Acid-catalyzed hydrolysis of hesperidin at elevated temperatures

Article abstract of DOI:10.1016/S0008-6215(00)00081-1

Dilute sulfuric acid was used as a catalyst for hydrolysis of hesperidin suspensions in water at temperatures ranging from 25 to 180 °C. Significant acceleration of the reaction was observed at 120 °C and higher temperatures. This increase in the rate of hydrolysis can be attributed to increased solubilization of hesperidin in water at higher temperatures. Partial hydrolysis of hesperidin at 140 °C was used for the preparations of hesperetin-7-glucoside, which has a value in the synthesis of dihydrochalcone sweeteners. Simple separation of hesperetin and hesperetin-7-glucoside by extraction with dry acetone or lower alcohols has been developed. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00081-1

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Carbohydrate Research 328 (2000) 141–146
Acid-catalyzed hydrolysis of hesperidin at elevated
temperaturesꢀ
Karel Grohmann *, John A. Manthey, Randall G. Cameron
US Citrus and Subtropical Products Laboratory, 600 A6e. S., N.W., Winter Ha6en, FL 33881, USA
Received 12 October 1999; accepted 16 March 2000
Abstract
Dilute sulfuric acid was used as a catalyst for hydrolysis of hesperidin suspensions in water at temperatures ranging
from 25 to 180 °C. Significant acceleration of the reaction was observed at 120 °C and higher temperatures. This
increase in the rate of hydrolysis can be attributed to increased solubilization of hesperidin in water at higher
temperatures. Partial hydrolysis of hesperidin at 140 °C was used for the preparations of hesperetin-7-glucoside,
which has a value in the synthesis of dihydrochalcone sweeteners. Simple separation of hesperetin and hesperetin-7-
glucoside by extraction with dry acetone or lower alcohols has been developed. © 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Hesperidin; Hesperetin; Hesperetin-7-glucoside; Rhamnose; Acid hydrolysis
immature fruit, which can contain more than
1. Introduction
40% hesperidin on a dry-weight basis, but the
concentration still exceeds 2% of dry weight of
Hesperidin,
(S)-7-[a-rhamnopyranosyl-
peel, rag and pulp in mature sweet oranges,
while the concentration in juice is only 0.02–
0.06% [2,3].
(1“6)-b- -glucopyranosyloxy]-2,3-dihydro-
D
5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-
4H-1-benzopyran-4-one, a tasteless rham-
noglucoside (rutinoside) of hesperetin, is one
of the most available flavonoid compounds. It
is present in fruit, leaves and in the bark of
orange (Citrus sinensis) and other citrus spe-
cies [1]. Its concentration is especially high in
The bulk of hesperidin (\90%) is thus
present in processing byproducts, and a minor
amount is in the juice [2]. The citrus process-
ing industry in Florida alone can potentially
supply \10,000 tons of hesperidin on an
annual basis, with additional hesperidin avail-
able from the processing industry in Brazil
and elsewhere. Suspended solids in citrus peel
juice and molasses are enriched in hesperidin
and other insoluble flavonoids [3], but the
bulk of hesperidin would have to be extracted
from peel and other solid byproducts [2].
Hesperidin can be a source of valuable
chemicals and chemical intermediates. Rham-
ꢀ South Atlantic Area, Agricultural Research Service, US
Department of Agriculture. Mention of a trademark or propri-
etary product is for identification only and does not imply a
guarantee or warranty of the product by the US Department
of Agriculture. The US Department of Agriculture prohibits
discrimination in all its programs and activities on the basis of
race, color, national origin, gender, religion, age, disability,
political beliefs, sexual orientation, and marital or family
status.
nose (6-deoxy- -mannose) is a sweet sugar
L
* Corresponding author. Tel.: +1-863-2934133; fax: +1-
863-2998678.
