Cytotoxic and hypoglycemic activity of triterpenoid saponins from camellia oleifera abel. Seed pomace

Article abstract of DOI:10.3390/molecules22101562

One new and three known triterpenoid saponins were isolated and identified from Camellia oleifera seeds through IR, NMR, HR-ESI-MS and GC-MS spectroscopic methods, namely oleiferasaponin A₃, oleiferasaponin A₁, camelliasaponin B

Full text of DOI:10.3390/molecules22101562

molecules
Article
Cytotoxic and Hypoglycemic Activity of Triterpenoid
Saponins from Camellia oleifera Abel. Seed Pomace
Tai-Mei Di 1, Shao-Lan Yang 1, Feng-Yu Du 2, Lei Zhao 1, Tao Xia 3
and Xin-Fu Zhang 1,
*
ID
ID
1
College of Horticulture, Qingdao Agricultural University, Qingdao 266109, China;
dtmtea@163.com (T.-M.D.); shaolanyang@126.com (S.-L.Y.); zhaolei_tea@163.com (L.Z.)
College of Chemistry and Pharmacy, Qingdao Agricultural University, Qingdao 266109, China;
fooddfy@126.com
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036,
China; xiatao62@126.com
2
3
*
Correspondence: zxftea@163.com; Tel.: +86-0532-880-30231
Received: 19 July 2017; Accepted: 15 September 2017; Published: 21 September 2017
Abstract: One new and three known triterpenoid saponins were isolated and identified from
Camellia oleifera seeds through IR, NMR, HR-ESI-MS and GC-MS spectroscopic methods, namely
oleiferasaponin A3, oleiferasaponin A1, camelliasaponin B1, and camelliasaponin B2. The structure of
oleiferasaponin A3 was elucidated as 16
28-dihydroxymethylene-olean-12-ene-3
(1 2)- -D-galactopyranosyl-(1 3)]- -D-gluco-pyranosiduronic acid. Camelliasaponin B1 and
α
-hydroxy-21
β
-O-angeloyl-22
α
-O-cinnamoyl-23α-aldehyde-
β
-O-[ -D-galactopyranosyl-(1
β
→
2)]-[ -D-xylopyranosyl-
β
→
β
→
β
camelliasaponin B2 exhibited potent cytotoxic activity on three human tumour cell lines (human
lung tumour cells (A549), human liver tumour cells (HepG2), cervical tumour cells (Hela)). The
hypoglycemic activity of oleiferasaponin A1 was testified by protecting pancreatic
high-glucose damage.
β-cell lines from
Keywords: Camellia oleifera; triterpenoid saponin; oleiferasaponin A3; cytotoxic activity;
hypoglycemic activity
1. Introduction
Triterpenoid saponins are vital plant secondary metabolites that have been applied to
cosmetics [1,2], agriculture [3], and medicine [4,5] for their diverse biological and pharmacological
activities. Camellia oleifera was named for its seeds with plentiful edible oil. Tea seed pomace—the
byproduct of oil manufacture—contains about 8% saponins, which have historically been wasted
without full use [
and activity identification of saponins obtained from Camellia oleifera seed have been published.
There are 11 novel triterpenoid saponin compounds obtained from Camellia oleifera seed [ 13].
Meanwhile, their cell protective activity [14], antioxidant activity [10 15], anti-fungal activity [16 17],
cytotoxic activity [11 13 18] have been reported, indicating that the different activities depend on
6]. In recent years, some research works concerning the extraction, structures,
6
–
,
,
–
,
the different compound structures. Approximately 30 types of saponin were indicated by liquid
chromatography–mass spectrometry (LC-MS) analysis in the seed pomace of Camellia oleifera [17].
Therefore, it is significant to continue extracting, identifying, and exploring the biological and
pharmacological activities of saponins in Camellia oleifera seed pomace.
One new triterpenoid saponin (oleiferasaponin A3) and three known saponins (oleiferasaponin A1,
camelliasaponin B1, and camelliasaponin B2) were isolated from the tea seed pomace of Camellia oleifera
in our study. We observed that camelliasaponin B1 and camelliasaponin B2 significantly inhibited the
proliferation of human lung cancer cells (A549), human liver cancer cells (HepG2), cervical cancer cells
(Hela), especially A549 cell lines. In addition, a glucose-stimulated insulin secretion (GSIS) experiment
Molecules 2017, 22, 1562; doi:10.3390/molecules22101562
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indicated that oleiferasaponin A1 possessed protective activity on pancreatic
β-cell lines injured by
high-glucose. Our results will promote further application of oleanane-type saponins in pharmacology.
