Food Chemistry 134 (2012) 11991204

Contents lists available at SciVerse ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Identication and quantication of constituents of Gardenia jasminoides Ellis

(Zhizi) by HPLC-DADESIMS
M.C. Bergonzi , C. Righeschi, B. Isacchi, A.R. Bilia
University of Florence, Dept. of Pharmaceutical Sciences, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy

a r t i c l e

i n f o

Article history:
Received 21 July 2010
Received in revised form 25 October 2011
Accepted 26 February 2012
Available online 6 March 2012
Keywords:
Gardenia jasminoides Ellis (Zizhi)
HPLC-DADMS
Qualitative and quantitative analysis
Iridoids
Crocins
Caffeoyl quinic acid derivatives
Method validation

a b s t r a c t
A simple, rapid and specic HPLC method was carried out for the analysis of characteristic constituents in
Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C18 column by gradient elution with mixtures of methanol and
water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic
acid derivatives and 438 nm for crocins.
The analytical method was validated and the quantication of active compounds, namely iridoids, was
performed. Linearity, precision, repeatability, stability, accuracy, limit of detection (LOD) and limit of
quantication (LOQ) were also reported. This assay was successfully applied for qualitative and quantitative analysis of ve commercial samples of G. jasminoides Ellis.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
The fruits of Gardenia jasminoides Ellis (Rubiaceae) (Gardeniae
Fructus, Chinese name Zhizi) are widely used in the Traditional
Chinese Medicine. Numerous properties are reported for this herbal drug and its preparation, namely in the treatment of irritability
in febrile diseases, jaundice, acute conjunctivitis, epitasis, haematuria, pyogenic infections and ulcers of the skin, and also, externally,
sprains and painful swellings due to blood stasis (Chang & But,
1987; Tang & Eisenbrand, 1992; Ukita, Yamasaki, Ino, Kawamoto,
& Saito, 1994; Wang, Tseng, Huang, & Tsai, 2004). Recent studies
have also attributed antiangiogenic properties to Zhizi (Park, Joo,
Kim, & Lim, 2003).
In addition to Gardeniae Fructus, Gardeniae Fructus Preparatus
is also present on the market, obtained by processing Fructus
Gardeniae which is stir-baked or broken into pieces in a hot pot
with middle heat until it becomes burnt-brown or burnt-black
externally and the inner surface and seed coats yellowish-brown
or dark brown (Pharmacopeia of Peoples Republic of China,
2005).
The major constituents of Gardenia fruits are iridoid glycosides
including geniposide, gardenoside, genipin-1-O-b-gentiobioside,
geniposidic acid, acetylgeniposide, scandoside methyl ester,
Corresponding author. Tel.: +39 055 4573678; fax: +39 055 4573680.
E-mail address: mc.bergonzi@uni.it (M.C. Bergonzi).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2012.02.157

shanzhiside and gardoside. Many authors have assumed that these

compounds are the molecules responsible for the biological
activities of this herbal drug and its preparations (Li et al., 2000;
Miura, Nishiyama, Ichimaru, Moriyasu, & Kato, 1996; Park et al.,
2003; Ukita et al., 1994; Yamauchi, Fujimoto, Kuwano, Inoue, &
Inoue, 1976).
Gardenia fruit is also widely used in Asian countries as a natural
colourant because of the presence of crocins, a series of mono- and
di-glucosyl esters of crocetin, crocin-1, -2, -3 (Pster, Meyer, Steck,
& Pfander, 1996; van Calsteren et al., 1997).
Numerous studies have reported that even crocins possess a
variety of biological effects, principally the antioxidant properties
(Ahmad et al., 2005; Escribano, Alonso, Coca-Prados, & Fernndez,
1996; He et al., 2005; Ochiai et al., 2004; Shen & Qian, 2006; Tseng,
Chu, Huang, Shiow, & Wang, 1995; Xiang et al., 2006).
Ubiquitous quinic acid derivatives have also been identied in
the fruit of G. jasminoides (Nishizawa & Fujimoto, 1986; Nishizawa,
Izuhara, Kaneko, & Fujimoto, 1987; Wenhao et al., 2010).
The aim of this study was to establish a reliable HPLC method
for simultaneous determination of these three classes of constituents in Gardenia jasminoides Ellis, namely iridoids, crocins and caffeoyl quinic acid derivatives.
Some chromatographic methods have been previously reported
(He, Cheng, Chen, & Zhou, 2006; Xu, Cao, Wang, & Luo, 2003) but
none of them can be applied to investigate quality control of the
commercial products.

