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Food Research International 44 (2011) 2289–2294

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Food Research International


j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Isolation and characterization of pigment from Cinnamomum burmannii' peel


Ming-xiong Tan a,b, Dian-hua Gan a, Liu-xin Wei a, Ying-ming Pan a, Shao-qing Tang a, Heng-shan Wang a,⁎
a
The Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Chemical Engineering of Guangxi Normal University, Guilin, 541004, PR China
b
Department of Chemistry and Biology of Yulin Normal College, Yulin, 537000, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Cinnamomum burmannii is cultivated for use as a spice, and as an ornamental tree. With the aim to develop a
Received 25 February 2010 natural colorant for use in cosmetics and food additives from a new source, in the present study, pigment
Accepted 31 May 2010 derived from C. burmannii' peel (CBP) was isolated by alkaline extraction, acid hydrolysis, repeated
precipitation and purification by Sephadex G-75 with a total yield of 0.06 g/100 g (wet weight basis). The
Keywords:
color values of the pigment was E1% 1 cm 278 nm = 65.21. Physical and chemical properties revealed that CBP
Cinnamomum burmannii' peel
Pigment
presses similar properties as most natural pigment. It was scarcely soluble in both water and all common
Purification organic solvents, and was soluble only in alkaline aqueous and DMSO. It was stable under ultraviolet light or
Physical and chemical properties room-light, stable in the range of 25–100 °C, relatively stable in alkaline aqueous and reducer, but was
bleached by strong oxidants (KMnO4, H2O2 and NaOCl). Sodium benzoic acid, tartaric acid, table salt and
cane sugar affected it slightly. Spectroscopic analysis of CBP in relation with its structure was also discussed.
This is the first report on the characterization of pigment obtained from C. burmannii 'peel.
© 2010 Elsevier Ltd. All rights reserved.

1. Introduction Cinnamomum burmanii (Lauraceae) is native to Southeast Asia and


Indonesia (Elliott & Brimacombe, 1987), which was cultivated for use
There is a growing interest in the development of colorants as a spice, and as an ornamental tree in China (Zhang et al., 2008). It is
from natural sources as additives for use in the food industry, which the source of the flavor called Cassia, similar to cinnamon. Cassia is an
has been encouraged by a strong consumer demand for natural ingredient in Chinese five-spice as a flavor agent for confectionary,
products. Natural colorants have become increasingly popular with desserts, and pastries. Also, it is used to treat nausea and flatulence. The
consumers because synthetic colorants are frequently perceived as extract from C. burmannii (leaf, bark and root) is also used as an
undesirable or harmful (Hendry & Houghton, 1996; Wissgott & acesodyne and as a folk medicine for the treatment of acne (Chen et al.,
Bortlik, 1996; Giusti et al., 1998; Orzechowski, Ostaszewski, Jank, & 1992). C. burmannii, using only the bark or stick, has dark purple
Berwid, 2002; Winterhalter, 2007). Melanin is one of the largest berries and is considered to be an agricultural waste produced in large
classes of natural pigments extracted from plants (Sava, Yang, Hong, amounts (Wang, Pan, Tang, & Huang, 2006). It would be desirable from
Yang, & Huang, 2001; Wang, Pan, Tang, & Huang, 2006), animals an ecological and economical point of view to identify biological
(Hong & Simon, 2005), and microorganisms (Koroleva et al., 2007). activities and promote effective use of the berries (Iqbal, Haleem,
They are dark-brown or black pigments with high molecular weight Akhtar, Zia-ul-Haq, & Akbar, 2008; Sylvia et al., 2009). Previous studies
formed by oxidative polymerization of phenolic or indolic compounds of its biochemical and physiological activities mostly focused on the
in organisms. Melanin from natural sources possesses a broad bark and leaves (Luo, Zheng, & Xie, 2005; Sun, Zhao, Rao, Cai, & Zheng,
spectrum of biological activities, including protecting organisms 2005; Chen et al., 1992), and there are few report addressed on the
against UV-radiation, enzymatic lysis, and oxidants (Montefiori & extract of its fruit as natural colorants (Jiang, Liu, & Lu, 2009) and the
Zhou, 1991). Melanin as natural colorants for use in food and extract of its seed in relation to the chemical components (Gan et al.,
cosmetics has been encouraged by a strong consumer demand, due 2009). Moreover, the melanin pigments occupy a unique position
to its safety and pharmacological activities, particularly as substitutes among the natural pigments as their involvements in human pigment.
for synthetic dyes (Orzechowski et al., 2002; Winterhalter, 2007). Based on diversity of biological sources for pigments, there is an
increasing interest in investigating the melanin pigments extraction
and characterization from various sources and its application (Hung,
Sava, Makan, et al., 2002; Aghajanyan et al., 2005; Selvakumar,
Rajasekar, Periasamy, & Raaman, 2008; Chen et al., 2008).
In the present study, the isolation and characterization of pigment
⁎ Corresponding author. Tel./fax: +86 773 2120958. from C. burmannii' peel were carried out and its potential as a
E-mail address: wang_hengshan@yahoo.com.cn (H. Wang). substitute for synthetic colorants was examined.

