Two new diketopiperazines and a new glucosyl sesterterpene from Alternaria alternata, an endophytic fungi from Ceratostigma griffithii

Article abstract of DOI:10.1016/j.phytol.2015.10.024

Two diketopiperazine derivatives, altenarizines A (1) and B (2), and a new glucosyl sesterterpene, 24-α-d-glucosyl-(-)-terpestacin (3), together with two known phytotoxic sesterterpenes, (-)-terpestacin (4) and fusaproliferin (5), were isolated from the fermentation broth of an endophytic fungus Alternaria alternata, which was obtained from the fresh root of Ceratostigma griffithii. Structures of all the isolates were identified by spectroscopic data.

Full text of DOI:10.1016/j.phytol.2015.10.024

Phytochemistry Letters 14 (2015) 260–264
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Two new diketopiperazines and a new glucosyl sesterterpene from
Alternaria alternata, an endophytic fungi from Ceratostigma griffithii
Da-Le Guoa,c, Min Zhaoa,c, Shi-Ji Xiaob, Bing Xiaa, Bo Wana, Yu-Cheng Gud,
Li-Sheng Dinga, Yan Zhoua,
*
a
Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041,
PR China
b
Pharmacy School, Zunyi Medical College, Zunyi 563000, PR China
University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, PR China
Syngenta Jealott’s Hill International Research Centre, Berkshire RG42 6EY, UK
c
d
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 21 August 2015
Received in revised form 24 October 2015
Accepted 27 October 2015
Available online 10 November 2015
Two diketopiperazine derivatives, altenarizines A (1) and B (2), and a new glucosyl sesterterpene, 24-a-D-
glucosyl-(ꢀ)-terpestacin (3), together with two known phytotoxic sesterterpenes, (ꢀ)-terpestacin (4)
and fusaproliferin (5), were isolated from the fermentation broth of an endophytic fungus Alternaria
alternata, which was obtained from the fresh root of Ceratostigma griffithii. Structures of all the isolates
were identified by spectroscopic data.
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Ceratostigma griffithii
Alternaria alternata
Diketopiperazine
24-a-D
-glucosyl-(ꢀ)-terpestacin
1. Introduction
It is well known that Alternaria sp. has the ability to produce
various of terpenoids, including diterpenoids (Visconti et al.,1992),
Alternaria species belong to Deuteromycetes, have been
identified as a prolific fungal source of secondary metabolites
such as steroids, terpenoids, pyrones, quinones, phenolics and
sesquiterpenoids (Gamboa-Angulo et al., 2002), triterpenoids
(Gamboa-Angulo et al., 1997) and mixed terpenoids which have
a multiple biogenesis (Ichihara et al., 1983; Kjer et al., 2009; Pero
et al., 1973; Thomas, 1961). However, to the best of our knowledge,
there are rare reports on sesterterpeniods from Alternaria sp. This
phenomenon makes our group take the strain’s habitat into
account. Qinghai-Tibet Plateau owns a special environment with
high altitude, strong radiation, and large temperature difference
between day and night. Thus, plants and endophytes which occupy
this area should have special survival strategies including
production of unique secondary metabolites (Fig. 1).
nitrogen-containing compounds, which exhibit
a variety of
biological activities (Lou et al., 2013). For instance, alternariols
and altenuenes, which were typical components from the genus
Alternaria, were examined to have cytotoxicities (Aly et al., 2008)
and antimicrobial (Jiao et al., 2006) properties. In the course of our
ongoing research on the unique compounds from plant endo-
phytes, Alternatia alternata as an endophytic fungi has been
isolated and identified from Ceratostigma griffithii. The chemical
investigation of its fermentation broth led to the isolation of two
new diketopiperazine derivatives namely altenarizine
A
(1),
2. Results and discussion
altenarizine B (2) and a new glucosyl sesterterpene 24-
a-D-
glucosyl-(ꢀ)-terpestacin (3), along with two known phytotoxic
sesterterpene, (ꢀ)-terpestacin (4), fusaproliferin (5). Details of the
isolation, structure elucidation, and antibacterial activity of these
compounds are discussed below.
