Innovative Food Science and Emerging Technologies 10 (2009) 253259

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Innovative Food Science and Emerging Technologies

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 / i f s e t

Production of coumaric acid from sugarcane bagasse

S.Y. Ou , Y.L. Luo, C.H. Huang, M. Jackson
Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China

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

Article history: Phenolic acids were released from sugarcane bagasse by alkaline hydrolysis at 30 C for 4 h; The alkaline
Received 17 September 2008 hydrolysates were ultraltrated, the permeates puried with anion exchange resin. The phenolic acids bound
Accepted 23 October 2008 by the resin were desorbed by a mixture of waterethanolHCl solution (36: 60: 4) after washing the resin
Editor proof receive date 2 December 2008 with water, ethanol and dilute HCl respectively. The combined eluents were concentrated for crystalization,
and the crystals ltered and washed using 1% (v/v) HCl. After this purication process, the purity of products
Keywords: reached 89.7% based on coumaric acid. Results of HPLC/MS, HPLC using standard coumaric acid and ferulic
Sugarcane bagasse acid showed that the main component of the puried bagasse hydrolysate was p-coumaric acid rather than
Phenolic acids ferulic acid. The puried products showed the same antioxidant activity, reducing power and free radical
Coumaric acid scavenging capacity as the standard p-coumaric acid.
Production
The technology could be applied on industrial scale.
Industrial relevance: This research presents a technology to produce coumaric acids from sugarcane bagasse.
The rst step is to release coumaric acid by alkaline hydrolysis. The second step is to remove the viscous
polysaccharides and protein by ultraltration. The third step is to purify coumaric acid from the permeate of
ultraltration by anion chromatography, and the alkaline could be reused to hydrolyze the bagasse. The
technology showed potential application on industrial scale.
2008 Elsevier Ltd. All rights reserved.

1. Introduction acid has been shown to inhibit indoleamine 2, 3-dioxygenase (IDO)

Sugarcane bagasse, a waste by-product of the sugar and alcohol
industries, is generated in large quantities, about 54 million tons of
bagasse being produced annually throughout the world (Sun, Sun,
Sun, & Su, 2004). The main components of bagasse are carbohydrates
(40%45% cellulose and 30%35% hemicelluloses) and lignin (Xu, Sun,
Liu, & Sun, 2006). Bagasse also contains phenolic acids, especially
trans-ferulic acid and coumaric acid linked to hemicellulose and
lignin, 1.29% and 1.76% respectively on a dry matter basis (Xu et al.,
2005).
Ferulic acid has many physiological functions, including antiox-
idant, antimicrobial, anti-inammatory, anti-thrombosis, and anti-
cancer activities (Ou & Kwok, 2004). It also protects against coronary
disease, lowers serum and hepatic cholesterol, and increases sperm
viability (Ou & Kwok, 2004). P-coumaric acid exerts antioxidant
activity both in vitro and in vivo, and showed a protective effect from
UV-B-induced oxidative damage in SIRC cells (Lodovici, Raimondi,
Guglielmi, Gemignani, & Dolara, 2003) and doxorubicin-induced
oxidative stress in rat's heart (Abdel-Wahab et al., 2003). P-coumaric
Corresponding author.
E-mail address: tosy@jnu.edu.cn (S.Y. Ou).
expression in IFN--activated macrophages and prolactin (PRL)
secretion (Kim et al., 2007). IDO, a key enzyme that catalyses the
initial and rate-limiting step in the degradation of tryptophan, is
simultaneously expressed both in murine dendritic cells and macro-
phages stimulated with interferon- (IFN-). And PRL, one of ele-
ments in the immune-neuroendocrine network, is mainly synthesized
in and secreted from the anterior pituitary gland and lymphoid organs
(Kim et al., 2007), suggesting that p-coumaric acid plays role in
immune regulation in human.
However, animal tests showed that the bioavailability of phenolic
acids bound by ber is relatively low in spite of their high levels in
ber and the bioavailability was not improved by feed adaptation.
Bound ferulic acid, especially diferulic acids, may not improve but,
rather, limit the bioavailabilities of non bound phenolic acids in vivo,
suggesting that ingestion of free phenolic acids is more effective than
bound phenolic acids for conveying their physiological functions to
the body (Zhao, Egashira, & Sanada, 2005).
In order to prepare value-added products from sugarcane bagasse
and to remove the components that toxify yeast during ethanol
production (Chandel, Kapoor, Singh, & Kuha, 2007), purication of
phenolic acids from the alkali-hydrolysate of sugarcane bagasse was
carried out by anion exchange chromatography in this study. The
antioxidant activity of the puried compounds from bagasse was also
investigated.

