Hydrolysis of the extract of rosa roxburghii tratt catalyzed by imidazolium ionic liquids

Article abstract of DOI:10.14233/ajchem.2013.14598

Rosa roxburghii tratt (Cili) is a kind of fruit and medicinal plant in China, which is rich in vitamins and flavonoids. In the study, nine Bronsted acidic ionic liquids were first used as catalysts in the preparation of quercetin directly from the extract of rosa roxburghii tratt. Through rapid screening under the same reaction conditions, [PSMIM][HSO₄] was found to be the best candidate catalyst. Then the sequential experiments were carried out with [PSMIM][HSO₄] as catalyst. The reaction conditions were further optimized by response surface methodology and the hydrolytic dynamics was also investigated. Reusing and recovering of the ionic liquid were also studied with fairly good results. It is proved that this green catalyst is ideal for developing new hydrolysis process for crude extract of total natural flavonoids as the substitute of traditional inorganic acids.

Full text of DOI:10.14233/ajchem.2013.14598

Asian Journal of Chemistry; Vol. 25, No. 13 (2013), 7327-7331
http://dx.doi.org/10.14233/ajchem.2013.14598
Hydrolysis of the Extract of Rosa roxburghii Tratt Catalyzed by Imidazolium Ionic Liquids
1
1
1
2,*
CHANG YANG , HAO ZENG , HANG SONG , SHUN YAO1,* and HONGYAN MA
1Department of Pharmaceutical and Biological Engineering, Sichuan University, Chengdu 610065, P.R. China
2School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, P.R. China
*Corresponding author: Fax: +86 28 85403397;Tel: +86 28 85405221; E-mail: cusack@scu.edu.cn; 747487678@qq.com; hongyan203@hotmail.com
(Received: 20 September 2012;
Accepted: 24 June 2013)
AJC-13701
Rosa roxburghii tratt (Cili) is a kind of fruit and medicinal plant in China, which is rich in vitamins and flavonoids. In the study, nine
Bronsted acidic ionic liquids were first used as catalysts in the preparation of quercetin directly from the extract of rosa roxburghii tratt.
Through rapid screening under the same reaction conditions, [PSMIM][HSO4] was found to be the best candidate catalyst. Then the
sequential experiments were carried out with [PSMIM][HSO4] as catalyst. The reaction conditions were further optimized by response
surface methodology and the hydrolytic dynamics was also investigated. Reusing and recovering of the ionic liquid were also studied with
fairly good results. It is proved that this green catalyst is ideal for developing new hydrolysis process for crude extract of total natural
flavonoids as the substitute of traditional inorganic acids.
Key Words: Ionic liquids, Rosa roxburghii tratt, Catalysis, Flavonoids, Quercetin.
INTRODUCTION
In recent years, more and more new resources of medicinal
plants are attracting the attention from relative researchers.
Rosa roxburghii tratt (Cili), also known as sweet chestnut rose
and the new king of vitamin C, is a wild plant found only in
certain high mountain ranges of Mainland China (Fig. 1) and
used as traditional Chinese medicine from ancient to nowadays.
It is also widely used as a natural food for humans and animals
alike and has been found effective in treatment of the longevity,
cancer, immunity and atherosclerosis1. Cili contains a lot of
components with antioxidant activity, which may contribute
to its health benefits. The major antioxidant constituents
includes rutin (Fig. 2) also known as vitamin P, 59.8-128.9
mg per gram fresh weight and vitamin C (7.9-23.9 mg per
Fig. 1. Cili and its distribution in China
gram fresh weight) and a few methods were developed to
extract total flavonoids which was mainly composed of rutin2.
Quercetin (Fig. 2), 3,3',4',5,7-pentahydroxylflavone, is the
aglycone of rutin and is used as an important bioactive ingre-
dient in supplements, beverages drugs and foods. Recent
researches have revealed that it can be used as efficient anti-
tumor drug and platelet aggregation inhibitor3 and its effects
of antioxidation, vasodilatation and antitumor are all stronger
than rutin. However, as a matter of fact, the studies on its prepa-
ration from natural resources have fallen behind bioactivity
researches and applications of quercetin in recent years.
On the other hand, the study on ionic liquids (ILs) is a
globally hot field in these years. Ionic liquids are molten salts

7328 Yang et al.
Asian J. Chem.
