosa chemical database. Simple phenolic compounds included catechol (93) and pyrogallol (95), common occurring products in the hydrolysable tannins pathway [35]. Benzoic acid derivatives with frequent [M − H − 44]⁻ corresponding to the neutral loss of CO₂, comprised protocatechuic (96) and vanillic (97) acids [37]. The amino derivatives aminocatechol (94) and amino protocatechuic acid (98) were also detected [52]. The chromone detected at m/z 193, was identified as trihydroxy-chromone (99) and had its molecular formula confirmed by HRMS.

The jasmonic acid (100) and its sulphated derivative 5′-hydroxy-sulphonyloxy jasmonic acid (105), earlier isolated from the L. racemosa twigs and leaves [23], could be found at m/z 209 and 305, respectively. ESI/TOF MS data confirmed the proposed compounds. Ordinary oleic and linoleic fatty acids (102-104), jasmonic acid biosynthetic precursor, were further encountered. Another sulphated derivative isolated from L. racemosa leaves [26] was found at [M – H]⁻ at m/z 707 and was identified as integracin D (107) [26]. Due to concentration limits, the compound could not be detected in the ESI/TOF MS analysis and structure was not fully confirmed.

3. Materials and Methods

3.1. Chemical Reagents and Solvents

Preparation of extracts was carried out with analytical grade solvents from Tedia Brazil (Rio de Janeiro, Brazil). LC-ESI/TOF-MS/MS analyses used HPLC grade solvents from Tedia Brazil (Rio de Janeiro, Brazil). HPCCC separations were performed with analytical grade solvents from Fisher Chemicals (Loughborough, UK). ESI-MS/MS analyses were done with HPLC grade solvents from VWR Chemicals (Radnor, PA, USA). NMR analyses used deuterated solvents from Cambridge Isotope Laboratories (Tweksbury, MA, USA) and TMS as internal standard. All aqueous solutions were prepared with pure water produced by Milli-Q water (18.2 MΩ) system (Thame, UK).

3.2. Preparation of the Extract

Laguncularia racemosa (3 kg) was collected at Guaratiba Biological and Anthropological Reserve (Rio de Janeiro, Brazil) in November 2010. Specialist researchers from the Nucleus of Mangrove Studies (University of the State of Rio de Janeiro) helped in the localization, identification and collection of the plant. The leaves were dried and grounded in a laboratory mill (Laboratory Retsch mill, Haan, Germany) and 1800 g were submitted to maceration with ethanol–water 8:2 (v/v) in 10 cycles of 24 h. The solvent was evaporated under reduced pressure at 50 °C and the crude extract (255 g) was partitioned between water and organic solvents, affording different extracts: n-hexane (4 g), dichloromethane (8 g), ethyl acetate (15 g) and aqueous (215 g).

3.3. Thin Layer Chromatography

Preliminary analyses of EtOAcPart, solvent system evaluation tests and CCC fraction analyses were done by thin layer chromatography (TLC) on normal phase silica gel TLC plates (SiO₂-60, F254, Merck, Darmstadt, Germany, gel 60 RP-18, F254S) developed with EtOAc–acetone–H₂O 25:15:10 (v/v/v), and acetonitrile-H₂O 1:1 (v/v) for reversed phase C18-plates (RP18W, Macherey and Nagel, Düren, Germany). Results were visualized by using spray-reagent H₂SO₄ (10% m/v) in methanol with vanillin 5% in ethanol and flash heating on a hot plate 105 °C.