E-mail address: grohmann@citrus.usda.gov (K. Grohmann).
present in many plants in the form of rham-
nosides. It is a precursor for the synthesis of a
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00081-1

142
K. Grohmann et al. / Carbohydrate Research 328 (2000) 141–146
flavoring agent furaneol [4-hydroxy-2,5-
dimethyl-3(2H)-furanone], an important
investigated the acid-catalyzed hydrolysis of
hesperidin in aqueous suspensions at tempera-
tures from 25 to 180 °C. Results of these
investigations are described in this communi-
cation.
aroma component of some fruits (e.g., pineap-
ple, raspberry and strawberry) and processed
foods [4]. Hesperetin, (S)-2,3-dihydro-5,7-di-
hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-
1-benzopyran-4-one, has potential medicinal
applications and can be used for preparation
of dyes [2] and sweeteners [5,6]. Hesperetin-7-
2. Experimental
b-
D
-glucoside, (S)-7-(b-
D
-glucopyranosyloxy)-
Hesperidin (97% pure) and hesperetin
(\95% pure) were obtained from Sigma-
Aldrich (Milwaukee, WI). An authentic sam-
2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxy
phenyl)-4H-1-benzopyran-4-one, an interme-
diate in the acid-catalyzed hydrolysis of hespe-
ridin, is potentially even more valuable,
because it can be converted into an intensely
sweet dihydrochalcone by simple hydrogena-
tion in alkaline media, whereas hesperidin di-
hydrochalcone is tasteless [5,6]. Controlled
hydrolysis of hesperidin to produce rhamnose
ple of hesperetin-7-b- -glucoside was pre-
D
pared by R.M. Horowitz and B. Gentili,
USDA, (Pasadena, CA). Other chemicals were
of reagent grade and were obtained from
Sigma-Aldrich. or Fisher Scientific (Pittsburg,
PA).
Acid treatment.—Hesperidin was suspended
in deionized water in a 2-L stirred pressure
reactor constructed from acid-resistant alloy
Carpenter 20 Cb (Parr Instrument Co., Mo-
line, IL). The reactor was equipped with an
acid injection device constructed from an acid-
and pressure-resistant sample cylinder (Whit-
ney Co., Highland Heights, OH) by adding a
valve on each end and a tubing connecting one
end of the cylinder to the reactor and the other
one to a pressurized nitrogen supply. The
diluted acid in the injection device was pre-
heated to a proximity of the desired tempera-
ture by an electric tape heater.
The reactor charge was preheated to a de-
sired temperature, the required amount of di-
lute sulfuric acid was injected by nitrogen
overpressure, and the timing of the reaction
was initiated. The temperature during the reac-
tion was controlled to 91 °C, and the reac-
tion was quenched by immersing the reactor in
an ice-water bath. A portion of cooled reaction
mixture was filtered through a glass fiber filter
(1.2 mm pore size) and washed with deionized
water for determination of dry weight loss.
The combined filtrate and washes were neu-
tralized by adding sodium bicarbonate, and
these were used for determination of sugars
released by the acid treatment. Samples of
solid residues from the reactor were dissolved
in Me2SO and analyzed by thin-layer or re-
versed-phase liquid chromatography as de-
scribed below. The solid residues were also
and hesperetin-7-b- -glucoside could lead to
D
production of valuable chemical intermediates.
Such hydrolyses are hampered by the very low
solubility of hesperidin in water, which is esti-
mated to be 20 ppm or less [2,7,8] as well as in
many organic solvents. The low solubility of
hesperidin in water causes an unusual resis-
tance to acid- and enzyme-catalyzed hydrolysis
and has led to the development of several
hydrolytic mixtures containing acid catalysts
in organic solvents [9–12]. Low solubility of
hesperidin in hot water is also indicated by the
use of this solvent to remove impurities from
precipitated hesperidin [13].
Wilson [14] overcame the insolubility of hes-
peridin in aqueous acids by dissolving hespe-
ridin in aqueous alkali and converting it into a
salt of hesperidin chalcone. When this solution
was added to a hot aqueous acid at pHB1,
hydrolysis occurred faster than reformation
and precipitation of hesperidin. A disadvan-
tage of this approach is a high consumption of
acid for neutralization of the base and forma-
tion of inorganic salts as a byproduct.
Since solubility of a related citrus flavonoid
naringin, (S)-7-[a-
L
-rhamnopyranosyl-(1“2)-
b -
D
- glucopyranosyloxy] - 2,3 - dihydro - 5-
hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopy-
ran-4-one, in water rapidly increases at tem-
peratures higher than 50 °C [15], we felt that
hesperidin may exhibit similar behavior at
temperatures higher than 100 °C. We therefore

K. Grohmann et al. / Carbohydrate Research 328 (2000) 141–146
143
sistant reactor. The reactor was closed and
heated under stirring to 140 °C. Preheated
0.5% (v/v) H2SO4 (100 mL) was then injected,
hesperidin was hydrolyzed for 1 h at 1409
1 °C and the reaction was stopped by immers-
ing the reactor in an ice-water bath. The
reactor and its contents were stored in the
cold room at 2 °C for 2–12 h to allow precip-
itation of hesperetin and hesperetin glycosides
from the aq phase. The clear aq layer could
then be poured off, with only minor (0.2–0.3
g) losses of total hesperetin derivatives.