2. Results
2.1. The Characterization of the Oleiferasaponin A3
One new and three known oleanane-type saponins were obtained, the structures of which were
further deduced mainly by the data of IR, NMR, HR-ESI-MS and GC-MS experiments (Figure 1), the
spectrums can be found in supplementary materials.
R1
S1
S2
S1
S1
R2
H
R3
oleiferasaponin A1
oleiferasaponin A3
camelliasaponin B1
camelliasaponin B2
trans-2-hexenoyl
cinnamoyl
angeloyl
tigloyl (trans-angeloyl)
O-angeloyl
H
H
Figure 1. Structure of compounds.
The molecular formula C67H96O28 of oleiferasaponin A3 was determined from the HR-ESI-MS
[M − H]− ion peak at m/z 1347.5980. The IR spectrum of oleiferasaponin A3 showed absorption bands
at 3371 and 1618 cm−1, ascribable to hydroxyl and olefinic groups, and broad bands at 1042 cm−1
suggestive of an ether functional group. The NMR (Table 1) data of oleiferasaponin A3, along with the
HSQC spectrum, showed the correlation between the anomeric proton signals of sugar units at δH, 4.38
,
0
0000
00
(1H, d, J = 7.7 Hz, H-1 ), 4.53 (1H, d, J = 6.1 Hz, H-1 ), 5.05 (1H, d, J = 7.9 Hz, H-1 ), 5.07 (1H, d, J = 7.7
000
Hz, H-1 ), and
δ
c 103.4 (C-1’ of glucuronopyranosyl, GlcA), 106.2 (C-1”” of xylopyranosyl, Xyl), 101.2
00
000
(C-1 of galactopyranosyl, Gal), 100.2 (C-1 of Gal), respectively, which indicated the presence of four
sugar residues. In addition, a cinnamoyl group (δH 6.49, 7.72 (1H, d, J = 16.0 Hz, Cin-H-2, Cin-H-3),
7.42, 7.42, 7.42, 7.60, 7.60 (1H, m, Cin-H-6, 7, 8, 9, 5), 117.4 (Cin-C-2), 127.8 (Cin-C-5, 9), 128.6 (Cin-C-6,
8), 130.1 (Cin-C-7), 134.4 (Cin-C-4), 145.3 (Cin-C-3), and δC 167.6 (Cin-C-1)), and an angeloyl group (δH
6.02 (1H, q, 22-O-Ang-3), 1.85 (3H, d, J = 7.2 Hz, 22-O-Ang-4), 1.82 (3H, s, 22-O-Ang-5)), which are also
present in isotheasaponins B3 isolated from the leaves of the tea plant Camellia sinensis var. sinensis [19].
The remaining 1H and 13C-NMR signals, corresponding to a triterpene aglycon, showed the presence
of six methyls (δH 0.93, 0.97, 1.05, 1.14,1.19, and 1.53 (3H, each, all s, H3-29, 26, 25, 30, 24, 27)), eight
methylenes (δH 0.95, 1.55, 1.15, 1.73, 1.25, 2.68, 1.29, 1.68, 1.39, 1.72, 1.83, 2.09, 1.95, and 2.00 (2H, both

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m, H2-6, 1, 19, 7, 15, 2, 11), 3.02, 3.33 (2H, d, J = 10.8 Hz, H2-28)), including an oxygenated one, seven
sp3 methines (four are oxygenated) (δH 1.38, 1.82, 2.71, and 3.90 (1H, each, all m, H1-5, 9, 18, 3), 5.62,
6.04 (1H, d, J = 10.2 Hz, H1-22, 21), 4.06 brs of H1-16), six sp3 quaternary carbon (δC 35.4, 35.6, 39.9,
41.1, 47.2, and 55.0 (C-20, 10, 8, 14, 17, 4), one tri-substituted double bond at δC 141.6 of C-13), and an
aldehyde carbonyl (δH 9.50 (1H, s, H-23)). The positions of the Ang, Cin group and sugar components
in oleiferasaponin A3 were clarified by an HMBC experiment (Figure 2), which showed a correlation
between δH 6.04 (10.2) (H, d, H-21) and δC 168.1 of Ang-C-1, δH 5.62 (10.2) (H, d, H-22) and δC 167.6
of Cin-C-1, as well as GlcA-H-10 and δC 84.7 (C-3 of the aglycone), Gal-H-100 and δC 77 (GlcA-C-20),
Gal-H-1000 and δC 81.6 (GlcA-C-30), Xyl-H-10000 and δC 82.4 (Gal-C-2’).
