Chromatographic Techniques for the Separation of

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Chapter 1

Chromatographic Techniques for the Separation of Polymethoxyflavones from Citrus Ram M. Uckoo, G. K. Jayaprakasha, and Bhimanagouda S. Patil* Vegetable and Fruit Improvement Center, Department of Horticultural Sciences, 1500 Research Parkway Ste A120, Texas A&M University, College Station, TX 77845 *E-mail: [email protected]

Polymethoxyflavones (PMF’s) are naturally occurring flavonoids present in Rutaceae family in the Citrus genus. Due to the potential use of PMF’s as a chemopreventive agent and functional food, a rapid reproducible method for large scale isolation is critical. Isolation of PMFs from citrus is a challenging task due to complex sample matrices. Recent advances in chromatographic techniques led to several isolation and identification methods of PMF’s from citrus. The present chapter discusses about the various techniques used for extraction and isolation of PMFs from different citrus species. The identification of these compounds using 1H NMR and 13C NMR is also described.

Introduction Citrus is one of the most consumed and cultivated fruit crop. Apart from its savory taste, citrus consumption is also correlated with various health benefits due to the presence of several bioactive compounds. Some of these bioactives include limonoids (1, 2), flavonoids (3, 4), carotenoids, phenolic acids (5), organic acids (6), furocoumarins (7, 8), and amines (9). Due to rapid hybridization and mutations coupled with polyploidy nature of citrus led to development of several varieties. These genetical variations might have resulted in characteristic changes in the levels of bioactive compounds. Based on the levels of bioactive compounds, chemotaxonomy was also proposed for the classification of the citrus genus based on the variation of limonoids (10). Similarly, the variation in composition of © 2012 American Chemical Society In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


polymethoxyflavones was also used as a basis for chemotaxonomic classification of the Citrus genus (11). Polymethoxyflavones are a group of flavonoids with two or more methoxy groups. There are more than 25 PMFs reported from citrus among which the major occurring are tangeretin (5,6,7,8,4′-pentamethoxyflavone), heptamethoxyflavone (3,5,6,7,8,3’,4’-heptamethoxylflavone), nobiletin (5,6,7,8,3,4’-hexamethoxyflavone), tetramethoxyflavone (5,6,7,4’tetramethoxyflavanone) and sinensitin (5,6,7,3’,4’-pentamethoxyflavone) (Fig. 1 & 2). While limited literature is available on the evolution of PMFs in citrus, methoxylation of flavone or flavanone aglycones was proposed as a pathway for biosynthesis of PMF’s in oranges (12). They occur in leaves, peel and juice but are mainly localized in the peels of the citrus fruits. Studies suggest that the concentration of PMF’s varies based on maturity and species of citrus (13, 14). In plants, PMF’s are considered to be protective against disease causing pathogens (15–17). On human health perspective, PMF’s were investigated since early 19th century and implicated in several health benefits such as antiproliferative (18), anticancer (19–22), anti-inflammatory (23), antilipogenic and antimutagenic (24) activity. A comprehensive review explaining the multitude health benefits of PMF’s was reported by Li et al., (25). Due to their relevant role in health benefits, PMF’s were isolated from different species and parts of citrus (Table 1). The isolation of PMF’s was achieved by using several extraction and isolation methodologies.

Extraction Methods Solvent Extraction Polymethoxyflavones are low polar compounds and can be extracted using non polar solvents such as hexane and polar solvents including water (18), ethanol and methanol (26–28). Moreover, these compounds were extracted from various parts of citrus such as peel, leaves and cold pressed oil. Raman et al. (29), reported extraction of C. reticulata peels using non polar hexane solvent followed by treating with 10% sodium hydroxide solution. The mixture was later extracted with diethyl ether, washed with water and subjected to adsorptive separation using cation exchange resin Dowex 50WX2 to yield nobiletin and tangeretin. Chaliha et al. (30), reported extraction of C. jambhiri peels using petroleum ether solvent in a Soxhlet apparatus for separation of PMF’s. Jayaprakasha et al. (31), reported extraction of C. reticulata (Blanco Coorg Mandarin) using hexane and chloroform successively in a Soxhlet apparatus. The extracts were subjected to further separation using silica gel column chromatography for isolation of desmethylnobiletin, nobiletin and tangeretin. Miyake (32) reported the extraction efficiency of PMF’s using ethanol and aqueous solution of ethanol at various proportions (5%, 25%, 50%, 75% and 100%). Among the evaluated ratios, 75% ethanol in water and 100% ethanol resulted in 100% extraction efficiency. Moreover, extraction of PMF’s was influenced by the temperature of the solvent. Extraction of citrus fruits using hot 25% ethanol in water resulted in higher content of PMF’s in comparison to water, 5% and 25% ethanol aqueous 4 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


