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Rhoeo spathacea (Swartz) Stearn leaves, a potential natural food colorant Joash Ban Lee Tan, Yau Yan Lim*, Sui Mae Lee School of Science, Monash University Sunway Campus, Bandar Sunway, 46150 Petaling Jaya, Selangor, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history:
Anthocyanins, a popular choice for natural food colorants, are unstable at low acidity or
Received 30 October 2013
neutral pH. Thus, anthocyanins that are less affected by pH, such as those with multiple
Received in revised form
acylated groups, are highly desirable. One such source is from the leaves of Rhoeo spathacea
7 January 2014
(Swartz) Stearn, an herbal plant traditionally decocted and consumed orally. This study
Accepted 10 January 2014
tests the stability of the purple-red colour of R. spathacea extract from pH 1.0 to 11.5 stored
Available online 6 February 2014
under refrigerated and room temperature conditions, in the presence and absence of light over a period of 60 days. The colour stability was also monitored in a solid food model (jelly)
Keywords:
and a liquid food model (barley water) for a month. The colour was found to be remarkably
Rhoeo spathacea
stable in acidic pH, and completely stable in the food models. The primary anthocyanin in
Anthocyanin
R. spathacea leaves was isolated and identified by NMR to be rhoeonin.
Food colorant
1.
Introduction
Food colorants play an important role in improving the marketability of foods and beverages by improving their aesthetic appearance. However, the questionable safety of these colorants has led to a reduction in the number of permitted synthetic colorants, while increasing the demand for natural colorants. One such class of natural colorant is the anthocyanins, due to their attractive red, purple and orange colours, solubility in water and comparatively low toxicity (Bakowska-Barczak, 2005). Anthocyanins are one of the most widespread natural pigments (Fernandes, Calhau, & Mateus, 2013) and found in a large variety of fruits, vegetables and flowers (Norberto et al., 2013). They have been reported to exhibit cytotoxicity against cancerous cells (Barrios et al., 2010), anti-inflammatory (Chen, Xin, Zhang, & Yuan, 2013), antimicrobial (Hwan, Dae, & Choung, 2012), neuroprotective (Norberto et al., 2013) and cardioprotective properties, in addition to being antioxidants, thus improving the nutritional value of the food (Bakowska-Barczak, 2005). These traits have
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made anthocyanin studies an increasingly popular field in food research (Hwan et al., 2012). Unfortunately, a major pitfall of most anthocyanins is their sensitivity to pH, with most anthocyanins being unstable at low acidity or neutral pH. However, acylated anthocyanins are considerably more stable than unacylated anthocyanins under these conditions, as the stacking of the acyl groups with the pyrylium ring protects it from nucleophilic attacks and subsequent decolorization (BakowskaBarczak, 2005; Patras, Brunton, O’Donnell, & Tiwari, 2010; Pe´rez-Gregorio, Garcı´a-Falco´n, Simal-Ga´ndara, Rodrigues, & Almeida, 2010). Therefore, this makes extracts of acylated anthocyanins highly desirable. One such extract is from Brassica oleracea (red cabbage), which is currently one of the most valuable forms of stable anthocyanin-based food colorants commercially available due to its diacylated anthocyanins (Bakowska-Barczak, 2005). Members of the Commelinaceae family often contain otherwise-rare and exceptionally stable trisubstituted anthocyanins (Saito & Harborne, 1983). A member of this family,
* Corresponding author. Tel.: +60 355146103; fax: +60 355146364. E-mail address:
[email protected] (Y.Y. Lim). 1756-4646/$ - see front matter 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jff.2014.01.012
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Rhoeo spathacea (Swartz) Stearn, is an herbal plant traditionally used as a functional food particularly in South America where the leaves are boiled and taken orally daily for the treatment of cancer (Rosales-Reyes et al., 2008) and gonorrhea (Halberstein, 2005). Studies also show that aqueous crude R. spathacea leaf extracts exhibit in vivo antitumoral activity in rats (Rosales-Reyes et al., 2008) while ethanolic leaf extracts are antigenotoxic (Gonza´lez-Avila et al., 2003) and antimutagenic, by being able to prevent DNA damage caused by alkylation (Arriaga-Alba et al., 2011) and reactive oxygen species (Gonza´lez-Avila et al., 2003). In a previous study by our research group, boiling aqueous extracts of the leaves were found to have good antioxidant activity and broad-spectrum antibacterial activity (Tan, Lim, & Lee, 2013). Its leaf decoction is also believed, according to Chinese folklore, to be good for relieving cough, cold and nose bleed (Goh, 2000). The daily consumption of the leaf decoctions in Southeastern Mexico (Rosales-Reyes et al., 2008) and scientific studies supporting its ethnobotanical values qualify it as a ‘‘functional food’’ under the latest definition by the Academy of Nutrition and Dietetics (formerly the American Dietetic Association), which defines a functional food as ‘‘whole foods along with fortified,
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enriched, or enhanced foods that have a potentially beneficial effect on health when consumed as part of a varied diet on a regular basis at effective levels based on significant standards of evidence’’ (Crowe & Francis, 2013). The primary anthocyanin in this plant, a triacylated anthocyanin called rhoeonin, had previously been reported based on fast atomic bombardment mass spectrometry (Idaka, Ogawa, Kondo, & Goto, 1987; Tatsuzawa et al., 2010) but its structure was not fully established. Hypothetically, the presence of rhoeonin, and possibly other triacylated anthocyanins in R. spathacea leaves may make it a potential treasure trove as an anthocyanin-based food colorant. To test this hypothesis, the in vitro stability of an anthocyanin-rich partially purified sub-fraction stored for 60 days under different pH, temperature and lighting conditions was measured. The colour stability of the extract was also tested in two model foods: one solid (jelly) and one liquid (barley water) by monitoring the stability of the colour’s hue and tone based on CIE L*a*b* (CIELAB) coordinates over a period of 15 days. This was done to determine the viability of R. spathacea extracts as a food colorant in actual practical application. The acute toxicity of the extract was also analyzed on rats
Fig. 1 – % Stability of 70% methanol sub-fraction colour at pH 1.0–6.0 at 25 C (dark), 25 C (light) and 8 C (dark).
