Transformation of geniposide into genipin by immobilized β-glucosidase in a two-phase aqueous-organic system

Article abstract of DOI:10.3390/molecules16054295

Genipin is the bioactive compound of geniposide and a natural cross-linking agent. In order to improve the preparation process of genipin, the hydrolysis of geniposide to genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was studied. β-Glucosidase was immobilized by the crosslinking-embedding method using sodium alginate as the carrier. The optimum reaction temperature, pH value and time were 55 °C, 4.5 and 2.5 h, respectively. To reduce genipin hydrolysis and byproduct production the reaction was carried out in an aqueous-organic two-phase system comprising ethyl acetate and sodium acetate buffer. The product was analyzed by HPLC, UV, IR, and NMR. The yield of genipin was 47.81% and its purity was over 98% (HPLC).

Full text of DOI:10.3390/molecules16054295

Molecules 2011, 16, 4295-4304; doi:10.3390/molecules16054295
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Transformation of Geniposide into Genipin by Immobilized
β-Glucosidase in a Two-Phase Aqueous-Organic System
Yi-Shun Yang 1,2, Tong Zhang 1,*, Shai-Cheng Yu 2, Yue Ding 1, Li-Ying Zhang 1, Chen Qiu 2
and Duo Jin 2
1
Experiment Center of Teaching & Learning, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China; E-Mail: becky105@163.com (Y.-S.Y.)
2
College of Chemical Engineering, East China University of Science and Technology, Shanghai
200237, China; E-Mail: scyu@ecust.edu.cn (S.-C.Y.)
* Author to whom correspondence should be addressed; E-Mail: zhangtdmj@yahoo.com.cn;
Tel.: +86 21 5132 2318; Fax: +86 21 5132 2332.
Received: 15 February 2011; in revised form: 3 May 2011 / Accepted: 13 May 2011 /
Published: 24 May 2011
Abstract: Genipin is the bioactive compound of geniposide and a natural cross-linking
agent. In order to improve the preparation process of genipin, the hydrolysis of geniposide
to genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was
studied. β-Glucosidase was immobilized by the crosslinking-embedding method using
sodium alginate as the carrier. The optimum reaction temperature, pH value and time were
55 °C, 4.5 and 2.5 h, respectively. To reduce genipin hydrolysis and byproduct production
the reaction was carried out in an aqueous-organic two-phase system comprising ethyl
acetate and sodium acetate buffer. The product was analyzed by HPLC, UV, IR, and NMR.
The yield of genipin was 47.81% and its purity was over 98% (HPLC).
Keywords: immobilized β-glucosidase; geniposide; genipin; aqueous-organic two-phase
system
1. Introduction
Fructus Gardeniae is the dried ripe fruit of Gardenia jasminoides Ellis (Rubiaceae). It is widely
used in Traditional Chinese Medicine because of its cholagogue, sedative, diuretic, anti-inflammatory,

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and antipyretic effects [1]. Geniposide, a natural iridoid glycoside, is one of the major effective
compounds of Fructus Gardeniae. It has been reported that geniposide has many important curative
effects, such as hepatic-protective, cholagogic effects [2], antithrombotic effects [3], and diabetes
curative effects [4].
Studies have shown that geniposide has hepatic-protective and choleretic effects by oral
administration, but these effects are absent if given by injection. That is because geniposide is
hydrolyzed to its bioactive compound genipin by human intestinal microflora enzymes, and it is
genipin that has the hepatic-protective and choleretic effects [5-7]. Therefore, if genipin is given
directly to patients, the differences between intestinal metabolic activities of geniposide in individuals
can be avoided, and the dose of genipin can be precisely controlled and this should improve its
curative effects and decrease its toxicity.
It is reported that genipin has many other medicinal effects, such as anti-inflammatory [5],
anticancer [6], antithrombotic [3], antibacterial [7], gastritis curative [8], diabetes curative [9],
neurotoxicity inhibition [10], and antidepressant-like effects [11]. In addition, genipin is a natural
cross-linking agent in biological applications [12,13]. It is also used to prepare a series of blue
pigments (gardenia blue) used in the food industry [14]. As the content of genipin in Fructus Gardeniae
is very low [15], genipin (2) is prepared mainly from hydrolysis of geniposide (1) by β-glucosidase
(Scheme 1) [16]. However, the low productivity and high product cost of this method have limited the
application of genipin.
Scheme 1. Hydrolysis of geniposide by β-glucosidase.
In this paper, we aimed to improve the preparation of genipin. The hydrolysis of geniposide to
genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was studied. Because
of its hemiacetal ring-structure, genipin is unstable in water, and it probably hydrolyzes to a double-
aldehyde structure 4 (Scheme 2) [17], which is similar to the cross-linking agent glutaraldehyde.
Scheme 2. Presumed hydrolysis of genipin.
The hydrolysis of genipin decreases its yield. Therefore, an aqueous-organic two-phase system was
used so the genipin produced can be extracted into the organic phase during the reaction, but the
organic solvent may destroy the enzyme. Moreover, genipin crosslinks with amines, amino acids and

