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Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb. through an apoptotic signaling pathway in vitro

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Objective: To investigate the biochemical constituents of the fruits of Terminalia bellerica (Gaertn.) Roxb. (hereafter termed T. bellerica) and estimate the anti-cancer activity of different polar extracts. Methods: To rapidly screen and identify the biochemical constituents of ethyl acetate (EA) extracts of T. bellerica, ultra performance liquid chromatography-electrospray ionization/mass spectrometry (UPLC-ESI-MSn) was done. The CellTiter-Blue™ cell-viability assay was used to ascertain the anti-cancer activity of different polar extracts in 10 human cancer cell lines. Results: Forty polyphenols of the EA extract of T. bellerica were characterized tentatively. The EA extract exhibited significant anti-cancer activity against ZR-75-1 cells (half-maximal inhibitory concentration = 27.33 (0.98) μg/mL) and Colo-205 cells (39.65 (2.99) μg/mL) in vitro. Treatment of ZR-75-1 cells with 20 and 60 μg/mL of the EA extract elicited dose-dependent apoptosis percentages at an early stage of 17.58 (0.74) % and at a late stage of 29.20 (1.22) %; Colo-205 cells at the same concentration of EA extract had values of 21.33 (1.03) % and 40.55 (0.34) %, respectively. Western blotting suggested that ZR-75-1 and Colo-205 cells treated with the EA extract showed a similar increasing tendency for expression of cleaved anti-poly adenosine diphosphate ribose polymerase I. Conclusion: We identified a total of 40 chemical constituents, of which 11 were first obtained from the Terminalia Linn. genus using UPLC-ESI-MSn. Meanwhile, we observe that the EA extract of T. bellerica possesses anti-cancer activity, especially against breast and colon cancers. Keywords: Terminalia bellerica, Anti-cancer activity, UPLC-ESI-MSn, CellTiter-blue
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Anti-cancer activity of an ethyl-acetate
extract of the fruits of Terminalia bellerica
(Gaertn.) Roxb. through an apoptotic
signaling pathway in vitro
Shi Li, Ting Ye, Linjin Liang, Wenyi Liang, Ping Jian, Kun Zhou,
Lanzhen Zhang*
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China
Received 11 October 2018; received in revised form 18 November 2018; accepted 18 November 2018
Available online ---
KEYWORDS
Terminalia bellerica;
Anti-cancer activity;
UPLC-ESI-MS
n
;
CellTiter-blue
Abstract Objective: To investigate the biochemical constituents of the fruits of Terminalia
bellerica (Gaertn.) Roxb. (hereafter termed T. bellerica) and estimate the anti-cancer activity
of different polar extracts.
Methods: To rapidly screen and identify the biochemical constituents of ethyl acetate (EA) ex-
tracts of T. bellerica, ultra performance liquid chromatography-electrospray ionization/mass
spectrometry (UPLC-ESI-MS
n
) was done. The CellTiter-Bluecell-viability assay was used to
ascertain the anti-cancer activity of different polar extracts in 10 human cancer cell lines.
Results: Forty polyphenols of the EA extract of T. bellerica were characterized tentatively.
The EA extract exhibited significant anti-cancer activity against ZR-75-1 cells (half-maximal
inhibitory concentration Z27.33 (0.98) mg/mL) and Colo-205 cells (39.65 (2.99) mg/mL)
in vitro. Treatment of ZR-75-1 cells with 20 and 60 mg/mL of the EA extract elicited dose-
dependent apoptosis percentages at an early stage of 17.58 (0.74) % and at a late stage of
29.20 (1.22) %; Colo-205 cells at the same concentration of EA extract had values of 21.33
(1.03) % and 40.55 (0.34) %, respectively. Western blotting suggested that ZR-75-1 and Colo-
205 cells treated with the EA extract showed a similar increasing tendency for expression of
cleaved anti-poly adenosine diphosphate ribose polymerase I.
Conclusion: We identified a total of 40 chemical constituents, of which 11 were first
obtained from the Terminalia Linn. genus using UPLC-ESI-MS
n
. Meanwhile, we observe that
the EA extract of T. bellerica possesses anti-cancer activity, especially against breast and co-
lon cancers.
* Corresponding author.
E-mail address: zhanglanzhen01@126.com (L. Zhang).
Peer review under responsibility of Beijing University of Chinese Medicine.
+MODEL
Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
https://doi.org/10.1016/j.jtcms.2018.11.006
2095-7548/ª2018 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC
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an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/
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Introduction
Terminalia bellerica (Gaertn.) Roxb. (hereafter termed T.
bellerica) belongs to the Terminalia Linn. genus of the
Combretaceae family. It is a type of traditional Tibetan
medicine named “Maohezi” in Chinese and “Pilile” in Ti-
betan. T. bellerica is located mainly in India, Myanmar, Sri
Lanka and Malaysia, with some distribution in Yunnan
Province in China.
1
According to the classic Tibetan book Crystal Pearl of
Materia Medica (“Jingzhu Bencao” in Chinese),
2
T. beller-
ica fruits have been used as medicines for thousands of
years for clearing heat, resolving toxins, and inducing
diuresis and regulating Rlung ( ), Mkhris pa ( ), and
Bad kan ( ) in Tibetan medicine theory recorded in
The Four Medical Tantras (rGyud-bzhi) in China.
3
Pharma-
cologic investigations have reported anti-oxidation, anti-
bacterial, anti-convulsive, hepatoprotective, cholesterol-
reducing, hypoglycemic, anti-myocardial-necrosis, and
anti-cancer effects.
4,5
Phytochemical investigations have
shown abundant compounds in the pitted fruits of T. bel-
lerica, such as non-volatile constituents (e.g., polyphenols,
triterpenes, cardiac glycosides, lignans) and volatile con-
stituents (e.g., fatty acids, vitamins).