0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.05.022
2290 M. Tan et al. / Food Research International 44 (2011) 2289–2294

2. Experimental section purification of the pigment against carbohydrates and proteins.


Organic solvents were used to wash away lipids. Multiple precipita-
2.1. Materials tions were employed to sequester the pigment from low molecular
weight polyphenols and to improve homogeneity. Each precipitation
In our experiments, mature C. burmannii' peel were collected from included acidification of the pigment solution by the addition of HCl to
Guilin City, China, and taken immediately to the laboratory where pH 2.5 and filtered (Sava et al., 2003).
they were manually peeled and subjected to the pigment extraction. The samples of the pigment were hydrolyzed with 7 mol/L HC1 at
All chemicals used were of analytical grade. 100 °C for 2 h (Harki, Talou, & Dargen, 1997), filtered, and the filter
residue was washed with distilled water. The non-hydrolysable
2.2. Measurements pigment was washed sequentially with chloroform, ethyl acetate
and ethanol and then dried. The solid matter of each sample was re-
Elemental analyses (C, H, N, and O) were carried out on a Perkin- dissolved in 1 mol/L NH3–H2O and the mixture was filtered. The
Elmer Series II elemental analyzer. Infrared spectra were obtained on supernatant was acidified with 1 mol/L HC1 and the filter residue was
a Perkin-Elmer FTIR Spectrometer. Ultraviolet–visible absorption washed with water. The re-precipitation was repeated four times. The
spectrum of the pigment solution was recorded on a UV-1100 filter residue was washed with water until the reaction for chloride
spectrophotometer (Beijing Rayleigh Analytical Instrument Co., Ltd., ions was negative (Wang et al., 2006).
Beijing, China), 0.015 g/100 g pigment sample solutions in the The final purification by chromatography on Sephadex G-75
phosphoric acid buffer. JASCO V-530 UV–vis spectrophotometer column was carried out in 50 mM phosphate buffer (pH 7.5) at a
(Tokyo, Japan). The Raman spectra for the solid pigment was obtained flow rate of 0.5 mL/min, to yield the fractions containing similar high
with a confocal laser scanning microscope Raman spectrometer molecular weight pigments, the fractions collected were monitored
(Renishaw). by UV absorbance at 275 nm (Hung, Sava, Juang, et al., 2002; Hung,
Sava, Makan, et al., 2002; Sava, Galkin, et al., 2001; Sava, Yang, et al.,
2.3. Isolation and purification of the pigment 2001).
The pigment solution was prepared by the following procedure.
Isolation of the pigment was performed according to a previously The filter residue was suspended in distilled water made slightly
reported procedure (Sava, Hung, Blagodarsky, Hong, & Huang, 2003; alkaline by 0.5 mol/L NH3–H2O to pH 9, incubated at 50 °C for 1 h and
Wang et al., 2006) employed with minor adjustments (Fig. 1). First, filtered. The ammonia was removed by a rotary evaporator under
the raw materials of C. burmannii' peel were washed with running reduced pressure to a final pH of 7.5 (Hung, Sava, Juang, et al., 2002;
water at a volume ratio of 1:50 (raw materials/water) for 5 min Sava, Galkin, et al., 2001; Sava, Yang, et al., 2001; Wang et al., 2006).
followed by sequestering of solid matter, and then immersed in water
(25 °C) at volume ratio 1:10 (wet peel/water), and 10 g/100 g 2.4. Physico-chemical properties of the pigment
NH3·H2O was added to adjust pH value to 10.5 (a final concentration
was about 2 g/100 g). After 24 h incubation at room temperature, the 2.4.1. The pigment solubility
mixture was passed through a 0.45-mm nylon filter, and then filtered 0.1 g of the pigment was added to 10 mL of water, aqueous acid,
to obtain the pigment extract. The extract was acidified to pH 2.5 with alkali (such as Na2CO3, NaOH, and NH3–H2O solution), or organic
2 mol/L HCl solutions for 10 h at room temperature, and the crude solvents (such as benzene, chloroform, ethyl acetate, ethanol,
pigment obtained after filtration. methanol, acetic acid, aether, petroleum ether, hexane, acetone and
The extracted pigment was purified (Fig. 1) by acid hydrolysis, DMSO etc.) with stirring at 25 °C for 1 h, and stood for 0.5 h. then
organic solvent (chloroform, ethyl acetate and ethanol) treatment filtered and the absorption of the solutions were recorded at λmax to
and repeated precipitation. The acid hydrolysis was employed for attain solubility of the pigment. Melanin pigment from Osmanthus
fragrans' seeds prepared in our lab was used in comparison (Wang
et al., 2006).

