Synthetic and antitumor comparison of 9-O-alkylated and carbohydrate-modified berberine derivatives

Article abstract of DOI:10.1007/s13738-020-01985-0

Abstract: Berberine is a naturally occurring isoquinoline alkaloid with moderate antitumor effect. It has been proved that berberine can affect the microenvironment of cancer cells, which is critical for cancer therapy and improving human immunity. Therefore, structural modification of berberine to obtain derivatives with high antitumor effect attracts the great attention of scientists. This paper describes the synthesis and in vitro antitumor investigation of some 9-O-alkylated and carbohydrate-modified berberine derivatives using high efficient strategy. The cytotoxicity investigation was accomplished on different cancer cell lines by the well-optimized MTT assay, and the results indicated that 9-O-hydrophobic modified berberine derivative 9 with long lipid chain showed promising in vitro antitumor activity with the IC₅₀ value as low as 4.87 ± 0.24?μM. Percentage of apoptosis was measured by a Hoechst 33342/PI staining strategy on HeLa cells, which revealed that compound 9 could induce 84% of cell apoptosis at very low concentration (5?μM). Flow cytometry analysis indicated that compound 9 might inhibit HeLa cell proliferation by G2 phase arrest. The expression of various cell apoptosis proteins was measured by western blot analysis, and the results indicated that compound 9 could induce over-expression of Caspase-3, Parp, and Bax and down-regulate the expression of P53 and PCNA. All these data indicated the high potential of compound 9 for developing novel anticancer drugs. Graphic Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s13738-020-01985-0

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-01985-0
ORIGINAL PAPER
Synthetic and antitumor comparison of 9‑O‑alkylated
and carbohydrate‑modifed berberine derivatives
Rongchun Wang1,2 · Stoyka Rostyslav3 · Xiaobin Li1,2 · Houwen Lin4 · Xuanming Zhang1,2 · Shanshan Zhang1,2
Kechun Liu1,2 · Lizhen Wang1,2
·
Received: 25 September 2019 / Accepted: 19 June 2020
© Iranian Chemical Society 2020
Abstract
Berberine is a naturally occurring isoquinoline alkaloid with moderate antitumor efect. It has been proved that berberine can
afect the microenvironment of cancer cells, which is critical for cancer therapy and improving human immunity. Therefore,
structural modifcation of berberine to obtain derivatives with high antitumor efect attracts the great attention of scientists.
This paper describes the synthesis and in vitro antitumor investigation of some 9-O-alkylated and carbohydrate-modifed
berberine derivatives using high efcient strategy. The cytotoxicity investigation was accomplished on diferent cancer cell
lines by the well-optimized MTT assay, and the results indicated that 9-O-hydrophobic modifed berberine derivative 9
with long lipid chain showed promising in vitro antitumor activity with the IC50 value as low as 4.87 0.24 μM. Percentage
of apoptosis was measured by a Hoechst 33342/PI staining strategy on HeLa cells, which revealed that compound 9 could
induce 84% of cell apoptosis at very low concentration (5 μM). Flow cytometry analysis indicated that compound 9 might
inhibit HeLa cell proliferation by G2 phase arrest. The expression of various cell apoptosis proteins was measured by west-
ern blot analysis, and the results indicated that compound 9 could induce over-expression of Caspase-3, Parp, and Bax and
down-regulate the expression of P53 and PCNA. All these data indicated the high potential of compound 9 for developing
novel anticancer drugs.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-020-01985-0) contains
supplementary material, which is available to authorized users.
* Kechun Liu
wlzh1106@126.com
* Lizhen Wang
liukechun2000@163.com
1
Biology Institute, Qilu University of Technology (Shandong
Academy of Sciences), Jinan 250103, Shandong Province,
China
2
Engineering Research Center of Zebrafsh Models for Human
Diseases and Drug Screening of Shandong Province, Qilu
University of Technology (Shandong Academy of Sciences),
Jinan 250103, Shandong Province, China
3
Department of Regulation of Cell Proliferation
and Apoptosis, Institute of Cell Biology, National Academy
of Sciences of Ukraine, Lviv, Ukraine
4
Research Center for Marine Drugs, State Key Laboratory
of Oncogenes and Related Genes, Department of Pharmacy,
School of Medicine, Ren Ji Hospital, Shanghai Jiao Tong
University, Shanghai 200127, China
Vol.:(0123456789)
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Graphic Abstract
Keywords Berberine · HeLa cell · Antitumor · Alkylated · Carbohydrate
Introduction
Berberine is a naturally occurred isoquinoline alkaloid
isolated from various Berberis plants [1, 2] and has been
extensively used in China for the treatment of gastroenteritis
diseases and bacterial infection for hundreds of years [3, 4].