6e8
Of these constit-
uents, polyphenols along with polymers and derivatives of
gallic acid and ellagic acid, are the primary constituents of
T. bellerica.
We wished to search for the bio-active species of tradi-
tional Tibetan medicines, primarily those with anti-cancer
activity. Conventional methods for the characterization of
herbal components involve repeated isolation and purifica-
tion, which is time-consuming, expensive and does not allow
low-concentration compounds to be identified. Here, we
employed a rapid and novel method to reach our aim.
Materials and methods
General reagents
Most biologic reagents were purchased from Thermo Fisher
Scientific (Waltham, MA), including media (Dulbecco’s
modified Eagle’s medium [DMEM], RPMI-1640, L-15, McCoy’s
5A), fetal bovine serum (FBS) and penicillin/streptomycin
(100 U/mL). Insulin was purchased from Shanghai Hengfei
Shengwu Keji Youxian Gongsi (Shanghai, China). Mass-
spectrometric and analytical-grade reagents were ob-
tained from Beijing Chemical Works (purity >98% by high-
performance liquid chromatography; Beijing, China), such
as ethanol (EtOH), dichloromethane (CH
2
Cl
2
), ethyl acetate
(EA) and dimethylsulfoxide (DMSO).
Plant material
T. bellerica fruits were purchased from Beijing Tibetan
Hospital (Beijing, China). The plant was collected in Nepal
in May 2016, and identified by Professor Chunsheng Liu of
the Beijing University of Chinese Medicine (Beijing, China).
A voucher specimen (20160419) has been deposited in the
herbarium of the Beijing University of Chinese Medicine.
Extraction procedure
The dried fruits of T. bellerica (2.53 g) were reduced to
powder and extracted thrice with 70% EtOH (100 mL, v/v)
under ultrasound for 1 h at 25 C. After filtration, the sol-
vent was removed under reduced pressure to give a residue
(240.22 mg), which was suspended in water and partitioned
successively with CH
2
Cl
2
and EA, respectively. Thus, the
solvent was evaporated to dryness in vacuo under 40 C,
and three extracts were obtained in turn: CH
2
Cl
2
(28.34 mg), EA (131.91 mg) and H
2
O (45.62 mg). Then, one
part of each three extracts was dissolved in methanol for
ultra performance liquid chromatography-electrospray
ionization/mass spectrometry (UPLC-ESI-MS
n
). The other
part of each three extracts was dissolved in DMSO at 1/1000
of the medium, respectively, and diluted with DMEM or
RPMI-1640 to the final concentration.
UPLC-ESI-MS
n
of EA extracts
UPLC was carried out on an Ultimate 3000 UPLC system
(Dionex, Sunnyvale, CA) coupled with a binary pump and
auto-sampler with an Acquity UPLC BEH C18 column
(2.1 100 mm i.d.; Waters, Milford, MA). ESI-MS spectra
were obtained using an LTQ OrbitrapXL mass spectrom-
eter (Thermo Fisher Scientific) operating in negative-ion
mode. Data were collected and analyzed according to the
specified software of the manufacturer.
The EA extract was dissolved in methanol and passed
through a 0.22-mm membrane. Aliquots (4 mL) were injec-
ted for UPLC-ESI-MS
n
. The mobile phase consisted of
methanol (A) and 0.2% acetic acid (B) with a linear gradient
elution at a flow rate of 0.3 mL/min. The elution program
was: 5e15% A (0e5 min); 15e25% A (5e10 min); 25e60% A
(10e15 min); 60e90% A (15e25 min); 90e90% A
(25e30 min). Detection wavelengths were set at 270 and
280 nm. The mass detector was optimized to obtain
maximum yields of [M-H]
, [M-H-H
2
O]
, [2M-H]
, [M-2H]
,
[3M-H]
, [M-3H]
, [M-CO]
or [M-CO
2
]
ions of the com-
pounds. In addition, full-scan MS was done in the range of
m/z 100e1500.
Cell culture
Ten human cell lines were used. BT-474 cells (breast ductal
carcinoma) were cultured in DMEM supplemented with
20 mg/mL of insulin. Colo-205 (colorectal carcinoma), HT-29
(colon carcinoma), MCF-7 (breast carcinoma), ZR-75-1
(ductal carcinoma) and LnCap (prostatic carcinoma) cells
were cultured in RPMI-1640. SW480 cells (colon carcinoma)
2 S. Li et al.
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Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
were cultured in L-15 medium and RPMI-1640. T-47D cells
(breast carcinoma) were cultured in RPMI-1640 supple-
mented with 20 mg/mL of insulin. Hec-1-A cells (endome-
trial carcinoma) were cultured in McCoy’s 5A and DMEM. SH-
SY5Y cells (neuroblastoma) were cultured in DMEM.
All cell lines were grown in a cell-culture incubator in
their media supplemented with 10% FBS and penicillin/
streptomycin at 37 C in humidified air containing 5% CO
2
.
Measurement of anti-cancer activity
(CellTiter-Blue(CTB) cell-viability assay)
The anti-cancer activity of the three extracts of T. bellerica
was determined by the CTB assay (Promega, Fitchburg, WI).
The principle of the CTB assay is the reduction of resazurin
to resorufin by metabolically active cells, which can be
measured indirectly. Cells were seeded in 96-well plates at
2000 cells/well in 200 mL of medium for 24 h before incu-
bation with various concentrations of the EA for 72 h. Af-
terwards, 20 mL of CTB solution was added to each well
and incubation allowed for 2 h. Then, fluorescence was
measured at an excitation wavelength of 560 nm and
emission wavelength of 590 nm with a micro-plate reader
(SpectraMax
M2; Molecular Devices, Silicon Valley, CA).