2.4.2. Temperature effect on the stability of the pigment


The heat stability of 0.005 g/100 g pigment solution was measured
after treatment in a thermostatically controlled bath at 25, 50, 75 and
100 °C. The samples were held at each temperature for specific times
and then cooled immediately in an ice bath. Subsequently the absorp-
tion of the solutions was recorded at λmax.

2.4.3. Light effect on the stability of the pigment


The 0.005 g/100 g pigment solution were held under natural light,
at dark place or under the ultraviolet-light far from 30 cm for specific
time and the absorbance was determined at λmax.

2.4.4. Oxidant and reducer effect on the stability of the pigment


The determination was performed according to the basic proce-
dure designed with minor modifications (Paim, Linhares, Magrich, &
Martin, 1990). 10 mL of 0.005 g/100 g pigment solution and 50 mL of
different concentrations of KMnO4, K2Cr2O7, NaOCl, H2O2, and Na2SO3
were mixed, and then the absorbance of the homogenate was deter-
mined at λmax.

2.4.5. Sugar and salt effects on the stability of the pigment


The 0.5 g/100 g solutions of sugar (such as glucose and cane
Fig. 1. The procedure for extraction and purification of CBP. sugar), and salt were prepared, and then mixed in 10 mL of 0.005 g/
M. Tan et al. / Food Research International 44 (2011) 2289–2294 2291

100 g pigment solution. The absorbance of the solutions was mea-


sured every 20 min.

2.4.6. Determination of the color value of the pigment


The color value of the pigment was determined according to the
basic procedure designed with minor adjustment (Tian, He, Liu, Bian,
& Miao, 2002), natural colorants were expressed with the absorbance
under certain density. The absorbance was measured with 0.2 g/100 g
density, l cm color tube under the wavelength 278 nm. The color value
was expressed in the form of E1% 1 cm 278 nm of absorbance.

2.4.7. Statistical analysis


All experimental results were centered at using three parallel
measurements of mean ± standard deviation. Analysis of variance
was performed by ANOVA. Duncan's new multiple-range test was
used to determine the differences of means. P b 0.05 was regarded as
significant; P b 0.01 was considered very significant.
Fig. 2. The UV spectra of CBP.

3. Results and discussion


ratio and O:N ratio in purified pigment indicated that acid hydrolysis
3.1. Pigment isolation and purification and washing with organic solvents led to a loss of aliphatic chains,
hydrocarbons and sugar group. A comparison of C/H ratio in both
CBP was isolated from C. burmannii' peel by alkaline extraction, pigments (Table 1) allows for a conclusion on a greater content of
acid hydrolysis, repeated precipitation and purification by Sephadex aromatic rings or heterocyclic group in CBP.
G-75. This treatment gave a better purification by the infrared
spectrum and the results of elemental analysis of the “pure” pigment 3.2.2. UV–vis absorption spectroscopy of pigment
(Harki et al., 1997). The average yield of the pigment obtained after In Fig. 2 the ultraviolet–visible absorption spectrum (200–500 nm)
acid hydrolysis, extraction with organic solvents, and repeated of CBP solutions. It exhibits strong optical absorbance in a spectral
precipitation was 0.34 g/100 g (wet weight basis). Final separation range from 210 to 230 nm, and medium strong absorbance range from
using Sephadex G-75 gave a yield of 18 g/100 g purified pigment 230 to 450 nm. The absorption spectrum of CBP shows a typical peak
(from the products before chromatography), the total yield of purified at 278 nm, which was similar to the melanin pigment from black tea
CBP from C. burmannii' peel was 0.06 g/100 g (wet weight basis). leaves and synthetic melanin pigment (Sava, Galkin, et al., 2001; Sava,
Yang, et al., 2001), suggesting the presence of phenol or heterocyclic
3.2. Spectroscopic analysis of the pigment group.