Recently, many research groups have revealed that berberine
exhibited moderate antitumor efects [5, 6], such as inhib-
iting the proliferation of many cancer cell lines, inducing
the apoptosis and diferentiation of many species of tumor
Fig. 1 Structural modifcation of berberine on diferent positions
cells, and restraining the invasion and metastasis of tumor
cells. It has been well proved that berberine can bind with
the G-quadruplexes, an important structure unit in human
DNA and RNA for transcriptional modulations and telom-
erase inhibition, which results in cancer cell senescence
and apoptosis [7]. Berberine can also regulate the expres-
sion of some Bcl-2 proteins (Bax, Bim, Bcl-2, and Bcl-xL)
and afect the mitochondrial membrane potential, which
are critical for tumor cell apoptosis [8, 9]. Moreover, the
high anti-infammatory and antioxidant efects of berberine
can improve the microenvironment of cancer cells, thereby
leading to the cell cycle arrest and inhibition of cancer cell
proliferation [10]. Therefore, berberine is an attracting lead
molecule utilized for developing novel antitumor agents.
During the past several years, many research groups have
focused on the structural modifcation of berberine (Fig. 1).
For example, modifcation of berberine on its C-13 position
with hydrophobic aromatic and lipid groups can enhance its
binding ability with DNA or RNA, thus resulting in obvious
improvement on its antitumor activity [9, 11]. The C-12 modi-
fcation of berberine with Mannich bases can give berberine
derivatives with both anticancer and antioxidant activities [1,
12]. However, the structural modifcation of berberine on its
C-13 and C-12 positions is quite limited, due to the low reac-
tivity of the hydrogen atom in these two positions. In con-
trast, the 9-O-modifcation of berberine is easily achievable
to provide derivatives with antitumor, anti-infammation, and
antioxidant activities [11, 13, 14]. More importantly, the key
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Experimental
General procedures
Berberine was commercially available from Tokyo Chemi-
cal Industry (TCI). Other solvents and reagents were
bought as analytical grade and used without any further
purifcation. 1H NMR spectra were measured on a Bruker
AV 400 spectrometer, and high-resolution mass spectra
(HRMS) were measured on an Agilent QTOF 6520 mass
spectrometer. HeLa cell lines were purchased from the
American Type Culture Collection and cultured in DMEM
medium (Invitrogen) containing 10% fetal bovine serum,
10 units/mL penicillin-G, and 10 mg/mL streptomycin.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) was bought from Sigma. Hoechst 33342/PI
double staining kit was obtained from Genview (China).
Anti-PCNA, anti-Caspase-3, anti-Parp, anti-Bax, anti-
BCL 2, anti-P53, and anti-GAPDH antibodies were bought
from Cell Signaling Technology.
Scheme 1 Synthesis of 9-O-alkylated berberine derivatives 6–9
intermediate 9-OH pseudoberberine 1 (Scheme 1) can be eas-
ily obtained through pyrolysis of berberine with high yield
and selectivity [15, 16] and then will be conveniently used
for introducing various functional groups on its 9-O-position.
Consequently, many 9-O-modifed berberine derivatives had
been synthesized in the past several years, and some of them
showed high antitumor activity [17]. However, the antitumor
activity of these synthesized berberine derivatives is still not
strong enough for developing novel antitumor agents, despite
the remarkable advances made in this area. Therefore, the
structural modifcation and functionalization of berberine still
remains necessary and is of great importance.
General procedure for synthesis of 9‑O‑alkylated
berberines 6–9
Berberine (10 g, 27 mmol) was heated at 190 °C under
reduced pressure for 30 min to give a crude solid, which
was subjected to column chromatography using dichlo-
romethane/methanol (5/1) as the eluent to aford com-
pound 1 (7.6 g, yield = 79%) as a purple solid.