solution under cold and hot water and 5% ethanol in water. Similarly, these compounds were extracted from peels of C. reticulata Blanco cv. Ponkan by refluxing with 75% ethanol for 3 h (33). The ethanol solution was concentrated and further extracted with dichloromethane to yield PMF’s rich fraction. Wang et al., (34) reported extraction of PMF’s from dried peel powder of C. reticulata by refluxing with 75% (v/v) ethanol for 10 h. The extract was concentrated and extracted with chloroform to yield mixture of PMF’s. Individual PMF’s were separated by column chromatography using chloroform:acetone (9:2, v/v). Chen and Montanari (35) reported extraction of PMF’s from leaves of Dancy tangerine using methanol:chloroform (1:1). The extract was further subjected to separation using combination of flash C18 column chromatography and C18 preparative HPLC for separating individual PMF’s. In a recent report (36) from our lab hexane was suggested as a better solvent in comparison to chloroform and methanol solvents for Soxhlet extraction of dried peel powder of Cleopatra mandarin (C. reshni). Soxhlet extraction by hexane yielded extract with low occurance of flavonoid glucosides and higher content of PMF’s. Supercritical Fluid Extraction Apart from citrus peel and leaves, cold pressed oil is a rich source of PMF’s. Extraction of these compounds from the precipitate of winterized (storing the oil at -20º C for long duration of time) citrus peel oil was commonly reported (37–40). These compounds were also extracted from peel oil extract using super critical fluid extraction (37). Recently, the optimum conditions for extraction of nobiletin and tangeretin from C. depressa Hayata by supercritical CO2 was developed by comparing various combinations of pressure and percentage of modifier ethanol solvent. Optimum extraction was achieved by ethanol (85%) as a modifier in supercritical CO2 maintained at 80 °C and 30 MPa of pressure. Also, the % yield of PMF’s by SFE was 107% as compared to conventional solvent extraction yielding 100% (41).

Figure 1. Structures of polymethoxyflavones isolated from citrus (see Appendix A for larger version of figure). 5 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 2. Structures of hydroxylated polymethoxyflavones isolated from citrus (see Appendix A for larger version of figure).

Yao et al., (42) reported extraction of PMF’s from dried peels of C. sinensis Osbeck by enzymatic hydrolysis. The dried peel powder (100 g) was extracted exhaustively using 95% ethanol (1,500 mL) and 5% cellulase at 60º C for 2.5 h. The extract was concentrated and treated with diethyl ether (200 mL × 3) and washed with 0.4% sodium hydroxide solution until the extract turned colorless. The clear diethyl ether extract was collected, concentrated, and freeze-dried to obtain crude PMFs (564 mg). In addition to the traditional extraction methodologies, an advanced technology such as microwave-assisted extraction has also been reported for extraction of PMF’s. Dried peels of C. yuko Hort. ex Tanaka were refluxed using microwave for 2.5 min to 5 min with methanol yielding 0.12% and 0.10% of tangeretin and nobiletin respectively (26).

