Supercritical Carbon Dioxide-Induced Hydrolysis of ... - ISSF 2012

Supercritical Carbon Dioxide-Induced Hydrolysis of Hesperidin
in Subcritical Water
ABSTRACT
Armando T. Quitain 1 , Duangkamol Ruen-ngam 2 , Wahyudiono 1 , Masahiro Tanaka 3 ,
Mitsuru Sasaki 1 and Motonobu Goto 2*
1 Graduate School of Science and Technology,
2 Bioelectrics Research Center, Kumamoto University
3 ASCII Corporation
* Corresponding Author: mgoto@kumamoto-u.ac.jp, Fax: +81-96-342-3665
Hesperidin (hesperetin-7-O-rutinoside), a flavanone glycoside is found abundantly in immature citrus fruits.
Clinical studies on hesperidin had shown antiatherosclerotic, anti-inflammatory, antitumor, antithrombogenic,
antiosteoporotic and antiviral effects. This tasteless compound can be converted into more useful chemical
compounds such as hesperetin, its aglycone and hesperetin-7--D-glucoside by hydrolysis. These products are
thought to be more valuable than hesperidin because these can be easily converted into an intensely sweet
compound by simple alkaline hydrogenation and can be used as starting materials for the preparation of dyes and
sweeteners that has value-added medicinal functions such as analgesic, anti-inflammatory, antioxidant and
anti-atherogenic properties. Conventional method of hesperidin hydrolysis to produce these valuable compounds
uses sulfuric acid as catalyst under elevated temperatures. To avoid the use of this harmful catalyst, supercritical
carbon dioxide-induced hydrolysis in subcritical water is proposed.
The synergistic effects of these two most promising green solvents gave better yields of the two target
degradation products without employing any harmful acid catalysts at a pressure range of 10-25 MPa and
temperature of 110-140C. The effect of operating conditions such as pressure, temperature, reaction time and
addition of cosolvent such as ethanol were found to affect the hydrolysis rate. Owing to an increase in formation
of carbonic acid, which serves as a catalyst for the reaction, hydrolysis rate increased at elevated pressure.
Moreover, the reaction was also found to be positively dependent on temperature, obtaining high yield of the
main products, hesperetin--glucoside and hesperetin. Addition of ethanol as a cosolvent might increase
solubility of hesperidin, however, it only inhibited the reaction and formation of carbonic acid. At the maximum
temperature of 140 o C and pressure of 25 MPa investigated in this work, the highest concentration of
hesperetin--glucoside of 4.2 mol/l was obtained in reaction time of 2 h, whereas for hesperetin the highest was
6.9 mol/l obtained in 3 h. Among the hydrolysis methods investigated, the proposed method was the most
selective towards formation of the target compounds.
INTRODUCTION
Bioflavonoids are present in substantial amount in citrus, especially the immature ones, and even in their
processing by-products [1-5]. These useful compounds possess bioactivities including preventive or therapeutic
effects on the treatment of numerous diseases such as cardiovascular-related illness and cancer [6-7]. Clinical
studies had also shown antiatherosclerotic, anti-inflammatory, antitumor, antithrombogenic, antiosteoporotic and
antiviral effects. The mechanisms of action and potential applications of these flavonoids had been reviewed by
Nijveldt el al. [4].
Hesperidin (hesperetin-7-O-rutinoside, hereby referred to as HPD), having a chemical structure shown in Fig.
1 (a), is one of many bioflavonoids that can be found mostly in immature citrus fruits, its composition can reach
more than 40% on a dry-weight basis. This tasteless compound can be a good source of more useful chemical
compounds such as hesperetin, its aglycone and hesperetin-7--D-glucoside. Hesperetin-7--D-glucoside
(hereby referred to as HBG) as shown in Fig. 1 (b), an intermediate hydrolysis product. This is known to be more
valuable than HPT because it can be easily converted into an intensely sweet compound by simple alkaline
hydrogenation. Clinical test conducted on this compound showed that it could increase circulation limitation
time in digestion process and could also decrease toxicity [4]. Another useful compound that can be obtained
from the hydrolysis of hesperidin is hesperetin (hereby referred to as HPT) as shown in Fig. 1 (c) can be used as
a starting material for the preparation of dyes and sweeteners that has value-added medicinal functions such as

analgesic, anti-inflammatory and antioxidant properties [8-11].