1200

M.C. Bergonzi et al. / Food Chemistry 134 (2012) 11991204

In the present work, we have developed and validated a

HPLC-DADMS method suitable for identifying 12 compounds. A
comprehensive validation study was also carried out and covered
linearity, precision, repeatability, stability, accuracy, limits of
detection and quantication of the iridoids which represent the
main molecules responsible for the activity of Zhizi. The phenolic
compounds were also quantied using chlorogenic acid as external
standard. Only qualitative analysis of crocins is reported because of
the lack of suitable standards on the market for their quantication.
The developed HPLC method would be helpful for the quality
control of G. jasmonoides Ellis, its preparata and related Chinese
remedies.

2. Experimental procedures
2.1. Apparatus for HPLC-DADESIMS analysis
The HPLC system consisted of a HP 1100L instrument equipped
with a HP 1040 diode-array detector (DAD), an automatic injector,
an auto sampler and a column oven and managed by a HP 9000
workstation (Agilent Technologies, Palo Alto, CA, USA). The HPLC
system was interfaced with a HP 1100 MSD API-electrospray
(Agilent Technologies, Palo Alto, CA, USA).
The reverse-phase column was a Luna C18 (250  4.6 mm, 5 lm,
Phenomenex) maintained at 27 C. The mobile phase was a twostep linear gradient CH3OH (A)/H2O (pH 3.2, HCOOH) (B) at a ow
rate of 1 ml/min: 0.104.0 min 10% A and 90% of B; 4.040 min
90% B to 10% B. Before the HPLC analysis, each sample was ltered
through a cartridge-type sample ltration unit with a polytetrauoroethylene (PTFE) membrane (d = 13 mm, porosity 0.45 lm, Lida
manufacturing Corp.) and immediately injected. The injected volume of sample was a 20-ll solution. UVVis spectra were recorded
in the range 210500 nm, and chromatograms were acquired at 240
(iridoid glycosides), 315 (quinic acid derivatives), 438 nm (crocins).
The HPLC system was interfaced with a HP 1100 MSD APIelectrospray (Agilent Technology, Palo Alto, CA, USA). The interface
geometry, with an orthogonal position of the nebulizer with respect
to the capillary inlet, allowed the use of analytical conditions similar
to those of the HPLC-DAD analysis. The same column, mobile phase,
time period and ow rate were used. Mass spectrometry operating
conditions were optimised in order to achieve maximum sensitivity
values; gas temperature 350 C at a ow rate of 10 l/min, nebulizer
pressure 30 p.s.i., quadrupole temperature 30 C, and capillary voltage 3500 V. Full scan spectra from m/z 100 to 800 in the positive ion
mode were obtained (scan time 1 s). Mass spectra were performed
in negative and positive ion mode, setting the fragmentation energy
between 80 and 120 V and applying the same chromatographic conditions described previously.

Name
Zhizi
Gardeniae
Gardeniae
Gardeniae
Gardeniae

Code
(Fructus)
(Fructus)
(Fructus)
(Fructus)

(Zhizi)
(Zhizi) (Hebei)
Preparata (Chaozhizi) (Hebei)
Preparata (Jiaozhizi) (Hebai)

25845
32496
33031
33024
32990

The sample 32496 was used for the validation study.

2.4. Preparation of sample solutions
Ten millilitres of 50% methanol solution was added to 1 g of the
powdered herbal drug; this hydroalcoholic extract was ultrasonicated for 40 min, and ltered (Wagner, 2004). The extraction was
repeated three times. The ltrate was evaporated until dryness
(Drug-extract-ratio, D.E.R. 3.0-3.6:1) and the residue, precisely
weighed, was dissolved in methanol to prepare the solutions for
method validation and analyses.
3. Results and discussion
3.1. Optimisation of chromatographic conditions
This study concerns the HPLC-DADESIMS characterisation of
extracts of Gardeniae Fructus (Zhizi). The wavelengths of 240, 315
and 438 nm were selected for acquiring chromatograms of iridoid
glycosides, quinic acid derivatives and pigments, respectively. In
order to achieve better chromatographic separation, various linear
gradients of acetonitrilewater and methanolwater were investigated at a ow-rate of 1.0 mL min 1. Finally, the gradient programme described in the experimental part was chosen because
all the peaks in the chromatogram were clearly separated. The
HPLC-DAD proles of the methanolic extract at the wavelengths
of 240, 315 and 438 nm are reported in Figs. 13, respectively. The
compounds identied are reported in Table 1. The method allowed
the separation of the iridoids, of quinic acid derivatives and four
crocins.
The identication of compounds was carried out by comparing
the characteristic UV absorption spectra, retention time and MS
data of standard compounds to those already present in literature.
The peaks indicated in Fig. 2 by are caffeoylquinic acid derivatives, identied by their UV absorption spectra and the presence
of different diagnostic fragments in their MS spectrum, such as
[quinic acid H] m/z 191, [quinic acid H H2O] m/z 173,
[caffeoyl H COO] m/z 135.
This HPLC method was also applied in the determination of the
Fructus Gardeniae constituents in ve commercial samples, as reported in Section 3.3. All the analyses were repeated in triplicate.