Cell viability was calculated with the data obtained from
the wells that contained known numbers of viable cells.
Then, the half-maximal inhibitory concentration (IC
50
) was
calculated. Data are the mean of five parallel samples, and
each sample was assayed in triplicate. Inhibitory
rate was calculated using the following formula: Inhibitory
rate (%) Z(A
control
eA
sample
)/A
control
100%.
Flow cytometry
Flow cytometry with annexin-V staining was carried out on
the EA extract. During the plated exponential growth phase
of ZR-75-1 and Colo-205 cells, 20.0 and 60.0 mg/mL of the
EA extract was added and left for 72 h. Then, we collected
and washed all the cells stained with fluorescein
isothiocyanate-labeled annexin-V (annexin V-FITC) and
propidium iodide (PI) and incubated them for 10e20 min at
25 C in the dark. Finally, samples were subjected to flow
cytometry (BD Bioscience, San Jose, CA).
Western blotting
ZR-75-1 and Colo-205 cells were incubated with the EA
extract (0, 6.0, 20.0, 60.0 and 200.0 mg/mL) for 72 h. Cells
were prepared by direct addition of NP40 lysis buffer
(150 mM of NaCl, 1% w/v of NP-40, 50 mM of TriseHCl (pH
8.0) and 1 mM of protease inhibitor) on ice. Then, we
collected adherent cells and washed them twice with
phosphate-buffered saline. Cell lysates were combined
with the appropriate amount of 6 sodium dodecyl sulfate
(SDS) loading buffer (10% SDS, 5% b-mercaptoethanol, 10%
glycerol, 0.2% bromophenol blue and 0.125 M of TriseHCl,
pH 6.8) and heated for 10 min at 100 C. After centrifuga-
tion 21 918gfor 10 min, supernatants were collected and
subjected to SDS-polyacrylamide gel electrophoresis for
1e2 h at 100 V. This was followed by transfer to poly-
vinylidene difluoride (PVDF) membranes and blockade with
5% skimmed milk in TBST (20 mmol/L of TriseHCl, pH 7.6,
137 mmol/L of NaCl, and 0.1% Tween 20) for 1 h at 4 C.
Finally, PVDF membranes were probed with primary anti-
bodies and incubated with secondary antibodies for 2 h at
25 C and visualized with ECL Western blotting Substrate. b-
actin was used as a loading control. The primary antibodies
were anti-poly adenosine diphosphate ribose polymerase
(PARP) I (1:1000 dilution) and anti-actin (1:5000); second-
ary antibodies were goat anti-rabbit (1:5000) and goat anti-
mouse (1:5000). Densitometry was done by calculating the
number of pixels per band using ImagePro (Media Cyber-
netics, Rockville, MD). Data referred to the number of
pixels of the protein band relative to the pixels of the
corresponding b-actin band.
Statistical analyses
Data are expressed as the mean (standard deviation (SD)).
Four independent experiments were done in triplicate.
Statistical analyses were carried out SPSS v17.0 (IBM,
Armonk, NY). The Student’s t-test was applied in mea-
surement of cell viability. One-way analysis of variance was
used for data obtained from Western blotting. P<.05 was
considered statistically significant.
Results
Analyses of the EA extract of T. bellerica by UPLC-
ESI-MS
n
According to MS data, the retention time, assumed molec-
ular formulae, calculated exact masses and published
constituents or available reference compounds, 40 poly-
phenols were characterized tentatively (Fig. 1). A summary
of compounds together with characteristic information
on MS and MS/MS spectra are presented in Table 1 and
their chemical structural formulae are displayed in
Supplementary Fig. 1. For constituents that may have
several isomeric structures with the same exact mass value
(e.g., different positions of galloyl substituents), unam-
biguous structures could not be determined in each case.
Gallic acid and derivatives
Simple galloyl esters of glucose were the main compounds.
They were gallic acid, gallate easer, and glucose derivatives
with substituent groups, such as di-(1e6), tri-(7e15), tetra-
(16e18), and penta-O-galloyl (19e20), which had a com-
mon feature of a main fragment ion at m/z 169, 313 or 331
that corresponded to a galloyl moiety or a galloylhexose
unit with or without a H
2
O molecule.
9e13
Compounds all had
multiple peaks, which supposedly contained several iso-
mers with the same molecular-ion peak and fragment ions
at different retention times, as reported previously.
14
Ellagic acid and derivatives
Ellagic acid and derivatives contain a lactonized form of
hexahydroxydiphenoyl (HHDP) when released by hydroly-
sis.
15,16
An HHDP group was typically a fragment ion of 301
Anti-cancer activity of ethyl-acetate extract of the fruits of T. bellerica 3
+MODEL
Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
derived from two galloyl groups. Punicalagin (21) was based
on the [M-H] ion at m/z 1083 and other ion fragments at m/
z781 [M-H-HHDP]
, 601 [M-H-glucose-HHDP]
, 451 [M-H-
galloyl -glucose-HHDP]
and 301 [M-H-galloyl-galloyl-
glucose-HHDP]
.
17
Several related compounds were inves-
tigated by UPLC-MS
n
, such as mono- (22e29) or dimethyl
ellagic acids (30e32) or even their glycosides, with pentose
and hexose being determined and analyzed, with a common
MS fragment at m/z 301 [M-H]
, 257 [M-H-CO
2
]
and 185 [M-
H-H
2
O-2CO
2
]
.
