6th Thailand-Taiwan Academic Cooperation Conference on “Food and Agricultural Innovation:
Going Global” 15 November 2010 at Kasetsart University, Bangkok, Thailand
Effect of seed germination of Cajanus cajan on pinostrobin and
cajaninstilbene acid quality
Chen Chih-Yuan a Lai Chun-Yen b Huang Tzou-Chi b*
a
Graduate Institute of Bioresources , National Pingtung University, 912, Pingtung, Taiwan
b Department of Food Science, National Pingtung University, 912, Pingtung, Taiwan
Corresponding author: tchuang@mail.npust.edu.tw
ABSTRACT
The guandu bean or pigeon pea (Cajanus cajan (L.) Millsp.) is a kind of leguminous plant of the Fabbaceae family and is grown in Asia, Africa and South America. With
the improvement of the life quality, disease prevention and health care, Cajanus cajan has been paid more attentions. Seeds were soaked in water for 24 hours and
germinated. The sprouts of Cajanus cajan were freeze-dried and divided into root, stem and leave. Microwave-assisted extraction using ethanol-water (60:40) as solvent was
applied to extract the major components from the dried samples. LC-MS were used to characterize the chemical structure. Two major compounds, pinostrobin and
cajaninstilbene acid were found in the leave of spouts but not in the seeds. The concentration of these two compounds increased with increased cultivation time.
Keywords: Cajanus cajan, seed germination, microwave-assisted extraction, LC-MS, pinostrobin, cajaninstilbene acid
Introduction
Pigeon pea (Cajanus cajan (L.) Millsp.) is a famous and multi-use grain crop in semi-tropical and tropical
developing countries. Pigeon pea is an ideal source of protein and Vitamin B in human diet, especially in the
vegetarian population. It has been used for many years for treating diabetes, hepatitis, measles, jaundice and
dysentery, expelling bladder stones and stabilizing the menstrual period (Fu et al., 2006). Chemical composition
investigations revealed that there are a number of phenolic compounds present in pigeon pea leaves, stems and roots,
including flavonoids, stilbenes, etc.(Duker-Eshun et al., 2004). In previous investigation, the ethanol extracts of
pigeon pea leaves as well as the main constituents vitexin, orientin, pinostrobin and cajaninstilbene acid exhibited
moderate antioxidant activity (Wu et al., 2009). Therefore, pigeon pea is a beneficial food and potential source of
natural antioxidants.
Using standards for pinostrobin and cajaninstilbene acid, retention times, UV–Vis spectra, and MS–MS
spectra matched pinostrobin and cajaninstilbene acid from Pigeon pea extract. The variation in contents of
pinostrobin and cajaninstilbene acid compounds in pigeon pea during seedling growth period was shown in Fig.
2. Two major compounds, pinostrobin and cajaninstilbene acid were found in the leave of spouts but not in the
seeds. The concentration of these two compounds increased with increased cultivation time. It was observed that
Seedlings are vulnerable to pathogen attacks and for efficient protection, chemical defenses must be deployed
and accumulated very early in the seedling development (Ceballos et al., 1998), hence, much secondary
metabolites are generated. The variation in contents of phenolic compounds correlates with the metabolic balance
of plants and external environmental disturbances.
RT: 0.00 - 60.00
40.62
900000
A
3.74
O
600000
uAU
O
OH
500000
O
49.68
400000
Matured pigeon pea seeds were bought from JIANSHIH township, HSINCHU county, TAIWAN. After immersion
in water for 24 h, the pigeon pea seeds were planted in the same laboratory.
Cajaninstilbene acid
47.45 min
9.99
700000
Germination procedure
44.91
Pinostrobin 41.67 min
800000
Materia and Method
NL:
9.58E5
WaveLengt
h1:280,
UV 1w-leaf
43.17
OH
OH
O
O
300000
38.68
200000
21.84
35.23
24.04
24.81
15.97
25.89
100000
Extraction of compounds
50.79
32.11
52.41
0
0
10
15
20
25
30
Time (min)
35
40
45
50
55
60
RT: 0.00 - 60.01
NL:
9.22E5
WaveLengt
h1:280,
UV
non-bean
4.13
900000
B
850000
800000
750000
700000
650000
600000
550000
uAU
Pigeon pea seedlings were separated into leaves, stems and roots, respectively. After sampling, two samples of fresh
pigeon pea were weighed, one was used to determine the moisture content; the other was used for extraction.
Sample (1 g) was homogenised for 2 min with 20 ml of aqueous-ethanol (40:60, v/v) solution, the mixture was
subjected to solvent extraction in a microwave-assisted extraction system (Microwave Start E, Milestone, USA) for
10 min. The irradiation power was 700W and the extraction temperature was 75 C. After vacuum filtered. All
solutions and samples prepared for LC–MS were filtered through 0.45 m nylon membranes (Millipore, MA, USA)
prior to use.
5
500000
450000
400000
19.80
350000
13.40
300000
32.65
250000
21.00
200000
22.35
24.30
150000
LC-MS conditions
100000
35.98
27.95
17.40
43.78
44.20
38.20
44.62
53.41
46.65
50000
54.78
0
0
5
10
15
20
25
30
Time (min)
35
40
45
50
55
60
RT: 0.00 - 60.00
3.76
NL:
4.13E5
WaveLengt
h1:280,
UV 1w-root
400000
C
350000
300000
250000
uAU
Chromatographic analyses were performed by using LCQ series LC-MS System(Thermo Fisher Scientific ,USA).