1466-8564/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2008.10.008
254 S.Y. Ou et al. / Innovative Food Science and Emerging Technologies 10 (2009) 253259
2. Materials and methods
2.1. Materials
Sugarcane bagasse was obtained from Overseas Chinese Sugar
Processing Company (Taishan, Guangdong Province, China). Styrene
anion exchange resin 717 (Strong basis, with exchange capacity
3.6 mmol/g), was purchased from Tianyuan Group Shanghai Resin
Factory Co., LTD (Shanghai, China). Ferulic acid, p-coumaric acid,
thiobarbituric acid, 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH)
were purchased from Sigma-Aldrich (St. Louis, Mo). All other
chemicals and solvents used were of analytical grade.
2.2. Alkaline hydrolysis of sugarcane bagasse
Sugarcane bagasse was hydrolyzed using 1% NaOH solution ac-
cording to our previous report (Ou, Luo, Xue, & Huang, 2007) with
some modication. The bagasse was rst oven dried to 4% moisture
content and then ground to pass a 45-mesh sieve. Triplicates of 2000 g
batches of sugarcane bagasse were mixed with 20 L of a solution of
10 g/L NaOH and 100 mg/L NaHSO3 (to prevent phenolic acid
oxidation) in a 30-L reactor and hydrolyzed at 40 C, 160 rpm for 4 h.
After hydrolysis, the mixture was centrifuged in a basket centrifuge at
4000 g and the residue washed with 2 2000 mL of water. The ltrates
were combined and ultraltrated to about 1000 mL using a NUF model
hollow ber ultraltrator (Wuxi Ultraltrating Equipment Company,
Jiangsu Province, China) with a 5000 Da molecular cutoff. The
ultraltration permeates were collected and the concentration of
phenolic acids was determined spectrophotometrically at 320 nm in a
1 cm cuvette with a UV-2101 PC UV/VIS Scanning Spectrophotometer
(Shimadzu), ferulic acid as a standard.
2.3. Separation of phenolic acids from ultraltration permeates by anion
exchange chromatograph
2.3.1. Determination of exchange capacity of styrene anion exchange
resin for phenolic acids in ultraltration permeates
Styrene anion exchange resin 717 was pre-treated according to the
manufacturer and then 1 mL of styrene anion exchange resin 717 was
mixed with 100 mL of permeate in a 250-mL ask and stirred at
200 rpm for 4 h at room temperature. The ion-exchange capacity was
calculated according to the difference of concentration of phenolic
acids (based on ferulic acid) before and after adsorption.
2.3.2. Determination of optimal ow rate of ultraltration permeates in
resin column
10 mL of pre-treated styrene anion exchange resin 717 and 100 mL
ultraltration permeate were loaded into a glass column (i.d. = 2.8 cm)
and a series of permeates eluted at rates of 1.0, 2.5, 5.0 mL/min.
Phenolic acid concentration (based on ferulic acid) in the eluates was
determined spectrophotometrically at 320 nm.
2.3.3. Effect of concentration of phenolic acids in ultraltration
permeates on adsorption quantity
The ultraltration permeates were diluted by 2, 3, 4, 5 times using
1% NaOH solution. 10 mL of styrene anion exchange resin 717 and
100 mL of diluted ultraltration permeates were loaded into a glass
column (i.d. = 2.8 cm) and eluted at 1.0 mL/min. Phenolic acid concen-
trations (based on ferulic acid) in the permeates were determined
spectrophotometricately at 320 nm.
2.3.4. Optimization of eluent solution
10 mL of the phenolic acids-adsorbed resins were eluted using the
solutions in Table 3 at 1 mL/min. Optimal eluent solution mixture was
determined according to the optimal recovery of phenolic acids in the
eluents.
10 mL of phenolic acids-adsorbed resin was eluted with 150 mL of
H2O/Ethanol/HCl (36:60:4) at 1 mL/min. Phenolic acids (A320, based
on ferulic acid) were determined on 10 mL fractions of eluent.
2.3.5. Batch tests (Fig. 1)
250 mL of pretreated styrene anion exchange resin 717 were
loaded into in a column (i.d. = 8 cm). 20 L of ultraltration permeates
were passed through the resin at a constant rate of 10 mL/min by a
peristaltic pump. After the hydrolyzed bagasse permeate was bound
to the column, the resin was washed with successive portions of
1000 mL of deionized water at 50 C, 300 mL of ethanol and then
300 mL 2% HCl at 20 mL/min. Phenolic acids were then desorbed from
the column using 1500 mL H2O/Ethanol/HCl (36:60:4) at 10 mL/min.
The rst 200 mL of eluents was discarded and the next 1300 mL of
eluents were collected and evaporated under reduced pressure with a
rotary evaporator (model RE52-3 Shanghai Huxi Instrumental
Company, Shanghai, China) at 40 C. The residue was crystallized at
4 C for 4 h, the crystals collected by ltration, carefully washed with
100 mL 0.1% HCl solution, and then lyophilized at 20 C and 10
100 Pa for 20 h (FD-1 model lyophilizer Beijing Boyikang Instrument
CO. Ltd., Beijing, China) (Fig. 1).
Color and phenolic acid concentration in all the elutes were
determined spectrophotometrically at 520 nm and 320 nm (ferulic
acid as a standard) respectively, the color value (U/mL) was calculated
as OD520 dilution factor.
2.3.6. Determination of phenolic acids by HPLC
The analytical HPLC system consisted of an Agilent 1100 Series
high-performance liquid chromatograph (Waldbronn, Germany)
equipped with a diode array detector. The HPLC pumps, autosampler,
column oven, and diode array system were monitored and controlled
using the HP Chem Station computer program. The injection volume
was 10 L. Phenolic acid separation was done with an Eclipse XDB-C18
(4.6 mm 250 mm, 5 m) and the temperature of the column oven was
set at 40 C. Elution was carried out using an isocratic system consisting
of 1% acetic acid: methanol (72:28) at 1 mL/min. Phenolic acids were
detected at 313 nm, with ferulic acid and coumaric acid as standards.
2.3.7. Determination of phenolic acids by LC/MS
An API-2000 LCMS/MS triple quadrupole mass spectrometer
equipped with a Turbo Ion SprayTM ionization source (Applied
Biosystems/MDS Sciex, Toronto, Canada) was used for mass spectro-
metry. The analytical column was a SS Exsil ODS C18 (4.6 150 mm, 5 m).
The mobile phases were 1% acetic acid: methanol (72:28) at 200 l/min.
The source dependent parameters optimized were nebuliser gas (gas 1):
20 psig, heater gas (gas 2): 30 psig, source temperature: 150 C.
Compound dependant parameters set were declustering potential (DP)
78 V, entrance potential (EP) 10 V and focusing potential (FP) 300 V.
2.4. Antioxidant test
A ferric thiocynate (FTC) method and a thiobarbituric acid (TBA)
method were used to investigate phenolic acid antioxidant activity in
a linolic acid system according to Shanmugasundaram and
Fig. 1. Batch process diagram for preparation of phenolic acids from sugarcane bagasse.
 S.Y. Ou et al. / Innovative Food Science and Emerging Technologies 10 (2009) 253259 255