18,19
Chebulic acid and chebulic ellagitannins
Chebulic acid and chebulic ellagitannins have a bridging n-
O-HHDP unit instead of two galloyl residues. This feature
was confirmed with the MS fragments at m/z 651, 633 and
481. MS fragments at m/z 337, 314, 293 and 275 corre-
sponded to neutral loss of H
2
OorCO
2
, such as chebulagic
acid (1-O-galloyl-2,4-O-chebuloyl-3,6-O-HHDP-b-D-glucose),
and its methyl derivative (methyl neochebulagate) (33) had
main ions at m/z 953 [M-H]
, 801 [M-H-galloyl]
, 985 [M-H]
and 783 [M-H-galloy-CH
3
O
]
, respectively, which had
common ions at m/z 633 [M-HHDP]
and 463 [M-HHDP-
galloyl]
.
20
The same situation was noted for chebulinic acid (1,3,6-
tri-O-galloyl-2,4-O-chebuloyl-b-D-glucose) and methyl
neochebulinate (34). They were identified preliminarily to
be based on the ions at m/z 955 [M-H]
and 987 [M-H]
,
respectively, with characteristic ions at m/z 937 [M-H-
H
2
O]
, 785 [M-galloyl]
, 617 [M-2galloyl-H
2
O]
, 465 [M-
3galloyl]
and 275 [M- H-2galloyl-H
2
O-chebuloyl]
in the
Figure 1 UPLC-ESI-MS
n
total ion current profile of an EA extract of T. bellerica.
4 S. Li et al.
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Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
Table 1 UPLC-ESI-MS
n
and nano-ESI-MS
n
data (m/z) for constituents identified from the EA extract of T. bellerica.
Peak Rt (min) Molecular formula [M-H]
MS/MS (m/z) Error (Dppm) Tentative identification
1e6 7.02 C
20
H
20
O
14
483.08 465,331,313,169 1.1 Di-galloyl-glucose
7.54 C
20
H
20
O
14
483.08 331,313,169 1.3
8.07 C
20
H
20
O
14
483.08 465,313 1.6
8.26 C
20
H
20
O
14
483.08 331,313, 1.6
8.82 C
20
H
20
O
14
483.08 331,313,169 1.5
9.19 C
20
H
20
O
14
483.08 331,313,169 1.4
7e15 7.74 C
27
H
24
O
18
635.09 617,465, 313,169 0.2 Tri-galloyl-glucose
8.19 C
27
H
24
O
18
635.09 617,465,313,169 0.5
8.74 C
27
H
24
O
18
635.09 617,465,331,313 1.0
9.47 C
27
H
24
O
18
635.09 483,465,313,169 1.1
9.89 C
27
H
24
O
18
635.09 617,483,465,313 0.4
10.11 C
27
H
24
O
18
635.09 617,465,313,169 0.7
10.26 C
27
H
24
O
18
635.09 617,465,313,169 1.9
10.56 C
27
H
24
O
18
635.09 465,313,169 1.1
11.18 C
27
H
24
O
18
635.09 465,313,169 1.3
16e18 11.31 C
34
H
28
O
22
787.10 635,617,465,313 0.5 Tetra-galloyl-glucose
11.45 C
34
H
28
O
22
787.10 635,483,465,313 0.4
11.77 C
34
H
28
O
22
787.10 635,617,483,313 1.3
19e20 12.48 C
41
H
32
O
26
939.11 787,635,465,313 1.2 Penta-galloyl-glucose
13.41 C
41
H
32
O
26
939.11 787,635,465,313 1.7
21 5.69 C
48
H
28
O
30
1083.06 781,601,451,301 0.8 Punicalagin
22 14.94 C
15
H
8
O
8
315.01 300,271,243,151 3.0 3-O-methyl ellagic acid
23 13.04 C
19
H
14
O
12
433.04 301 3.7 Ellagic acid pentose
24 14.30 C
20
H
17
O
12
478.06 315,301 3.6 3-O-methyl-(4‘-O-a-D- arabinfuranosyl) ellagic acid
25 14.28 C
20
H
16
O
12
447.06 432,315,301 1.7 4-O-(a-L-rhamnosyl) ellagic acid (eschweilenol C)
26 12.80 C
21
H
19
O
13
478.07 315,300,271 4.4 3-O-methyl-(4‘-O-b-D-glucopyranosyl) ellagic acid
27 14.69 C
21
H
18
O
12
461.07 446,328,313 1.0 3-O-methyl-(4‘-O-a-L- rhamnosyl)ellagic acid
28 16.11 C
20
H
16
O
12
447.06 432,315,300 0.3 3‘-O-methyl-(4-O-b-D-xylopyranosy) ellagic acid
29 15.83 C
27
H
20
O
16
599.07 447,429,301,169 1.1 4-O-(4“-O-galloyl-a-L-rhamnosyl)ellagic acid
30 16.14 C
16
H
10
O
8
329.03 314,299,285,271, 0.6 3,3‘-di-O-methyl ellagic acid
31 16.17 C
28
H
22
O
16
613.08 429,329, 283,169 0.8 3,3‘-di-O-methyl-4-O- (2“-O-galloyl-b-D-xylopyranosy) ellagic acid
32 15.47 C
28
H
22
O
16
613.08 429,329,314,169 1.0 3,3‘-di-O-methyl -4-O- (3“-O-galloyl-b-D-xylopyranosy) ellagic acid
33 11.84 C
42
H
34
O
28
985.12 953,909,783,633,463,301 0.1 Methyl neochebulagate
34 12.88 C
42
H
36
O
28
987.13 955,937,785,635,617,465,275 0.9 Methyl neochebulinate
35 8.97 C
28
H
28
O
20
683.11 351,331,314 0.5 Methyl neochebulanin
36 11.28 C
34
H
28
O
23
803.09 651,633,481,465,337,293,275,169 0.4 1,6-di-O-galloyl-2,4- chebuloyl-b-D-glucopyranoside
37 12.67 C
34
H
28
O
23
803.09 651,633,465,337,319,293,275 0.2 1,3-di-O-galloyl-2,4- chebuloyl-b-D-glucopyranoside
38 10.81 C
27
H
22
O
18
633.07 481,463,331 0.2 Corilagin
39 8.38 C
34
H
26
O
22
785.08 633,483,481,301, 1.3 Tellimagrandin
40 10.56 C
41
H
30
O
26
937.10 785,635,481,329 1.8 Tellimagrandin
Anti-cancer activity of ethyl-acetate extract of the fruits of T. bellerica 5
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Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
mass spectrum. Likewise, chebulanin (1-O-galloyl-2,4-O-
chebuloyl-b-D-glucose) and methyl neochebulanin (35) had
fragment ions at m/z 651 [M-H]
and 683 [M-H]
, respec-
tively.