Alternarizine A (1) was isolated as a yellow gum. Its molecular
formula was assigned as C24H28N2O3 on the basis of the HRESIMS
([M + H]+ at m/z 393.2171, calcd. for 393.2173). The IR spectrum
showed the presence of two amide carbonyl signals at 1675 and
1670 cmꢀ1; characteristic IR absorption bands indicated methylene
(2934 cmꢀ1) and benzene (1634 cmꢀ1) groups. The 1H and 13C NMR
spectra (Table 1) in combination with 1H-1H COSY and HSQC
recorded for 1 indicated the presence of a N-methylated phenylala-
nine (Phe) residue [dH 7.40 (2H, t, J = 7.4 Hz), 7.31 (1H, t, J = 7.4 Hz),
* Corresponding author. Fax: +86 028 828 908 10.
E-mail address: zhouyan@cib.ac.cn (Y. Zhou).
http://dx.doi.org/10.1016/j.phytol.2015.10.024
1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

D.-L. Guo et al. / Phytochemistry Letters 14 (2015) 260–264
261
Fig. 1. New compounds 1–3, and known compounds 4–5 isolated from Alternaria alternata.
Table 1
NMR data of aternarizine A (1) and B (2).
Position
1
2
dH (J in Hz)
dC
dH (J in Hz)
dC
1
2
3
4
5
6
7
–
–
–
–
–
165.8
57.1
–
–
166.0
56.9
–
3.78 dd (11.7, 2.9)
5.55 s
3.83 d (11.6)
5.71 s
–
165.6
63.2
39.8
–
165.5
63.5
39.7
4.22 dd (3.5, 4.5)
2.82 dd (13.6, 2.9)
4.17 t (3.7)
2.90dd (13.6, 2.9)
0.66 dd (13.5, 11.7)
0.85 dd (13.5, 11.6)
8
–
128.0
130.3
115.3
158.1
64.8
119.6
138.2
26.1
–
127.8
130.1
115.1
158.1
64.8
119.5
138.4
26.0
9, 13
10, 12
11
14
15
6.78 m
6.78 m
6.83 m
6.83m
–
–
4.44 d (6.7)
5.46 m
4.46 d (6.7)
5.46 t (6.6)
Fig. 2. The 1H-1H COSY (bold lines) and key HMBC (H ! C) correlations of 1.
16
17
–
–
1.79 s
1.79 s
18
1.73 s
18.1
1.73 s
18.4
19
3.31 dd (14.0, 3.5)
3.17 dd (14.0, 4.5)
–
36.7
3.22 dd (14.0, 3.5)
3.11 dd (14.2, 4.5)
35.8
20
134.8
130.6
129.2
127.9
33.2
–
126.7
130.7
114.5
159.2
33.5
21, 25
22, 24
23
N-Me
23-OMe
7.19 d (7.4)
7.40 t (7.4)
7.31 t (7.4)
3.12 s
6.92 d (8.5)
7.09 t (8.5)
–
3.10 s
3.74 s
55.4
400 MHz for 1H and 100 MHz for 13C, in CDCl3.
7.19 (2H, d, J = 7.4 Hz), 4.22 (1H, dd, J = 3.5,4.5 Hz), 3.31 (1H, dd,
J = 14.0, 3.5 Hz), 3.17 (1H, dd, J = 14.0, 4.5 Hz), 3.12 (3H, s); dC 165.8,
134.8, 130.6, 129.2, 127.9, 63.2, 36.7, 33.2]; a tyrosine (Tyr) unit [dH
6.78 (4H, m), 5.55 (1H, br. s), 3.78 (1H, dd, J = 11.7, 2.9 Hz), 2.82 (1H,
dd, J = 13.6, 2.9 Hz), 0.66 (1H, dd, J = 13.5, 11.7 Hz); dC 158.1, 130.3,
128.0,115.3, 57.1, 39.8]andaisopentenylgroup[dH 5.46(1H,m),4.44
(2H, d, J = 6.7 Hz), 1.79(3H, s), 1.73(3H, s); dC 138.2, 119.6, 64.8, 26.1,
18.1]. The HMBC cross peaks were observed between the N- methyl
protonsignal(dH 3.12)andthecarbonsignalsofC-6(dC 63.2)andC-2
Fig. 3. The key NOESY correlations of 1.