Resinous material which coated vessel walls
and other interior surfaces was dissolved in
500 mL of warm (40–45 °C) acetone contain-
ing 50 mL of deionized water. The acetone
solution was cooled to rt, filtered and evapo-
rated to dryness under reduced pressure. Ap-
proximately 26–28 g of beige powdery residue
was recovered and dried overnight in the vac-
uum oven at 40 °C. Dry solids were broken
into small pieces and stirred with 100 mL of
dry, warm (4095 °C) acetone for 2–3 h. A
light beige powdery residue was filtered off,
washed twice with 50 mL of dry acetone,
dried in the stream of air and finally dried in
the vacuum oven. Approximately 8–13 g of
essentially pure (95%) hesperetin-7-glucoside
were obtained in this manner. An additional
2–3 g of hesperitin-7-glucoside were obtained
by repeating the procedure described above,
using 50 mL of warm dry acetone to dissolve
the bulk of the dry residue from the first
filtrate, filtering the insoluble solids and wash-
ing the filter with 50 mL of dry acetone.
Hesperetin-7-glucoside was thus obtained in
approximately 26–42% of theoretical yield.
Additional removal of hesperetin can be ac-
complished by suspending dry hesperetin-7-
glucoside in 100 mL of dry, hot (60–80 °C)
1-butanol or 1-propanol and filtering the
slurry. Hesperetin-7-glucoside is practically in-
soluble in these solvents, but hesperetin forms
approximately a 3 and 5% solution in 1-
propanol at 65 and 80 °C, respectively.
Purified hesperetin-7-glucoside was sometimes
contaminated by charred particles from the
reactor. In these cases hesperetin-7-glucoside
was further purified by dissolving it in DMF
to 10–20% concentration. Black particles were
removed by centrifugation, hesperetin-7-glu-
extracted with hot lower aliphatic alcohols or
acetone to investigate separation of hes-
peretin, hesperetin-7-glucoside, and hespe-
ridin.
Analytical procedures.—Rhamnose and glu-
cose were separated and determined by ion-ex-
change chromatography on a 4×250 mm
CarboPac PA-1 anion-exchange column
(Dionex Corp., Sunnyvale, CA) using dilute
NaOH and NaOH–sodium acetate gradient
as eluants [16] and a pulsed amperometric
detector
(Dionex).
2-Deoxy- -galactose
D
(Sigma-Aldrich) was used as an internal stan-
dard, and the detector response was calibrated
for each individual sugar. Hesperidin, hes-
peretin-7-b- -glucoside and hesperetin were
D
usually separated by thin-layer chromatogra-
phy on 20 cm cellulose plates (Sigma-Aldrich,
100 mm thick) using 15% AcOH–water as the
solvent system [12]. Plates were dried in a
stream of air, developed by exposure to am-
monia fumes, and observed under UV light
[17] to estimate relative amounts of each com-
pound in various fractions.
Separation and quantitative determination
of flavonoids in selected samples was obtained
by HPLC using a C18 phase bonded to silica
gel (Partisil 5ODS3 analytical column, What-
man Lab Sales, Hillsboro, OR) as described
previously [3].
Authentic samples of hesperidin, hesperetin-
7-b- -glucoside and hesperetin were used as
D
external standards for identification and quan-
titative determination of the respective com-
pounds in both chromatographic systems.
Solubility of hesperidin.—Solubility of hes-
peridin in water at elevated (120, 130 and
140 °C) temperatures and pressures was esti-
mated by placing increasing concentrations of
hesperidin powder in water into thick-walled,
screw-capped glass vials (Reacti-Vial™, Pierce
Chemical Co., Rockford, IL) and heating a set
of vials to a desired temperature in a heating
block. Even distribution of heat was assured
by partially filling the heating block wells with
glycerol.