HPLC analysis was performed with an LC-20AT pump
(Shimadzu, Kyoto, Japan), a Welchrom C18 column, 5 µm,
250 mm × 4.6 mm i.d. (Welch Materials, Maryland, USA), an
HCT-360 LC column cooler/heater (Hengao Tech & Dev,
Tianjin, China) and an SPD-M20A PDA detector (Shimadzu,
Kyoto, Japan). A Class-VP workstation (Shimadzu, Kyoto,
Japan) was used for data acquisition. Using methanol/water/
TFA (70:30:0.5 v/v/v) as mobile phase at the flow rate of 0.4
mL/min with the column temperature was set at 35 ºC. The
injection volume was 10 µL and the samples were detected at
254 nm. The pH meter was provided by Shanghai Shinuo
Physical Optical Instruments Co., Ltd. (Shanghai, China).
Sonication was performed on the KQ2200DE model numerical
controlled ultrasonic cleaner provided by Kun Shan Ultrasonic
Instruments Co., Ltd. (Jiangsu, China).
Physical and structure characterization instruments used
are listed as follows: XRC-1 Micro melting point apparatus
(uncalibrated, Sichuan University, China); AVATAR 360-FT
infrared spectrometer (Nicolet, USA); Varian Mercury 400
NMR (Varian, USA); Finnigan-TRANCE MS (Finnigan,
USA).
Extraction of total flavonids: The pieces of Cili (200 g)
were powdered and extracted with 65 % ethanol (2 L) three
times at 60 ºC. Then the extract was evaporated to form the
syrup. The syrup was then dissolved in water by sonication
and partitioned with n-butanol of equal volume three times.
The combined n-butanol solution was vacuum evaporated at
65 ºC and ca. 6.8 g of residue was obtained.
Preparation and identification of quercetin: The 100
mL three neck flask was charged with aqueous ionic liquid
(50 mL) and then it was heated to the temperature set before
hand. Then the flavonoids extract (0.78 g) was added in. During
the reaction, the reaction mixture was sampled at regular
intervals and the samples were concentrated to obtain the solid
substances for HPLC analysis. The mixture reacted under mild
stirring for hours at constant temperature and finally it was
cooled down to room temperature. The suspension was filtered
and the remaining yellow solid was washed with 30 mL water.
Further purification was performed by recrystallization in 50 %
ethanol. Finally the crystallized product was dried at 100 ºC.
Physical and structure characterization data about the crystal
are given below:
Fig. 2. Structure of quercetin (1) and rutin (2)
which are consisted of bulky organic cations with organic/
inorganic anions. Most of them are liquid around room tempe-
rature. Nowadays, they are widely used in chemical synthesis
as solvents and/or catalysts. As catalysts, they are promising
to replace many catalysts worked in traditional reactions such
as alkylation, esterification, Michael addition, oligomerization
and rearrangement4-8. BrÖnsted acidic ionic liquids give great
promise of using ionic liquids as green catalysts in hydrolysis
reactions for their strong acidity and water solubility. Their
application in acid-catalyzed hydrolysis would overcome the
shortcomings in traditional hydrolysis process catalyzed by
inorganic acids to some extent, such as equipment corrosion,
long reaction time, sewage emission and carbonization of the
product under high temperature. But it has not been reported
about its application in direct hydrolysis of natural extract.
Based on the above background, there should be a new
way to utilize natural resource of Cili besides the direct
extraction of rutin, which is the preparation of quercetin from
the crude extract by hydrolysis with the new and environment-
friendly catalyst. Meanwhile, the combination of green
chemistry and medicinal phytochemistry is desirable. So in
the following research, nine Brønsted acidic ionic liquids
replaced common inorganic acids were first used to prepare
quercetin from the crude extract of Cili. Through the compa-
rison of conversion ratios of the hydrolysis reaction, the best
catalyst was selected for further study. Then the optimization
of conditions was performed with the response surface method
and the dynamical property under the best reaction condition
was also studied. Finally, recovering and reusing of ionic liquid
were investigated and obtained fairly good results.