Previous research revealed that the introduction of lipo-
philic groups on leading molecules could facilitate the penetra-
tion of antitumor drugs into cell membrane and enhance their
cytotoxicity against many cancer cell lines [18, 19], which
indicated that lipidation of berberine should be a promising
modifcation strategy. On the other hand, the hydrophilic car-
bohydrate groups have also been proved critical for enhancing
the antitumor activity of lead compounds through improving
their bioavailability and/or receptor-binding afnity [20–25].
For example, some rhamnose-modifed saponins showed high
cytotoxicity against human cancer cell lines, which might be
caused by the rhamnose-binding lectin expressed by cancer
cells that could promote the transportation of saponins [26].
Hence, the carbohydrate modifcation might have some posi-
tive efect on the antitumor activity of berberine, while there
is no work to compare the antitumor activity of the lipid and
carbohydrate-modifed berberine derivatives till now. This
paper synthesized some 9-O-alkylated berberine derivatives
with diferent lengths of lipid chain, and their cytotoxicity was
measured in vitro on HeLa cell lines. As a comparison, the
cytotoxicity of some carbohydrate-modifed berberine deriva-
tives was also measured on the same cell lines.
A solution of compound 1 (500 mg, 1.4 mmol), bromo-
hydrocarbon (4.2 mmol) and K2CO3 (580 mg, 4.2 mmol)
in 5 mL of DMF was stirred at 80 °C for 3 h. After TLC
(dichloromethane/methanol 7/1) showed the conversion of
the starting materials, the mixture was diluted with 50 mL
of dichloromethane and washed with water (20 mL × 3).
The organic layer was dried with absolute sodium sulfate
and evaporated under reduced pressure. The resultant resi-
due was purifed by column chromatography using dichlo-
romethane/methanol (10/1 → 5/1) as the eluent to provide
9-O-alkylated berberines 6–9.
9-O-ethyl berberine chloride (6). Compound 1 was
reacted with bromoethane 2 by the general procedure
described above to give product 6 as a yellow solid
1
(458 mg, yield = 85%). H NMR (400 MHz, DMSO-
d6): δ 9.82 (s, 1 H, CH = N), 8.95 (s, 1 H, ArH), 8.20 (d,
J = 8.8 Hz, 1 H, ArH), 8.00 (d, J = 9.2 Hz, 1 H, ArH), 7.80
(s, 1 H, ArH), 7.10 (s, 1 H, ArH), 6.18 (s, 2 H, –OCH2O–),
4.96 (t, J = 6.0 Hz, 2 H, CH2), 4.36 (9, J = 6.8 Hz, 2 H,
OCH2CH3), 4.06 (s, 3 H, OCH3), 3.21 (t, J = 6.4 Hz, 2
H, CH2), 1.46 (t, J = 6.8 Hz, 3 H, CH2CH3); 13C NMR
(100 MHz, DMSO-d6): δ 151.00, 150.29, 148.16, 145.90,
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Journal of the Iranian Chemical Society
143.07, 137.94, 133.48, 131.18, 127.13, 123.81, 122.36,
120.94, 120.67, 108.89, 105.90, 102.56, 70.41, 57.52,
55.72, 26.82, 15.84. ESI-TOF HRMS (m/z): calcd for
DMSO-d6): δ 150.89, 150.30, 148.17, 145.78, 143.37,
137.94, 133.51, 131.17, 127.20, 123.75, 122.15, 120.94,
120.70, 108.90, 105.90, 102.56, 74.73, 57.53, 55.79, 31.73,
29.95, 29.27, 29.16, 26.82, 25.75, 22.56, 14.44. ESI-TOF
+
C21H20NO4 , [M–Cl]+, 350.1387; found, 350.1390.