21
The presence of compounds (36) and (37) showed
chebulic glycosides with two galloyl units, which were the
basis of ions at m/z 803 [M-H]
, 651 [M-H-galloyl]
, 481 [M-
H-2galloyl-H
2
O]
and 337 [M-H- 2galloyl-glucose]
.
22
Other compounds
In the present study, known substances of this type, which
were corilagin (38), tellimagrandin I (39) and II (40), were
confirmed on the basis of the fragment ions at m/z 633 [M-
H]
, 785 [M-H]
and 937 [M-H]
, respectively.
23
The frag-
ments corresponded to loss of one HHDP unit (m/z 331, 483
and 635, respectively) or loss of one or two or three galloyls
(m/z 481, 633 and 329, respectively).
Cytotoxicity assays
Several studies have shown that polyphenols from many
types of plants possess potent activity against various
cancer cell lines.
24e29
We used 10 human cell lines to
investigate the toxicities of increasing concentrations of
the three EA extracts for 72 h. Viability was determined by
the CTB assay. ZR-75-1 and Colo-205 cells were selected
specifically to evaluate toxicity (Fig. 2A2B). The underly-
ing mechanisms of anti-cancer actions were studied by
observing apoptosis (via flow cytometry) and expression of
essential target proteins (via Western blotting). Low doses
of the EA extract could inhibit the growth of cultured ZR-
75-1 and Colo-205 cells in a dose-dependent manner. ZR-
75-1 cells were more sensitive to exposure to the EA extract
than Colo-205 cells treated under identical conditions, with
IC
50
values of 27.33 (0.98) mg/mL and 36.63 (2.99) mg/mL,
respectively. Inhibitory concentration of the ZR-75-1 and
Colo-205 cells in IC
50
by extracts at different positions is
displayed in Table 2.
Data for CPT are shown for clarity purposes. Error bars
represent the standard error of the mean.
Induction of apoptosis
The EA extract can trigger the apoptosis of human cancer
cells.
30e33
ZR-75-1 (Fig. 3A) and Colo-205 cells (Fig. 3B)
were treated with 20 and 60 mg/mL of the EA extract for
72 h. As the dose escalation progressed, cells in the EA-
treated group exhibited the morphologic features associ-
ated with apoptosis (Fig. 3). At low doses, evidence of
early-stage apoptosis was seen (FITC fluorescence),
whereas annexin-V was intact and bound to impermeable
cell membranes. With increasing doses, more late-stage
apoptosis/necrotic cells became evident that caused FITC
and PI to be positive due to staining of nuclear material.
Treatment of ZR-75-1 cells with 20 and 60 mg/mL of the EA
extract resulted in apoptosis rates of 17.58 (0.74) % and
29.20 (1.22) %, respectively, which were significantly
different from those of the blank control (PZ.00 and 0.01,
respectively). Treatment of Colo-205 cells with the EA
extract provided similar results for early- and late-stage
apoptosis, with 20 and 60 mg/mL eliciting apoptosis rates
21.33 (1.03) % and 40.55 (0.34) %, respectively. We
concluded that the EA extract induced dose-dependent
apoptosis in these two cell lines.
Western blotting
PARP is a multifunctional protein found in most eukaryotic
cells, and is activated by recognition of damaged DNA
fragments. Hence, PARP is considered to denote DNA
Figure 2 Optical-density profiles of ZR-75-1 and Colo-205 cells treated with H
2
O, EA and CH
2
Cl
2
extracts, respectively. Note: OD:
optical density; EA: ethyl-acetate; CH
2
Cl
2
: dichloromethane; CPT: camptothecin.
Table 2 Inhibitory concentration of extracts at different positions on ZR-75-1 and Colo-205 cells.
H
2
O(mg/mL) EA (mg/mL) CH
2
Cl
2
(mg/mL) CPT (mM/L)
ZR-75-1 (IC
50
) / 27.33 (0.98) 233.30 (4.24) 0.06 (0.02)
Colo-205 (IC
50
) 58.43 (0.91) 36.63 (2.99) 255.86 (4.61) 1.87 (0.20)
Note: IC
50
: half maximal inhibitory concentration; EA: ethyl-acetate; CH
2
Cl
2
: dichloromethane; CPT: camptothecin.
Data are expressed as mean (SD). N Z4.
6 S. Li et al.
+MODEL
Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
damage. PARP is a cleavage substrate for the caspase
family, and also has an important role in apoptosis. Western
blotting showed changes in levels of cleaved PARP I (Cle-
PARP) in lysates from two cell lines treated with various
concentration of the EA extract.