3.4. LC-ESI/TOF MS Preliminary Analysis

The EtOAcPart was also analysed by LC–ESI/TOF-MS with a 1200 Series LC-chromatograph (Agilent, Palo Alto, CA, USA) coupled with a MicrOTOF II time-of-flight mass spectrometer (Bruker Daltonics, Inc., Billerica, MA, USA). 5 µL injection was performed with an autosampler on a Poroshell EC-C18 column (100 × 2.1 mm; 2.7 µm, Agilent, Palo Alto, CA, USA). The source temperature was set at 200 °C, the drying gas (nitrogen) flow rate was 10.0 L/min and the nebulizer gas (nitrogen) pressure was 4 bar. Data were acquired in negative mode in the range of m/z 100–1500. The capillary voltage was 3.8 kV, the capillary exit voltage was −150 V, the skimmer 1 and 2 voltages were 50 V and 23 V, respectively, the hexapole 1 voltage was set to −23 V, the hexapole RF voltage was 120 Vpp, lens 1 transfer was 68 μs and lens 1 pre plus stage was 7 μs. Mass calibration was achieved by infusing ammonium formate in an isopropanol–water mixture (1:1, v/v) as an external standard. All data were analysed using Bruker Daltonics ESI Compass Data Analysis Version 4.0 SP 1 (Bruker Daltonics Inc., Billerica, MA, USA). The mobile phase consisted of spectroscopic grade methanol (B) and ultrapure water (A) containing 0.05% (v/v) formic acid. The linear gradient elution was set from 10% to 100% of B in 90 min at a flow rate of 0.3 mL/min.

3.5. High Performance Countercurrent Chromatography

3.5.1. Equipment

CCC separations were performed on a semi-preparative HPCCC system (model Spectrum, Dynamic Extractions Ltd., Gwent, UK) equipped with two counter-balanced bobbins with perfluoroalkoxypolymer (PFA) tubing (1.6 mm i.d.) wound in multi-layer coiled-columns, resulting in 143.5 mL total volume (V_C). The rotation speed was adjusted to the maximum velocity of 1600 rpm (240 g). Solvent phase systems were delivered by a constant flow pump (Agilent HP1200, Palo Alto, CA, USA) to the HPCCC system. A semi-preparative sample loop (7.15 mL) was used to inject the dissolved sample over a low-pressure valve (Upchurch Model V-450, with 1.6 mm i.d. fittings) to the chromatographic system. Fractions were collected by a fraction collector (Agilent HP1200, Palo Alto, CA, USA).

3.5.2. Three-Phase Solvent System Test Evaluation

The three-phase solvent systems were composed of n-hexane–methyl acetate –acetonitrile–water and n-hexane–tert-butyl methyl ether–acetonitrile–water [11,53,54]. For the experiments for solvent system evaluation, 2 mg of the EtOAcPart were dissolved in a test tube containing 2 mL of each phase of the thoroughly equilibrated solvent systems. The test tubes were shaken vigorously for compound partition. After the phase layers had completely separated and distribution equilibrium was established, the resulting phase layers were analyzed by TLC (Supplementary Figure S4).

3.5.3. Solvent System and Sample Preparation

The selected solvent system n-hexane–tert-butyl methyl ether–acetonitrile–water (2:3:3:2, v/v/v/v) was thoroughly equilibrated in a separatory funnel at room temperature. The three phases were separated shortly before use and degassed by ultra-sonication for 5 min. The sample solution was prepared by dissolving the sample at fixed concentration (100 mg/mL) and coil-volume (5% V_C) in the lower aqueous phase only.

3.5.4. HPCCC Separation Procedure

Separation was performed in a normal step-gradient elution mode. The more aqueous lower phase was used as the stationary phase while organic upper and middle phases were used as mobile phases as shown in Figure 3. The system was completely filled with the lower aqueous stationary phase. Rotation was set to 1600 rpm. For the separation, the upper organic mobile phase was pumped at 4.0 mL/min. After reaching hydrodynamic equilibrium, the sample was injected to the HPCCC column. For the first elution step, 214.5 mL mobile phase (1.5 V_C) of upper phase was pumped through. For the second elution step, 2 V_C (286 mL) of the middle phase was pumped through the HPCCC system. Fractions were collected at 1 min intervals. For the extrusion step, rotation was reduced to 200 rpm and the column contents were pushed out of the system by lower phase at 8.0 mL/min and fractions were collected at 30 s intervals. The temperature control was maintained at 30 °C.

Figure 3.

HPCCC three phase solvent system step-gradient procedure.