(
dC 165.8), which indicated the methyl group was at N-1. The
correlations from the H-6 (dH 4.22) to C-5 (dC 165.6) and N-metheyl
dC 33.2) and the correlation from the H-3 (dH 3.78) to C-2 (dC 165.8)
relevant protons as shown in Fig. 3. The presence of the NOE
interactionbetweenH-3 (dH 3.78) andH-6 (dH 4.22)indicateda boat
formofthepiperazineringandthecisrelationshipbetweenH-3and
H-6. The value of its negative optical rotation was close to those of
(3S,6S)-3-benzyl-6-(4-hydroxybenzyl)-2,5-piperazinedione (Zeng
et al., 2005), which suggested the 3S and 6S configuration of 1. A
Marfey’s analysis had been done and the absolute configuration of
(
confirmed the dikepiprezine core skeleton. The proton H-14 (dH
4.44) showed correlation peaks with the carbon singals of C-11 (dC
158.1) and C-10, 12 (dC 115.3), suggesting the connection of the
isopentenyl group to C-11 via a oxygen atom. Thus, the primary
structure of 1 was established as shown in Fig. 2. The relative
stereochemistry of 1 was established by NOESY correlations of

262
D.-L. Guo et al. / Phytochemistry Letters 14 (2015) 260–264
N-methyl-phenylalanineresiduewas confirmedasL(Marfey,1984).
Therefore, the absolute configuration of 1 was established as
(3S,6S)-3-benzyl-6-(4-isoprenyloxybenzyl)-2,5-piperazinedione,
and it was named alternarizine A (1).
CHꢀꢀCH2ꢀꢀCH-(D) [dH 5.43 (1H, m), 2.81(1H, dd, 14.4, 7.0), 2.40
(1H, m), 1.90 (1H, m), 1.55 (3H, s); dC 137.4, 130.1, 50.4, 30.3, 10.6],
ꢀꢀCHꢀꢀCH2-(E) [dH 3.89 (1H, dd 9.5, 3.7), 3.73 (1H, dd 9.5, 6.8), 2.84
(1H, m); dC 71.4, 36.3] and a set of typical
a-glucosyl signals (F).
Alternarizine B (2) was obtained as a yellow gum, and its
molecular formula was determined as C25H30N2O4 by the HRESIMS
([M + H]+ at m/z 423.2283, calcd. 423.2278) with 12 degrees of
unsaturation. IR absorptions implied the presence of aromatic ring
(1300, 827 cmꢀ1) and amide carbonyl (1676, 1663 cmꢀ1). The NMR
data was similar to alternarizine A (1), and the differences were the
methoxyl instead of a proton at C-23 which created a group of
typical para-substituted benzene signals [dH 6.92 (2H, d, J = 8.5 Hz),
7.09 (2H, t, J = 8.5 Hz); dC 114.5, 126.7, 130.7, 159.2] and a oxymethyl
signal [dH 3.74 (3H,s); dC 55.4]. The final structure was established
by 2D NMR experiments including HSQC and HMBC. The absolute
configuration of 2 was established as 3S, 6S by the similarity of
optical rotation of 1 and 2’s.
These were linked together with the help of HMBC data (Fig. 4). The
correlations between H-5 (dH 2.29, 2.04) and C-4 (dC 138.7),
between H-9 (dH 2.06, 1.77) and C-8 (dC 133.9), between H-11 (dH
3.99) and C-12 (dC 137.4), and between H-15 (dH 2.81) and C-1 (dC
50.2) linked and cyclized spin systems A,B, C, and D, in sequence.
Both HMBC cross peaks between H-15 (dH 2.81) and C-16 (dC 151.8)
and between H-19 (dH 0.95) and C-18 (dC 210.5) connected spin
system D, A, and 2-hydroxyacryl ketone and created another 5-
membered carbocyclic ring system. Those correlations between H-
23 and C-16 connected spin system E and 2-hydroxyacryl ketone
and finally generated a terpestacin-type sesterterpenoid. The
HMBC correlation between the H-24 (dH 3.89, 3.73) and the
terminal carbon (dC 100.5) confirmed the location of the glycosyl.
The absence of the NOE interaction between H-15 (dH 2.81) and H-
19 (dH 0.95) indicated that they were not in the same orientation.