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Fig. 2 – % Stability of 70% methanol sub-fraction colour at pH 6.5–8.1 at 25 C (dark), 25 C (light) and 8 C (dark).
as an animal model to scientifically determine its safety despite its traditional use as an orally-consumed decoction. Lastly, the structure of rhoeonin, the primary anthocyanin contributing to the colour, was elucidated based on 13C nuclear magnetic spectrometry (NMR) study.
2.
Materials and methods
2.1.
Samples
Fresh R. spathacea leaf samples were obtained from Petaling Jaya, Malaysia. The identity of the plant was confirmed by Dr. Wong Khoon Meng, former professor of Botany, Institute of Biological Sciences, University of Malaya.
2.2.
Extraction of samples
Fresh leaves were washed thoroughly with water and dabbed dry and weighed before being subjected to liquid nitrogenaided crushing with a mortar and pestle. The crushed leaves were then extracted with 70% aqueous acetone +1% formic
acid thrice at a ratio of 10 mL solvent per 1 g of leaf sample. This crude extract was then dried at 30 C with a rotary vacuum evaporator (Eyela Tokyo Rikakikai Co. Ltd., Tokyo, Japan) and freeze-dried overnight in a Christ Alpha 1–4 freeze dryer (Osterode am Harz, Germany). An open glass column was packed with Diaion HP-20 at a ratio of 10.0 g of stationary phase for every 1.0 g of dry crude extract. The samples were dissolved in minimal water and loaded onto the stationary phase. Isocratic elution was used, starting with water at three times the volume of the stationary phase. This was followed by an equal volume of 70% methanol to obtain an anthocyanin-rich 70% methanol subfraction. This sub-fraction was dried at 40 C with a rotary vacuum evaporator before being freeze-dried overnight.
2.3. Colour and stability of sub-fraction in buffered solutions Buffer solutions of 17 different pH values were prepared according to Cabrita, Fossen, and Andersen (2000). Similarly, the measurement of colour and stability were also based on
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Cabrita et al. (2000) with minor modifications. The dried subfraction (obtained from Section 2.2) was dissolved in each buffer at a concentration of 14 mg/mL. These prepared stock solutions were then split into three aliquots, stored under one of three different conditions: 10 C dark in the cold room, 25 C dark and 25 C light, both in 25 C incubators with the lights off and lights on respectively, for a total of 60 days, parafilmed and under air atmosphere. UV/Vis spectra were measured between 240 and 700 nm on a Shimadzu UVmini1240 spectrometer, with the pure buffer as the blank. These measurements were conducted 1 h after dissolution, and then after 1, 2, 5, 8, 15 and 60 days. The colour intensities were measured at kmax, and colour stability was expressed as the percentage of absorbance remaining after a certain time interval for that specific treatment.
2.4.
