Article pubs.acs.org/JAFC
Chemometric Analysis of Chromatographic Fingerprints Shows Potential of Cyclopia maculata (Andrews) Kies for Production of Standardized Extracts with High Xanthone Content Alexandra E. Schulze,†,# Dalene de Beer,*,§ André de Villiers,‡ Marena Manley,† and Elizabeth Joubert†,§ †
Department of Food Science and ‡Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch) 7602, South Africa § Post-Harvest and Wine Technology Division, Agricultural Research Council (ARC), Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa S Supporting Information *
ABSTRACT: Cyclopia species are used for the production of honeybush tea and food ingredient extracts associated with many health benefits. A species-specific high-performance liquid chromatography (HPLC) method for Cyclopia maculata, developed and validated, allowed quantification of the major compounds in extracts from “unfermented” and fermented C. maculata. Two xanthones were tentatively identified for the first time in a Cyclopia species, whereas an additional four compounds were tentatively identified for the first time in C. maculata. “Fermentation” (oxidation) decreased the content of all compounds, with the exception of vicenin-2. Similarity analysis of the chromatographic fingerprints of unfermented C. maculata aqueous extracts showed extremely low variation (r ≥ 0.97) between samples. Some differences between wild-harvested and cultivated seedling plants were, however, demonstrated using principal component analysis. Quantitative data of selected compounds confirmed the low level of variation, making this Cyclopia species ideal for the production of standardized food ingredient extracts. KEYWORDS: chromatographic fingerprint analysis, food ingredient extract, herbal tea, honeybush, HPLC-DAD, LC-MS, method development, phenolic compounds, similarity analysis, PCA
■
material,14 could serve as a chemical marker of quality. Analysis of the aqueous extract used in the antiobesity 6 and antidiabetic14 studies revealed the presence of isomangiferin, eriocitrin, hesperidin, hesperetin, scolymoside, iriflophenone-3C-β-glucoside, and phloretin-3′,5′-di-C-glucoside in addition to mangiferin.6 As C. maculata extracts may in the future be used as nutraceutical ingredients, chromatographic fingerprint analysis would allow a broader evaluation of samples, especially when all peaks are not quantifiable, and would highlight small differences between samples that cannot be determined by quantification of individual compounds.15 Chromatographic fingerprint analysis is a technique that is gaining use in the quality control of natural products and has been accepted for this purpose by the World Health Organization.16 Such fingerprints, combined with pretreatment of data (i.e., alignment of peaks, removal of background noise, and normalization), allow an alternative approach to compare the complete chromatographic profile, using multivariate techniques.17 However, for effective chromatographic fingerprint analysis efficient resolution of peaks is required. The “generic” HPLC method,18 applied for the analysis of C. maculata, suffers from a number of disadvantages, those being elution of highly polar compounds in the void volume, limited resolution of minor compounds,
INTRODUCTION Herbal tea prepared from several endemic South African Cyclopia spp., also known as honeybush, was “rediscovered” during the 1990s1 and has since become popular on the global herbal tea market.2 The herbal tea product is available in both “fermented” (oxidized) and “unfermented” or “green” form. Other uses of honeybush include food ingredient extracts (i.e., hot water extracts) for products such as iced teas and confectionary, which take advantage of increasing consumer awareness of the health benefits associated with tea and herbal teas. Studies demonstrating the potential health-promoting properties of aqueous extracts of Cyclopia spp., those being antimutagenic, anticancer, phytoestrogenic (as reviewed by Joubert et al.3), antidiabetic,4,5 and antiobesity,6,7 recognized the role of their phenolic compounds as bioactive compounds. To fully understand the health-promoting potential of Cyclopia spp. and identify bioactive compounds for future quality control purposes, greater insight into the phenolic composition of the various species is required. From in-depth investigation of the phenolic composition of Cyclopia intermedia and Cyclopia subternata,8−11 two of the major commercialized species, differences relating to species were evident. In the present study the focus was on Cyclopia maculata, as this species is currently under investigation for commercial cultivation to meet the growing demand for honeybush. Demonstration of antiobesity6,7 and antidiabetic properties12 for an aqueous extract of C. maculata intensified interest in this species for production of nutraceutical extracts. Mangiferin, known for its antidiabetic properties13 and a major constituent of the plant © XXXX American Chemical Society