34e38
Accurate estimation
of the molecular size of bands was verified by running pre-
stained molecular-weight standards with each gel, and the
same membranes were also probed with an antibody
against b-actin as a loading control. The ratio of expression
of Cle-PARP I to b-actin suggested that treatment with the
Figure 3 Apoptosis as detected using flow cytometry. A: effects of the EA extract on apoptosis induction in ZR-75-1 cells; B:
effects of the EA extract on apoptosis induction in Colo-205 cells; C: quantitative analyses of early and late apoptosis in ZR-75-
1 cells induced by different doses of the EA extract; D: quantitative analyses of early and late apoptosis in Colo-205 cells induced by
different doses of the EA extract. Note: Data suggest activation of apoptosis by the EA extract due to the presence of two annexin-
V-positive populations representing early-stage (bottom right) and late-stage (top right) apoptosis. Data are expressed as mean
(SD). N Z4. ** represents P<.01 vs blank control.
Anti-cancer activity of ethyl-acetate extract of the fruits of T. bellerica 7
+MODEL
Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
EA extract showed a similar increasing tendency for the
Cle-PARP I level in ZR-75-1 and Colo-205 cells (Fig. 4A).
Upon treatment with 200.0 mg/mL of EA, ZR-75-1 cells
showed expression of Cle-PARP I of 53.44% and Colo-
205 cells showed expression of Cle-PARP I of 84.06%
compared with the blank control (P<.01) (Fig. 4B). These
findings suggested that increasing expression of Cle-PARP I
might be involved in the apoptosis of ZR-75-1 and Colo-
205 cells after treatment with the EA extract.
Discussion
Cancer is a very serious threat to human health. The side
effects of surgery and chemotherapy are also harmful.
Hence, searching for novel anti-cancer drugs is important.
It is possible that such drugs could be discovered among the
many herbal extracts used in traditional Chinese medicine.
T. bellerica is a traditional Tibetan ingredient in Sanguo
Tang which consists of the other two herbs of Terminalia
chebula Retz. and Phyllanthus emblica L., and exerts ef-
fects on clearing heat, resolving toxins, dispelling wind and
eliminating dampness, diffusing the lung, resolving phlegm,
and cooling blood. A mong these three ingredients, only the
pharmacologic effects of T. bellerica have not been studied
in depth. Previously, 13 high-content compounds were
isolated from T. bellerica, most of which were poly-
phenols.
59
In the present study, UPLC-ESI-MS
n
revealed 40 poly-
phenols in an EA extract of T. bellerica. Approximately 50%
were in the form of gallic acid and its simple derivatives,
followed by ellagic acid and its derivatives 25% (Fig. 1). Our
study provides important data on the chemical constituents
of T. bellerica, and will help basic research in traditional
Tibetan medicine.
Then, using cytotoxicity assays, flow cytometry and
western blotting, we found that the EA extract T. bellerica
exerted the strongest anti-cancer activity in vitro among
the three isolated extracts (CH
2
Cl
2
, EA and H
2
O), particu-
larly for ZR-75-1 and Colo-205 cells (Fig. 2). Furthermore,
the EA extract mediated and executed apoptotic cell death
at early and late stages (Fig. 3) by cleaving/inactivating the
essential target proteins required for the growth and divi-
sion of cells, such as PARP I (Fig. 4).
The toxicity seen in ZR-75-1 and Colo-205 cells provides
support for the notion that polyphenols in the EA extract
warrant further study for discovery of anti-cancer agents,
especially against breast and colon cancer.
36e38
In addition,
investigations and further tests (including separation and
purification) of the polyphenols in the EA extract of the
Figure 4 Effects of the EA extract on expression of PARP I and Cle-PARP I in ZR-75-1 and Colo-205 cells. A: total expression and
phosphorylation of PARP I were analyzed by Western blotting in cells, and b-actin was used as a loading control; B: ratio of
expression of Cle-PARP I/b-actin was analyzed in the two cells. Note: Data are expressed as mean (SD). N Z3. ** represents P<.01
vs blank control.
8 S. Li et al.
+MODEL
Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
fruits of T. bellerica are in progress in our research team to
evaluate the bio-active potential of individual constituents.
We shall continue to investigate the constituents of the EA
extract of T. bellerica to ascertain their mechanism of
action.
Conclusions
We identified 11 chemical constituents of Terminalia Linn.
genus for the first time using UPLC-ESI-MS
n
. EA extract of T.
bellerica possesses anti-cancer activity, especially against
breast and colon cancers. By studying the chemical con-
stituents of the fruits of T. bellerica, we have laid the
foundation for research of the anticancer activity of
different extracts. Hopefully, our study will provide a reli-
able basis for the development of innovative anticancer
drugs.
Funding
This study was supported by National Natural Science
Foundation of China: Surface Project (81274187) and Lon-
gitudinal Research Project of the Beijing University of
Chinese Medicine (2020072120043).
Conflicts of interest
No competing financial interests exist.
CRediT authorship contribution statement
Lanzhen Zhang: Conceptualization, project administra-
tion, supervision, writing review & editing. Shi Li: Writing
original draft, methodology, data curation, and formal
analysis. Ting Ye: Data curation. Linjin Liang: Data cura-
tion. Wenyi Liang: Formal analysis. Ping Jian: Resources.
Kun Zhou: Software.
Acknowledgement
We thank Dr. Chunguo Wang of the UPLC-ESI-MS
n
Labora-
tory, Beijing Institute of Traditional Chinese Medicine,
Beijing University of Chinese Medicine (Beijing, China) for
his great help with MS measurement.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jtcms.2018.11.006.
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10 S. Li et al.
+MODEL
Please cite this article as: Li S et al., Anti-cancer activity of an ethyl-acetate extract of the fruits of Terminalia bellerica (Gaertn.) Roxb.
through an apoptotic signaling pathway in vitro, Journal of Traditional Chinese Medical Sciences, https://doi.org/10.1016/
j.jtcms.2018.11.006
... Therefore, these structural differences between phytochemicals of two extracts could explain the differences between the biological activities. Gallic acid antiviral and antioxidant activities, anticancer activity by inducing apoptosis, downregulating genes involved in cell cycle and angiogenesis, and stimulating a cellular immune response [24,[70][71][72][73] 5 ...