2.2. Standards and reagents

3.2. Method validation

Geniposide CRS provided by Chinas National Institute for the

Control of Pharmaceutical and Biological Products (NICPBP, Beijing,
China), purity 95.63% by elemental analysis. Chlorogenic acid
(P99%) was from Extrasynthese, Genay Cedex, France.
MeOH (HPLC grade) was purchased from Merck (Darmstadt,
Germany); 85% formic acid was provided by BDH AnalaR. Water
was puried by a Milli-Qplus system from Millipore (Milford, MA,
USA).

The method was validated according to ICH guidelines (ICH,

1994, 1996). The method was found to be specic and suitable for
the routine analysis because of its simplicity, sensitivity, accuracy
and reproducibility. It can be conveniently used for the quantication of iridoids in G. jasminoides Ellis commercial samples.

2.3. Samples
Five commercial samples of Gardenia Fructus were supplied by
PLANTASIA Heinrich Handel-Mazzettiplaz 1, A-5110 Oberndorf,
Austria.

3.2.1. Linearity
Linearity range of response was determined for geniposide
(the main iridoid) reference standard CRS, chlorogenic acid and
G. jasminoides Ellis extract. Geniposide was used to optimise the
extraction of the herbal drug, because it is the main iridoid of the
extract.
Linearity was determined on ve levels of concentration with
three injections for each level. Geniposide and chlorogenic acid

1201

M.C. Bergonzi et al. / Food Chemistry 134 (2012) 11991204

mAU

2500

2000

1500

1000

3
2

500

5
0
5

10

15

20

25

min

Fig. 1. HPLC prole at 240 nm of Gardenia jasminoides Ellis extract; iridoids (15) are listed on Table 1.

mAU
450

400

350

300

250

200

*
*

150

100

*
*

50

0
0

10

15

20

25

30

35

min

Fig. 2. HPLC prole at 315 nm of Gardenia jasminoides Ellis extract; caffeoyl quinic derivatives (68) are listed on Table 1.

showed a linear response from 1.0 to 1000 lg/ml and all the curves
had coefcients of linear correlation P0.998.
A linearity of response in the range from 0.07 to 1.86 mg/ml of
G. jasminoides Ellis extract (concentrations used: 0.07, 0.14, 0.28,
0.57, 1.86 mg/ml in methanol) was observed for the main constituent geniposide. Additionally, the value of R2 was from 0.9994 to
0.9990, with the slope R.S.D. values lower than 1.5%, which indicated a high accuracy of the method.

ratio. A signal-to-noise ratio 3:1 is generally considered acceptable

for estimating the detection limit. The sample that produces a signal-to-noise ratio of approximately 10:1 corresponds to the concentration at which the analyte can be reliably quantied. Geniposide
at a concentration of 0.192 lg/ml (injected 5 ll, 0.76 ng) produces
a signal-to-noise ratio of approximately 3.2; while geniposide at a
concentration of 0.532 lg/ml (injected 10 ll, 5.32 ng) produces a
signal-to-noise ratio of approximately 12.1.

3.2.2. Limit of detection (LoD) and limit of quantitation (LoQ) of

geniposide
The detection limit (LoD) and the quantitation limit (LoQ) of
geniposide was determined by calculation of the signal-to-noise

3.2.3. 48-Hour extract stability

Reference compounds and the G. jasminoides Ellis methanolic
extract (1.80 mg/ml) were solubilised in methanol before the

1202

M.C. Bergonzi et al. / Food Chemistry 134 (2012) 11991204

9

mAU

1400

1200

1000

800

600

400
10

11

12

200

0
0

10

15

20

25

30

35

min

Fig. 3. HPLC prole at 438 nm of Gardenia jasminoides Ellis extract; crocins (912) are listed on Table 1.