Table 1. NMR spectroscopic data for oleiferasaponin A3 (in methanol-d4).
Position
δC
δH
Position
δC
δH
1
2
3
4
5
6
7
8
38
24.3
84.7
55
1.15 m, 1.73 m
1.83 m, 2.09 m
3.90 m
21-O-Ang
Ang-1
Ang-2
Ang-3
Ang-4
Ang-5
22-O-Cin
Cin-1
168.1
128
137.2
14.6
19.5
6.02, q (7.2)
1.85, d (7.2)
1.82
47.4
19.8
31.8
39.9
46.6
35.6
23.2
123.6
141.6
41.1
33.4
68.1
47.2
39.4
46.4
35.4
78.4
73.8
209.3
9.4
1.38 m
0.95 m, 1.55 m
1.29 m, 1.68 m
167.6
117.4
145.3
134.4
127.8
128.6
130.1
103.4
77
81.6
69.6
75.6
174.9
101.2
73.6
75.1
69.6
76.4
63
100.2
82.4
75.5
69.1
76.4
61.2
106.2
74.9
76.9
70.3
65.9
9
1.82 m
Cin-2
Cin-3
Cin-4
6.49 d (16.2)
7.72 d (16.2)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.95 m, 2.00m
5.44 m
Cin-5, 9
Cin-6, 8
Cin-7
7.60 m
7.42 overlap
7.42 overlap
4.38 (7.8)
3.79 overlap
3.90 overlap
3.56 overlap
3.64 overlap
0
1.39 m, 1.72 m
4.06 brs
GlcA-1
GlcA-2’
GlcA-3’
GlcA-4’
GlcA-5’
GlcA-6’
2.71 m
1.25 m, 2.68 m
00
6.04 d (10.2)
5.62 d (10.2)
Gal-1
Gal-2
Gal-3
Gal-4
Gal-5
Gal-6
Gal-1
Gal-2
Gal-3
Gal-4
Gal-5
Gal-6
Xyl-1
Xyl-2
Xyl-3
Xyl-4
5.05 d (7.8)
3.51 m
3.82overlap
3.84 overlap
3.33 m
00
00
9.50 s
1.19 s
1.05 s
0.97 s
00
00
15
00
15.9
26.3
63.1
28.2
18.8
3.02 d (10.8), 3.30 m
5.07 d (7.8)
3.68 overlap
3.57 overlap
3.83 m
000
1.53 s
3.02 d (10.8), 3.33 d (10.8)
0.93 s
000
000
000
1.14 s
000
3.65 overlap
3.71 overlap, 3.81overlap
4.53 d (7.8)
3.31 m
000
0000
0000
0000
3.90 m
3.55 m
3.99 m, 3.21 m
0000
0000
Xyl-5
1H (δ ppm, J in Hz, s: Single peak; d: Double peaks; m: Multipeaks) and 13C-NMR (δ ppm).

Molecules 2017, 22, 1562
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Figure 2. Key HMBC correlations of oleiferasaponin A3.