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Among conventional treatment methodologies, surgery, hyperthermia, radiation, and chemotherapy have become integral components of treatment for most cancers. Radiation therapy in the treatment of many malignancies is always the better choice over surgery and chemotherapy. Ionizing radiation produced as a consequent of using these radiations have always been a concern in these treatment methods. Synthetic radio-protectors with their inherent limitations are being used till date to reduce the mortality of these radiations; still, it compromises the clinical efficacy of these administrations. Hence, investigations for alternative methods, including natural resources such as plant and fruit extracts are being explored to treat radiation-mediated ailments. The present review article endeavors to provide a comprehensive, updated, and chronological account of these most promising plants and fruit extracts and their bioactive principles as radio-protectors. We present the merits and demerits of radiation therapy; cell stress generation of reactive oxygen species (ROS) associated with radiation need and availability of radio-protectors. Finally, we discuss green-based bioactive compounds which have radioprotective properties.
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Background Terminalia bellirica (Gaertn.) Roxb. is one of the oldest medicinal herbs of India, Pakistan, Nepal, Bangladesh and Sri Lanka as well as South-East Asia. Its medicinal utility has been described in the different traditional medicinal systems, such as Ayurveda, Unani, Siddha, and traditional Chinese medicine. Purpose The present study is aimed at providing a comprehensive overview on the traditional medicinal use, major phytoconstituents, biological and pharmacological activities and related mechanisms of actions and clinical studies of T. bellirica. Another objective is to describe current limitations and future direction of T. bellirica-related research. Methods PubMed, ScienceDirect, Scopus, Cochrane Library, and EBOSCO host databases were selected to explore literature published between 1980 and 2020 (till February). Keywords used in various combinations comprised of Terminalia bellirica, phytoconstituents, health effects, pharmacological activities, molecular targets, in vitro, in vivo, clinical studies, and disease prevention. Results A broad spectrum in vitro and in vivo studies suggested various biological and pharmacological effects, including antioxidant, anti-inflammatory, immunomodulatory, antimicrobial, hepatoprotective, renoprotective, antidiabetic, anti-hyperlipidemic, and anticancer activities. Diverse bioactivities of T. bellirica have been ascribed to the presence of many bioactive phytochemicals, such as glucoside, tannins, gallic acid, corilagin, ellagic acid, ethyl gallate, gallyl glucose, chebulagic acid, and arjunolic acid. Conclusion Preclinical and clinical studies have suggested that T. bellirica plant and its phytoconstituents have immense potential for prevention and treatment of various diseases. Additional in vivo studies and clinical trials are warranted to realize the complete medicinal attributes of this plant.
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This study was designed to search for novel anti-cancer compounds from natural plants. The 70% ethanolic extract from the rizhomes of Cimicifuga dahurica (Turcz.) Maxim. (Ranunculaceae) was found to possess significant in vitro anti-proliferative effects on MCF-7 breast cancer cells. A phytochemical investigation using assay-guided fractionation of the ethanolic extract of C. dahurica resulted in the isolation of one new phenolic amide glycoside 3, two new lignan glycosides 4 and 7, one new 9,19-cycloartane triterpenoid glycoside 6, and thirteen known constituents 1, 2, 5, and 8–17. The structures of 3, 4, 6, and 7 were established using contemporary NMR methods and from their HRESIMS data. The anti-proliferative effects of isolated compounds were evaluated using the BrdU-proliferation kit. Five among the 17 isolated compounds showed significant anti-proliferative effects (p � 0.05), wherein compound 7 showed the most significant anti-proliferative and cell cycle arresting effect (p �0.05) which followed a dose dependent manner. Western blot protein expression analysis showed a down expression of c-Myc and cyclin D1 which further elucidated the anti-proliferation mechanism of compound 7 while apoptotic effects were found in association with Bcl-2 family protein expression variations. Conclusively this study reports the isolation and identification of 17 compounds from C. dahurica, including four novel molecules, in addition to the fact that compound 7 possesses significant anti-proliferative and apoptotic effects in vitro that may require further exploration.
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In the search for new antibacterial agents from natural sources, we revealed that a crude methanol extract of Sapium baccatum was highly active against Ralstonia solanacearum, a causal agent of a serious disease called bacterial wilt of tomato. The bioassay-guided fractionation of this extract resulted in the isolation of seven known active compounds, including gallic acid, methyl gallate, corilagin, tercatain, chebulagic acid, chebulinic acid, and quercetin 3-O-α-L-arabinopyranoside. Their chemical structures were determined by electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. An in vitro antibacterial bioassay using a broth microdilution method revealed that, except for quercetin 3-O-α-L-arabinopyranoside (MIC = 250 μg/mL), the isolated compounds exhibited strong antibacterial activity against R. solanacearum (MIC = 26–52 μg/mL). Among the seven compounds, methyl gallate exhibited the strongest broad-spectrum activity against most of the plant pathogenic bacteria tested (MIC = 26–250 μg/mL). In the in vivo experiments, the crude extract of S. baccatum at 2000 and 1000 μg/mL reduced the development of tomato bacterial wilt by 83 and 63%, respectively, under greenhouse conditions after 14 days of infection. The results suggested that the extracts of S. baccatum or isolated tannins could be used as natural bactericides for the control of bacterial wilt of tomato.