Table 1
Compounds identied in the Gardenia Fructus extract.
Peak

tR

MS

UVmax nm

Compound

1
2
3
4
5
6
7
8
9
10
11
12

7.51
9.03
13.68
16.17
24.45
25.85
24.38
24.76
27.15
29.26
35.06
35.48

[M+Na]+ m/z 427

[M+Na]+ m/z 427
[M+Na]+ m/z 573 [M + H]+ m/z 551
[M+Na]+ m/z 411
[M+Na]+ m/z 453
[M+Na]+ m/z 609
[M H] m/z 659
[M H] m/z 559
[M H] m/z 975
[M H] m/z 813
[M H] m/z 489
[M H] m/z 327

240
240
240
240
240
250, 355
315
315
438
438
438
438

Scandoside methylestere
Gardenoside
Genipin gentobioside
Geniposide
Acetylgeniposide
Quercetin-3-rutinoside
3,4-Dicaffeoyl-5-(3-hydroxy-3-methylglutaroyl) quinic acid
Caffeoyl sinapoylquinic acid
Crocin 1
Crocin 2
Crocin 3
Crocetin

Table 2
%D/Z (D = determined concentration and Z = determined concentration on hour-zero)
values over 48 h stability of standards and geniposide in the extract.
Time
(h)

Standard
geniposide

Standard clorogenic
acid

Geniposide in the
extract

0
4
8
12
16
20
24
28
32
36
40
44
48

100
98.58
98.87
98.89
99.62
98.81
99.17
99.05
99.01
98.97
98.59
98.71
98.99

100
99.93
99.49
99.05
98.97
98.43
98.41
98.21
97.94
97.67
97.29
96.98
96.82

100
99.30
99.60
99.74
99.32
99.15
98.99
98.83
98.03
98.04
97.98
97.87
97.87

analyses and their stability at room temperature was evaluated

every 4 h, up to 48 h. The standards and the extract were found
to be stable in methanol solution at room temperature for at least
48 h. In Table 2 the %D/Z (D = determined concentration and
Z = determined concentration on hour-zero) values over the course
of the stability time period was reported.

3.2.4. Reproducibility of the injection integration

The reproducibility of the injection integration procedure was
determined for geniposide (0.96 mg/ml) and the G. jasminoides Ellis
extract. The solutions were injected ten times and the relative
standard deviation (R.S.D.) values were calculated.
R.S.D. of extract at the concentration of 1.14 mg/ml were: geniposide 1.07%; scandoside methylesthere 1.72%, gardenoside 1.87%,
genipin gentiobioside 1.23%; acetylgeniposide 0.64%, 3,4-dicaffeoyl-5-(3-hydroxy-methylglutaroyl) quinic acid 1.41% and
caffeoyl sinapoylquinic acid 0.98%.
3.2.5. Repeatability of the method
In order to evaluate the repeatability of the method, three
solutions at different concentrations (0.57, 1.14 and 1.86 mg/ml of
G. jasminoides Ellis dry extract in methanol) were prepared. Each
solution was injected three times. The contents of iridoids and
quinic acid derivatives were calculated in order to estimate the
R.S.D. Geniposide (R.S.D. 0.72%, 0.27% and 1.02% respectively),
scandoside methylesthere (R.S.D. 0.40%, 1.3%, 1.17%), gardenoside
(R.S.D. 1.71%, 1.51%, 1.41%), genipin gentiobioside (R.S.D. 1.39%,
1.76%, 1.35%), acetylgeniposide (R.S.D. 1.0%, 0.98%, 0.40%) (3,4-dicaffeoyl-5-(3-hydroxy-methylglutaroyl) quinic acid (R.S.D. 1.6%,
0.17%, 0.60%) and caffeoyl sinapoylquinic acid (R.S.D. 1.57%, 0.48%,
0.94%).

1203

M.C. Bergonzi et al. / Food Chemistry 134 (2012) 11991204

Fig. 4. The overlay of 3 UV-spectra (240 nm) at the beginning, at the apex and at the end of the peak geniposide at 16.17 min.