(a) Hesperidin (b) Hesperetin--glucoside (c) Hesperetin
Figure 1. Chemical structure of (a) hesperidin (HPD) (b) hesperetin--glucoside (HBG)
and (c) hesperetin (HPT)
Hydrolysis of HPD was first carried out way back in 1881 by Tiemann et al. as reported in the patent
application of Wingard [12]. The method employed aqueous sulfuric acid at elevated temperatures in the
presence of an alcohol cosolvent. To date, no significant modification to the method has been applied, and most
of the reported techniques by modern researchers utilize sulfuric acid as catalyst. Grohmann et al. [3] utilized
dilute sulfuric acid at a concentration of about 0.05% to hydrolyze hesperidin under elevated temperature
obtaining highest yield of glucose and rhamnose as products from hydrolysis reaction at 140C. Acid-catalyzed
hydrolysis under subcritical water conditions at the temperature range of 150 to 320 o C has also been applied to
the decomposition of some biochemical compounds including monosaccharides, disaccharides and cellulosic
biomass [13-18]. However, with the health risk posed by the use of such acid especially if the products are
intended for human consumption, alternative safe methods are currently being explored.
Utilizing supercritical carbon dioxide (ScCO2) to induce hydrolysis of hesperidin under hydrothermal
conditions without employing any harmful catalyst could be a better alternative. This method employs the idea
that the formation and dissociation of carbonic acid from the reaction of H2O and CO2 especially at elevated
temperatures and pressures serves as a catalyst for the reaction, thereby, enhancing hydrolysis rate [19-22]. The
catalytic effect of carbonic acid on hydrolysis was first investigated by Van Walsum et al. [23] on hot water
treatment of corn stover up to a pressure of 5.5 MPa and temperature range of 180-220C. Addition of carbonic
acid enhanced the occurrence of xylose and furan in hydrolysate. Instead of carbonic acid, Rogalinski et al. [22]
used supercritical carbon dioxide for the hydrolysis of biopolymers in subcritical water at temperatures of
240-280C, taking the liquefaction of cellulose as an example. The obtained experimental results up to a
temperature of 260 o C showed an enhancement in liquefaction rates of cellulose in hydrothermal-ScCO2 mixed
solvents at elevated pressure compared with only water. This result implied that the cleavage of glucosidic bond
of disaccharides could occur under acidic condition at elevated temperature pressurized by carbon dioxide. Only
limited information about the application of the technique to hydrolysis of natural compounds is available in
literatures, and nothing has been reported so far on the hydrolysis of citrus bioflavonoids.
In this work, the proposed technique was applied to the hydrolysis of HPD into above mentioned more useful
compounds (i. e. HBG and HPT). The effects of various parameters such as reaction time, temperature, pressure
and addition of cosolvent such as ethanol were investigated.
MATERIALS AND METHODS
Chemicals and reagents
Most chemicals and reagents used in the experiments, including standard samples of hesperidin (92%) and
hesperetin (97%) for quantitative analysis of the reaction products, and solvents such as acetic acid (99.9%) and
acetonitrile were purchased from Wako Pure Chemical Industries Ltd. (Kyoto, Japan). Carbon dioxide (99.9%)
and nitrogen (99.99%) gases were purchased from Uchimura-Sanso Co. Ltd. (Kumamoto, Japan).
