Isolation and Characterization of Anthocyanins from Hibiscus

Article pubs.acs.org/jnp

Isolation and Characterization of Anthocyanins from Hibiscus sabdarif fa Flowers Claudia Grajeda-Iglesias,† Maria C. Figueroa-Espinoza,† Nathalie Barouh,‡ Bruno Baréa,‡ Ana Fernandes,§ Victor de Freitas,§ and Erika Salas*,⊥ Montpellier SupAgro and ‡CIRAD, UMR 1208 Ingénierie des Agro-polymères et Technologies Émergentes, 2 Place Viala, F-34060 Montpellier, France § REQUIMTE − Laboratório Associado para a Química Verde, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal ⊥ Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito Universitario s/n, Campus Universitario No. 2, CP 31125, Chihuahua, México †

ABSTRACT: The intense red-colored Hibiscus sabdarif fa flowers are an inexpensive source of anthocyanins with potential to be used as natural, innocuous, and health-beneficial colorants. An anthocyanin-rich extract from hibiscus flowers was obtained by ultrasound-assisted extraction. By a single-step process fractionation using a Sep-Pak C18 cartridge, the main hibiscus anthocyanins, delphinidin-3-O-sambubioside (Dp-samb) and cyanidin-3-Osambubioside (Cy-samb), were separated and then characterized via NMR and HPLC-ESIMS data. Since Dp-samb was the most abundant anthocyanin identified in the extract, its colorant properties were studied by the pH jumps method, which allowed the calculation of the single acid−base equilibrium (pK′a 2.92), the acidity (pKa 3.70), and the hydration constants (pKh 3.02). Moreover, by using size-exclusion chromatography, new cyanidin-derived anthocyanins (with three or more sugar units) were successfully identified and reported for the first time in the hibiscus extract.

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potential natural source of anthocyanins, which confer the brilliant red color to the petals and make this plant an attractive and cheap source of natural food colorants. The chemical structures of the main anthocyanins in hibiscus were clarified by Du et al.,7 who identified delphinidin-3-O-sambubioside (Dpsamb) and cyanidin-3-O-sambubioside (Cy-samb) as the major anthocyanins. Since then, several reports have confirmed these anthocyanins structures.8−12 Recently, Borrás-Linares et al.13 reported the chemical composition and the antioxidant and antibacterial activities of 25 major varieties of roselle harvested in Mexico. These authors agreed on the identification of Dpsamb as the most abundant hibiscus anthocyanin, independently of the variety, and suggested its major contribution to the color of the calyces. Structurally, anthocyanins correspond to the glucosidic forms of the anthocyanidins, cyanidin (e.g., in berries, black beans),14 delphinidin (e.g., in blackcurrant, hibiscus flowers),10,15 and pelargonidin (e.g., in strawberries)16 being the most widely naturally distributed, from a total of 31 monomeric anthocyanidins properly identified.17 Anthocyanins belong to a larger group of polyphenols, the flavonoids, which are capable of absorbing light in both the UV and visible (from yelloworange to bluish-green) regions.18 With only one chromophore,

ince ancient times, color has been considered as one of the most important sensory properties in plants. In the early 1900s, the origin of color in nature was addressed by Richard Willstätter,1 who concluded that colors displayed in flowers depended on the presence of a combination of different anthocyanins, in their different structural forms and concentrations, which could interact with the intracellular medium as well as with other molecules.2 Color is the first attribute to be perceived in foods and beverages and is usually positively correlated with standards of quality by the consumer.3 Since anthocyanins constitute a major flavonoid group responsible for the color diversity found in the plant kingdom,4 several anthocyanin-rich flowers and fruits have become the focus of intensive research to evaluate their potential as natural colorant sources, in light of the increasing demand to replace synthetic colorants in food, cosmetics, and other products. Hibiscus sabdarif fa L. (commonly known as hibiscus flower, roselle, bissap, jamaica flower) is an annual bush of the Malvaceae family that grows in regions where tropical and subtropical weather prevails.5 Countries such as Thailand, India, Senegal, and Mexico, among others, are important producers of this plant, where it has a long history of edible and medicinal uses; however, it is still not considered an important product at an economical level.6 Hibiscus flowers are consumed worldwide as a cold beverage or a hot drink after infusion of the dried sepals and calyces. The resulting exhausted flower is a © 2016 American Chemical Society and American Society of Pharmacognosy