+
9-O-butyl berberine chloride (7). Compound 1 was
reacted with bromobutane 3 by the general procedure
described above to give product 7 as a yellow solid (434 mg,
yield=75%). 1H NMR (400 MHz, DMSO-d6): δ 9.75 (s, 1 H,
CH=N), 8.95 (s, 1 H, ArH), 8.19 (d, J=9.2 Hz, 1 H, ArH),
8.00 (d, J=9.2 Hz, 1 H, ArH), 7.80 (s, 1 H, ArH), 7.10 (s, 1
H, ArH), 6.18 (s, 2 H, –OCH2O–), 4.96 (t, J=6.0 Hz, 2 H,
CH2), 4.30 (t, J=6.8 Hz, 2 H, OCH2CH2CH2CH3), 4.06 (s, 3
H, OCH3), 3.21 (t, J=6.0 Hz, 2 H, CH2), 1.90-1.83 (m, 2 H,
OCH2CH2CH2CH3), 1.57-1.47 (m, 2 H, OCH2CH2CH2CH3),
0.99 (t, J = 7.6 Hz, 3 H, OCH2CH2CH2CH3); 13C NMR
(100 MHz, DMSO-d6): δ 150.87, 150.30, 148.16, 145.75,
143.36, 137.94, 133.51, 131.18, 127.20, 123.74, 122.14,
120.93, 120.70, 108.89, 105.91, 102.56, 74.43, 57.54, 55.80,
32.02, 26.82, 19.03, 14.24. ESI-TOF HRMS (m/z): calcd for
HRMS (m/z): calcd for C27H32NO4 , [M–Cl]+, 434.2326;
found, 434.2330.
General procedure for synthesis
of carbohydrate‑modifed berberines 17–22
The solution of alkynylated berberine derivative 10 (100 mg,
0.25 mmol), azido sugars 11–16 (0.25 mmol), CuSO4·5H2O
(33 mg, 0.13 mmol), sodium ascorbate (26 mg, 0.13 mmol)
and methanol (3 mL) in 3 mL of DMF was heated to 50 °C
and stirred at ambient temperature for 3 h. After TLC
showed the conversion of the starting materials (dichlo-
romethane/methanol/H2O, 10/3/1), the solvent was evap-
orated and the resultant residue was subjected to column
chromatography to give carbohydrate-modifed berberine
derivatives 17–22. The H and C NMR spectra are shown in
supporting information.
+
C23H24NO4 , [M–Cl]+, 378.1700; found, 378.1703.
9-O-hexanyl berberine chloride (8). Compound 1 was
reacted with bromohexane 4 by the general procedure
described above to give product 8 as a yellow solid (549 mg,
Cell viability assay
1
yield = 89%). H NMR (400 MHz, DMSO-d6): δ 9.76 (s,
1 H, CH = N), 8.95 (s, 1 H, ArH), 8.19 (d, J = 8.8 Hz, 1
H, ArH), 7.98 (d, J = 8.8 Hz, 1 H, ArH), 7.80 (s, 1 H,
ArH), 7.10 (s, 1 H, ArH), 6.18 (s, 2 H, –OCH2O–), 4.96 (t,
J=6.0 Hz, 2 H, CH2), 4.28 (t, J=6.8 Hz, 2 H, OCH2CH-
2CH2CH2CH2CH3), 4.05 (s, 3 H, OCH3), 3.21 (t, J=5.6 Hz,
2 H, CH2), 1.92-1.84 (m, 2 H, OCH2CH2CH2CH2CH2CH3),
1.52-1.45 (m, 2 H, CH2), 1.39-1.29 (m, 4 H, 2×CH2), 0.91
(t, J=6.8 Hz, 3 H, OCH2CH2CH2CH2CH2CH3); 13C NMR
(100 MHz, DMSO-d6): δ 150.89, 150.30, 148.16, 145.77,
143.37, 137.93, 133.51, 131.17, 127.19, 123.76, 122.15,
120.94, 120.70, 108.89, 105.91, 102.56, 74.74, 57.53, 55.79,
31.53, 29.92, 26.82, 25.41, 22.56, 14.42. ESI-TOF HRMS
HeLa cells (2 × 106/well) were cultured for 24 h at 37 °C
and 5% CO2 atmosphere in 96-well plates, using DMEM
medium containing 10% fetal bovine serum (FBS), 10 units/
mL penicillin-G, and 10 mg/mL streptomycin, 2 mM glu-
tamine, and 2% sodium pyruvate. The medium was then
replaced with DMEM medium containing 0.5% DMSO and
diferent concentrations of berberine derivatives (3.125,
6.25, 12.5, 25, 50, and 100 μM). The DMEM medium con-
taining 0.5% DMSO was employed as control well. After
24 h of incubation, 10 μL of MTT (5 mg/mL) was added to
each well and the cells were cultured for another 4 h. There-
after, the supernatant in each well was removed and 200 μL
of DMSO was added. After 10 min of swirling, the optical
density (OD) values of the plates were measured on a MR
spectroscopy at 570 nm. The relative viability of treated
cells was expressed as percentage of control untreated cells.