Experimental apparatus and methodology
The schematic diagram of the experimental apparatus is shown in Fig. 2. It consists of a high-pressure pump
that delivers liquefied carbon dioxide into a 30-ml high pressure vessel that serves as a reactor. Six heating
probes were attached to the reactor. A thermocouple was also attached at the center of the reactor and connected
to a temperature controller to monitor and control the reaction temperature. Carbon dioxide was preheated prior
to its introduction into the reactor. The pressure of the system was controlled by a back-pressure regulator

(hereby referred to as BPR).
CO2 cylinder
cooler
CO2 pump
Figure 2. Schematic diagram of the experimental apparatus.
In a typical experiment, about 1 mg of hesperidin was dissolved in 20 ml distilled water (50 mg/l), then
loaded into a high-pressure 30-ml batch reactor with a Teflon-coated stirrer chip placed inside. The reactor was
closed and placed on top of a magnetic stirrer as shown in the schematic diagram in Fig. 2. While heating to the
set reaction temperature in the range of 100-140C, liquefied CO2 was pumped into the reactor until the desired
pressure of 10-25 MPa was reached. The temperature was controlled automatically by temperature controller,
while the pressure was adjusted using BPR when necessary. The content of the reactor was kept at the desired
reaction temperature and pressure for up to 4 h of reaction time at constant stirring. After the reaction time has
elapsed, the reactor was gradually cooled down to room temperature (25C). The samples were taken from the
reactor for analysis thereafter. If subsequent analysis could not be carried out right after each experimental run,
samples were stored in a refrigerator at a temperature of about 5C. Three trials were performed at each
experimental condition to check repeatability of the results.
Analysis of products
Quantitative amount of hesperidin (HPD), hesperetin-β-glucoside (HBG) and hesperetin (HPT) in the
samples were analyzed using a high performance liquid chromatography (HPLC) apparatus coupled with a diode
array detector (DAD) (Shimadzu Corporation, Japan). Samples were pretreated using a 0.45 m filter, then
placed in vial and arranged in a sample rack for automatic injection of about 10 µl. Separation was carried out
using an Inertsil®ODS-3 column (250 × 4.6 mm i.d., 4 m particle size) (GL Sciences Inc., Japan) at a
temperature of 35C. Gradient elution was performed using a mobile phase solvent A consisting of 0.1% acetic
acid in water, and nevlost B consisting of 0.1% acetic acid in 75% acetonitrile at a flow rate of 1.0 ml/min.
During the course of analysis, the ratios of solvent A and B were adjusted as follows: 0 min (78:22), 10 min
(72:28), 17 min (62:38) 30 min (52:48), 36 min (32:68), 40 min (0:100) 45 min (0:100), 60 min (78:22).
Compounds were detected at a wavelength of 285nm. The amount of hesperidin and hesperetin in the samples
were quantified using a standard curve prepared by dissolving standard compounds in methanol. The retention
time for hesperetin-β-glucoside was determined using the compound synthesized by the methods of Grohmann et
al. [3]. This was further verified by comparing the UV spectra of the peak with those reported in literatures [3,
24]. The calibration curve for HBG was estimated to be similar to that of HPD.
RESULTS AND DISCUSSION
heater
heater
Solubility and stability of hesperidin in H2O at elevated temperatures
Preliminary tests on solubility of HPD in water at elevated temperatures in the range of 110-140C were
carried out to determine suitable concentration for the experiments. About 1 to 200 mg of hesperidin standard
was dissolved in 20 ml of water, and then heated to reach the above mentioned temperatures. After heating, the
solution was allowed to cool down gradually to room temperature, and then kept overnight in the refrigerator at a
temperature of 5C prior to analysis. Based on the results, a concentration of 50 mg/l was chosen to be the
concentration of hesperidin in the succeeding hydrolysis experiments. This concentration, at which no
precipitates formed even after cooling at 5C, is close to 53 mg/l used by Grohmann et al. [3] in the study of
hesperidin conversion into hesperetin--glucoside. Moreover, stability tests on 1 mg of hesperidin in 20 ml of
water showed no decomposition of hesperidin took place when the mixture was kept at the above mentioned
P
T
Magnetic stirrer
BPR

temperatures for 4 h. This implies that hesperidin is stable at this temperature range, and reaction would not
proceed without adding suitable catalyst.