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proteins, and forms a kind of pigment (gardenia blue) [12]. As β-glucosidase is a kind of protein,
genipin crosslinks with it during the hydrolysis of geniposide. Therefore, we immobilized the enzyme
to protect it from both the organic solvent and genipin.
2. Results and Discussion
2.1. Optimize Immobilization Conditions of β-glucosidase by Orthogonal Experiment
Based on univariate experiments, L9 (34) was used to design the orthogonal experiment. The design
and results were shown in table 1.
Table 1. The design and results of optimizing immobilization conditions of β-glucosidase
by orthogonal experiment. (A) Concentration of sodium alginate (w/v); (B) Concentration
of glutaraldehyde (v/v); (C) Concentration of CaCl2 (w/v).
Factor
B (%)
0.1
Immobilization
efficiency (%)
30.12
Entry
A (%)
C (%)
0.5
1
1
2
1
1
0.5
30.6
3
4
5
6
7
8
9
1
2
2
2
3
0.8
0.1
0.5
0.8
0.1
0.5
0.8
1.5
1
36.44
30.68
25.92
35.48
31.24
35.88
38.32
1.5
0.5
1.5
0.5
1
3
3
K1
K2
K3
R
32.39
30.69
35.15
4.46
30.68
30.8
36.75
6.07
33.83
33.2
31.2
2.63
As Table 1 shows, the influence was B > A > C within the range of our experiments. The optimum
immobilization conditions were confirmed as below (A3B3C1): the sodium alginate (carrier),
glutaraldehyde (cross-linking agent) and β-glucosidase were mixed to a final concentration of 3%
(w/v), 0.8% (v/v) and 5 U/mL, respectively. The mixture was crosslinked in room temperature for 2 h.
Then it was added dropwise to 0.5% (w/v) calcium chloride solution and left standing for 2 h. Based
on the optimum immobilization conditions, the immobilization efficiency of β-glucosidase
was 40.92%.
2.2. Optimum Reaction Temperature and pH Value
The reaction rate of free and immobilized β-glucosidase was assayed in different reaction
temperatures (40, 45, 50, 55, 60, 65 °C). As Figure 1(A) shows, the optimum reaction temperature of
free and immobilized β-glucosidase were 50 °C and 55 °C, respectively. The relative enzyme activity
of immobilized β-glucosidase was obviously higher than free β-glucosidase within the temperature

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range of 40~65 °C. This proves that immobilization increases the heat resistance of β-glucosidase, and
the immobilized enzyme can adapt to the temperature variation better than the free enzyme.
Figure 1. (A) Influence of reaction temperature to relative reaction rate. The relative
reaction rate is equal to relevant reaction rate at different temperature divide by the highest
reaction rate within 40~65 °C. (B) Influence of reaction pH value to relative reaction rate.
The relative reaction rate is equal to relevant reaction rate at different pH value divide by
the highest reaction rate within pH 3.5~5.5.
The reaction rate of free and immobilized β-glucosidase was assayed in different reaction pH values
(3.5, 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5.0, 5.5). As Figure 1(B) shows, the optimum reaction pH value of free
β-glucosidase as well as immobilized β-glucosidase was 4.5. The relative enzyme activity of
immobilized β-glucosidase was obviously higher than free β-glucosidase within pH value of 3.5~5.5. It
proves that immobilization reduces the impact of pH changes on enzyme activity, and increases the
reaction stability of β-glucosidase at different pHs and its pH tolerance.
2.3. Hydrolysis of Geniposide by Immobilized β-glucosidase in One-phase System
The optimum reaction time was determined according to the method described in Section 3.6. As
Figure 2(A) shows that when there was almost no geniposide in the reaction solution at 3.5 h, the
concentration of genpin reached a peak value. Based on the above results, genipin was prepared
(Section 3.8). The yield was 48 mg (31.90%). This is much lower than the theoretical yield. There
were probably two reasons. First, genipin reacted with enzyme and formed gardenia blue during the
reaction. We found that the reaction solution became dark blue after several hours of reaction.
Secondly, genipin hydrolyzes in water, which will reduce its yield, too.
2.4. Hydrolysis of Geniposide by Immobilized β-glucosidase in Two-phase System
Because the yield of genipin transformed from geniposide by immobilized β-glucosidase in
one-phase system was very low, a two-phase system was considered. It was comprised of 50% ethyl
acetate (organic phase) and 50% sodium acetate buffer (water phase). Genipin can be extracted from