Received: October 26, 2015 Published: June 17, 2016 1709

DOI: 10.1021/acs.jnatprod.5b00958 J. Nat. Prod. 2016, 79, 1709−1718

Journal of Natural Products

Article

Scheme 1. Structural Transformations of Delphinidin-3-O-sambubioside (Dp-samb) Flavylium Cation in Strongly Acidic to Alkaline Aqueous Media22,29

Figure 1. HPLC chromatogram from Hibiscus sabdarif fa extract (HE) recorded at (A) 280 and (B) 520 nm. 1: protocatechuic acid (PA); 2: chlorogenic acid (CGA); 3: delphinidin-3-O-sambubioside (Dp-samb); 4: cyanidin-3-O-sambubioside (Cy-samb). (C) HE fractionation by Sep-Pak C18 cartridge. The compounds isolated in each fraction (from F1 to F4) are indicated in A. Each fraction is symbolized by a different background and separated by a dotted line. n.i. = not identified.

the flavylium nucleus, these pigments can provide a large range of colors, due to their interactions with compounds existing in aqueous medium (copigmentation, metal complexation).4 Also, anthocyanins are regarded as important nutraceuticals since

they play a potential role in the prevention of various diseases associated with oxidative stress.19−21 Nevertheless, anthocyanin color is highly unstable and depends on factors such as pH.22 The network of chemical 1710



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Figure 2. Full-scan (+) ESIMS of the main compounds from the hibiscus extract (HE) obtained in fractions: F2 (A) delphinidin-3-O-(2″xylosyl)glucoside (Dp-samb) (inset: MS2 at m/z 597.1 gave the fragment ion of delphinidin-3-O-glucoside at m/z 303.1); and F3 (B) cyanidin-3-O(2″-xylosyl)glucoside (Cy-samb) [inset: MS2 at m/z 581.2 and at m/z 449.1 gave the fragment ion of cyanidin-3-O-glucoside (Cy-glc) at m/z 287.1].

retrochalcone (Ctrans) results from deacetalization of hemiketal B, and the cis-retrochalcone (Ccis), from isomerization of Ctrans.23,24 Interconversion of these species produces large changes in color and stability. The colored species are AH+ (red), at low pH values, and A (purple/blue), which is not thermodynamically stable.2 Copigmentation and metal complexation are the most widespread anthocyanin-stabilizing mechanisms in plants.4,18 The term copigmentation refers to the phenomenon that influences color intensity and stability of the anthocyanins via their hydrophobic association with the planar unsaturated parts

reactions that take place from the anthocyanins and related compounds in aqueous solutions, according to different acid− base, hydration, and tautomeric reactions, was established by Brouillard et al.23 In this paper, delphinidin-3-O-sambubioside (Dp-samb) is used to represent the species found in acidic and neutral aqueous solutions, shown in Scheme 1. The flavylium cation (AH+) is the predominant species in the equilibrium under strongly acidic conditions.2 With an increase in pH, AH+ undergoes two parallel reactions: deprotonation, to form the quinoidal base (A), and hydration at C-2 (or C-4) followed by proton loss, to give the hemiketal (B).20,21 The trans1711



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Figure 3. Extract from the 1H NMR (400 MHz) spectra in methanol-d4/TFA-d1 (2%) (Bruker Avance III) of (A) F2 purified fraction [delphinidin3-O-sambubioside (Dp-samb), suggested] and (B) F3 purified fraction [cyanidin-3-O-sambubioside (Cy-samb), suggested].

of, for example, flavonoids and phenolic acids, which usually have no color by itself, but when added to an anthocyanin solution, it noticeably enhances the color of the solution.25−27 A great number of colorless natural compounds (hydroxylated benzoic and cinnamic acids and hydroxyflavones, among others) are able to act as copigments.2 Therefore, factors such as structure, pH, temperature, and the presence of other molecules (copigments) are crucial for anthocyanin color in solution. In the present work, the isolation by a single-step process and the characterization of the main anthocyanins found in the hibiscus flower extract (HE) are reported, with the objective to provide new information to support the potential use of this product as a natural source of colorants. The color properties (kinetic and thermodynamic constants) of the main anthocyanin identified in HE, Dp-samb, were determined for the first time by the pH jumps technique.28,29