Preliminary tests on catalytic role of carbon dioxide at elevated pressure
As previously mentioned in the introduction, the proposed method employs the concept that the formation
and dissociation of carbonic acid from the reaction of H2O and CO2 according to eq. 1 especially under elevated
temperature and pressure serves as a catalyst for the reaction [22, 25].
- (1)
Carbonic acid, even though considered as a weak acid, may promote hydrolysis of HPD consisting of glycosidic
bonds that seems to be weaker than those of cellulose. Brito-Arias [26] had confirmed that phenolic glycosides
can be decomposed even in dilute acid solution, close to the acidic conditions being employed in this work. It
was also reported that glucosidic bond could be cleaved under carbonic acid catalyzed subcritical water [15, 22].
Similarly, cleavage of rhamnosidic and glucosidic bonds of HPD might also take place obtaining main products
such as HBG and HPT, respectively.
In this regard, the catalytic effect of adding carbon dioxide at elevated pressures on reaction under
hydrothermal condition was first verified by carrying out experiments at a pressure and temperature of 10 MPa
and 140C, respectively. The results were compared with those obtained using N2, an inert gas, instead of carbon
dioxide and in water without carbon dioxide. The catalytic effect of adding carbon dioxide is evident from the
obtained chromatograms in Fig. 3 (a). The peaks of the products (i. e. HBG and HPT at residence times of 23
and 38 min, respectively) are noticeably large in the presence of carbon dioxide compared to that of water alone
in Fig. 3 (c) or even when pressurized with an inert gas such as N2 at 10 MPa in Fig. 3 (b). This indicates that
hydrolysis of hesperidin took place in the presence of carbon dioxide at high pressure, confirming the catalytic
effect of its addition.
(a) water + CO2 at 10 MPa
hesperetin-β-glucoside
(b) water + N2 at 10 MPa
(c) water
hesperidin
hesperitin
Figure 3. Comparison of HPLC-DAD chromatographs of products obtained at 140C and
reaction time of 2 h
Dependency of hydrolysis reaction on carbon dioxide pressure
The solubility data of carbon dioxide in water at a pressure range of 10-25 MPa and temperature range of
110-150C, as reported by Takenouchi and Kenney [20], showed that the solubility of carbon dioxide increases
with increasing pressure. Even at a low pressure of 10 to 25 MPa, the solubility of CO2 in water may increase up
to 60%. This increase in solubility of carbon dioxide at elevated pressures enhances the formation of carbonic
acid, which serves as a catalyst for the reaction [20-22, 24].
Experiments at 140C and reaction time of 2 h were performed at various pressures up to 25 MPa.
Preliminary results show decreasing trend of HPD concentration, with increasing pressure. This indicates a
positive dependency of hydrolysis rate with increasing carbon dioxide pressure, evident from the increase in
products concentration.
The conversion of HPD was close to 52%, while the reaction was more selective towards HBG formation. At
a higher pressure of 25 MPa, the conversion increased, but the selectivity of HBG decreased due most likely to
its further decomposition to HPT as a result of the cleavage of the glucosidic bond.

Concentration 10 5 (mol/l)
Relation of hydrolysis reaction on temperature and time
Reactions under hydrothermal conditions are highly dependent on temperature and time. It is expected that at
a relatively lower temperatures the hydrolysis would proceed slowly, thus it would take longer time for the
reaction to reach equilibrium or completion. In contrast, at higher temperatures, the reaction rate is fast obtaining
high yield even in short time.
This behavior is evident from the results of the experiments at a pressure of 25 MPa conducted on the effect of
time up to 4 h and temperature of 110-140C on the amount of each compound in hydrolysate as shown in Fig. 4
(a) - (c). The initial rate of reaction indicates by the slope of the graph for the change in concentration of
hesperidin with time at various temperatures increases with increasing temperature. As a result, the formation of
two main products, especially HPT, increases with increasing temperatures from 110 to 140C.