24-
a
-
D
-Glucosyl-(ꢀ)-terpestacin (3) was isolated as a yellow
gum. The molecular formula C31H48O9 was confirmed by HRESIMS
([M + Na]+ at m/z 587.3196, calcd. for 587.3191) and indicated
8 units of unsaturation. The IR spectrum showed absorption band
at 3429 cmꢀ1, which suggested the presence of hydroxyl group in
the molecule. Its 1H NMR spectrum (Table 2) exhibited signals due
The sugar unit and aglycone component was determined to be D-
glucopyranosyl and (ꢀ)-terpestacin by TLC analysis of acid
hydrolyzed 3 (Wu et al., 2015). Thus, the absolute configuration
of 3 was deduced as 24-
a
-
D
-glucosyl-(ꢀ)-terpestacin and was
to three olefinic protons [
a set of typical -glucosyl signals [
3.63 (1H, m), 3.37 (1H, m), 3.29 (1H, m), 3.67 (1H, m), 3.80 (1H, dd,
11.8, 2.3)], three vinylic methyl singlets [ 1.67 (6H, s), 1.55 (3H, s)],
and two methyl singlets [
1.32 (3H, d, 7.0), 0.95 (3H, s)]. The 13C
d
5.43 (1H, m), 5.34 (1H, m), 5.18 (1H, m)],
reinforced on the positive sign of the specific rotation ½a D
ꢁ
20 = +38
a
d
4.78 (1H, d, 3.7), 3.58 (1H, m),
for 3 and ½a D
ꢁ
20 = ꢀ21.1 for (ꢀ)-terpestacin (Jin and Qiu, 2012).
Compounds 1–5 were tested against Candida albicans, Fusarium
graminearum, Fusarium vasinfectum, Saccharomyces cerevisiae, and
Aspergillus niger. However, the compounds were inactive even at
d
d
NMR spectrum (Table 2), displayed 31 carbon signals, and HMQC
data indicated the presence of five CH3 (including three vinylic
the high concentrations 100
mg/mL.
methyl carbons at
d
10.6, 15.4, 15.5), eight CH2 (including a
62.7), eleven CH (including three olefinic
123.5,126.1,130.1 and five -glucosyl carbons at 71.9,
3. Experimental
a
-glucosyl carbons at d
carbons at
d
a
d
3.1. General
73.7, 74.2, 75.3, 100.5), and seven quaternary carbons (including
five olefinic carbons at 133.9, 137.4, 138.7, 149.3, 151.8 and a keto
carbon at 210.5). These data suggested that compound 3 is a
bicyclic sesterterpenoid with a 2-hydroxyacryl ketone (three
carbons at
151.8, 149.3, 210.5). 1H-1H COSY spectra suggested
the presence of the spin systems ꢀꢀCH2ꢀꢀCH C(CH3)-(A) [dH 5.34
(1H, m),1.77 (1H, m), 2.34 (1H, m),1.67 (3H, s); dC 138.7,123.5, 40.4,
C(CH3)-(B) [dH 5.18 (1H, m), 2.31 (1H, m),
d
Optical rotations were determined on a JASCO P-1020 polarim-
eter at room temperature. UV spectra were recorded on a
PerkinElmer Lambda 35 UV–vis spectrophotometer. IR spectra
were measured by PerkinElmer one FT-IR spectrometer (KBr). 1D
and 2D NMR were carried out on a Bruker-Ascend-400 MHz
instrument at 300 K, with TMS as internal standard. HRESIMS was
d
d
¼
15.5], ꢀꢀCH2ꢀꢀCH2ꢀꢀCH
¼
recorded on
Preparative HPLC was performed on a Waters 2545 equipped
m) using a
a Bruker MicrO TOF-Q II mass spectrometer.
2.29 (1H, m), 2.10 (1H, m), 2.04 (1H, m), 1.67 (3H, s); dC 133.9, 126.1,
41.6, 25.0, 15.4], ꢀꢀCH2ꢀꢀCH2ꢀꢀCH(OH)-(C) [dH 3.99 (1H, m), 2.06
with a Kromasil RP-C18 column (10 ꢂ 250 mm, 5
m
(1H, m), 1.77 (2H, m), 1.64 (1H, m); dC 77.1, 36.1, 30.9], C(CH3)
¼
Waters 2489 UV detector. Column chromatography (CC) was
performed with silica gel and Sephadex LH-20. All the solvent used
were of analytical grade.
Table 2
24-a-D
-Glucosyl-(ꢀ)-terpestacin (3).