Color and stability of extract in food models
The two types food models prepared for this section, barley water and jelly, are known local household applications for
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the leaves of R. spathacea, where the decocted leaf is used as a colorant. To emulate this, 300 g of fresh R. spathacea leaves were decocted in 500 mL of water for 1 h under constant heat. This was left to cool for 2 h, then freeze dried for two days. The barley water was prepared by boiling 1 L of water with 6 g of freeze-dried decoction and 50 g of barley for 30 min. The jelly was prepared by boiling 500 mL of water with 3 g of freeze-dried decoction, 125 g of jelly powder and 125 g of sugar for 30 min, before being left to harden for 2 h in the cold room (10 C). Both samples were stored in a dark cold room. CIELAB measurements were taken with a HunterLab ColorFlex EZ colorimeter 2 h after preparation, and then after 1, 2, 5, 8, and 15 days. Colour stability was expressed in terms of L* (lightness of colour), a* (positive values representing a magenta hue) and b* (negative values representing a blue hue). The Total Colour Difference, DE*, was calculated with the following formula: 2 0:5
DE ¼ ðDL2 þ Da2 þ Db Þ
Fig. 3 – % Stability of 70% methanol sub-fraction colour at pH 8.6–11.5 at 25 C (dark), 25 C (light) and 8 C (dark).
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2.5.
Isolation of rhoeonin
An open glass column was packed with 15–25 lm ODS Chromatorex (Fuji Silysia) at a ratio of 10.0 g of stationary phase for every 1.0 g of the dried sub-fraction from Section 2.2. The samples were dissolved in minimal water and loaded onto the stationary phase. A solvent gradient of 100% water to 100% methanol in 20% increments was used, followed by 100% methanol to 100% acetone in 25% increments. The volume for each solvent was equal to the volume of the stationary phase. Fractions were collected at every tenth of the overall volume used for a specific solvent. Fractions were pooled based on their RP-HPLC profile. RP-HPLC analysis was performed using Agilent Technologies 1200 Series with quaternary pump (model G1311A) and a G1315B diode array detector. The stationary phase used was an Agilent Eclipse XDB C18 column (4.6 · 250 mm, 5 lm), with the two mobile phases being water + 0.1% trifluoroacetic acid (Solvent A) and methanol + 0.1% trifluoroacetic acid (Solvent B), with a linear gradient of 95% Solvent A to
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100% Solvent B in 15 min, and maintained at 100% Solvent B for an additional 5 min. The mobile phase was delivered through the column with the flow rate of 1.0 mL/min during the running of the samples. The samples were analyzed at four wavelengths: 210, 254, 280 and 365 nm.
2.6.
LC–MS analysis
LC–MS spectra were recorded on a Thermo Finnican model LCQ Deca coupled mass spectrometer. Electron spray ionization was used under the following conditions for both positive and negative ion modes: capillary voltage, 2.7 kV; source temperature, 100 C; desolvation temperature, 350 C; cone gas flow, 30 L/h; desolvation gas flow, 700 L/h. The column used was ACQUITY UPLC BEH C18 1.7 lm, 2.1 · 50 mm column, with an ACQUITY PDA (ACQ-PDA) detector. The mobile phase consisted of water +0.1% formic acid (solvent A) and acetonitrile +0.1% formic acid (solvent B). Gradient elution was performed as follows: from 0 to 5 min, linear gradient of 5% solvent B to 30% solvent B; from 5 to 6 min, linear gradient
Table 1 – CIELAB values for agar–agar and barley prepared with R. spathacea decoction as a colorant. Sample
Time (day)
L*
a*
b*
Agar–agar
0 1 2 5 8 15 DE15days
21.5 ± 1.4 20.4 ± 0.7 20.0 ± 1.6 18.7 ± 1.6 20.9 ± 2.0 19.7 ± 1.5 2.5 ± 1.6
10.9 ± 0.9 11.4 ± 0.2 11.8 ± 1.1 10.6 ± 0.3 9.4 ± 1.1 10.6 ± 0.9
9.6 ± 0.5 10.3 ± 0.2 9.8 ± 0.3 10.0 ± 0.2 9.5 ± 0.6 9.7 ± 0.7
Barley
0 1 2 5 8 15 DE15days
26.0 ± 0.6 31.9 ± 0.1 32.4 ± 0.1 33.0 ± 0.2 32.6 ± 0.1 32.7 ± 0.1 7.1 ± 0.4
16.3 ± 0.0 16.7 ± 0.1 16.8 ± 0.2 16.5 ± 0.1 16.3 ± 0.2 15.6 ± 0.1
11.0 ± 0.0 11.8 ± 0.1 11.2 ± 0.1 10.1 ± 0.0 9.9 ± 0.1 8.8 ± 0.1
Results are expressed as mean ± S.D. (n = 3).
Fig. 4 – Mass spectrum of rhoeonin in the (A) positive mode and (B) negative mode.
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of 30% solvent B to 100% solvent B; from 6 to 8 min, isocratic at 8 min. The software used was Waters MassLynx 4.1.
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C NMR spectra were recorded at 150 MHz in CD3OD with a Bruker AVANCE III 600 MHz FT-NMR system (Bremen, Germany). Chemical shifts are reported relative to CD3OD. Data was analyzed with Top Spin version 3.0 software
experimentation). With free access to food and water, behavioral parameters such as convulsion, hypo- or hyperactivity, vomiting, and increased or decreased respiration were observed for 1, 4 and 24 h after the treatment. The treated rats were further observed for up to 14 days for any signs of toxicity and mortality. On the 14th day post oral treatment, the rats were sacrificed via cervical dislocation. The liver, kidney and stomach were collected for macroscopic examinations of any possible deformities as indications of toxicity.