EXPERIMENTAL
Yellow needle crystal. m.p. 313-314 ºC. EI-MS (rel. int.):
m/z 302 [M]+, 274 (M-28), 273 (M-29), 245 (M-57), 153,
152, 142, 137, 124, 123, 109, 77. IR (KBr, νmax, cm-1): 3500-
Rosa roxburghii tratt (Cili) was collected from suburbs of
Liangshan Yi Autonomous Prefecture, Sichuan Province,
China. For collection at the initial stage of maturity, only full
yellow fruits were picked up in October, 2009. Then they were
cut into pieces and dried in a ventilated drying oven and stored
in plastic bags at low temperature and protected from light
before their extraction.
1
3000, 1672, 1618, 1520, 1513, 1430, 1361, 1316; H NMR
(300 MHz, acetone-d6): δ (ppm): 6.27 (1H, d, J = 2 Hz, H-6),
6.53 (1H, d, J = 2 Hz, H-8), 7.00 (1H, d, J = 8.5 Hz, H-5'),
7.70 (1H, dd, J = 8.5 Hz, 2.1 Hz, H-6'), 7.81 (1H, d, J = 2.1
Hz, H-2').
Rutin (purity of 99.3 %) was purchased from HongYi
Bio-engineering Co. (Chengdu, Sichuan, China). Quercetin
(purity of 98.1 %) was purchased from the National Institute
of Food and Drug Control (Beijing, China). Methanol, ethanol,
n-butanol and trifluoroacetic acid (TFA) were purchased from
Chengdu KeLong chemical reagent factory (Chengdu, Sichuan,
China) with the purities up to 99.5 %. Water is redistilled. Ionic
liquids were synthesized referred to the previous reports9-14, their
purities were determined by HPLC and were all greater than 96 %.
Analysis of the samples: First of all, the standard curves
of rutin and quercetin were established on the basis of a series
of methanol solution of rutin and quercetin with the concen-
trates of 1.000, 0.800, 0.500, 0.200, 0.100, 0.050, 0.020, 0.010
mg/mL and 0.500, 0.250, 0.100, 0.050, 0.025, 0.010 mg/mL,
respectively. The obtained equation for the standard curve of
rutin was
Y = 405908x – 47392
(1)

Vol. 25, No. 13 (2013)
Hydrolysis of the Extract of Rosa roxburghii Tratt Catalyzed by Imidazolium Ionic Liquids 7329
R2 = 0.9997, the linear scope was 0.1-8.0 µg; the equation for
the standard curve of quercetin was
TABLE-1
STRUCTURES AND pH VALUES OF THE IONIC LIQUIDS
pH
value*
No
A
IL
Cation
+NH
N
Anion
y = 913183x – 629557
(2)
R2 = 0.9996, the linear scope was 0.1-5.0 µg.
where y was the integral area of relative peak of rutin or
quercetin and x was the corresponding amount of rutin or
qucertin. Then we analyzed the samples. They were analyzed
under the same conditions stated above with the reference
substances. The dry solid samples were dissolved in methanol
and diluted to suitable concentrates and then they were filtered
through a 0.2 µm film and injected into HPLC. The conversion
ratios of the samples were calculated by eqns. 1 and 2 given
above and the formula of conversion ratio according to the
integral areas of relative peaks.
[HMIM][HSO4]
HSO4-
1.2
1.6
5.6
+NH
N
•
BF4-
B
C
D
E
[HMIM][BF4]
[HMIM][p-TSA]
[HMIM][TfO]
+NH
N
p-TSA-
RESULTS AND DISCUSSION
Comparison of catalytic performance of ionic liquids:
Since acidity is one of the most important factors that influ-
ence the conversion ratio in the acid-catalyzed hydrolysis of
rutin with traditional methods, we chose nine BrÖnsted acidic
ionic liquids as our experiment objects. Table-1 shows their
structures and pH values.
+NH
N
•
CF3SO3-
5.7
4.9
+NH
N
•
The catalytic activity of the selected ionic liquids was
compared under the same molar concentrations (20 mmol/
100 mL aqueous solution) by the conversion ratios of rutin at
100 ºC for 4 h. Fig. 3 shows the curves of the conversion ratios
of different ionic liquids. It was obvious that ionic liquids
[HMIM][HSO4], [HMIM][BF4], [PSMIM][H2PO4] and
[PSMIM][HSO4] successfully promoted the reaction with
relatively high efficiency and the yields were all up to 90 %.