extractions, allows plant extracts to diffuse across cell walls to the solvent due to ultrasound, causing cell rupture over a shorter period. It is also advantageous because of lower solvent consumption.30,31 Ethanol (20%, v/v) was used in order to decrease the total extracted polysaccharides and increase the percentage of phenolic compounds in the extract. Accordingly, it was previously reported that a hibiscus aqueous extract was richer in total saccharides than the EtOH extract (60%, v/v), which, instead, was richer in anthocyanins, but, in contrast, a greater phenolic compound extraction was obtained when using EtOH−H2O mixtures than EtOH alone.32,33 Thus, using a solvent mixture of EtOH and H2O could be more effective for the extraction of phenolic compounds, especially anthocyanins, from plants. HPLC analysis of the HE (Figure 1A and B) showed the presence of two major peaks at 520 nm, which is the commonly used wavelength for anthocyanin analysis.17 On the basis of their mass spectra, these anthocyanins were identified as delphinidin-3-O-(2″-xylosyl)glucoside (delphinidin-3-O-sambubioside, Dp-samb) (Figure 2A) and cyanidin-3-O-(2″-xylosyl)glucoside (cyanidin-3-O-sambubioside, Cy-samb) (Figure 2B), with [M+] at m/z 597.1 and 581.2 and retention times of 13.7 and 16.2 min, respectively. Dp-samb corresponded to the most

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RESULTS AND DISCUSSION Extract Characterization. H. sabdarif fa L. dry calyces were subjected to ultrasound extraction to obtain an anthocyaninrich extract (HE). This method, in contrast to conventional 1712



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Figure 4. Fractionation of F3 from HE in a Toyopearl HW-40(S) glass column (250 × 16 mm). Full-scan (+) ESIMS of the identified compounds in the subfractions: (a) cyanidin-3-O-trisaccharide m/z 713.2; (b) cyanidin-3-O-sambubioside m/z 581.2; (c and d) cyanidin-3-O-glucoside m/z 449.1.

obtained from F2, which was isolated with a purity of 96.6%. Two peaks are observed, the base peak at m/z 597.1 corresponding to Dp-samb and a minor peak at m/z 303.1, which corresponds to its respective aglycone (delphinidin, Dp), indicating that the molecule is being fragmented in the source and that the sugar units are connected at the same position to the aglycone. This fragmentation pattern helped to confirm the sambubiose unit [−3-O-(2″-xylosyl)glucoside] as the corresponding glycosyl unit linked to the anthocyanidins.10,11,44,45 Further MS2 experiments on the base peak m/z 597.1 showed an intense fragment ion for the anthocyanin aglycone at m/z 303.1 (Figure 2A inset). In contrast, from the ESIMS of the compound purified from F3 (Figure 2B), three abundant ions were observed in the full-scan spectra, [M+] at m/z 581.2, 449.1, and 287.1. The ion at m/z 581.2 was assigned to the Cysamb, and that at m/z 287.1, to its respective aglycone (cyanidin, Cy), showing the same fragmentation pattern as the Dp-samb (F2). The MS2 fragmention at m/z 449.1 (Figure 2B inset) gave the same ion at m/z 287.1 under the same analysis conditions. This indicated the presence of a second anthocyanin in the same fraction (F3), cyanidin 3-O-glucoside (Cy-glc), which was reported to be present in the hibiscus extract in minor quantities.12,46 Considering that the NMR method is especially valuable for structure elucidation of anthocyanins and the determination of the nature and sites of substitution of the sugar moieties,17 NMR analysis of the purified compounds was done to corroborate the information obtained from the HPLC and ESIMS analyses, and the results were compared with published

abundant compound (46% of total area) detected at 520 nm in the HE (25% for Cy-samb), which was in agreement with published data.10−12,33−35 Fractionation of Hibiscus Extract and Anthocyanins Characterization. HE was fractionated in order to separate the identified anthocyanins from other phenolic and nonphenolic compounds. Fractionation was carried out using a Sep-Pak C18 cartridge (Figure 1C), which has been successfully used to separate non-polyphenolic substances in some polyphenolic extracts.36−42 The two major anthocyanins present in the HE were obtained in different fractions, by varying the polarity of the solvent system (F2 and F3, Figure 1C). All fractions were characterized by HPLC-ESIMS. F1 was rich in compounds that absorbed at 280 and 327 nm. The major peaks in this fraction were identified as protocatechuic acid (7.5 min, Figure 1A), which presents maximum wavelengths at 260 and 294 nm, and chlorogenic acids (8.9 and 12.9 min, Figure 1A), which also presents a maximum wavelength at 327 nm. The presence of these phenolic acids in HE was previously reported.11,43 In F2 a major peak was assigned to Dp-samb (13.7 min, Figure 1B). The peak corresponding to Cy-samb (16.2 min, Figure 1B) was detected in F3. Finally, F4 contained only traces of other compounds. Afterward, F2 and F3 were subjected to preparative HPLC analysis for further purification. Detection was set at 280 nm, to ensure that no other compounds would be collected in the final fraction. The anthocyanin-rich fractions obtained were analyzed by means of ESIMS. Figure 2A shows the full-scan ESIMS in positive mode of the anthocyanin 1713