3.2.6. Intermediate precision

The same samples described in the Section 3.2.5. were injected
six times on three different days for the purpose of evaluating
intermediate precision relative to geniposide, used as compound
representative of the extract.
Precision intraday, n = 6 R.S.D. 0.93%, 2.50%, 0.59%; precision
interday n = 18 R.S.D. 1.86%, 0.76%, 1.32%.
3.2.7. Precision of the sample preparation
To evaluate the precision of the sample preparation, three solutions at concentration about 0.57 mg/ml (0.57, 0.58, 0.60 mg/ml) of
dry extract in methanol were prepared. Each solution was injected
three times. The contents of iridoids and quinic acid derivatives
were calculated in order to estimate the R.S.D. All R.S.D. values of
the main constituents resulted between 0.55% and 1.2%.
3.2.8. Accuracy
In order to calculate the biases for the linearity data, the accuracy
of the method was determined by analysing the percentage recovery
of geniposide into the three preparations of G. jasminoides Ellis
extract. Three independent solutions of extract were prepared
(0.57, 1.14 and 1.86 mg/ml) and each was injected three times.
The average percentage recovery was calculated for each level of
concentration. The accuracy was determined by spiking 50 lg/ml
of geniposide separately to the three batches of the extract. The
percentages recovery of geniposide standard spiked into three
preparations were 93.63 2.15%; 96.21 1.04% and 97.27 2.55%,
respectively.
3.2.9. Specicity
The peak purity was investigated by inspecting the UV-spectra
and MS spectra at the beginning, at the apex and at the end of the
peaks of each constituent of the extract. No deviations were seen.
As an example, Fig. 4 showed the overlay of 3 UV-spectra at the

beginning, at the apex and at the end of the peak geniposide

(240 nm).
3.3. Sample analysis
The variability of the constituents in the different samples is
stressed, with special emphasis on iridoids, crocins and quinic acid
derivatives. The quantitative determination of iridoids was
performed at 240 nm, using a geniposide CRS as external standard.
Table 3 reports the content of iridoids, expressed as geniposide,
found in each Gardeniae Fructus sample.
No qualitative differences among the diverse samples were
found but quantitative differences in the constituents were found
and were attributable to different sources or different methods of
processing of the herbal drug. Sample 25845 consisted of the whole
herbal drug (Zhizi) and was the richest sample in iridoids content.
Considering the two samples 32496 and 33031, the different prole
could be attributed to their different sites of collection. The two
samples of Gardeniae Fructus Preparata (33024 Chaozhizi; 32990
Jiaozhizi) have qualitative and quantitative proles of iridoids
which are similar to those of Gardenia Fructus indicating that the
process obtained by heating the herbal drug does not inuence
particularly the composition of iridoids in the drug, according to
the results previously reported in the literature (Sheu & Hsin, 1998).
The quantitative analysis of quinic acid derivatives was performed at 315 nm, using chlorogenic acid as external standard.
The results were summarised in Table 4. Their content ranged from
23.16 to 44.39 mg/g of extract. Sample 33031 had the poorest content of these compounds, as well as for the iridoids.
From our studies it seems that the variability of the constituents content could be related to the geographic area of origin
rather than the processing of the plant material.
The chromatographic proles of the crocins were the same for all
ve samples: the derivatives identied were crocetin, crocin-1,

Table 3
Content (mg/g and %) of iridoids found in Gardeniae Fructus extracts.
Sample

Scandoside methylesthere mg/g of

extract, (%)

Gardenoside mg/g of
extract, (%)

Genipin gentiobioside mg/g of

extract, (%)

Geniposide mg/g of
extract, (%)

Acetylgeniposide mg/g of
extract, (%)

25845
32496
33031
33024
32990

8.76 1.09
8.60 0.48
1.77 0.24
4.64 1.25
4.92 0.46

20.72 0.98 (0.21%)

10.29 0.79 (1.03%)
1.70 0.56 (0.17%)
16.90 0.55 (0.17%)
11.25 2.92 (1.12%)

20.07 1.98 (2.00%)

10.57 0.28 (1.06%)
9.23 0.07 (0.92%)
16.90 0.55 (1.69%)
7.73 2.16 (0.77%)

213.31 2.37 (21.33%)

155.30 0.94 (15.53%)
125.49 1.60 (12.55%)
152.36 1.98 (15.23%)
157.94 13.85 (15.79%)