They were much higher than those reported values that were
lower than 80 %15. This proved that acidity played a key role
in this reaction, which was determined by both anions and
cations. [H2PO4]– is a weaker acidic group with nearly no
secondary ionization compared to [HSO4]–, so it could produce
much less H+ that worked as catalyst in the reaction. The acidity
of the cations follows the order: [PSMIM]+ > [HMIM]+ >
[BMIM]+, for [PSMIM]+ has a sulfonic group which may
cooperate with the anion to provide a stronger acidity and
[BMIM]+ cannot ionize while [HMIM]+ can.
MeSO3-
[HMIM][MeSO3]
+N
N
•
H2PO4-
F
[BMIM][H2PO4]
6.1
SO3H
SO3
H
H
H
+N
N
•
HSO4-
G
H
I
[PSMIM][HSO4]
[PSMIM][HSO4]
[PSMIM][H2PO4]
1.6
1.0
1.5
N
N
SO3H
SO3
+N
N
•
HSO4-
N
N
SO3H
SO3
+N
N
H2PO4-
N
N
*pH values were measured in the 20 mmol/100 g aqueous solution at
30 ºC by pH meter.
Optimization of reaction conditions:After [PSMIM][HSO4]
was chosen as the candidate ionic liquid for further study,
further investigation was carried out about the influence of
temperature, time and the amount of ionic liquid on the reaction.
It was found that temperature was the most important factor
among them. When the temperature was lower than 80 ºC, no
product was detected even if the reaction time was extended.
Secondly, a certain amount of ionic liquid is necessary for the
reaction. The hydrolysis reaction did not occur without the
addition of ionic liquids. Finally, the conversion ratios were
time-dependent which could be concluded from Fig. 3. These
three factors were all considered and optimized in the optimi-
zing experiments.
Fig. 3. Catalytic performance of ionic liquids under the standard reaction

7330 Yang et al.
Asian J. Chem.
Response surface methodology (RSM) is a collection of
mathematical and statistical technique, which becomes an
effective tool to optimize the experimental conditions with
the least experiment time in the research of medicinal plants16.
The following experiments were designed in Box-Behnken
Design (BBD) mode of RSM. The data were processed with
Design-expert 7.0.0. The predicted optimal reaction conditions
were as follows: T = 98.5 ºC, mIL = 0.19 mol/L, t = 1.05 h. The
conditions were no severer than the best hydrolysis conditions
in the reported method in which the reaction mixture was
refluxed in 0.5 % (weight percentage) of aqueous H2SO4 for
1 h17. And they were validated by the corresponding experi-
ments. Table-2 shows the designed experiments and the
corresponding results. The analysis of variance is valid. And
the final equation in terms of coded factors was
0.1
-0.8
-1.7
-2.6
-3.5
2.00
1.63
100.00
95.00
90.00
1.25
85.00
0.88
80.00
0.50
Fig. 4. Response surface of the optimizing experiments (R1 is the
conversion ratio)
where x0 is the initial concentrate of rutin, x is the concentrate
of rutin at time t, k1 and k2 are the rate constants, C1 and C2 are
constants. Through simple calculation on the basis of the HPLC
data, the values of ln (x0 – x) were correlated with time by a
linear mode and the equation was obtained as
TABLE-2
DESIGNED EXPERIMENTS AND THE
CORRESPONDING RESULTS OF [PSMIM][HSO4]
Time
(h)
Temp.
(ºC)
Molar fraction of [PSMIM] Response
Run
[HSO4] (mol/L)
value
y = –1.678x – 4.400
(6)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1.25
1.25
2.00
1.25
0.50
0.50
2.00
1.25
2.00
2.00
0.50
0.50
1.25
1.25
1.25
1.25
1.25
100
80
90
90
90
100
80
90
90
100
90
80
90
100
90
80
0.10
0.20
0.10
0.15
0.20
0.15
0.15
0.15
0.20
0.15
0.10
0.15
0.15
0.20
0.15
0.10
0.9075
0.1131
0.6257
0.5464
0.3555
0.8263
0.1537
0.5501
0.8848
0.9344
0.1120
0.0339
0.5474
0.9416
0.5528
0.0933
0.5481
where y was equal to ln (x0 – x) and x was the reaction time
and the correlation coefficient (R) was 0.9953. It showed that
the hydrolysis reaction was a first order reaction in which the
rate constant (k1) was 1.678 h-1.