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the sodium acetate buffer into ethyl acetate in the two-phase system during the reaction. Thus, genipin
hydrolysis and byproduct formation were reduced.
Figure 2. (A) Hydrolysis of geniposide by immobilized β-glucosidase. (B) TLC analysis of
hydrolysis of geniposide by immobilized β-glucosidase in two-phase system. (a) Buffer
layer; (b) Ethyl acetate layer; (c) Standard genipin.
The optimum reaction time was determined according to the method described in Section 3.7. It can
be seen in Figure 2(A) that the concentration of genipin was the highest when reacted for 2.5 h, while
the yield was 51.16%. Based on the above results, genipin was prepared (Section 3.9). The yield was
95 mg (63.08%). The purity of genipin was over 98% (HPLC).
2.5. Reaction Stability of Immobilized β-glucosidase in Two-phase System
We repeated the preparation of genipin five times according to Section 3.9, and the immobilized
enzyme was recycled and reused. The yields of the five repetitions were 61.69%, 57.17%, 56.71%,
47.15%, 46.61%, respectively. The yield of genipin decreased with time. There were probably three
reasons. Firstly, the ethyl acetate may reduce the activity of β-glucosidase as it may destroy the spatial
configuration of the enzyme. Secondly, it is known that enzyme activity decreases with time, and
immobilized enzyme is no exception. Thirdly, part of enzyme reacted with genipin, and formed the
gardenia blue byproduct. Therefore, the immobilized enzyme gel became blue after use, and the gel
became darker when used many times. The yield of the fifth run decreased to 75.57% of that of the
first time, which was in an acceptable extent.
3. Experimental
3.1. Materials
Sodium alginate (CP); glutaraldehyde (25% solution, BR); geniposide (extracted from gardeniae
fruits, purity was 90% by HPLC); DNS reagent (3,5-dinitrosalicylic acid 1%, phenol 0.2%,
sodium sulfite 0.05%, NaOH 1%, sodium potassium tartrate 20%); geniposide standard (NICPBP,

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0749-200007) and genipin standard (Seebio Biotechnology, 6902-77-8); β-glucosidase (from
Trichoderma reesei, enzyme activity was 867 u/g, Shanghai Baofeng biochemistry Ltd.);
p-nitrophenyl-β-D-glucoside (p-NPG, Alfa Aesar).
3.2. Determination of β-glucosidase Activity
The activity of β-glucosidase (per unit) was defined as moles of p-NPG converted by g free or
immobilized enzyme per minute [20]. The determination method of free β-glucosidase was as follows:
free β-glucosidase (0.1 mL), 4 mM p-NPG solution (1 mL), sodium acetate buffer (pH 4.5, 0.9 mL)
were mixed and incubated in water bath (50 °C) for 10 min. Then the reaction was stopped by adding
1 mol/L Na2CO3 solution (1 mL). The p-nitrophenol released was monitored at 400 nm by UV-Vis
Spectrophotometer (WFZ-UV-2000, Unico Instrument, China). The activity of the immobilized
β-glucosidase was determined by the same method except that 0.1 mL free enzyme solution was
replaced by appropriate amount of immobilized enzyme gel and 0.1 mL sodium acetate buffer.
3.3. Immobilization of β-glucosidase
β-Glucosidase was immobilized by the crosslinking-embedding method using sodium alginate
as the carrier [21]. Sodium alginate was dissolved in distilled water (20 mL). β-Glucosidase and
cross-linking agent glutaraldehyde were added to the alginate solution and stirred immediately, then
the alginate solution was added dropwise into CaCl2 (100 mL) from a height of 10 cm and left standing
for 2 h. The gel beads were fixed. They were washed with distill water and stored in refrigerator (4 °C).
3.4. Determination of Reaction Rate
The content of glucose released from hydrolysis of geniposide is equal to that of genipin (Scheme 1).
Thus the reaction rate was determined as the amount of glucose released from hydrolysis of geniposide
per minute:
Δ[glucose ] (mg/mL)
Reaction rate (%) ＝
× 100%
(1)
Δt (min)
Free β-glucosidase: free β-glucosidase (0.1 mL), 4 mM geniposide solution (1 mL), sodium acetate
buffer (pH 4.5, 0.9 mL) were mixed and incubated in water bath (50 °C) for 10 min. Then the reaction
was stopped by adding 1 mol/L Na2CO3 solution (1 mL). DNS reagent was added for coloration [22].
Then the solution was monitored at 550 nm. Immobilized β-glucosidase: the method was the same
with free enzyme except that 0.1 mL free enzyme solution was replaced by appropriate amount of
immobilized enzyme and 0.1 mL sodium acetate buffer.
3.5. Determination of Geniposide and Genipin
High Performance Liquid Chromatography (HPLC, Agilent) was used to determine the content of
geniposide and genipin [16]. The mobile phase consists of acetonitrile and water with a ratio of 15:85
and a flow-rate of 1 mL/min. The samples were filtered by 0.45 µm microporous filtering membrane
(MEMBRANA) before injection. The assay was carried out after injecting 20 µL sample to HPLC