Separation Methods Tangeretin was the first PMF isolated from Tangerine (Citrus nobilis deliciosa) oil by Nelson (43). Nobiletin was isolated from the peels of Chinese mandarin oranges (C. nobilis) by Tseng (44). Tetramethoxyflavone and heptamethoxyflavone were reported and identified by Swift (45) in the neutral fraction of orange peel oil. Sinensitin was, isolated by Born (46), and named by Swift (47). Although all major PMFs were isolated by the late 60’s, extensive isolation was triggered by the implication of these compounds in the several health beneficial properties and consumers’ interest in natural products. PMFs were separated using thin layer chromatography (TLC), preparative high performance liquid chromatography (prep-HPLC), supercritical fluid chromatography (SFC), and high speed counter current chromatography (HSCCC). 6 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Table 1. Polymethoxyflavones isolated from citrus species using different chromatographic methods Species

Isolated from

Isolation method

Isolated PMF’s*

Ref

C. sinensis L.

Peel oil

FC/prep HPLC

3, 10

(50)

C. sinensis

Peel oil

FC/SFC

3, 5, 10, 16

(37)

C. sinensis

Peel oil

OC/Flash

16

(66)

C. sinensis

Peel oil

Precipitation

10

(67)

C. sinensis

Molasses

SEC

3, 5, 7, 10, 14, 16,

(68)

C. sinensis Osbeck

Peel extract

OC/TLC/FC

3, 5, 7, 10, 14, 16, 20, 21, 22

(69)

C. kinokuni Hort. ex Tanaka

Peel extract

OC/prep-TLC

5, 7, 10, 16, 26

(70)

C. nobilis

Juice

OC

5, 10

(71)

C. reticulata

Leaves

OC/FC/prep-HPLC

5, 7, 10, 22, 29

(35)

C. reticulata Blanco

Peel extract

HSCCC

5, 10, 16, 22

(54)

C. reticulata Blanco cv. ponkan

Peel extract

HSCCC

4, 7, 8, 10

(33)

C. reticulata (Blanco Coorg mandarin)

Peel extract

OC

5, 10, 22

(31)

C. sunki Hort. ex Tanaka

Peel extract

Semi-prep HPLC

1, 4, 5, 7, 8, 10, 20, 22

(18)

C. aurantium

Not mentioned

OC

3, 7, 10

(24)

C. paradisi

Peel oil

HSCCC

5, 10, 16

(72)

C. aurantifolia

Peel oil

HSCCC

5, 10, 16

(72)

C. jambhiri Lush

Peel extract

OC

5, 20

(30)

C. hassaku

Peel extract

OC/prep-TLC

1, 4, 5, 7, 8, 10, 12, 16

(49)

C. yuko Hort

Peel extract

HPLC

5, 10

(26)

* The identification and the structure of the isolated PMF’s are given in Fig 1 and Fig 2 with the corresponding numerical. Abbreviations: Flash chromatography (FC); Preperative high performance liquid chromatography (Prep HPLC); Supercritical fluid chromatography (SFC); Open column chromatography(OC); Size exclusion chromatography (SEC); Thin layer chromatography (TLC); High speed counter current chromatography (HSCCC).