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data.44 1H NMR spectra are shown in Figure 3. The 1H NMR data for the compound purified from F2 (Figure 3A) corresponded to the structure of the Dp-samb, as suggested from the previous ESIMS analysis, and agreed with published data.44,47 Nevertheless, in the case of F3, even when the compound obtained showed only one peak after the preparative HPLC purification, the NMR spectrum exhibited an unusual aromatic region. The singlet at 9 ppm is typical for H-4 of most anthocyanins; however, in our sample, the 1H NMR spectrum showed two singlets in the same region (Figure 3B), confirming the hypothesis that two different anthocyanins were present in the F3 sample, as was established by the ESIMS data. For this reason, F3 was subjected to a second purification step (Experimental Section). As was previously described, F2 was rich in Dp-samb, which was purified and characterized. In contrast, in the case of F3, where Cy-samb was the most abundant anthocyanin identified, a second purification step was needed, since the presence of a second anthocyanin, Cy-glc, which coeluted with the Cy-samb, was confirmed by NMR and ESIMS data. Therefore, F3 was subjected to size exclusion chromatography, for a final purification step, with acidified H2O as the mobile phase (Experimental Section). This technique permitted the separation of four well-defined red-colored rings (subfractions a−d, Figure 4), corresponding to the different compounds present in F3, which were characterized by HPLC-ESIMS (Figure 4). Since Toyopearl functions as a size exclusion gel, it was possible to separate the different anthocyanins in F3 according to their molecular weight, which was not possible with the Sep-Pak C18 cartridge (absorption/desorption phase). Thus, Cy-samb and Cy-glc were not the only anthocyanins identified by HPLCESIMS in the subfractions obtained from F3. Following the fragmentation pattern shown by the anthocyanin-3-sambubiosides, and also because they were the first to elute (subfraction a, Figure 4), the presence of more complex polyglycosylated anthocyanins derived from a cyanidin unit was suggested (Scheme 2). The anthocyanins identified showed the same fragmentation pattern as Cy-samb. The intense ions observed at m/z 713.2 and 287.1 could correspond to the cyanidin glycosylated with a trisaccharide [cyanidin-3-O-(dixyloxy)-

glucoside] and to the Cy-aglycone, respectively. In this case, it is assumed that the sugar units are linked directly to the same position in the anthocyanidin unit since only the ion corresponding to the aglycone was observed in the MS data. Moreover, the ions at m/z 743.2, 845.2, 875.2, and 977.3 could correspond to the proposed cyanidin-based compounds cited in Scheme 2. Similar polyglycosylated anthocyanins have been previously identified in some varieties of berries.48,49 Cabrita et al.50 reported the structure of three anthocyanin-3-Otrisaccharides isolated from the edible berries of Vaccinium padifolium, the 3-O-(6″-O-α-rhamnopyranosyl-2″-O-β-xylopyranosyl-β-glucopyranosides) of cyanidin, petunidin, and peonidin, which were identified before in blue berries and constituted less than 2% of the total anthocyanin content. These anthocyanin tri-, tetra-, or even pentasaccharides have not been previously detected in the hibiscus flower. This is likely because they are present in such a complex anthocyanin mixture, making it difficult to identify and properly characterize their structures. However, this is an interesting finding because, by using size exclusion chromatography with acidified water as solvent, it was possible to separate the anthocyanins present in the cyanidin-rich fraction (F3), which are also contributing to the intense red color that characterizes the hibiscus flowers. A more accurate analysis is needed to completely elucidate their structures. Anthocyanin Color Properties Evaluation. In Nature, anthocyanins are molecules responsible for the red to blue/ purple colors of flowers and fruits. In spite of their chemical structure, based on the flavylium chromophore, the stability of anthocyanins largely depends on chemical and physical parameters including temperature, presence of metals and other compounds, and pH values.2,29 In fact, the color of anthocyanins is particularly highly dependent on pH.22 As already mentioned, under acidic aqueous solutions, it is possible to distinguish at equilibrium five anthocyanin species (Scheme 1). The thermodynamic equilibrium of the system can be reached in a time scale from a few minutes to days, starting from the flavylium cation, and depending on the flavylium substituent.29 According to Scheme 1, the kinetics of the flavylium structural transformations can be defined by the following equations: Ka