Received: June 20, 2014 Revised: October 2, 2014 Accepted: October 8, 2014
A
dx.doi.org/10.1021/jf5028735 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Table 1. UV−Vis and MS Characteristics (LC-MS and -MSE in Negative Ionization Mode) of Phenolic Compounds Identified in Freeze-Dried Aqueous Extracts of Green and Fermented Cyclopia maculata peak
a
tR (min)b
c
λmax (nm)
accurate massd
error (ppm)
proposed molecular formula
1
3.38
235, 280, 316
423.0927
0.0
C19H19O11
2
5.75
293
407.0978
−0.2
C19H19O10
3
6.26
317,
871.1915
2.1
C40H39O22
4
8.55
317,
421.0768
−0.7
5 6
8.84 9.05
327 316,
437.0716 421.0767
7
9.24
263, 276, 366 239, 257, 366 237, 259, 239, 255, 365 235, 271,
329
8 9
9.61 10.20
10 11
fragments 423 (32), 333 (16), 303 (100), 223 (7), 193 (27), 165 (6), 125 (5), 109 (5) 407 (48), 317 (24), 287 (100), 245 (10), 193 (5)
phenolic compound maclurin-3-C-βglucoside iriflophenone-3-C-βglucoside unidentified
C19H17O11
871 (100), 751 (17), 691 (13), 557 (24), 539 (5), 421 (53), 403 (5), 331 (8), 301 (13), 269 (7)e 421 (100), 331 (19), 301 (27), 271 (5), 259(5)
mangiferin
−0.9 −0.9
C19H17O12 C19H17O11
437(100), 347 (54), 317 (83), 288 (23), 274 (9) 421 (100), 331 (35), 301 (50), 271 (7), 258 (9)
hydroxymangiferin isomangiferin
593.1498
−1.3
C27H29O15
593 (100), 503 (5), 473 (14), 383 (17), 353 (24), 297 (7)
449.1080 437.0727
0.9 −1.6
C21H21O11 C19H17O12
449 (83), 287 (100), 151 (89), 135 (48) 437 (100), 347 (6), 317 (43), 289 (15)
10.39 11.78
281f 236, 255, 272, 323 282f 279
apigenin-6,8-di-Cglucoside (vicenin-2) eriodictyol-O-hexoside hydroxyisomangiferin
449.1087 613.1779
0.7 1.6
C21H21O11 C27H33O16
12
12.70
281, 330sh
595.1660
−0.5
C27H31O15
449 (67), 287 (100), 151 (84), 135 (41), 107 (10) 613 (100), 595 (34), 493 (29), 475 (6), 433 (66), 403 (36), 373 (65), 331 (8), 297 (19), 269 (17), 209 (14), 135 (16) 595 (89), 287 (100), 151 (70), 135 (20), 107 (6)
13
13.76
256, 264, 350
593.1519
2.2
C27H29O15
593 (100), 285 (87)
14
14.18
280
597.1826
1.2
C27H33O15
15
16.73
283, 328sh
609.1814
−0.8
C28H33O15
597 (100), 477 (21), 459 (5), 417 (9), 387 (45), 357 (62), 345 (7), 315 (22), 285 (13), 209 (11) 609 (56), 301 (100)
eriodictyol-O-hexoside 3-hydroxyphloretin3′,5′-di-C-hexoside eriodictyol-7-Orutinoside (eriocitrin) luteolin-7-O-rutinoside (scolymoside) phloretin-3′,5′-di-Cglucoside hesperetin-7-Orutinoside (hesperidin)
a
Peak numbers correspond to peaks in Figure 1. bRecorded during LC-DAD-MS analysis using a Waters Acquity UPLC instrument. csh, shoulder. Accurate mass determined experimentally. eData acquired in MS/MS mode. fTypical flavanone shoulder at ca. 330 nm not detected due to low concentration. d
side), Extrasynthese (Genay, France; mangiferin, eriocitrin, and eriodictyol-7-O-glucoside), Phytolab (Vestenbergsgreuth, Germany; vicenin-2), and Chemos (Regenstauf, Germany; isomangiferin). Iriflophenone-3-C-glucoside (purity ≥94%), isolated from Cyclopia genistoides,19 was obtained from the Post-Harvest and Wine Technology Division, ARC Infruitec-Nietvoorbij (Stellenbosch, South Africa). Deionized water, prepared using an Elix water purification system (Millipore, Milford, MA, USA), was further purified to HPLC grade using a Milli-Q Academic water purification system (Millipore). Plant Material and Extract Preparation. Two sets of plant material were used in the present study. All plants were identified by M. Joubert, a honeybush cultivation expert from the ARC Honeybush Breeding Program (Cultivar Development Division, ARC InfruitecNietvoorbij, Stellenbosch, South Africa) on the basis of the classification of Schutte.20 Plant material for sample set 1 comprised nine batches of plant material, harvested during summer (December 2009) from bushes forming part of the same natural population growing in marsh land on Welgedacht farm (Riversdale, South Africa). The shoots from two to three bushes made up a batch. Each plant material batch (n = 9) was processed as follows: Thick stems with little or no side stems were removed, and the plant material was mechanically shredded (≤3 mm), mixed, and divided into two subbatches for preparation of unfermented and fermented plant material (n = 9 each). The unfermented plant material was prepared by drying one sub-batch without delay in a cross-flow drying tunnel at 40 °C for 6 h (