Reusing and recovering of [PSMIM][HSO4]: In the
study of reusing, the filtrate was reused directly for another
nine times under the optimized conditions. The results were
satisfied in terms of conversion ratio as shown in Fig. 5. The
conversion ratio did not decrease severely and the total reaction
time needed for full conversion also did not change.
90
0.15
ln (R1) = -0.63 + 0.53A + 1.17B + 0.22C - 0.35AB
- 0.20AC - 0.28A2 - 0.51B2 (3)
where R1 was the conversion ratio of rutin,A was time, B was
temperature and C was the amount of ionic liquid. The impor-
tant parameters met the required standard and Table-3 shows
the analysis results of variance. The response surface is depicted
in Fig. 4. Since temperature and time have much more effect
on the conversion ratio than that of the amount of ionic liquid,
they were chosen as x and y axis in the response surface. The
diagram showed that the natural logarithm of conversion ratio
increased with the increase of temperature and time. The
conversion ratio was prone to be constant when the temperature
was close to 100 ºC and the time used was around 2 h. While
extending of reaction time and increasing temperature are at
least not energy-efficient.
Fig. 5. Conversion ratio for the reuse of [PSMIM][HSO4]
Then in the study of recovering, the filtrate that came from
the post-process of the reaction mixture was firstly distilled
under vacuum to remove water. The residual was diluted by
anhydrous ethanol and filtrated to remove sugars. Then the
filtrate was concentrated in vacuum and washed with three
portions of acetone and subsequently disposed of the residual
acetone in vacuum. Finally a brown viscous liquid was obtained.
Then it was used for the next reaction and the reaction rate did
not decrease.
The chemical reaction dynamics was also investigated
under the optimal hydrolysis conditions. As we all know, in a
first order reaction:
ln (x0 – x) = -k1t + C1
(4)
While in a second order reaction:
1
= k2t + C2
x0 − x
(5)

Vol. 25, No. 13 (2013)
Hydrolysis of the Extract of Rosa roxburghii Tratt Catalyzed by Imidazolium Ionic Liquids 7331
TABLE-3
ANALYSIS OF VARIANCE
Source
Model
A
B
C
Sum of squares
15.81
2.28
Df
7
1
1
1
1
1
1
1
9
5
4
16
Mean square
2.26
F Value
P-value/Prob > F
50.45
50.84
246.35
8.37
10.77
3.65
7.49
24.09
–
< 0.0001
< 0.0001
< 0.0001
0.0178
0.0095
0.0884
0.0229
0.0008
–
2.28
11.03
0.37
0.48
0.16
11.03
0.37
0.48
0.16
0.33
1.08
0.40
0.40
AB
AC
A2
0.34
1.08
0.045
0.081
0.00002132
–
B2
Residual
Lack of fit
Pure error
Cor total
3777.71
–
< 0.0001
–
0.0000853
16.21
–
–
Note: A for time, B for temperature and C for amount of [Psmim]HSO4.
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Conclusion
In this study, [PSMIM][HSO4] was found to be a promising
catalyst for the hydrolysis of crude extract from Rosa roxburghii
tratt. The hydrolysis conditions of rutin with [PSMIM][HSO4]
as catalyst were optimized by response surface methodology.
With the participation of green chemical solvent, the best
reaction conditions were no severer than that of the traditional
methods and the yield was higher than that of the traditional
methods. The reusability and recyclability were validated with
fairly good results. As ionic liquids possess so many advan-
tages compared to traditional catalysts is believed that this
preparative method has great potential and broad prospects in
the chemical study of fruit and natural functional foods.
ACKNOWLEDGEMENTS
14. Y.Y. Wang, W. Li and L.Y. Dai, Chem. Pap.,-Chemicke Zvesti, 62, 313
(2008).
15. Y.S. Li, Chem. Intermed., 8, 13 (2008).
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(2011).
17. G.Y. Huo, P. Zhu, N. Liu, S.Z. Zhang, R.H. Chen, X.M. Ding and J.H.
Zhao, Chem. J. Adhes., 31, 33 (2009).
Many thanks to Miss Ma of Guangdong Pharmaceutical
University for her work in the extraction of total flavonids,
structural characterization and writing of the paper. This project
was supported by the Youth Science Foundation (No.
JS20090719506921) of Sichuan University.
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