Table 1 Table 3
Effect of ow rate of ultraltration permeates (100 ml at total) on adsorption quantity Effect of different eluents on recovery of phenolic acids in eluents
by styrene anion exchange resin 717 (10 ml)
Test Ethanol Water 37% HCl HAc Citric acid Recovery
Flow rate (mL/ml resin/min) Exchange ratio (C0/Ce)a (mL) (mL) (mL) (mL) (g) (%)a
0.10 0.86 0.04 1 0 100 0 0 0 0.66 0.07
0.25 0.84 0.04 2 60 40 0 0 0 0.04 0.00
0.50 0.67 0.02 3 0 96 4 0 0 4.28 0.08
a 4 60 36 4 0 0 99.01 0.86
C0 and Ce are phenolic acid concentration in ultraltration permeates and resin
5 60 38 2 0 0 81.18 0.74
eluents respectively (n = 3).
6 60 38 0 2 0 73.56 0.80
7 60 40 0 0 2 86.89 0.83
a
Recovery = phenolic acids (mg) in eluents/phenolic acids adsorbed (mg) using
ferulic acid as a standard(n = 3).
Venkataraman (2006). A mixture of either 1 mg or 2 mg resin-puried
extract in 5 mL absolute ethanol, 5 mL of 2.5% linoleic acid in absolute Viladomat, Bastida, Poli, & Codina, 2008). Samples were dissolved in
ethanol, 10 mL of 0.05 M phosphate buffer (pH 7.0), 1 mL of 0.02 M methanol with different concentrations: 0.2 mL of sample mixed with
FeSO4, and 5 mL of water was placed in a test tube with a screw cap 5 mL of 2 10 4 M DPPH (A), 0.2 mL of sample mixed with 5 mL of 95%
and then placed in an oven at 40 C in the dark. The control (no methanol (B), 0.2 mL of deionized water mixed with 5 mL of 2 10 4 M
phenolics) and the standard were subjected to the same procedures as DPPH (blank 1) and 0.2 mL of deionized water mixed with 5 mL of 95%
the sample except that for the control, only the EtOH solvent was methanol (blank 2). These mixtures were reacted at 25 C for 30 min,
added, and for the standard, 1 and 2 mg samples of hydrolysate were centrifuged at 4000 rpm for 10 min, and supernatant absorbance
replaced with standard ferulic acid and p-coumaric acid respectively. measured at 517 nm. The nal concentrations of the extract were 200,
400, 600, 800 and 1000 g/mL respectively, with p-coumaric acid
2.4.1. FTC method for antioxidant activity standards.
To 0.1 mL of the reacted linoleic acid mixture, 9.7 mL of 75% ethanol The scavenging capacity for DPPH (P) was calculated as follows:
and 0.1 mL of 30% ammonium thiocyanate was added. Precisely 3 min  
after the addition of 0.1 mL of 0.02 M FeCl2 in 3.5% HCl, the absorbance A1 A2
P = 1 100
was measured at 500 nm for every 24 h for 7 d. A3
Where A1 = A517 of A; A2 = A517 of B; A3 = (A517 of blank 1 A517 of
2.4.2. TBA method for antioxidant activity
blank 2).
1 mL of 20% trichloroacetic acid and 2 mL of 0.3% TBA aqueous
solution (dissolved in 0.5% trichloroacetic acid solution) were added to
2.7. Statistical analysis
1 mL of linoleic acid sample solution. The mixture was placed in a
boiling water bath for 10 min, cooled, centrifuged at 3000 rpm for
Statistics with replicates were determined using SPSS 11.0 for
20 min and the absorbance of the supernatant measured at 532 nm.
Windows procedure.