The NOESY spectrum showed the cross peaks between H-22 at δH 5.62 and H-30 at δH 1.14, as
well as those between H-16 at δH 4.06 and H-28 at δH 3.02, 3.33, suggesting that H-22 and H-16 are
both
3.90 correlated with H-23 at δH 9.50 and H-21 at δH 6.04 correlated with H-29 at δH 0.93, indicating that
the glycosidic chain group at C-3 and Ang group at C-21 are -configured. The absolute configuration
β-oriented; that is, Cin group at C-22 and 16-OH group are both α-orientations. The H-3 at δH
β
of sugars of oleiferasaponin A3 was confirmed by acid hydrolysis and GC-MS analysis, which
revealed one unit of D-glucuronic acid (GlcA), two units of D-galactose (Gal) and one unit of D-xylose
(Xyl) [13
the structure of oleiferasaponin A3 as 16α-hydroxy-21β-O-angeloyl-22α-O-cinnamoyl-23α-aldehyde-
28-dihydroxymethylene-olean-12-ene-3 -O-[ -d-galactopyranosyl-(1 2)]-[ -d-xylopyranosyl-(1 2)-
-d-galactopyranosyl-(1 3)]- -d-gluco-pyranosiduronic acid. The other three known
compounds were oleiferasaponin A1 (22-O-cis-2-hexenoyl-A1-barrigenol 3-O-[ -d-galactopyranosyl
(1 2)][ -d-glucopyranosyl (1 2)- -l-arabinopyranosyl (1 3)]- -d-gluco-pyranosiduronic
acid) 10], camelliasaponin B1 (22-O-angeloyl-A1-barrigenol 3-O-[ -d-galactopyranosyl
(1 2)][ -d-glucopyranosyl (1 2)- -l-arabinopyranosyl (1 3)]- -d-gluco-pyranosiduronic
acid) and camelliasaponin B2 (22-O-trans-angeloyl-A1-barrigenol 3-O-[ -d-galactopyranosyl
(1 2)][ -d-glucopyranosyl (1 2)- -l-arabinopyranosyl (1 3)]- -d-gluco-pyranosiduronic acid) [20].
,20]. Synthesizing the above analysis of all the proton and carbon signals, we established
β
β
→
β
→
β
→
β
β
→
β
→
α
→
β
[
β
→
β
→
α
→
β
β
→
β
→
α
→
β
2.2. Anti-Proliferative Activity
Oleiferasaponin A1, oleiferasaponin A3, camelliasaponin B1 and camelliasaponin B2 obtained
from Camellia oleifera seed pomace were tested against three human tumour cell lines (A549, Hela,
HepG2) using cell proliferation bioassay (SRB). Camelliasaponin B1 and camelliasaponin B2 at the
concentration of 20
tested (Figure 3)—the inhibition ratios were more than 50%. Camelliasaponin B1 and camelliasaponin
B2 at the concentration of 10 M significantly inhibited the proliferation of human lung cancer cells
µM exhibited effective anti-proliferative activity on the human tumour cell lines
µ
(A549) (Figure 3)—the inhibition ratios were 94.44% and 79.12%, respectively. Our results indicated
that camelliasaponin B1 and camelliasaponin B2 possessed potent cytotoxic activity. There are some
reports about the structure–activity relationships of triterpenoid saponins [21,22].

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Figure 3. The effect of compounds on tumour cells proliferation. A1: Oleiferasaponin A1; A3:
Oleiferasaponin A3; B1: Camelliasaponin B1; B2: Camelliasaponin B2.
The structures of camelliasaponin B1 and camelliasaponin B2 are similar, except for the orientation
of C-22 angeloyl. Compared to previously reported results [11 13], it seems that the main groups
–
contributing cytotoxicity are the C-22 Ang group and the C-28 free hydroxy group. As a result, the
cytotoxic activity is a combined effect of sugar moieties and aglycone, rather than an isolated structural
effect. Oleiferasaponin A1 and oleiferasaponin A3 did not show cytotoxic activity.
2.3. Hypoglycemic Activity
Diabetes mellitus (DM) is the third most prevalent disease globally, and manifests as a disorder
of blood glucose caused by metabolic disorder, which can induce cardiovascular system diseases
and cancer, then threatening human health and life. Many studies regarding the cytotoxic activity
of triterpenoid saponins have been reported [11–13], while few have been conducted concerning
hypoglycemic activity [23]. Oleiferasaponin A1 and oleiferasaponin A3 did not exhibit cytotoxic
activity on three human tumour cell lines (A549, Hela, HepG2), so we carried out a hypoglycemic
activity study for further exploration of structure–activity relationship. Oleiferasaponin A1 and
oleiferasaponin A3 were tested for their protective effect on RIN-m5f (islet-
glucose. The insulin content of RIN-m5f cells upon treatment under 16.7 mmol/L glucose are shown
below (Figure 4). With higher oleiferasaponin A1 concentration, the insulin levels of RIN-m5f (islet-
β cells) injured by high
β
cells) was enhanced, which indicates that oleiferasaponin A1 has potential hypoglycemic activity
against the damage induced by high glucose, and oleiferasaponin A1 may be a therapeutic agent
for hyperglycemia treatment. Regarding the oleiferasaponin A3 group, no improvement effect on
insulin levels was found in RIN-m5f (islet-β cells) injured by high glucose, even at a concentration of
100 M. The difference of bioactivity between oleiferasaponin A1 and oleiferasaponin A3 is due to the
µ
different structure, including aglycone and sugar moieties (Figure 1). Compared with oleiferasaponin
A3, we infer that the trans-2-hexenoyl group of oleiferasaponin A1 at C-22 may influence the activities,
cooperating with sugar moieties.