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(LC-20A, Shimadzu) with the chromatographic column (Welchrom-C18, Welch) temperature of 30 °C
and the detection wavelength of 238 nm.
Actual yield of genipin (mol)
Productivity of genipin (%)＝
×100%
(2)
Theoretical yield of genipin (mol)
Thin layer chromatography (TLC) was used to detect geniposide and genipin [21]. Spots sampled
with a capillary tube were place on GF254 TLC plate. The plate was dipped into a solvent comprising of
ethyl acetate, chloroform, and methane acid (with the ratio of 5:4:1). Then it was detected under UV
light (254 nm).
3.6. Determination of Optimum Reaction Time in One-phase System
Immobilized β-glucosidase (1 U) and geniposide (0.05 g) was added to sodium acetate buffer (pH
4.5, 10 mL). The mixture was incubated in water bath (50 °C) for 3.5 h. During the reaction, reaction
solution (100 mL) was sampled with HPLC every half an hour to determine the concentration of
geniposide and genipin.
3.7. Determination of Optimum Reaction Time in Two-phase System
Immobilized β-glucosidase (1 U) and geniposide (0.05 g) were added into two-phase system
composed of sodium acetate buffer (pH 4.5, 10 mL) and ethyl acetate (10 mL). Then the mixture was
stirred and formed an emulsion in 55 °C water bath for several hours. During the reaction, 100 μL
organic phase and water phase liquid were sampled respectively with HPLC every half an hour to
determine the concentration of geniposide and genipin. The total content of geniposide (or genipin)
in two-phase system was equal to the sum content of geniposide (or genipin) in organic phase and
water phase.
3.8. Preparation of Genipin by Immobilized β-glucosidase in One-phase System
Immobilized enzyme (5 U), geniposide (0.25 g) and sodium acetate buffer were mixed and
incubated in 50 °C water bath for several hours. The reaction liquid was separated from immobilized
enzyme gel by filtration. The solution was extracted by ethyl acetate (50 mL × 3). The above liquid
(ethyl acetate) was collected and dried by anhydrous sodium sulfate. Then the liquid was concentrated
to 5% (v/v) of the original volume. Put it in refrigerator (−5 °C) for 24 h and collected the crystals. The
crystals were dried in a vacuum drying oven for 1h, and then recrystallized from ethyl acetate.
3.9. Preparation of Genipin by Immobilized β-glucosidase in Two-phase System
Immobilized β-glucosidase (5 U) and geniposide (0.25 g) were added into two-phase system
composed of sodium acetate buffer (pH 4.5, 50 mL) and ethyl acetate (50 mL). Then the mixture was
stirred and formed an emulsion in 55 °C water bath for several hours. The reaction liquid was
separated from the immobilized enzyme gel by filtration. Then the organic phase liquid and water
phase liquid were separated. The product was extracted from water phase liquid by ethyl acetate
(50 mL × 2). The extract and organic phase liquid were mixed and dried by appropriate anhydrous