3.2.1. Elemental analysis 3.2.3. Infrared spectroscopy of pigment


Elemental analysis was performed by conventional means and Infrared spectrometric techniques gave information on the main
included determination of C, H, and N contents. Analytical data of the functional groups in the melanin pigment structure (Harki et al.,
crude pigment and purified pigment studied are listed in Table 1. As 1997; Sava, Galkin, et al., 2001; Sava, Yang, et al., 2001). A detailed
shown in Table 1, crude pigment had a relatively small N% content of comparative analysis of the infrared spectra of the pigment studied
3.32%, higher C:N ratio of 15.87% and O:N ratio of 10.77%. After Acid (Fig. 3) may supply valuable information on the effect of each treat-
hydrolysis and washing with organic solvents, N% content of purified ment used to purify the pigment and the distinct functional groups
pigment increased and C:N ratio and O:N ratio reduced to 5.97% and prevailing in the various samples. The spectra display: (1) a strong,
4.12%, respectively. The N%, determined after acid hydrolysis, for the broad band at 3430 cm− 1, attributed to stretching vibrations of OH
different melanin pigments previously studied (Harki et al., 1997) and NH2 groups; (2) a strong band at 1650–1620 cm− 1 due to the
were 8.8% for bgammaN-glutaminyl4hydroxybenzene (GHB) melanin vibrations of aromatic C C, of amide I C O and/or of COO-groups; and
pigment, 6.3% for indole melanin pigment (DOPA dark pigment), 2–4% (3) a strong band at 1100 cm− 1 due to the stretching vibrations of C–
for heterogeneous melanin pigment and traces for catechol melanin
pigment. Some authors (Kukulyanskaya, Kurchenko, Kurchenko, &
Babitskaya, 2002) suggest that high nitrogen content in melanin
pigment appears from their melanoprotein nature. For the purified
pigment of C. burmannii, we have found the N% content to be 8.65
(Table 1). Thus it must be derived from a relatively high nitrogen-
containing precursor and it differs from catechol melanin pigment
(phenolic type). Accordingly, apart from proteins, other nitrogen-
containing compounds were captured into the melanin pigment
structure during cultivation.
The higher C:N ratio indicated that the crude pigment contains
aliphatic groups (Tu, Sun, Tian, Xie, & Chen, 2009). The reduced C:N

Table 1
The elemental analysis of the crude CBP and purified CBP.

Samples C (%) H (%) O (%) N (%)

Crude CBP 52.70 8.17 35.81 3.32


Purified CBP 51.7 5.86 33.72 8.65
Fig. 3. The IR spectra of the purified CBP (A) and crude CBP (B).
2292 M. Tan et al. / Food Research International 44 (2011) 2289–2294

Fig. 4. The Raman spectra of CBP.


Fig. 5. Effect of light on the stability of CBP. Error bars represent standard deviations of
the means (n = 3); P ≤ 0.05.