AH+ + H 2O ↔ A + H+; K a proton transfer

Scheme 2. Suggested Identities of the Anthocyanins Related to the Cyanidin Unit, Identified in Subfraction “a” from the Toyopearl Fractionationa

Kh

AH+ + H 2O ↔ B + H+; Kh hydration Kt

B ↔ Ctrans ; K t deacetalization Ki

Ctrans ↔ Ccis ; K i isomerization

(1) (2) (3) (4)

At equilibrium, the system behaves as a single acid−base equilibrium between the flavylium cation (AH+) and the conjugate base (CB), defined as the sum of the concentrations of the other species in the network; [CB] = [A] + [B] + [Ctrans] + [Ccis]. Thus, the complex equilibrium described by eqs 1−4 can be simplified in a single acid−base equilibrium, with acidity constant K′a (eqs 5 and 6): K ′a = K a + Kh + KhK t + KhK tK i

(5)

K ′a = [CB][H+]/AH+ ; CB a

= [A] + [B] + [Ctrans] + [Ccis]

Glc, glucose; Xyl, xylose. 1714

(6) DOI: 10.1021/acs.jnatprod.5b00958 J. Nat. Prod. 2016, 79, 1709−1718


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The equilibrium and the reaction rate constants of the anthocyanins can be determined by the application of relaxation techniques, pH and temperature jumps, introduced some decades ago,23 but which still continue to be the simplest methods to elucidate the kinetics of transformation of these compounds.29 By the drastic shift of the equilibrium of the system using an external influence, the metastable system thus formed is forced to shift to a new state of equilibrium.28 The use of pH jumps is more advantageous since it does not produce secondary physical effects. Placing the structural transformations of the anthocyanins by the pH shifts on a time scale, three main processes can be generally observed: (i) proton transfer is the fastest and occurs on a microsecond time scale; (ii) hydration takes place on a time scale from seconds (at pH 3−4) to several minutes (at pH closer to neutrality); and (iii) isomerization occurs in hours and is not dependent on pH.28,29 For this work, the chemical reactions involving the Dp-samb purified from HE were studied by perfoming direct pH jumps, from acidic to basic, in thermally equilibrated solutions, following the method reported by Fernandes et al.28 Thermal Equilibrium. The Dp-samb global apparent equilibrium constant, K′a, was obtained from the pH dependence of the absorption spectra of the equilibrated solutions (Dp-samb 6.0 × 10−5 M), displayed in Figure 5. These spectra

Table 1. Thermodynamic and Kinetic Constants Determined by the pH Jumps Method for the Transformations of Delphinidin-3-O-sambubioside (Dp-samb, 6.0 × 10−5 M): Comparison with the Constants Reported for Similar Compoundsa

pK′a pKa pKh kh (s−1) k‑h (M−1 s−1)

Dp-samb 6.0 × 10−5 M

Dp-glc 2.0 × 10−5 M (Leydet et al.52)

Cy-glc 6.0 × 10−5 M (Fernandes et al.28)

2.92 3.70 3.02 0.12 128.77

2.60 3.80 2.60 0.09 32.00

2.80 3.70 2.90 0.09 47.50

a

Dp-samb: delphinidin-3-O-sambubioside; Dp-glc: delphinidin-3-Oglucoside; Cy-glc: cyanidin-3-O-glucoside.

calculate the pK′a of the six most common anthocyanin-3-Oglucosides, e.g., 2.6, 2.3, 2.4, 2.5, 2.4, and 2.7, for delphinidin-, malvidin-, petunidin-, cyanidin-, peonidin-, and pelargonidin-3O-glucosides, respectively, described in detail by Leydet et al.52 The slightly higher value of the pK′a for the Dp-samb could suggest an influence of the second sugar moiety involved in the sambubioside unit. In contrast, in the case of the malvidin-3,5diglucoside, the pK′a of 1.7 is the lowest reported by the pH jumps method,29 showing that a C-5 sugar moiety does not provide higher stability to the flavylium cation. Proton Transfer. When a pH jump is made from pH < 1 to higher values, proton transfer from the flavylium cation is the first and faster reaction.28 The value of the proton transfer equilibrium constant, or acidity constant, Ka, can be obtained from the absorption spectra generated immediately after a pH jump from

Isolation and Characterization of Anthocyanins from Hibiscus

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