Procedure for preparing hesperetin-7-glu-
coside from hesperidin.—Hesperidin (50 g)
was suspended in 850 mL of deionized water,
and the slurry was poured into a pressure-re-

144
K. Grohmann et al. / Carbohydrate Research 328 (2000) 141–146
180 °C must have been very rapid, because
another hydrolysis at 140 °C for 5 min pro-
duced rhamnose in 92.3% and glucose in
51.5% yields, respectively. Unusually large ef-
fects of elevated temperatures on the rate of
hesperidin hydrolysis were confirmed by sev-
eral reactions performed at 60–100 °C and
longer reaction times. Results of these reac-
tions, summarized in Table 1, show that the
rate of hesperidin hydrolysis decreases dra-
matically at 100 °C and lower temperatures.
The yield of rhamnose was only 54.3% after
10 h of reaction at 100 °C. Hesperidin is also
quite resistant to acid-catalyzed hydrolysis at
room temperature, because no rhamnose or
glucose was released by treatment of 0.5%
hesperidin slurry with 0.5% H2SO4 for 5 days
at room temperature.
Increased solubilization of hesperidin in wa-
ter at elevated temperatures was confirmed by
a separate set of experiments described in
Section 2. The solubility of hesperidin in water
is approximately 0.1, 0.15 and 0.25 wt.% at
120, 130, 140 °C, respectively. These solubili-
ties compare very favorably with the ex-
tremely low (0.002%) solubility of hesperidin
in water at 60 °C [8] and are a probable
reason for accelerated acid-catalyzed hydroly-
sis of hesperidin at high temperatures. We
could not determine the solubility of hespe-
ridin at temperatures higher than 140 °C, due
to increased pressures and equipment limita-
tions, but even higher solubility can be ex-
pected at these temperatures.
Fig. 1. Yield of glucose and rhamnose during hydrolysis of
0.5% hesperidin slurry in 0.5% (v/v) sulfuric acid for 10 min
at 100–180 °C. – – glc, --ꢁ-- rha.
coside was precipitated by diluting the DMF
solution with 4–9 volumes of water with re-
covery of the material by filtration or centrifu-
gation. The resulting white precipitate was
washed with deionized water and dried at rt.
3. Results and discussion
An initial set of experiments designed to test
the hypothesis that the acid-catalyzed hydroly-
sis of hesperidin at elevated temperatures
should proceed at much higher rates is sum-
marized in Fig. 1. Using a low concentration
of hesperidin (0.5 wt.%), we observed a rapid
production of rhamnose and glucose at 120 °C
and higher temperatures. A sizable decrease in
the yields of rhamnose and glucose at 160–
180 °C can be attributed to the acid-catalyzed
decomposition of monosaccharides at elevated
temperatures [18]. The hydrolyses at 160 and
Since concentration of the acid catalyst af-
fects the relative rates of hydrolysis of individ-
ual glycosidic bonds, we performed several
investigations of hesperidin hydrolysis at 120
and 140 °C using different concentrations of
sulfuric acid. The results are summarized in
Figs. 2 and 3. The results summarized for
hydrolysis with 0.5% sulfuric acid at 120 and
Table 1
Hydrolysis of hesperidin at 60–100 °C
Temperature (°C)
Reaction time (min)
60
60
80
60
100
10
60
300
600
Rhamnose yield (% theor.)
Glucose yield (% theor.)
0
0
0.1
0
2.2
0.7
6.9
2.2
32.7
18.3
54.3
37.3

K. Grohmann et al. / Carbohydrate Research 328 (2000) 141–146
145
had been achieved. The molar ratio of hes-
peretin-7-glucoside to hesperetin is approxi-
mately 1:1 at this point with no remaining
hesperidin (data not shown). Additional hy-
drolysis of hesperidin-7-glucoside will produce
additional glucose and hesperetin, so the ratio
of rhamnose to glucose will ultimately become
1:1.
The experiments described above indicate
that optimal conditions for the preparation of
rhamnose and hesperetin-7-glucoside by hy-
drolysis of hesperidin in aqueous sulfuric acid
occur at 0.05–0.5% concentration of sulfuric
acid and 140–180 °C. A temperature range of
140–160 °C may be preferable, because we
observed significant darkening of the reaction
mixture hydrolyzed at 180 °C. Since several
publications describe purification and recov-
ery of rhamnose from hydrolyzates of hespe-
ridin and naringin [19–21], we did not
investigate recovery of this sugar. However,
only chromatographic purification of hes-
peretin-7-glucoside has been investigated [12],
which would make preparation of this com-
pound tedious and expensive. Therefore, we
have investigated purification of hesperetin-7-
glucoside by a simpler solvent extraction.