3.6. Metabolite Profiling by Offline Injections to ESI-MS/MS

The molecular weight profiles of the recovered HPCCC fractions were monitored by off-line ESI-MS/MS and were recorded in a single data file using the ion-trap mass-spectrometer HCT-Ultra ETD II (Bruker Daltonics, Bremen, Germany). Aliquots of 0.75 μL of odd numbered CCC fractions were directly filled to vials, dried and redissolved in 1.0 mL of methanol for conducting the ESI-MS analysis. Fractions were delivered to the ESI-MS/MS by a HPLC-pump (binary pump, G1312 A, 1100 Series, Agilent, Waldbronn, Germany) using the make-up solvent system with a flow rate of 0.5 mL/min composed of acetonitrile and water (1:1, v/v). ESI-MS/MS parameter settings were in the negative ionization mode, with scan-range between m/z 100–2000, where mostly deprotonated [M – H]⁻ ion signals were generated. An auto-MS/MS method fragmented the nine most intense peaks and to monitor and characterize co-eluting compounds. Drying gas was nitrogen (flow rate 10.0 L/min, 310 °C), and nebulizer pressure was set to 60 psi. Ionization voltage at HV capillary was 3500 V, HV end plate off set −500 V, trap drive 61.8, octupole RF amplitude 187.1 Vpp, lens 2 60.0 V, Cap Ex −115.0 V, max. accumulation time 200 ms, averages 5 spectra, trap drive level 120%, target mass range: m/z 500, compound stability 80%, Smart ICC target 70.000, ICC charge control ‘on’ and smart parameter setting ‘active’.

4. Conclusions

The combination of analysis of preparative HPCCC fractions with off-line injections to an ESI-MS/MS device was proven to be highly effective for a full metabolite chemical profile for polyphenols using the negative ionization mode. The use of a three-phase solvent system for HPCCC in a step-gradient elution mode was adequate to maintain equilibrium and chromatographic resolution while improving mobile phase strength. The ESI-MS/MS projection of the semi-preparative HPCCC experiment visualized over 100 compounds by selected single ion traces and was an adequate confirmation of the LC–ESI/TOF MS analysis. This study detected a variety of metabolites from different classes occurring in L. racemosa EtOAcPart and used chemotaxonomic data to guide the MS/MS putative structure elucidation.

Supplementary Materials

The following are available online, Figure S1: EtOAcPart preliminary analyses by LC-ESI-TOF-MS. The mobile phase was methanol (B) and water (A) containing 0.05% (v/v) formic acid. The linear gradient elution was set from 10% to 100% of B in 90 min. Figure S2: HPCCC off-line injection ESI-MS/MS profile of the the EtOAc Part by use of selected single ion traces for target compounds or classes. (a) Flavonoids, (b) hydrolysable tannins, (c) condensed tannins and (d) other compounds. Figure S3: TLC analysis of L. racemosa CCC combined fractions. On the left: reversed phase silica gel TLC plates developed with acetonitrile-H₂O 1:1 (v/v). On the right: Normal phase silica gel TLC plates developed with EtOAc-acetone-H₂O 25:15:10 (v/v/v). Visualization was done using 254 nm UV light and spray-reagent H₂SO₄ 10% and vanillin 5%. F means a group of jointed fractions according to TLC similarity. Figure S4: Three-phase solvent system test by TLC. CCC solvent system: n-hexane–MTBE–ACN-H₂O (1) 1-1-2-1, (2) 2-1-3-2, (3) 2-2-3-2, (4) 2-3-3-2 and (5) 3-5-5-2 (v/v); (U) upper, (M) middle and (L) lower phases. Normal phase silica gel TLC plates developed with EtOAc–acetone-H₂O 25:15:10 (v/v/v), visualized using λ 254 nm UV light and spray-reagent H₂SO₄ 10% and vanillin 5%.

Author Contributions

F.d.N.C. is responsible for the idea, execution and draft preparation; S.I. is responsible for supervision, funding administration and paper reviewing; G.J. is responsible for MS execution, MS data analysis and paper reviewing; P.H. is responsible for CCC execution and paper reviewing including English improvement; F.d.S.F. is responsible for MS data analysis, bibliography review and paper editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Newton Advanced Fellowship project financed by the Royal Society of the United Kingdom.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Please, contact the corresponding author for access to database.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples are not available.