2.8.
3.
2.7.
13
C NMR analysis
13
Acute toxicity testing
The method used to determine the safety consumption of the extract was in accordance to Lorke (1983) and Ong et al. (2011) with minor modifications. A single oral dose of the sub-fraction from Section 2.2 (5 g/kg) was administered to a group of 10 healthy male rats (80–120 g, fasted 24 h prior to
Table 2 –
Results and discussion
The samples buffered at pH 1.0–6.0 that were stored at 25 C showed over 80% colour stability after a period of 15 days (Fig. 1). In contrast, the stability of most anthocyanins typically started deteriorating at pH 2.0, losing their colour completely within 24 h at pH 4.0–5.0 (Francis, 1992).
13
C chemical shifts of rhoeonin. dC/ppm
Cyanidin Anthocyanidin 2 Anthocyanidin 3 Anthocyanidin 4 Anthocyanidin 5 Anthocyanidin 6 Anthocyanidin 7 Anthocyanidin 8 Anthocyanidin 9 Anthocyanidin 10 Anthocyanidin 1 0 Anthocyanidin 2 0 Anthocyanidin 3 0 Anthocyanidin 4 0 Anthocyanidin 5 0 Anthocyanidin 6 0 Ferulic acid (I) Ferulic acid (I) 1 Ferulic acid (I) 2 Ferulic acid (I) 3 Ferulic acid (I) 4 Ferulic acid (I) 5 Ferulic acid (I) 6 Ferulic acid (I) a Ferulic acid (I) b Ferulic acid (I) COOH Ferulic acid (I) 1 0 Ferulic acid (II) Ferulic acid (II) 1 Ferulic acid (II) 2 Ferulic acid (II) 3 Ferulic acid (II) 4 Ferulic acid (II) 5 Ferulic acid (II) 6 Ferulic acid (II) a Ferulic acid (II) b Ferulic acid (II) COOH Ferulic acid (II) 1 0
127.7 146.2 132.9 158.4 105.1 168.2 96.7 158.4 107.6 119.8 115.3 146.4 154.3 117.5 132.9 127.7 114.6 146.3 149.3 116.5 124.2 114.6 146.0 168.2 111.5 127.6 114.6 146.4 150.4 116.5 124.2 114.6 147.0 168.3 111.6
dC/ppm Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Ferulic acid (III) Glucose (3) Glucose (3) 1 Glucose (3) 2 Glucose (3) 3 Glucose (3) 4 Glucose (3) 5 Glucose (3) 6 Glucose (3) 4 Glucose (3) 5 Glucose (3) 6 Glucose (7) Glucose (7) 1 Glucose (7) 2 Glucose (7) 3 Glucose (7) 4 Glucose (7) 5 Glucose (7) 6 Glucose (3 0 ) Glucose (3 0 ) 1 Glucose (3 0 ) 2 Glucose (3 0 ) 3 Glucose (3 0 ) 4 Glucose (3 0 ) 5 Glucose (3 0 ) 6 Glucose (3 0 ) 1 Glucose (3 0 ) 2 Arabinose Arabinose 1 Arabinose 2 Arabinose 3 Arabinose 4 Arabinose 5
1 2 3 4 5 6 a b COOH 10
131.3 115.2 146.4 149.3 116.5 123.2 115.3 147.3 168.2 111.8 101.6 74.4 77.3 71.7 77.7 67.5 71.7 77.7 67.5 101.6 74.4 77.9 74.4 74.6 64.9 101.6 74.4 77.3 72.1 75.5 64.9 101.6 74.4 108.0 86.0 77.3 85.7 62.6
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Fig. 5 – Structure of rhoeonin.
Bathochromic shifts within the pH 1.0–6.0 range were also minimal, indicating that the red-coloured flavylium ion was still predominant. This flavylium ion form is the most stable of all anthocyanin states, and maintaining its presence is crucial in any attempt to increase colour stability by reducing pH-dependence (Francis, 1992). Consequently, the reddish colour of the extracts can be maintained even in food with lower acidity (pH 4.0–6.0) while most other stable anthocyanins, such as those from red cabbage, would convert into their purplish, predominantly-anhydrobase form at that pH range (Bakowska-Barczak, 2005). In R. spathacea, the anhydrobase form only became dominant between pH 6.5 and 8.1 (Fig. 2), and above which (Fig. 3) the unstable chalcone form was dominant. Chalcone form has no C-ring structure, which is present in flavylium ion, carbinol pseudobase and quinonoidal base forms at pH