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Figure 4. Cell protective effects of oleiferasaponin A1 and oleiferasaponin A3 on RIN-m5f cells injured
by high-glucose. The values are expressed as mean SD. * p < 0.05, ** p < 0.01 with respect to the
±
injured group.
3. Materials and Methods
3.1. General
HPLC was run on Agilent 1260 HPLC (Agilent, Palo Alto, CA, USA). IR (infrared) spectra was
recorded on Nicolet iN10 (Thermo Scientific Instrument Co., Boston, MA, USA) with KBr pellets. NMR
spectra was measured on an AVANCE III (600 MHz) spectrometer (Bruker, Fallanden, Switzerland)
using methanol-d4 (Sigma-Aldrich St. Louis, MO, USA) as solvent. HR-ESI-MS were determined on
an electrostatic field orbital trap mass spectrometer (Thermo Scientific, Bremen, Germany) using an
ESI source.
3.2. Plant Material
Tea seed pomace (Camellia oleifera) was collected from a factory in Shucheng, Anhui province,
China. The plant material was identified by one of the authors (Associate Prof. X.F. Zhang), and was
deposited in State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University.
3.3. Extraction and Isolation
The samples were extracted and isolated according to Zhang et al. [10]. The tea seed powder
◦
10 kg) was extracted three times with methanol at 60 C under reflux each for 3 h. Concentrated
solution (1.3 kg) was obtained after solvent evaporation under reduced pressure. The methanol extract
(1.0 kg) was suspended in water and purified by nanofiltration membrane (SJM, Hefei, Anhui, China).
Then, the purified solution (0.6 kg) was successively subjected to AB-8 macroporous resin column
(Bonc, Cangzhou, Herbei, China), ordinary-phase silica gel column to yield a high-purity fraction
(0.96 g), which was purified by HPLC (MeOH:H2O, 30:70) to furnish two saponin mixtures (Fr. 1,
0.13 g; Fr. 2, 0.17 g). The first faction was further purified by HPLC (acetonitrile-0.2% AcOH:H2O,
41:59, v/v) to afford oleiferasaponin A1 (8.7 mg) and oleiferasaponin A3 (3.9 mg). The second faction
was further purified by HPLC (acetonitrile-0.2% AcOH:H2O, 37:63, v/v) to camelliasaponin B1 (11.6 mg)
and camelliasaponin B2 (9.9 mg).
3.4. Acid Hydrolysis and GC-MS Analysis
Oleiferasaponin A3 was dissolved in 1 M HCI (Guoyao chemical reagent Co. Ltd, Beijing, China)
(1 mL) for 3 h at 90 ◦C, then extracted with chloroform (Guoyao chemical reagent Co. Ltd, Beijing,
China). The aqueous phase was evaporated under N2 flow. The residue was dissolved in 0.2 mL

Molecules 2017, 22, 1562
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pyridine (Aladdin Industrial Co. Shanghai, China) containing L-cysteine methyl ester hydrochloride
(10 mg/mL) and reacted at 70 ◦C for 1 h, then evaporated under N2 flow again. After concentrated,
0.2 mL trimethylsilylimidazole (Aladdin Industrial Co. Shanghai, China) was added for derivatization
◦
reaction, and reacted at 70 C for another 1 h. The reaction mixture was partitioned between n-hexane
and water. The organic phase was analysed by GC-MS (Agilent, Palo Alto, CA, USA) (injector
temperature at 280 ◦C; the initial oven temperature was 160 ◦C for 1 min, linearly increased to 200 ◦C
at 6 ◦C/min, then a further linear increase to 280 ◦C at 3 ◦C/min and held for 5 min). The standard
sugar samples were subjected to the same reaction and GC-MS conditions.