Separation of the analytes was achieved on a C18 reversed-phase column(2.0 mmX150 mm , 5μm, Phenomenex,
USA). The mobile phase consisted of methanol (A) and 0.08% formic acid aqueous solution (B) using the following
gradient elution program for separation: 0–30 min, 33–65% (A); 30–40 min, 65–90% (A); 40–50 min, 90% (A); 50–
60 min, 90–33% (A). The column temperature was maintained at 30℃, the flow rate 200 μl/min and the injection
volume was 10μl . The detection wavelength was 280 nm
23.94
200000
44.86
45.24
43.69
150000
Results and discussion
100000
35.97
43.17
47.76
36.31
50000
To further characterize individual components of the extract, MS and MS–MS spectra were
obtained. The pinostrobin and cajaninstilbene acid of ion scans are shown in Fig. 2. The MS–MS
spectrum of Fig. 1A matched the fragmentation spectrum of Cajaninstilbene acid (Fig.1C). The Fig.
1B matched the fragmentation spectrum of Cajaninstilbene acid (Fig.1D).
9.16
9.77
14.01
51.62
52.67
29.47
20.46
35.24
0
0
5
10
15
20
25
30
Time (min)
35
40
45
50
55
RT: 0.00 - 60.00
3.76
NL:
7.28E5
WaveLengt
h1:280,
UV
1w-stem
700000
D
650000
600000
550000
500000
450000
1w-leaf_100805020817 #1601 RT: 40.80 AV: 1 NL: 4.65E6
F: + c ESI Full ms2 271.00@cid35.00 [70.00-2000.00]
131.10
100
A
O
O
rHB
O
O
90
167.24
OH
OH
OH
m/z 271.096
80
O
O
OH
O
O
OH
i
O
OH
O
m/z 271.096
60
O
O
OH
O
m/z 271.096
O
40
10
0
44.78
uAU
250000
O
OH
200000
OH
Lib
m/z 337.145
O
150000
70
OH
47.67
m/z 293.155
9.29
O
23.94
100000
-H
OH
43.21
OH
13.98
50000
m/z 167.034
O
60
29.52
21.73
40.61
36.04
38.69
35.28
48.29
52.34
0
O
0
50
16.41
26.11
5
10
15
20
25
30
Time (min)
35
40
45
50
55
m/z 337.145
40
Fig. 2. Variation in contents of pinostrobin and cajaninstilbene acid in (A)leaves(B)seeds(C)roots(D)stems of pigeon
pea during seedling growth period.
30
20
229.01
103.22
165.12
253.02
193.28
98.95
200.84
120
140
160
180
200
m/z
225.30
220
References
10
243.07
237.97
240
260
280
300
pi-50x+ #1818 RT: 40.69 AV: 1 NL: 4.35E5
F: + c ESI Full ms2 271.00@cid30.00 [70.00-2000.00]
131.04
100
337.02
0
100
150
200
250
300
m/z
350
400
450
500
D
C
90
80
70
167.27
60
50
270.85
40
30
173.15
20
198.63
10
165.03
102.94
120
140
160
229.02
224.94
252.93
242.98 255.18
220
240
184.61
201.13
155.21
0
100
300000
O
184.73 198.72
100
350000
Lib
O
OH
173.16
20
O
80
i
OH
O
m/z 271.096
30
OH
-H
OH
rHR
50
O
400000
O
rHR
+H
B
OH
m/z 271.096
+H
70
292.90
100
Relative Abundance
90
1w-leaf #1938 RT: 47.70 AV: 1 NL: 1.12E6
F: - c ESI Full ms2 337.00@cid35.00 [90.00-2000.00]
180
200
m/z
260
280
300
Fig. 1. Ion mass spectrum of (A) pinostrobin in leaves of pigeon pea, (B) Cajaninstilbene acid in leaves of pigeon
pea, (C)Cajaninstilbene acid was purchased from Sigma,(D) Cajaninstilbene acid quote from Wei et al.,(2010).
1. Ceballos, L., Hossaert-McKey, M., McKey, D., & Andary, C. (1998). Rapid deployment of allelochemicals in exudates of
germinating seeds of Sesbania (Fabaceae): roles of seed anatomy and histolocalization of polyphenolic compounds in
antipathogen defense of seedlings. Chemoecology, 8, 141–151.
2. Duker-Eshun, G., Jaroszewski, J. W., Asomaning, W. A., Oppong-Boachie, F., & Christensen, S. B. (2004). Antiplasmodial
constituents of Cajanus cajan. Phytotherapy Research, 18, 128–130.
3. Xin Huaa, Yu-Jie Fua, Yuan-Gang Zu, Nan Wu, Yu Kong, Ji Li, Xiao Pengb, Thomas Efferth (2010) Plasma
pharmacokinetics and tissue distribution study of cajaninstilbene acid in rats by liquid chromatography with tandem mass
spectrometry. Journal of Pharmaceutical and Biomedical Analysis 52: 273–279.
4. Wei Liua, Yu Konga, Yuangang Zua, Yujie Fua, Meng Luoa, Lin Zhanga, Ji Lia (2010) Determination and quantification
of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography–tandem mass
spectrometry. Journal of Chromatography A 1217: 4723–4731.
5. Wei Liu, Yuan-Gang Zu, Yu-Jie Fu, Yu Kong, Wei Mac, Mei Yang, Ji Li, Nan Wua (2010) Variation in contents of phenolic
compounds during growth and post-harvest storage of pigeon pea seedlings. Food Chemistry 121: 732–739.
6. Yu-Jie Fu , Wei Liu, Yuan-Gang Zu, Mei-Hong Tong, Shuang-Ming Li, Ming-Ming Yan, Thomas Efferth, Hao Luo (2008)
Enzyme assisted extraction of luteolin and apigenin from pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Food Chemistry
111: 508–512.