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The high incidence of breast cancer in developed and developing countries, and its correlation to cancer-related deaths, has prompted concerned scientists to discover novel alternatives to deal with this challenge. In this review, we will provide a brief overview of polyphenol structures and classifications, as well as on the carcinogenic process. The biology of breast cancer cells will also be discussed. The molecular mechanisms involved in the anti-cancer activities of numerous polyphenols, against a wide range of breast cancer cells, in vitro and in vivo, will be explained in detail. The interplay between autophagy and apoptosis in the anti-cancer activity of polyphenols will also be highlighted. In addition, the potential of polyphenols to target cancer stem cells (CSCs) via various mechanisms will be explained. Recently, the use of natural products as chemotherapeutics and chemopreventive drugs to overcome the side effects and resistance that arise from using chemical-based agents has garnered the attention of the scientific community. Polyphenol research is considered a promising field in the treatment and prevention of breast cancer.
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One of the cancer molecular hallmarks is a deviant energetic metabolism, known as the Warburg effect, whereby the rate of glucose uptake is significantly increased and a high rate of glycolysis and lactic acid production occurs even when oxygen is present-"aerobic lactatogenesis". Accordingly, GLUT1 and MCT1, which are the main glucose and lactate transporters in cancer cells, respectively, have been proposed as oncogenes and are currently seen as potential therapeutic targets in cancer treatment. Polyphenols, commonly contained in fruits and vegetables, have long been associated with a protective role against cancer. Generally considered as nontoxic, dietary polyphenols are considered ideal chemopreventive and possibly chemotherapeutic agents. Several mechanisms of action of polyphenols in breast cancer cells have been proposed including modulation of intracellular signaling, induction of apoptosis through redox regulation or modulation of epigenetic alterations. Additionally, in vitro studies have shown that several polyphenols act as specific inhibitors of glucose transport in breast cancer cell lines and an association between their anticarcinogenic effect and inhibition of glucose cellular uptake has been described. Also, some polyphenols were found to inhibit lactate transport. Importantly, some polyphenols behave as inhibitors of both glucose and lactate cellular uptake by breast cancer cells and these compounds are thus very interesting in the context of a chemopreventive effect, because they deplete breast cancer cells of their two most important energy suppliers. So, the antimetabolic effect of polyphenols should be regarded as a mechanism of action contributing to their chemopreventive/chemotherapeutic potential in relation to breast cancer.
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The phenolic content of the ethanol extract of the stem bark of the Brazilian plant Schinopsis brasiliensis Engl. (Anacardiaceae) has been evaluated together with the antioxidant activity. The good antioxidant activity exhibited in the Trolox Equivalent Antioxidant Capacity (TEAC) assay (TEAC value = 3.04 mg/mL) encouraged us to investigate its constituents. An analytical approach based on LC-ESIMSⁿ was applied to rapidly obtain a metabolite profile of the ethanol extract of the stem bark of S. brasiliensis. Sixteen phenolic compounds, among which five galloyl derivatives, never reported before, have been isolated and their structures have been unambiguously elucidated by extensive spectroscopic methods, including 1D (¹H, ¹³C, TOCSY) and 2D (DQF-COSY, HMBC, and HSQC) NMR experiments. Moreover, the antioxidant activity of all the isolated compounds was evaluated, along with the cytotoxicity against the cancer cell lines A549 (human alveolar basal carcinoma) and Hela (human epitheloid cervix carcinoma). The previously undescribed compounds exhibited a high free-radical-scavenging activity, in the range of 1.10–1.86 mM.
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Nine hydrolyzable tannins, including three previously unknown and six artifacts, were isolated, together with thirty-nine known ones, from the fruits of Terminalia chebula Retz. (Combretaceae). They were identified as 1,2,3-tri-O-galloyl-6-O-cinnamoyl-β-d-glucose, 1,2,3,6-tetra-O-galloyl-4-O-cinnamoyl-β-d-glucose, 4-O-(2″,4″-di-O-galloyl-α-l-rhamnosyl)ellagic acid, 1′-O-methyl neochebulanin, dimethyl neochebulinate, 6′-O-methyl neochebulagate, dimethyl neochebulagate, dimethyl 4′-epi-neochebulagate, and methyl chebulagate by the spectroscopic interpretation. After evaluation for α-glucosidase inhibition of all isolated compounds, 1,2,3,6-tetra-O-galloyl-4-O-cinnamoyl-β-d-glucose and 4-O-(2″,4″-di-O-galloyl-α-l-rhamnosyl)ellagic acid showed significant inhibitory activities with IC50 values of 2.9 and 6.4 μM, respectively. In addition, inhibition kinetic studies showed that both compounds have mixed-type inhibitory activities with the inhibition constants (Ki) of 1.9 and 4.0 μM, respectively.
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Objective To determine the distribution of metabolites in the root barks of different tree peony cultivars for quality assessment. Methods Seven tree peony phenotypic cultivars with different colors were systematically analyzed using NMR-based metabolomics. Results A total of 16 metabolites from their methanol extracts were simultaneously identified and quantified, including one primary metabolite (sucrose) and 15 secondary ones (acetophenones, phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids). The quantitative data indicated that sucrose (90-180 mg/g) and acetophenones (15-100 mg/g), and non-phenolics, monoterpene glycosides, flavonoids, and unsaturated fatty acids (2-15 mg/g) were the major metabolites in these tree peony cultivars. The significantly increasing levels of paeonoside with bioactivity were observed in “Xiangyu”, “Wujinyaohui”, “Roufurong”, “Yaohuang”, “Zhaofen”, “Doulű”, and “Yingrihong” in order. Opposite trends in the levels of paeonoside and paeonol were observed in “Xiangyu” and “Yingrihong”, suggesting that the changes of the secondary metabolites in plants were influenced by primary metabolites, such as sucrose/glucose, and the different physiological processes occurred in different tree peony cultivars. Conclusion “Yingrihong” with red flower has the highest medicine quality whereas “Xiangyu” with white flower has the worst one based on the content of paeonoside.