O–C. However, spectra (A) of the residues before and after acid
hydrolysis showed bands that were strongly reduced or absent in the hexane, benzene, acetone, etc.), and also insoluble in acidity aqueous,
spectrum (B) of the purified pigment. These are (1) a strong band at but soluble in alkaline solutions (such as Na2CO3, NaOH) and slightly
2930 cm− 1 assigned to stretching vibration of aliphatic C–H; and (2) a soluble in DMSO. Therefore, the solubility of pigment is very similar to
band at 1450 cm− 1 attributed to bending vibration of aliphatic C–H. typical melanin pigment (Tu et al., 2009).
An obvious enhancement of the band at 1650–1620 cm− 1 can also be
observed in spectrum (B) in the composition of that in the spectrum
3.3.2. Effect of temperature on the stability of the pigment
(A). This band is due to stretching vibrations of numerous C O-
The effect of temperature on the pigment stability was determined
containing groups. It was confirmed by these IR data and the data of
to ascertain the potential use as a natural colorant. The pigment
the elemental analysis that in the purification processes of the crude
solutions were in a thermostatically controlled bath at 25, 50, 75,
pigment, acid hydrolysis and washing with organic solvents essen-
100 °C by incubating for 0.5, 1, 1.5, 2, 25 h, respectively. After being
tially led to a loss of aliphatic chains, hydrocarbons, and sugar group.
treated for 2.5 h from 25 °C to 100 °C, the absorbance of the pigment
The IR spectra of pigment are similar to those previously reported for
decreased slightly with a loss of 0.015, while only 0.01 was lost from
melanin pigment from Pleurotus cystidiosus (Selvakumar et al., 2008).
25 °C to 50 °C. And the color of the pigment solutions had no obvious
change. Thus, the results show that the pigment was stable to
3.2.4. Raman spectra of pigment
temperature (Kannan & Ganjewala, 2009).
The Raman spectrum of CBP is shown in Fig. 4. It can be observed
that the spectra of the pigment from C. burmannii' peel mainly
comprise two bands at 1583 and 1341 cm− 1 that are very similar to 3.3.3. Effect of light on the stability of the pigment
the Raman patterns observed natural pigment isolated from Sepia Fig. 5 showed that the absorbance of CBP solution slightly declined
officinalis (Centeno & Shamir, 2008). This can be interpreted as when put under natural illumination or UV-light for 10 days. After
originating from the in-plane stretching of the aromatic rings and the being treated with UV-light for 10 days the absorbance of the pigment
linear stretching of the C–C bonds within the rings, along with some decreased slightly with a loss of 0.0085, while only 0.0025 was lost in
contributions from the C–H vibrations in the methyl and methylene nature light. And the color of the pigment solutions had no obvious
groups (Huang et al., 2004). change. These indicated that light had less effect on the pigment
(Wang et al., 2006).
3.3. Physico-chemical properties of the pigment

3.3.1. The solubility of the pigment


The results (Table 2) indicated that CBP was scarcely soluble in
both water and most common organic solvents (such as chloroform,
ethyl acetate, ethanol, methanol, acetic acid, aether, petroleum ether,

Table 2
The solubility of pigment from the C. burmannii' peel.

Tests Pigment from the C.


burmannii' peel

Solubility in H2O at 25 °C −
Solubility in organic solvents (chloroform, ethanol, hexane, −
benzene, acetone, etc.)
Solubility in 1 mol/L NaOH at 100 °C (2 mg/5 mL) +++
Precipitation by 7 mol/L HC1 + at PH 2.5
DMSO +

+ Positive response.
+ + + Strong positive response. Fig. 6. Effect of oxidant and reducer on the stability of CBP. Error bars represent
− Negative response. standard deviations of the means (n = 3); P ≤ 0.05.
M. Tan et al. / Food Research International 44 (2011) 2289–2294 2293

Table 3 Acknowledgments
The absorbance of CBP with the increased concentration of Na2SO3.

Time Absorbance This study was supported by the Natural Science Foundation of
(h) Guangxi Province (No. 05112001-3B2, 20090106-4, 0832096, 0832024,
25 mg/L Na2SO3 50 mg/L Na2SO3 100 mg/L Na2SO3 200 mg/L Na2SO3
0991284 and 2010-013064), the Scientific Researches Foundation of
0 0.821 ± 0.06 0.821 ± 0.023 0.821 ± 0.071 0.821 ± 0.036
Educational Committee of Guangxi Province (200808LX407), and the
6 0.819 ± 0.043 0.822 ± 0.041 0.819 ± 0.059 0.818 ± 0.077
12 0.821 ± 0.01 0.819 ± 0.055 0.818 ± 0.077 0.817 ± 0.059 Project of the Key Laboratory of Medicinal Chemical Resources and
24 0.818 ± 0.033 0.817 ± 0.049 0.817 ± 0.054 0.816 ± 0.065 Molecular Engineering, Guangxi Normal University, China (0630006-
Values are mean ± s. d. (n = 3), P ≤ 0.05.
5D09), as well as Guangxi's Medicine Talented Persons Small Highland
Foundation (0808).

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