3.5. Cytotoxic Activity Assay
3.5.1. Cell Culture
Human lung tumour cell (A549) lines, human liver tumour cell (HepG2) lines, and cervical
tumour cell (Hela) lines were obtained from Qingdao Marine Biomedical Research Institute Limited
by Share Ltd Testing Center (Qingdao, Shandong, China). Cells were cultured in DMEM complete
medium supplemented with 10 % fetal bovine serum, 2 mM l-glutamine, 100 U·mL−1 penicillin, and
100
µg
mL−1 streptomycin at 37 ◦C in a 5% CO2 humidified atmosphere. The culture medium was
·
refreshed every other day. After 80% of the cells were fused, cells were kept in logarithmic phase by
trypsinization and subculturing.
3.5.2. Cell Viability Assay
Human tumour cell lines in logarithmic phase were seeded in a 96-well plate at 4
well (180 L per well), and incubated for 24 h. After 24 h, negative control without additions; solvent
control was supplied with 0.1% DMSO; positive control with 1 M adriamycin; 20 M, 10 M saponins
were added to trial group, all incubated for 72 h. Then, 50% (m/v) ice-cold trichloroacetic acid was
added to the medium for fixed cells. After staining by sulforhodamine B, tris solution (150 L per well)
×
103 cells per
µ
µ
µ
µ
µ
was added to culture medium. Absorbance values were measured at 540 nm using an enzyme-linked
immunosorbent reader (SpectraMax i3, Molecular Devices, San Francisco, CA, USA). The inhibition
rate of cell proliferation was calculated as:
Inhibition rate (%) = [(OD540 (control group) − OD540 (trial group)) / OD540 (control group)] × 100%
(1)
3.6. Hypoglycemic Activity Assay
Pancreatic
β-cell lines (RIN-m5f) were obtained from Qingdao Marine Biomedical Research
Institute Limited by Share Ltd. Testing Center (Qingdao, Shandong, China). Cells were cultured in
RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin
and 1% glutamine. Then, pancreatic
per well). Cells were set in four groups: normal group with 5.5 mmol L glucose; injured group with
16.7 mmol L−1 glucose; trial group with 16.7 mmol L−1 glucose and different concentrations (25, 50,
β
-cell lines (RIN-m5f) were seeded in a 96-well plate (1
×
104 cells
−1
100 µM) of oleiferasaponin A1; positive control group with 16.7 mmol/L glucose and 100 µM ZnSO4.
Each group was set three parallels and incubated for 48 h. Next, the medium was removed, cleaning
twice with polybutylene succinate (PBS). The cells were incubated in medium with 5.5 mmol L−1
glucose for 1 h. Then, the medium was replaced by medium with 33.3 mmol L−1 glucose and incubated
for 2 h. The supernatant was collected for insulin content detection using ELISA kit (CEA448Ra,
Cloud-Clone Corp, Houston, TX, USA).
4. Conclusions
Four triterpenoid saponins were isolated from Camellia oleifera Abel. seed pomace: oleiferasaponin
A3, oleiferasaponin A1, camelliasaponin B1, and camelliasaponin B2. The structure of oleiferasaponin
A3 was identified. Camelliasaponin B1 and camelliasaponin B2 exhibited potent cytotoxic activity

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on three human tumour cell lines (A549, HepG2, Hela). Oleiferasaponin A1 possessed potential
hypoglycemic activity protecting pancreatic β-cell lines from high-glucose damage.
Supplementary Materials: HR-ESR-MS and NMR spectra data of oleiferasaponin A3 can be accessed online.
Acknowledgments: This study was supported by the National Natural Science Foundation of China (Grant
No. 31301601). We gratefully acknowledge Fa-Li Bai and Ying Yang, Qingdao Institute of Bioenergy and Bioprocess
Technology, Chinese Academy of Sciences, for NMR and HR-ESI-MS measurements, as well as Fan-dong Kong,
Institute of Tropical Bioscience and Biotechnology, Chinese academy of tropical agricultural sciences and Guan-Hu
Bao, Anhui Agricultural University, for helping spectrum unfolding.
Author Contributions: In this paper, X.-F.Z and T.X. designed the experiments; T.-M.D., X.-F.Z., F.-Y.D. and S.-L.Y.
performed the experiments and analysed the data; L.Z. and S.-L.Y. contributed reagents and materials; T.-M.D.
wrote the paper. All authors approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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