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sodium sulfate. Then the liquid was concentrated to 5% of the original volume. Put the liquid in
refrigerator (−5 °C) for 24 h and collected the crystals. The crystals were dried in vacuum drying oven
for 1h, and then recrystallized by ethyl acetate.
3.10. Identification of Genipin
The product (genipin) was analyzed by UV (TU-1901), IR (Thermo) [18], NMR (Bruker,
400 MHz) [19]. The spectral data of the product was as follows: UV (MeOH) λmax 240 nm [6]. FT-IR
1
(KBr, cm−1): 3398.6, 3245.7, 1681.6, 1621.9, 1443.3, 1301.2 [18]. H-NMR (CDCl3) δ: 7.53 (s, 3H),
5.88 (s, 7H), 4.81 (d, J = 8.8 Hz, 1H), 4.43~4.19 (m, 2H), 3.25~3.18 (m, 1H), 2.92~2.85 (m, 1H),
13
2.58~2.52 (m, 1H), 2.10~2.03 (m, 1H) ppm. C-NMR (CDCl3) δ: 168.17, 152.74, 142.26, 131.13,
110.96, 96.56, 95.33, 61.53, 51.55, 48.38, 39.24, 36.22 ppm [19]. The data above were in accordance
with those reported before for genipin.
4. Conclusions
β-Glucosidase was immobilized by the crosslinking-embedding method using sodium alginate
as the carrier. The optimum reaction conditions of free and immobilized β-glucosidase were studied.
The optimum reaction temperature of free and immobilized β-glucosidase was 50 °C and 55 °C
respectively, while for both the optimum reaction pH value was 4.5. The immobilization increased the
heat resistance of β-glucosidase and reduced the impact of pH variation on its activity.
Ethyl acetate is a good solvent for genipin. In order to reduce genipin hydrolysis and byproduct
formation, the hydrolysis of geniposide was carried in an aqueous-organic two-phase system
comprised of ethyl acetate and sodium acetate buffer. Genipin can be extracted from sodium acetate
buffer into ethyl acetate during the reaction. The optimum reaction conditions were confirmed to be as
follows: a two-phase system comprising 50% ethyl acetate and 50% sodium acetate buffer (pH 4.5).
The reaction temperature and time were 55 °C and 2.5 h respectively. The yield of genipin in the two-
phase system reaction was higher than in one-phase system reaction. Compared with the one-phase
system, the yield of genipin increased 30% and the reaction time decreased 29% in the two-phase
system. The immobilized β-glucosidase can be recycled for long-term use and showed good stability in
this reaction. The method of genipin preparation established in this paper is simple, easy to realize and
with short production cycle. Moreover, it has solved the problem of enzyme recycling, decreased
production costs, simplified the isolation and purification procedure, and laid the foundation for
industrial production.
Acknowledgements
Support from National Natural Science Foundation of China (Project Number: 30500667), the
National Scientific and Technological Major Special Project of China (Project Number:
2009ZX09103-449), and Shanghai Municipal Education Commission Project (Project Number:
J51301, 10SG40) is gratefully acknowledged.