2.5. Reducing power

3. Results and discussion

The reducing ability of resin-puried extract was measured using

Phenolic acids, such as coumaric acid and ferulic acid are mainly
the ferric reducing/antioxidant power (FRAP) assay (Kong & Xiong,
esteried to hemicellulose and lignin in sugarcane bagasse (Xu et al.,
2006). 0.5 ml of sample solution were mixed with 5 mL of 0.2 M
2005), thus 1% NaOH solution was used to free them into the
phosphate buffer (pH 6.6) and 5 mL of 1% (w/v) K3Fe(CN)6 and left react
hydrolysates as an anion state. And ultraltration was applied to separate
at 50 C for 20 min in darkness. The reaction was stopped by adding
protein and viscous polysaccharides from the hydrolysate into the
2.5 ml 10% (w/v) of trichloroacetic acid (TCA) and the mixture
retentate and avoided adhesion of these macromolecules to anion
centrifuged at 4000 rpm for 10 min. 5 mL of the supernatant were
exchange resin.
mixed with 5 mL of distilled water and 1 mL of 0.1% (w/v) FeCl3, and the
absorbance measured at 700 nm. Samples were dissolved in ethanol
3.1. Separation of phenolic acids from ultraltration permeates by anion
with nal concentrations of 200, 400, 600, 800 and 1000 g/mL, and
exchange chromatograph
compared with p-coumaric acid standards.
3.1.1. Exchange capacity of styrene anion exchange resin for phenolic
2.6. Scavenging capacity of resin puried extract for
acids in ultraltration permeates
2,2-diphenyl-1-picrylhydrazyl free radical (DPPH)
The puried permeate by ultraltration contained 1612.8 mg/L phe-
nolic acids (according to absorbance at 320 nm using ferulic acid as a
The quenching of free radicals by extracts and puried fractions was
evaluated spectrophotometrically at 517 nm by the decolouration of a
methanol solution of DPPH (Backhouse et al., 2008; Faudale,

Table 2
Effect of phenolic acid concentration in ultraltration permeates on adsorption quantity
by styrene anion exchange resin 717 (n = 3)