5.14 0.35
7.43 0.06
3.97 0.76
6.95 0.19
6.12 0.83

(0.87%)
(0.86%)
(0.17%)
(0.46%)
(0.49%)

(0.51%)
(0.74%)
(0.39%)
(0.69%)
(0.61%)

1204

M.C. Bergonzi et al. / Food Chemistry 134 (2012) 11991204

Table 4
Content (mg/g and %) of caffeoyl quinic acid derivatives found in Gardeniae Fructus extracts.
Sample

3,4-Dicaffeoyl-5-(3-hydroxy3-methylglutaroyl) quinic acid

Caffeoyl sinapoylquinic acid

Other caffeoylquinic
derivatives (evidenced by in Fig. 2)

25845
32496
33031
33024
32990

4.89 0.22 (0.49%)

5.73 0.21 (0.57%)
3.76 1.11(0.38%)
5.97 0.58 (0.60%)
4.17 1.40 (0.42%)

20.43 0.18 (2.04%)

8.64 0.85 (0.86%)
9.25 0.038 (0.92%)
12.65 0.41 (1.26%)
6.96 0.81 (0.70%)

19.08 0.28
17.83 0.38
10.15 0.32
14.37 0.73
15.59 1.64

crocin-2, crocin3 (Fig. 3). The comparison of the AUCs of single peaks
in the different investigated commercial samples showed once
again that the lowest content was evidenced in sample 33031, collected from the district of Hebei, while the highest quantity was
present in the entire Zhizi (sample 25845).

4. Conclusions
The separation of the constituents of Gardenia fructus was successfully obtained with a C18 column using a gradient elution with
methanol and water as mobile phases. Detection wavelengths
were set at 240 nm for iridoid glycosides, 315 nm for quinic acid
derivatives and 438 nm for crocins. The method was validated
according to ICH guidelines, taking into account that iridoids represent the characteristic molecules responsible for the activity of
Zhizi. This HPLC assay was successfully used for the determination
of constituents for the qualitative and quantitative characterisation
of ve commercial samples of Gardeniae Fructus. The method was
found to be specic and suitable for the routine analysis because of
its simplicity, sensitivity, accuracy and reproducibility; it can be
conveniently used for iridoids in G. jasminoides Ellis commercial
samples. This HPLC method can be proposed for the quality control
of Gardenia jasmonoides Ellis and its related Chinese remedies. The
analytical method is also specic for other components of the extract, such as crocins and quinic acid derivatives.
References
Ahmad, A. S., Ansari, M. A., Ahmad, M., Saleem, S., Yousuf, S., & Hoda, M. N. (2005).
Neuroprotection by crocetin in a hemi-parkinsonian rat model. Pharmacology,
Biochemistry and Behavior, 81, 805813.
Chang, H. M., & But, P. P. H. (1987). Pharmacology and Applications of Chinese Materia
Medica, vol. 2. Singapore: World Scientic Publishing, p. 1005.
Escribano, J., Alonso, G. L., Coca-Prados, M., & Fernndez, J. A. (1996). Crocin, safranal
and picrocrocin from saffron (Crocus sativus L.) inhibit the growth of human
cancer cells in vitro. Cancer Letters, 100, 2330.
He, M. L., Cheng, X. W., Chen, J. K., & Zhou, T. S. (2006). Simultaneous determination
of ve major biologically active ingredients in different parts of Gardenia
jasminoides fruits by hplc with diode-array detection. Chromatographia, 64,
713717.
He, S.-Y., Qian, Z.-Y., Tang, F.-T., Wen, N., Xu, G.-L., & Sheng, L. (2005). Effect of crocin
on experimental atherosclerosis in quails and its mechanisms. Life Sciences, 77,
907921.
ICH, Text on validation of analytical proceduresICH Harmonised Tripartite
Guideline, 1994.
ICH, Validation of analytical procedures: methodologyICH Harmonised Tripartite
Guideline, 1996.