The highest amount of HBG of about 4.2 mol/l was obtained at reaction time of 2 h at a temperature of 140C
as shown in Fig. 4 (b). At longer reaction time, the amount decreased gradually due to its possible decomposition
to other compounds such as HPT as indicated by the results shown in Fig. 4 (c). The highest HPT concentration
of about 6.9 mol/l was obtained at reaction time of 3 h, attaining reaction equilibrium thereafter. The conversion
of HPD reached the maximum at 73.3% in 4 h, while the selectivity to HBG was high initially at 71.1% and
decreased with time reaching only about 39.8% in reaction time of 4 h.
10
9
8
7
6
5
4
3
2
1
Concentration 10 5 (mol/l)
10
9
8
7
6
5
4
3
2
1
0
0 1 2 3 4 5
Time (h)
0
0 1 2 3 4 5
Time (h)
(a) hesperidin
10
(b) hesperetin--glucoside (c) hesperetin
Concentration 10 5 (mol/l)
9
8
7
6
5
4
3
2
1
T ( C) Symbol
140
130
120
110
0
0 1 2 3 4 5
Time (h)
Figure 4. Effect of temperature and reaction time at 110-140C, 25 MPa
Based on these results, the hydrolysis of HPD is speculated to consist of complex reactions involving
consecutive reactions via HBG route to its final decomposition product – HPT. Parallel reaction involving direct
cleavage of the glucosidic bond in HPD to form HPT is also likely to occur. The degradation behavior can be
further explained by the fact that an increase in temperature decreases activation energy of the reaction, making

the hydrolysis faster. On other hand, at lower temperature of 110C, it is reported that HPD is quite resistant to
acid-catalyzed hydrolysis [2, 24], thus obtaining lower yield.
Based on these results, the overall degradation reaction of hesperidin follows a first-order rate kinetics, and the
concentration of hesperidin,

CONCLUSION
Hydrothermal hydrolysis of hesperidin (HPD), a bioflavonoid predominantly present in immature citrus fruits,
was investigated under hydrothermal conditions pressurized by supercritical carbon dioxide.
Hesperetin-β-glucoside (HBG) and hesperetin (HPT) were the main products obtained by the cleavage of
rhamnosidic and glucosidic bonds of HPD, respectively. These products are known to be more valuable than
HPD, and are used as precursors for the synthesis of functional compounds like sweeteners.
The catalytic effect of adding carbon dioxide under supercritical conditions was first verified, and the results
were further compared with those obtained using N2, an inert gas. Detailed experiments carried out in a
temperature range of 110-140 o C and pressures up to 25 MPa showed a positive dependency of hydrolysis rate
with increasing CO2 pressure owing to the increased formation of carbonic acid. The highest yields of HBG and
HPT were obtained at the maximum investigated pressure of 25 MPa and temperature of 140 o C. The highest
concentration of HBG of about 4.2 mol/l was obtained in 2 h of reaction time, whereas the highest concentration
of 6.9 mol/l of HPT was obtained in 3 h. The highest conversion of HPD was close to 52%. Addition of ethanol
(EtOH) as cosolvent might increase the solubility of HPD, but it inhibited hydrolysis reaction and the formation
of carbonic acid. Thus, addition of EtOH as cosolvent is not recommended in this method. Using the proposed
method, the reaction was also found more selective to the formation of the target compounds better than the
other hydrolysis methods reported in literatures.
The application supercritical carbon dioxide-induced hydrolysis of natural products without using any harmful
catalysts or organic solvents lays the groundwork for further development of this promising technology. Being
safe, clean and green, the outlook is bright for the application of this technology to food and pharmaceutical
industry. Future investigation includes elucidation of the mechanism of its action while widening the scope of its
possible applications.
ACKNOWLEDGEMENTS
This work was partly supported by the Ministry of Economy, Trade and Industry (Japan) and Kumamoto
University Global COE Program.
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