Phenolic acids concentration Phenolic acids concentration Phenolic acids

before adsorption (mg/L) in the eluents (mg/L) adsorbed (%)
1612.8 235.4 7.8 85.4
806.4 122.6 6.2 84.8
537.6 80.64 6.3 85.0
403.2 62.1 5.4 84.6
322.6 50.3 4.7 84.4
Fig. 2. Elution curve for phenolic acids-adsorbed anion exchange resin.
256 S.Y. Ou et al. / Innovative Food Science and Emerging Technologies 10 (2009) 253259

Fig. 3. HPLC of the puried sample (upper), p-coumaric acid (middle) and ferulic acid (below).

standard). The maximum exchange capacity of anion exchange resin 717 3.1.2. Effect of ow rate and concentration of phenolic acids in
for phenolic acids from sugarcane bagasse alkaline hydrolysates was ultraltration permeates on adsorption quantity
138.5 mg/mL (0.71 mmol/ml based on ferulic acid), lower than its theo- The ow rate of ultraltration permeates through the anion
retical exchange capacity (3.6 mmol/mL, rated by the resin manufacturer). exchange resin 717 signicantly affected the adsorption quantity
 S.Y. Ou et al. / Innovative Food Science and Emerging Technologies 10 (2009) 253259 257

Fig. 4. Mass spectrum of major puried product peak from HPLC.

(Table 1), when the ow rate increased from 0.1 to 0.5 mL per mL of
resin within 1 min, the exchange ratio decreased 22.1%. However, the
concentration of phenolic acids in the ultraltration permeates did not
affect the adsorption quantity (Table 2). When the concentration of
phenolic acids changed from 1612.8 to 322.6 mg/l, no signicant
change of percentage of adsorbed phenolic acids was observed. It is
possible that the unabsorbed absorbance at 320 nm (approximately
15%) may be contributed by other nonionic substances.
The advantage of using unneutralized sugarcane bagasse alkaline
hydrolysates to load onto the anion resin column is that NaOH could
be reused, thus greatly decrease release of pollutants to the envi-
ronment, and also to prevent the neutralized product, NaCl, from
decreasing the efciency of the anion resin.
3.1.3. Elution of phenolic acids from the resin
According to ion exchange principle, the adsorbed phenolic acids
at basic or neutral conditions should be eluted by acid solution.
However, 4% HCl only eluted less than 5% of the adsorbed phenolic
acids (Table 3). We speculate that two kinds of forces, namely ionaline
hydrolysates were ultraltrated, the permeates puion forces and
hydrophobic interactions between aromatic phenolic acids and the
resin contributed to the adsorption of phenolic acids. Thus, ethanol
was added to the acid eluent solution, including one strong acid HCL
and two weak acids, acetic acid and citric acid, and it was found that
addition of ethanol greatly increased desorption of phenolic acids
from the resin. The elution effect by mixture of ethanol with dilute HCl
was much better than that with two weak acids. When the water:
ethanol:HCl ratio was 36:60:4, almost all of adsorbed phenolic acids
were eluted (Table 3). This observation indicated that impurities and
colors adsorbed to the resin could be removed by sequentially
washing with water, HCl and ethanol before eluting phenolic acids
with the optimal solution.
The phenolic acid-adsorbed resin was eluted using the optimal
eluent, H2O/Ethanol/HCl (36:60:4) and the results were shown in
Fig. 2. Phenolic acids could be detected at 30 mL of eluent and the peak
appeared at 50 mL. After 80 mL of eluent, 87% of adsorbed phenolic
acids were washed out calculated according to the data in Fig. 2.
3.1.4. Batch tests results
Batch test showed that 80.25% of phenolic acids (determined
spechtrophotometrically at 320 nm based on ferulic acid) was ad-
sorbed by anion exchange resin. Color value in 1000 mL of deionized

Table 4
Antioxidant activity of alkali-released products, ferulic acid and coumaric acid against linoleic acid peroxidation (FTC method, n = 2)