7 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Preparative Thin Layer Chromatography (prep-TLC) Among the reported separation methods of PMF’s, prep-TLC is the most economical. It is relatively low in cost and does not require sophisticated instrumentation. However, this method is limited by the low amount of sample loaded and yield. Successive separations may be required for obtaining pure PMF’s. Del Rio et al., (48) reported separation of these compounds from peel oil of various Citrus fruits. Citrus oil was mixed with 2-propanol and distilled water in a decantation funnel and extracted with hexane. The 2-proponol/water phase was concentrated, mixed with water and liquid-liquid extraction was conducted using benzene. The organic phase was separated, concentrated and dehydrated by adding anhydrous sodium sulfate. The extract was placed on a TLC plate containing silica and eluted with benzene:acetone (3:1, v/v). The separated compounds were visualized by their fluorescence and the individual bands were collected and analyzed by HPLC and mass spectrometry. Machida and Osawa (49) reported the isolation of PMF’s from the peels of C. hassaku using a combination of column chromatography and prep-TLC. Citrus peels were extracted by ethanol under reflux and concentrated. The extract was partitioned between ether and water. The residue was separated on silica gel using benzene-acetone mixture. The components that tested positive in Mg-HCl test were further fractionated by prep-TLC yielding 8 different PMF’s. Preparative-HPLC Increased interest in investigating the biological activity of PMF’s and advancement in chromatographic techniques led to exploring isolation of PMFs using prep-HPLC. Chen at al., (40) reported the separation of these compounds from cold pressed Dancy tangerine peel oil solids using prep-HPLC. The procedure involved a combination of normal phase chromatography and C18 prep-HPLC. The dried tangerine oil solids were loaded to a open silica gel column and eluted with increasing polarity gradient of benzene/ethyl acetate, ethyl acetate, ethyl acetate/2-propanol and 2-propanol. The fractions with similar PMF’s were pooled and purified using C18 prep-HPLC with a gradient mobile phase of methanol/water and ethanol/water. The procedure was applied for separation of PMF’s from Dancy tangerine leaves leading to the isolation of pure compounds (35). However, use of solvents such as benzene for isolation studies should be avoided due to their carcinogenic and mutagenic properties. Li et al., (50) reported a gram-scale isolation method of nobiletin using a combination of normal phase flash chromatography and prep-HPLC. The procedure involved initial separation of orange peel extract using silica gel flash column eluted with a gradient solvent system of ethyl acetate and hexanes. The collected fractions containing nobiletin and 5,6,7,4′-tetramethoxyflavone were concentrated and further separated on a Regis chiral column connected to a prep-HPLC. The solvent system consisted of 35% ethanol and 65% hexanes with a flow rate set at 85 mL/min resulting in isolation of gram amounts of nobiletin and 5,6,7,4′-tetramethoxyflavone. Similar procedure was further applied for isolation of other PMF’s from cold-pressed orange peel oil (51). 8 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Supercritical Fluid Chromatography This method is one of the ideal methods for separation of PMF’s. This method involves use of pressure and temperature combinations maintained at critical point of the mobile phase used. Moreover, the absence of permanent adsorptive loss of sample on to the stationary phase which is commonly noticed in column chromatography makes this method advantageous. Among the various mobile phases used for SFC, CO2 along with methanol seems to be ideal for separation of PMF’s. This method was initially used for analyzing the authenticity of citrus oils by quantification of PMF’s (52). The separation of PMF’s was conducted using CO2 as mobile phase and methanol as a polar modifier. In an another report hydroxy- and methoxy-flavones were separated by supercritical CO2 chromatography on capillary columns using flame ionization and Fourier transform infrared (FT-IR) spectroscopy detection (53). Recently, a large scale isolation method of four PMF’s such as nobiletin, tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone and 5,6,7,4′-tetramethoxyflavone was reported using a combination of normal phase flash column separation and SFC separation (37). The raw material used for the separation was crude sweet orange peel extract. The extract was separated on a silica gel flash column using a gradient mobile phase. Individual fractions were analyzed by LC–ESI-MS and TLC and grouped into 6 groups. The groups that had high concentration of PMF’s were subjected to SFC separation using mobile phase of CO2 and methanol. The separated peaks were collected as individual fractions to obtain pure PMF’s. High-Speed Counter Current Chromatography This is a chromatography technique in which liquid–liquid partition is used as a strategy for separations and unlike other chromatographic techniques does not use any solid support matrix. Due to the characteristic absence of solid support matrix there is no loss of samples by adsorption. This method was first reported as efficient method for the preparative isolation and purification of polymethoxylated flavones from Tangereine peel extracts (54). Tangerine peels were extracted by light petroleum, concentrated and frozen. The sediment was dried and injected to the HSCCC in 15 mL sample injections. The separations were conducted using a two-phase solvent system composed of n-hexane, ethyl acetate, methanol and water (1:0.8:1:1) (v/v). The effluent was monitored with a UV detector at 254 nm and peak fractions were collected according to the elution profile. Similar peaks were pooled and four PMF’s nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, tangeretin and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone were isolated in milligram quantity. Flash Chromatography FC also called as medium pressure liquid chromatography which is a faster technique of column chromatography. The regulated application of medium pressure enables separation of compounds using large sample volumes, thereby yielding high quantity of pure compounds. Recent technological advances have 9 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