Three replicates were run for each concentration, and all
tests were repeated for 5 wells.
+
(m/z): calcd for C25H28NO4 , [M–Cl]+, 406.2013; found,
406.2015.
9-O-octanyl berberine chloride (9). Compound 1
was reacted with 1-bromooctane 5 by the general proce-
dure described above to give product 9 as a yellow solid
1
(480 mg, yield = 73%). H NMR (400 MHz, DMSO-
d6): δ 9.75 (s, 1 H, CH = N), 8.95 (s, 1 H, ArH), 8.19 (d,
J=9.2 Hz, 1 H, ArH), 8.00 (d, J=9.2 Hz, 1 H, ArH), 7.80
(s, 1 H, ArH), 7.10 (s, 1 H, ArH), 6.18 (s, 2 H, –OCH2O–),
4.95 (t, J = 5.6 Hz, 2 H, CH2), 4.28 (t, J = 6.8 Hz, 2 H,
OCH2CH2CH2CH2CH2CH2CH2CH3), 4.05 (s, 3 H,
OCH3), 3.21 (t, J =6.0 Hz, 2 H, CH2), 1.91-1.84 (m, 2 H,
OCH2CH2CH2CH2CH2CH2CH2CH3), 1.52-1.44 (m, 2 H,
CH2), 1.40-1.23 (m, 8 H, 4×CH2), 0.87 (t, J=6.8 Hz, 3 H,
OCH2CH2CH2CH2CH2CH2CH2CH3); 13C NMR (100 MHz,
Hoechst 33342/PI staining for cell apoptosis
Hoechst 33342/PI double staining was carried out to deter-
mine apoptosis in HeLa cells. The cells in 96-well plates
containing about 30,000 cells were incubated in binding
bufer containing Hoechst 33342 and PI for 15 min at 37 °C
in the dark. Fluorescence microscopic studies to detect
pro-apoptotic efects were carried out per well and using a
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BioTek Cytation 3 high content imager with a 20×objective.
The apoptosis cells were identifed based on positive stain-
ing with both propidium iodide (PI) and Hoechst 33342 and
absence of nuclear fragmentation. Each plate contained 3
replicates per condition, and two independent experiments
were performed. Three replicates were examined for each
concentration, and all tests were repeated in 3 wells.
was reacted with bromohydrocarbons 2–5 under the promo-
tion of potassium carbonate in DMF to provide the desired
berberine derivatives 6–9. The hydrophilic carbohydrate-
modified berberine derivatives 17–22 (Scheme 2) were
prepared by a similar procedure described previously [28]
from azido sugars 11–16 and alkynylated berberine deriva-
tive 10, which could be obtained by reaction of berberrubine
with propargyl bromide under mild conditions [28]. Previ-
ous work had been revealed that the 9-O-glucosyl-berberine
could improve the bioavailability of berberine, but showed
poor stability under both acidic physiological conditions and
high temperature [29]. To solve this problem, we use a tria-
zole linker to fnish the conjugation of berberine with car-
bohydrate, which can provide some N-glycosides with high
stability. Furthermore, the triazole linker has been proved to
play important role for enhancing the antitumor activity of
drugs, for example, improving the water solubility and the
receptor-binding afnity [30].
Cell cycle analysis
Cells were treated with diferent concentrations of com-
pound 9 (2.5, 5, and 10 μM) and cultured for 24 h. Then,
cells were harvested and washed with PBS, which was fol-
lowed by being fxed and permeabilized at −20 °C overnight
using ice-cold 75% ethanol. After that, cells were washed
with ice-cold PBS and treated with 100 μg/mL of RNase at
37 °C for half an hour. Finally, cells were stained with 1 mg/
mL PI at 4 °C for half an hour and subjected to cell cycle
analysis with a FACS Calibur analyzer (BD) and analyzed
with FlowJo software version 7.6.1 (Ashland, OR, USA).
Cells treated with solvent DMSO were used as the control
groups. Three replicates were examined for each concentra-
tion. Three replicates were examined for each concentration.