A500
Time(d) Control F.A.a (1 mg) F.A.a (2 mg) p-C.Aa. (1 mg) p-C.Aa (2 mg) P.B.Ha (1 mg) P.B.H.a (2 mg)
0 0.16 0.01 0.16 0.00 0.17 0.01 0.16 0.01 0.16 0.01 0.16 0.01 0.16 0.00
1 0.53 0.02 0.18 0.02 0.17 0.03 0.17 0.01 0.17 0.02 0.17 0.02 0.17 0.01
2 1.00 0.03 0.18 0.04 0.18 0.02 0.18 0.01 0.17 0.02 0.18 0.01 0.16 0.01
3 1.47 0.04 0.17 0.04 0.14 0.01 0.19 0.02 0.14 0.01 0.18 0.02 0.11 0.00
4 1.71 0.05 0.16 0.03 0.11 0.01 0.15 0.01 0.12 0.01 0.15 0.01 0.11 0.01
5 2.09 0.02 0.15 0.00 0.12 0.01 0.14 0.02 0.12 0.02 0.13 0.00 0.12 0.01
6 1.49 0.04 0.13 0.04 0.13 0.02 0.13 0.02 0.11 0.01 0.14 0.02 0.10 0.00
7 1.31 0.05 0.13 0.02 0.12 0.01 0.12 0.01 0.12 0.02 0.13 0.01 0.13 0.01
a
Ferulic acid (F.A.); p-Coumaric acid (p-C.A.); Puried bagasse hydolysate (P.B.H.).
258 S.Y. Ou et al. / Innovative Food Science and Emerging Technologies 10 (2009) 253259

Table 5
Antioxidant activity of alkali-released products, ferulic acid and coumaric against linoleic acid peroxidation (TBA method, n = 2)

A532
Time (d) Control F.A.a (1 mg) F.A.a (2 mg) p-C.A.a (1 mg) p-C.Aa (2 mg) P.B.Ha (1 mg) P.B.H.a (2 mg)
0 0.42 0.02 0.43 0.01 0.43 0.01 0.43 0.01 0.43 0.01 0.42 0.02 0.42 0.02
1 0.69 0.04 0.48 0.02 0.45 0.03 0.48 0.03 0.44 0.02 0.49 0.2 0.45 0.01
2 0.90 0.03 0.49 0.04 0.47 0.02 0.49 0.03 0.51 0.03 0.49 0.04 0.48 0.02
3 1.68 0.06 0.78 0.05 0.74 0.04 0.77 0.04 0.66 0.04 0.80 0.03 0.62 0.03
4 2.43 0.04 0.49 0.02 0.45 0.02 0.51 0.01 0.46 0.03 0.52 0.02 0.43 0.03
5 2.30 0.06 0.45 0.02 0.44 0.03 0.45 0.02 0.48 0.02 0.56 0.04 0.40 0.02
6 1.83 0.02 0.66 0.04 0.58 0.04 0.64 0.03 0.54 0.04 0.80 0.02 0.58 0.03
7 1.60 0.03 0.53 0.03 0.43 0.03 0.52 0.04 0.43 0.03 0.54 0.04 0.44 0.03
a
Ferulic acid (F.A.); p-Coumaric acid (p-C.A.); Puried bagasse hydolysate (P.B.H.).