(1.9%)
(1.8%)
(1.0%)
(1.4%)
(1.6%)

Li, Y., Kamo, S., Metori, K., Koike, K., Che, Q., & Takahashi, S. (2000). The promoting
effect of eucommiol from Eucommiae cortex on collagen synthesis. Biological &
Pharmaceutical Bulletin, 23, 5459.
Miura, T., Nishiyama, Y., Ichimaru, M., Moriyasu, M., & Kato, A. (1996).
Hypoglycemic activity and structureactivity relationship of iridoidal
glycosides. Biological & Pharmaceutical Bulletin, 19, 160161.
Nishizawa, M., & Fujimoto, Y. (1986). Isolation and structural elucidation of a new
lipoxygenase inhibitor from gardenia fructus. Chemical Pharmaceutical Bulletin,
34, 14191421.
Nishizawa, M., Izuhara, R., Kaneko, K., & Fujimoto, Y. (1987). 3-Caffeoyl-4sinapoylquinic acid, a novel lipoxygenase inhibitor from gardenia fructus.
Chemical Pharmaceutical Bulletin, 35, 21332135.
Ochiai, T., Ohno, S., Soeda, S., Tanaka, H., Shoyama, Y., & Shimeno, H. (2004). Crocin
prevents the death of rat pheochromocytoma (PC-12) cells by its antioxidant
effects stronger than those of a-tocopherol. Neuroscience Letters, 362, 6164.
Park, E. H., Joo, M. H., Kim, S. H., & Lim, C. J. (2003). Antiangiogenic activity of
Gardenia jasminoides fruit. Phytotherapy Research, 17, 961962.
Pster, S., Meyer, P., Steck, A., & Pfander, H. (1996). Isolation and structure
elucidation of carotenoid-glycosyl esters in Gardenia fruits (Gardenia
jasminoides Ellis) and saffron (Crocus sativus Linne). Journal of Agricultural and
Food Chemistry, 44, 26122615.
Pharmacopeia of Peoples Republic of China, vol. I, 2005, p. 95.
Shen, X.-C., & Qian, Z.-Y. (2006). Effects of crocetin on antioxidant enzymatic
activities in cardiac hypertrophy induced by norepinephrine in rats. Pharmazie,
6, 348352.
Sheu, S.-J., & Hsin, W.-C. (1998). HPLC separation of the major constituents of
Gardeniae Fructus. Journal of High Resolution Chromatography, 21, 523526.
Tang, W. & Eisenbrand, G. (1992). Chinese Drug of Plant Origin, SpringerVerlag,
Berlin, p. 539.
Tseng, T. H., Chu, C. Y., Huang, J. M., Shiow, S. J., & Wang, C. J. (1995). Crocetin
protects against oxidative damage in rat primary hepatocytes. Cancer Letters, 97,
6167.
Ukita, K., Yamasaki, T., Ino, T., Kawamoto, Y., & Saito, H. (1994). Pharmacological
evaluation of DS-4773 on sedative effect. Japanese Pharmacology and
Therapeutics, 22, 253268.
Van Calsteren, M.-R., Bissonnette, M. C., Cormier, F., Dufresne, C., Ichi, T., & LeBlanc,
J. C. Y. (1997). Spectroscopic characterization of crocetin derivatives from Crocus
sativus and Gardenia jasminoides. Journal of Agricultural and Food Chemistry, 45,
10551061.
Wagner H. Chinese drug monographs and analysis. Fructus Gardeniae Zhizi. 2004,
vol. 5, No. 22, ISSN 14308290.
Wang, S.-C., Tseng, T.-Y., Huang, C.-M., & Tsai, T.-H. (2004). Gardenia herbal active
constituents: applicable separation procedures. Journal of Chromatography B,
812, 193202.
Wenhao, H., Xuan, L., Honggao, X., Ying, G., Fang, Y., & Yanxiang, G. (2010). On-line
HPLC-ABTS screening and HPLC-DAD-MS/MS identication of free radical
scavengers in Gardenia (Gardenia jasminoides Ellis) fruit extracts. Food
Chemistry, 123, 521528.
Xiang, M., Qian, Z.-Y., Zhou, C.-H., Liu, J., Li, W.-N., & Li, W.-N. (2006). Crocetin
inhibits leukocyte adherence to vascular endothelial cells induced by AGEs.
Journal of Ethnopharmacology, 107, 2531.
Xu, Y., Cao, J., Wang, Y. M., & Luo, G. A. (2003). Simultaneous determination of 3
kinds of components in Gardenia by high-performance liquid chromatography
under different UVvis wave length. Yaoxue Xuebao, 38, 543546.
Yamauchi, K., Fujimoto, N., Kuwano, S., Inoue, H., & Inoue, K. (1976). The mechanism
of purgative action of geniposide, an iridoid glucoside of the fruit of Gardenia, in
mice. Planta Medica, 30, 3947.