Tests of solubility of hesperetin revealed that
Fig. 2. (a) Production of glucose and rhamnose during hy-
drolysis of 0.5% hesperidin slurry in 0.5% (v/v) sulfuric acid at
120 and 140 °C. –ꢁ– glc 120 °C, – – rha 120 °C, –ꢁ– glc
140 °C, – – rha 140 °C. (b) Production of glucose and
rhamnose during hydrolysis of 0.5% hesperidin slurry in 0.01
and 0.05% (v/v) sulfuric acid at 140 °C. –ꢁ– glc 0.01%
H2SO4, – – rha 0.01% H2SO4, –ꢁ– glc 0.05% H2SO4,
–
– rha 0.05% H2SO4.
140 °C (Fig. 2(a)) support rapid increase in the
rate of hydrolysis of both glycosidic bonds
with increasing temperature. A tenfold de-
crease in acid concentration led to only four-
fold increase in the time required for complete
hydrolysis of rhamnosyl groups (Fig. 2(b)),
but an additional decrease in acid concentra-
tion to 0.01% considerably decreased the reac-
tion rate.
The selectivity (i.e., ratio of rhamnose to
glucose produced) of acid-catalyzed hydrolysis
is illustrated in Fig. 3 for data from Fig. 2.
The selectivity was high during initial stages of
reaction, when concentration of hesperidin
was high, but it steadily decreased with in-
creasing degree of conversion and all data
converged at an approximate rha:glc ratio of
2:1 when almost complete release of rhamnose
Fig. 3. Molar ratio of rhamnose to glucose as a function of
rhamnose yield during hydrolysis of 0.5% hesperidin slurry in
0.01–0.5% (v/v) sulfuric acid at 120 and 140 °C. –ꢁ– 120 °C,
0.05% H2SO4; ··· ··· 140 °C, 0.01% H2SO4; –ꢂ– 140 °C.
0.05% H2SO4; ·–"–· 140 °C, 0.5% H2SO4.

146
K. Grohmann et al. / Carbohydrate Research 328 (2000) 141–146
it is quite soluble in dry acetone, forming
approximately 8% (w/v) solution at 25 °C,
11% at 40 °C and 13% at 50 °C, respectively.
Hesperetin is also soluble in methanol, form-
ing a 2% solution at 30 °C and in hot lower
alcohols, forming a 4% solution in methanol
at 55, ethanol at 65, 1-propanol at 70 and
2-propanol at 75 °C, respectively. It is less
soluble in 2-butanol, and glacial acetic acid,
forming 2% solution at 65 and 80 °C, respec-
tively, and ethyl acetate which produces ap-
proximately 1.5% solution at 50 °C. The
solubility of hesperetin-7-glucoside in dry ace-
tone is approximately 0.1% at room tempera-
ture; therefore, use of this solvent allows
efficient separation of relatively insoluble hes-
peretin-7-glucoside from hesperetin. Hespe-
ridin is practically insoluble in all solvents
listed above.
Hesperetin-7-glucoside and hesperetin can
be separated from hesperidin by dissolving the
first two compounds in aqueous acetone. Hes-
peretin-7-glucoside forms approximately 2%
solution in 60 and 80% acetone–water mix-
tures at room temperature and 5% solution in
both solvents at 50 °C. Solubility of hesperidin
in both solvents is less than 0.1% in the tem-
perature range of 25–50 °C, but hesperetin is
very soluble in 80% acetone, forming approxi-
mately 19 and 24% solutions at room temper-
ature and 50 °C, respectively. Hesperetin is
less soluble in 60% acetone, forming 4 and 8%
solutions at room temperature and 50 °C, re-
spectively. Of the solvent systems we investi-
gated, acetone, either dry or diluted with a
small amount of water, was the most efficient
solvent for the separation of hesperetin, hes-
peretin-7-glucoside and hesperidin.
tives, lower solvent costs, and decreased safety
and environmental problems.
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Hydrolysis of hesperidin in aqueous sulfuric
acid provides several advantages over previous
methods utilizing acid catalysts in organic sol-
vents, namely shorter reaction time, simple
separation of sugars from hesperetin deriva-
.