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References and Notes
1. Ni, H.Y.; Zhang, Z.H.; Fu, H.Z. Research and development of Fructus Gardeniae. China J. Chin.
Mater. Med. 2006, 31, 538-541.
2. Peng, J.; Qian, Z.Y.; Liu, T.Z.; Rao, S.Y.; Qu, B. Comparative studies on hepatic protective and
choleretic effect of geniposide and crocetin. Chin. J. New Drugs 2003, 12, 105-108.
3. Suzuki, Y.; Kondo, K.; Ikeda, Y.; Umemura, K. Antithrombotic effect of geniposide and genipin
in the mouse thrombosis model. Planta Med. 2001, 67, 807-810.
4. Xie, W.L.; Jin, Y.Z. Studies on the effects of geniposide in the lowering blood sugar of rat
diabetic model. Acta Acad. Med. CPAF 2008, 17, 580-581.
5. Wang, G.F.; Wu, S.Y.; Rao, J.J. Genipin inhibits endothelial exocytosis via nitric oxide in
cultured human umbilical vein endothelial cells. Acta Pharmacol. Sin. 2009, 30, 589-596.
6. Cao, H.; Feng, Q.; Xu, W.; Li, X.R.; Kang, Z.; Ren, Y.; Du, L. Genipin induced apoptosis
associated with activation of the c-Jun NH2-terminal kinase and p53 protein in HeLa cells. Biol.
Pharm. Bull. 2010, 33, 1343-1348.
7. Lelono, R.A.A.; Tachibana, S.; Itoh, K. Isolation of antifungal compounds from gardenia
jasminoides. Pak. J. Biol. Sci. 2009, 12, 946-956.
8. Lee, J.H.; Lee, D.U.; Jeong, C.S. Gardenia jasminoides Ellis ethanol extract and its constituents
reduce the risks of gastritis and reverse gastric lesions in rats. Food Chem. Toxicol. 2009, 47,
1127-1131.
9. Zhang, C.Y.; Parton, L.E.; Ye, C.P.; Krauss, S.; Shen, R.; Lin, C.T.; Porco, J.A.; Lowell, B.B.
Genipin inhibits UCP2-mediated proton leak and acutely reverses obesity- and high glucose-
induced b cell dysfunction in isolated pancreatic islets. Cell Metab. 2006, 3, 417-427.
10. Yamazaki, M.; Chiba, K.; Yoshikawa, C. Genipin suppresses A23187-induced cytotoxicity in
Neuro2a cells. Biol. Pharm. Bull. 2009, 32, 1043-1046.
11. Tian, J.; Cui, Y.; Hu, L.; Gao, S.; Chi, W.; Dong, T.; Liu, L. Antidepressant-like effect of genipin
in mice. Neurosci. Lett. 2010, 479, 236-239.
12. Fujikawa, S.; Nakamura, S.; Koga, K. Genipin,a new type of protein cross-linking reagent from
gardenia fruits. Agr. Biol. Chem. 1988, 53, 869-870.
13. Madhavan, K.; Belchenko, D.; Tan, W. Roles of genipin crosslinking and biomolecule
conditioning in collagen-based biopolymer: Potential for vascular media regeneration. J. Biomed.
Mater. Res. A 2011, 97A, 16-26.
14. Hou, Y.C.; Tsai, S.Y.; Lai, P.Y. Metabolism and pharmacokinetics of genipin and geniposide in
rats. Food Chem. Toxicol. 2008, 46, 2764-2769.
15. Cao, J.P.; Wang, Y.L.; Jia, Y.J. Simultaneous determination of geniposide and genipin in gardenia
jasminoides ellis by high performance liquid chromatography. J. Dalian Med. Univ. 2001, 23,
61-62.
16. Lu, Y.; Zhang, T.; Tao, J.S. Study on processing of hydrolyzing geniposide with β-glucosidase.
Acta Univ. Trad. Med. Sinensis Pharmacol. Shanghai 2008, 22, 76-78.
17. Yamano, T.; Tsujimoto, Y.; Noda, T.; Shimizu, M.; Ohmori, M.; Morita, S.; Yamada, A.
Hepatotoxicity of geniposide in rats. Food Chem. Toxicol. 1990, 28, 515-519.

Molecules 2011, 16
4304
18. Lu, Y.; Zhang, T.; Tao, J.S.; Ji, G.; Wang, S.M. Preparation, characterization, and pharmacokinetics
of the inclusion complex of genipin-β-cyclodextrin. Drug Dev. Ind. Pharm. 2009, 35, 1452-1459.
19. Xu, M.; Sun, Q.; Su, J.; Wang, J.; Xu, C.; Zhang, T.; Sun, Q. Microbial transformation of
geniposide in Gardenia jasminoides Ellis into genipin by Penicillium nigricans. Enzyme Microb.
Technol. 2008, 42, 440-444.
20. Zanoelo, F.F.; Polizeli, M.L.T.M.; Terenzi, H.F.; Jorge, J.A. β-glucosidase activity from the
thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS
Microbiol. Lett. 2004, 240, 137-143.
21. Zhao, L.G.; Li, L.J.; Wang, P.; Yu, S.Y. Immobilization of β-glucosidase by sodium alginate.
Chin. J. Bioproc. Eng. 2007, 25-31.
22. Zhao, K.; Xue, P.J.; Gu, G.Y. Study on Determination of Reducing Sugar Content Using 3,5-
Dinitrosalicylic Acid Method. Food Sci. 2008, 29, 534-536.
Sample Availability: Samples of the compounds 1 and 2 are available from the authors.
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