water, 300 mL of ethanol and 300 mL 2% HCl was 0.21, 0.65 and 0.12 U/
mL respectively, indicating that washing with warm water, ethanol and
HCl before desorption not only removed impurities bound by the resin,
but also decreased the color of the nal product. 13.56 g of phenolic
acids were obtained from 1000 g sugarcane bagasse after hydrolyza-
tion and eluting with H2O/Ethanol/HCl and crystallization.
3.2. Identication of phenolic acids in the puried sample
HPLC indicated one large peak (retention time 23.29 min) and two
small peaks in the puried samples, with the main component being
coumaric acid (retention time 23.37 min) instead of ferulic acid
(retention time 18.94 min) (Fig. 3). HPLC/MS analysis of the puried
samples (Fig. 4) showed three negative ions (163, 117, 145) in
accordance with mass spectra of p-coumaric acid which had three
positive ions (165, 119 and 147) according to Ryana, Robardsa, and
Laveeb (1999), further indicated that the main component of the
hydrolysate is p-coumaric acid. After purication by anion exchange
resin, the purity of products reached 89.7% quantitatively determined
by HPLC.
Ferulic acid is esteried to plant cell wall materials and can be
released by alkaline treatment (Liyana-Pathirana & Shahidi, 2006;
Martinez-Tome et al., 2004; Pan, Bolton, & Leary, 1998; Sun, Sun, &
Zhang, 2001), however, no ferulic acid was detected in the bagasse
hydrolysates by moderate alkaline hydrolysis conditions. One possible
reason is that ferulic acid and coumaric acid link to cell wall material in
different patterns, p-coumaric acid is principally associated with
lignin, whereas ferulic acid is mainly esteried with hemicellulose. The
linkage between ferulic acid and lignin largely depends on the raw
material identity (Torre, Aliakbarian, Rivas, Domnguez, & Converti,
2008). Another reason is that the bond between ferulic acid and
hemicellulose is stronger than the bond between coumaric acid and
hemicellulose, making the former more difcult to hydrolyze by
alkaline. If this is true, a two-step hydrolysis, where a higher concen-
tration NaOH hydrolysis follows a lower concentration hydrolysis
might be used to prepare ferulic acid and coumaric acid from sugar-
cane bagasse separately using anion exchange resin.
samples at two concentrations signicantly decreased A500 in a dose-
dependent manner compared to the control, its antioxidant activity
equaled to coumaric acid and ferulic acid. However, A500 increased at
an early stage and then decreased, indicating that peroxides decreased
with depletion of linoleic acid.
The formation of malonaldehyde in the TBA method is the basis for
evaluation of the extent of lipid peroxidation. Results in Table 5
showed that the puried bagasse hydrolysates had the same activity
for retarding oxidation of linoleic acid as ferulic acid and p-coumaric
acid.
Reducing power is one mechanism for the action of antioxidants; it
gives a value of electron-donating capacity of antioxidant (Garca-Sols,
Yahia, & Aceves, 2008; Karagzlera, Erdab, Emekb, & Uyguna, 2008).
The DPPH radical scavenging assay is commonly employed in
evaluating the ability of antioxidants to scavenge free radicals. The
change in absorbance at 517 nm is used as a measure of the scavenging
effect of a compound for DPPH radicals. The absorbance at 517 nm
decreases as the reaction between antioxidant molecules and DPPH
radical progresses. Hence, the more rapidly the absorbance decreases,
the more potent the antioxidant activity of the extract in terms of its
hydrogen atom-donating capacity (Alasalvar, Karamac, Amarowicz, &
Shahidi, 2006). Results in Table 6 showed that the puried sample
from sugarcane bagasse alkaline-hydrolysates had similar reducing
power and free radicals scavenging capacity to p-coumaric acid.
4. Conclusion
Soaking of sugarcane bagasse by suspending sugarcane bagasse in
ten volume of 1% NaOH (w/v) at 30 C for 4 h mainly released p-
coumaric acid among the bound phenolic acids. The p-coumaric acid
could be puried by anion exchange chromatography and the puried
sample showed the same antioxidant activity as standard p-coumaric
acid. NaOH and the resin could be reused in our experimental
technology, which showed potential application in commercial
production of natural p-coumaric acid from sugarcane bagasse.
Acknowledgment

3.3. Antioxidant activity, scavenging capacity for free radicals and We appreciate the Department of Education of Guangdong
reducing power of the prepared samples Province for supporting this research project (cgzhzd0709).

To investigate whether the product still retained the antioxidant

activity of coumaric acid after hydrolysis treatment and purication, Table 6
the antioxidant activity, reducing power and scavenging capacity for Reducing power and DPPH scavenging capacity of alkali-released products and
free radicals of the prepared samples was tested in vitro. coumaric acid (n = 3)
The FTC antioxidant method was used to determine the amount of Concentration Reducing power (A700) DPPH scavenging capacity (%)
peroxide generated at the initial stage of lipid peroxidation. During the (g/mL) Puried sample Coumaric acid Puried sample Coumaric acid
linoleic acid oxidation, peroxides formed and these compounds 200 0.053 0.008 0.052 0.007 43.2 0.8 44.2 0.7
oxidize Fe2+ to Fe3+. The Fe3+ ions form complex with SCN, which 400 0.105 0.010 0.112 0.009 67.4 1.2 66.4 1.3
has a maximum absorbance at 500 nm, the concentration of peroxide 600 0.184 0.014 0.191 0.013 79.1 0.9 81.3 1.1
decreases after addition of antioxidants (Shanmugasundaram & 800 0.357 0.012 0.362 0.015 82.3 1.3 82.6 0.9
1000 0.370 0.009 0.381 0.011 83.8 1.2 83.1 1.4
Venkataraman, 2006). Results in Table 4 showed that the prepared
 S.Y. Ou et al. / Innovative Food Science and Emerging Technologies 10 (2009) 253259
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ifset.2008.10.008.
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