also enabled conducting separations with online detection and robotic fraction collectors. These advancements have enabled in development of large scale separation of PMF’s. Dried peels of Cleopatra mandarin and Marrs sweet orange (C. sinensis L. Osbeck) fruits were powdered and extracted using a non polar solvent in a Soxhlet. The extract was concentrated, impregnated with silica gel and subjected to separation using flash chromatography. A gradient solvent system was used for separation and the eluent was monitored at wavelengths of 254 nm & 340 nm. Individual peaks were collected in fractions and pooled after analyzing by HPLC. The isolated compounds were identified as sinensitin, tetramethoxyflavone, nobiletin, and tangeretin using NMR and mass spectrometry (36, 55).

Identification and Structure Elucidation Identification of PMF’s is challenging due to their close similarity in structures and as well as molecular weight. This necessitates use of proper tools and techniques to determine their exact structure. Until late 70’s, infrared spectroscopic studies coupled with degradation and synthetic studies were commonly used for elucidating the structure of PMF’s. Although degradation and synthetic studies were used for structural analysis, IR analysis played an important role in confirmation of the structure of PMF’s. One such example can be given as the ambiguity in the flavonol constitution of a compound synthesized by Goldsworth and Robinson (56). The compound was considered identical to tangeretin as suggested by degradation and synthetic studies which was later proved to be different from that of the actual structure of tangeretin given as 5,6,7,8,4′-pentamethoxyflavone confirmed by the IR spectroscopy (57). These early investigations on the structure of isolated PMF’s were comprehensively reviewed by Sarin and Sheshadri (58). In modern era of chemistry, nuclear magnetic resonance (NMR) (28, 31, 34–36, 40, 49, 51, 59, 60) is used for accurate structure elucidation. The importance of 13C NMR and its application for identification of flavonoids was reviewed by Agrawal (61). The 1H NMR and 13C NMR of the reported PMF’s are summarized in Table 2 and 3 respectively. However, this method is limited by the requirement of large quantity of purified compounds. Other spectral analysis techniques used for identification of PMF’s were gas chromatography- mass spectrometry (GC-MS) (62, 63), and LC-MS (64, 65). These techniques provide valuable information in regards to the compounds molecular weight along with the fragment ions. The advantage of minimal sample requirement for identification provides a valuable tool for structure elucidation. Raman et al., (29) reported identification of nobiletin and tangeretin using mass spectrometry in negative electronspray ionization (ESI) mode. The structures were further elucidated by collisional activated dissociation (CAD) to generate fragmentation patterns of the deprotonated flavones. Wang and Zhang (64) reported positive electronspray ionization tandem mass spectrometry of PMF’s. Although NMR and MS studies provide structure information of individual isolated compounds, these methods are limited in application for identification of components in crude extracts. Recently, Weber et al., (38) reported the LC-NMR 10 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

method for identification PMF’s present in residues from molecular distillation of cold pressed peel oils of C. sinensis. The individual PMF’s were initially separated using HPLC followed by NMR analysis conducted in the stop-flow mode.


Table 2. 1H NMR of polymethoxyflavones isolated from citrus species (see Appendix A for larger version of table)


13C

NMR of polymethoxyflavones isolated from citrus species (see Appendix A for larger version of table)


Table 3.

Summary Several extraction methods, isolation techniques and identification strategies were reported for PMF’s. Overall review suggests that majority of the research on isolation of PMF’s is limited to only few species of citrus. Future efforts should be focused on exploring PMF’s in citrus species that have not been studied earlier. Moreover emphasis should be made to develop efficient large scale isolation methods of PMF’s using food grade solvents in order to benefit further in vivo studies on disease prevention.


Acknowledgments


The present research report is based on work supported by the United States Department of Agriculture Cooperative State Research, Education and Extension Service Initiative for Future Agricultural and Food Systems (USDA CSREES IFAFS) # 2009-34402-19831 and # 2010-34402-20875 ‘Designing Foods for Health’ through the Vegetable & Fruit Improvement Center, Texas AgriLife Research.

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