Position
dH (J in Hz)
dC
Position dH (J in Hz)
dC
3.2. Fungal material
1
2
3
4
5
6
7
8
–
50.2
40.4
123.5 19
138.7 20
41.6
25.0
126.1 23
133.9 24
17
18
–
149.3
210.5
17.2
15.5
15.4
10.6
1.77 m, 2.34 m
5.34 m
–
The title strain was isolated from the root of C. griffithii,
collected from the suburb of Lhasa. Tibet Autonomous Region,
People’s Republic of China. The culture was grown on potato
dextrose agar (PDA) and distinguished morphologically as
Alternaria sp., which was further reinforced by 18S rDNA sequence
with a 99% identity to A. alternata. The strain (GenBank acccession
No. KR632488) has been preserved at Chengdu Institute of Biology,
Chinese Academy of Sciences, China.
0.95 s
1.67 s
1.67 s
1.55 s
–
2.04, 2.29 m
2.10, 2.31 m
5.18 m
21
22
2.84 dd (11.3, 2.0) 36.3
–
3.89 dd (9.5, 3.7)
3.73 dd (9.5, 6.8)
1.32 d (7.0)
4.78 d (3.7)
3.58 m
3.63 m
3.37 m
3.29 m
3.80 dd (11.8, 2.3) 62.7
3.67 m
71.4
9,
1.77, 2.06 m
1.64, 1.77 m
3.99 dd (9.8, 3.5)
–
36.1
30.9
77.1
137.4 30
130.1 40
25
10
20
15.4
100.5
74.2
75.3
73.7
71.9
10
11
12
13
14
15
3.3. Fungal culture and extraction
5.43 m
1.90, 2.40 m
2.81 dd (14.4, 7.0) 50.4
30.3
50
60
This fungus was cultivated on 4.8 L scale using 500 mL
Erlenmeyer flasks containing 200 mL of the seed PDA medium
for three days then on 60 L scale using 1 L Erlenmeyer flasks
containing 10 mL seed PDA medium and 400 mL fermentation
medium (soluble starch 0.8%, peptone 0.5%, NaCl 0.2%, CaCO3 0.2%,
16
– 151.8
400 MHz for 1H and 100 MHz for 13C, in CD3OD

D.-L. Guo et al. / Phytochemistry Letters 14 (2015) 260–264
263
Fig. 4. The 1H-1H COSY (bold lines) and key HMBC (H ! C) correlations of 3.
MgSO4
ꢃ
7H2O 0.05%, K2HPO4 0.05%) for 14 days at 28 ꢄC on a rotary
1.8 mm particles, Waters) was eluted with a linear gradient of
50–98% (v/v) CH3OH–H2O containing 0.1% TFA for 8 min. The
standards gave the following retention times: 4.71 min for N-
shaker (250 rpm). The fermentation broth (60 L) of A. alternata was
filtered. The filtrate was firstly extracted with petroleum and
followed by EtOAc. The EtOAc solution was dried under vacuum
and yielded 16 g extract.
methyl-L-phenylalmine, and 5.95 min for N-methyl-D-phenylal-
mine. Compound 1 (1.1 mg) was hydrolyzed in 6N HCl (1 mL) and
heated at 120 ꢄC in a sealed vial for 12 h to yield the corresponding
amino acids. The cooled reaction mixture was evaporated to
dryness under reduced pressure, and HCl was removed from the
residual acid hydrolysate by repeated evaporation from frozen H2O
(1 mL). The amino acid mixture was then treated in the same
manner as the standards above (1% FDAA and 6% Et3N). The
mixture of FDAA derivatives was filtered, and the filtrate was
diluted with H2O and analyzed by HPLC. The FDAA derivative of the
amino acid liberated from 1 showed the peak at 4.71 min, matching
3.4. Fractionation and isolation
The EtOAc residue (16 g) was separated into 4 fractions by CC on
silica gel (300–400 mesh), eluting stepwise with CHCl3/MeOH
gradient (CHCl3, CHCl3/MeOH: 10:1(v/v), CHCl3/MeOH: 3:1(v/v),
MeOH). LC-MS analysis was performed on these fractions. The
fourth fraction (eluted with MeOH) was slected. Sephadex LH-
20 separation of this fraction (CHCl3/MeOH: 1:1, v/v) afforded 3
subfractions (Fr.1–Fr.3). Fr.2 was further purified on a Waters
preparative HPLC equipped with a Kromasil RP-C18 column
the retention time of N-methyl-L-phenylalmine.
(10 ꢂ 250 mm, ID ꢂ L; MeOH/H2O: 70:30, v/v) to afford
1
3.6. Acid hydrolysis
(8.3 mg), 2 (2.5 mg), 3 (4.6 mg), 4 (10.3 mg), and 5 (4.7 mg).
A solution of compound 3 (1.3 mg) in 2N aqueous HCl (5.0 mL)
was refluxed at 80 ꢄC for 2 h. The mixture was then diluted in water
(10 mL) and extracted with EtOAc (3 ꢂ 3 mL). The combined water
and EtOAc layers and evaporated to dryness to afford the glycoside
and aglycone. The residue was analyzed by silica gel TLC by
comparison with standard sugars and (ꢀ)-terpestacin. The solvent
systems were n-BuOH–MeOH (4:1) for glycoside and CHCl3–
CH3OH (10:1) for aglycone, and spots were visualized by spraying
with H2SO4/EtOH (1:9), and then heated at 110 ꢄC for 1 min. For the
sugar of 3, the Rf of glucose was 0.55, and the Rf of aglycone was
0.62 by TLC, respectively.
3.4.1. Altenarizine A (1)
Yellow gum, ½a D
ꢁ
20 = ꢀ96 (c = 0.01, MeOH); IR (KBr) 2934, 1675,
1670, 1456, 1250, 1033, 827 cmꢀ1; UV (MeOH) lmax 227.3 (4.12),
276.3 (2.93); HRESIMS m/z [M + H]+ 393.2171 (calcd. 393.2173); 1H
NMR and 13C NMR are seen in Table 1.
3.4.2. Altenarizine B (2)
Yellow gum, ½a D
ꢁ
20 = ꢀ124 (c = 0.01, MeOH); IR (KBr) 2935, 1675,
1662, 1456, 1249, 1032, 825 cmꢀ1; UV (MeOH) lmax 226.9 (4.17),
275.7 (2.96); HRESIMS m/z [M + H]+ 423.2283 (calcd. 423.2278); 1H
NMR and 13C NMR are seen in Table 1.
3.7. Antimicrobial assay
3.4.3. 24-
a
-
D
-Glucosyl-(ꢀ)-terpestacin (3)
The antibacterial activity was evaluated according to the
reported procedure with little modification (Shaaban et al.,
2012). Activity was determined using the Oxford cup method
with medium (dextrose 20.0 g/L, beef infusion 10 g/L, NaCl 5 g/L,
agar 17 g/L) respectively, inoculated with strains of Escherichia coli
Yellow gum, ½a D
ꢁ
20 = +38 (c = 0.01, MeOH); IR (KBr) 3429, 2922,
1632, 1384, 1026, 557 cmꢀ1; UV (MeOH) lmax 262 (3.82); HRESIMS
m/z [M + Na]+ 587.3196 (calcd. 587.3191); 1H NMR and 13C NMR are
seen in Table 2.
1.044 and Bacillus subtilis 1.079. To each cup was added 200
sample (compounds 1–5) dissolved in DMSO. Methicillin (5
m
L of
g/
3.5. Absolute configuration of 1
m
mL) and DMSO were used as positive and negative controls,
respectively. The plates were incubated at 37 ꢄC for 24 h. The
antimicrobial activity was evaluated by measuring the diameter
zone of growth inhibition against the test microorganism. MIC was
detected complying with the method described by Kubo et al.
(2004). All the test samples were dissolved in DMSO and the final
concentration of DMSO was not over 5% (v/v). The final range of
An amino acid (N-methyl-
phenylalmine standard, 1 mg, Sigma, St. Louis, MO, USA) was
dissolved in H2O (30 L) and treated with 1% 1-fluoro-2,4-
dinitrophenyl-5- -alanine amide (FDAA) in acetone (60 L) and
6% Et3N in 30
L of H2O at 40 ꢄC for 1 h. After cooling to room
temperature, the derivative was analyzed by UPLC with at UV
absorption of 340 nm. A HSS T3 column (50 mm ꢂ 2.1 mm i.d.,
L-phenylalmine or N-methyl-D-
m
L
m
m

264
D.-L. Guo et al. / Phytochemistry Letters 14 (2015) 260–264
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Acknowledgments
This work was financially supported a grant from the National
Natural Sciences Foundation of China (21572219, 21302180), the
State Key Laboratory of Phytochemistry and Plant Resources in
West China (No. P2015-KF09) and a Syngenta Postgraduate
Fellowship awarded to Da-Le Guo.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.phytol.
2015.10.024.
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