Cell viability study
The efect of the synthesized berberine derivatives on
HeLa cell lines was measured by MTT assay. Cells were
treated with diferent concentrations (0, 3.125, 6.25, 12.5,
25, 50, and 100 μM) of berberine derivatives for 24 h,
which was followed by the standard MTT analysis on
a microplate reader to give the results shown in Fig. 2.
Clearly, the cytotoxicity of berberine exhibited a dose-
dependent manner, and the cell viability was about 51% at
a concentration of 100 μM (Fig. 2a). Berberine derivative
6 with an ethyl group on its 9-O-position showed similar
cytotoxicity as berberine both at low and high concentra-
tions (Fig. 2b). In contrast, the cell viability of deriva-
tive 7 showed a slightly increase at low concentrations
(≤ 6.25 μM), followed by a dose-dependent decrease
(≥ 12.5 μM, Fig. 2c). Berberine derivative 8 showed
higher cytotoxicity than berberine at low concentrations
(≤50 μM, Fig. 2d), while its cell inhibition ability dramat-
ically increased when the concentration reached 100 μM,
which proved its potential antitumor activity at high con-
centrations. Interestingly, 9-O-octanyl berberine deriva-
tive 9 showed high cell inhibition ability both at low and
high concentrations (Fig. 2e). When the concentration was
12.5 μM, the cell inhibition ability of 9 was remarkably
higher than berberine, indicating its potential antitumor
activity. Compared with the lipidated berberine deriva-
tives 6–9, all hydrophilic carbohydrate-modifed berberine
derivatives exhibited low or similar cytotoxicity against
HeLa cell lines (Fig. 2f–k). This is because hydrophobic
modifcation of berberine might exhibit high permeability
into cell membrane, due to the presence of lipid bilayer in
Western blot
HeLa cells were cultured in 6-well plates and treated with
compound 9 (0.5, 2.5, 5, 10 μM) for 24 h. Then, HeLa cells
were collected and lysed by RIPA bufer. Protein concentra-
tion was determined by the BCA protein assay kit (Thermo
Scientifc, Rockford, IL, USA). Equivalent amounts of pro-
teins of each group were subjected to SDS-PAGE gel elec-
trophoresis and transferred onto nitrocellulose membranes
(Bio-Rad, Philadelphia, PA, USA). After being blocked with
5% non-fat milk in Tris-bufered saline bufer, the mem-
branes were incubated with primary antibodies (anti-PCNA,
anti-Caspase-3, anti-Parp, anti-Bax, anti-BCL 2, anti-P53
and anti-GAPDH), (1:1000) at 4 °C overnight, followed
by incubation with the corresponding secondary antibod-
ies (1:5000). Protein bands were visualized with an ECL
advanced Western blotting detection kit.
Results and discussion
Synthesis
The synthesis of lipidated berberine derivatives is depicted
in Scheme 1. Pyrolysis of berberine at high temperature
(190 °C) under reduced pressure (20-30 mm Hg) selec-
tively aforded 9-O-demethylated berberine 1 [27], which
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Journal of the Iranian Chemical Society
cell surface. In contrast, the hydrophilic modifed berber-
ines, despite having good water solubility, showed poor
permeability into the hydrophobic cell membrane, which
resulted in low ability on suppressing cancer cell growth.
Then, compounds 8 and 9 were subjected to a series
of cytotoxicity analysis against diferent cancer cell lines,
including human lung cancer (A549), human liver can-
cer (HepG2), breast (MCF7), human cervical carcinoma
(HeLa) and rat pheochromocytoma (PC12). The results
indicated that all these two compounds displayed high
cytotoxicity with IC50 values in the range of 4 to 10 μM
(Table 1), while berberine and cisplatin showed low
and moderate cytotoxicity against the cancer cell lines
described above. Compound 9 showed higher high cyto-
toxicity than 8, which proved that berberine derivative
with long lipid chain might exhibit high antitumor activ-
ity. In view of the above results, berberine derivative 9
was chosen as the leading compound for further biological
investigations.
[32]. As described in Fig. 3, the non-treated normal cells
(Con) with intact cell wall showed strong blue fuores-
cence which was generated by the cell wall penetrating
dye Hoechst 33342, and the slightly red fuorescence was
produced by the cell-impermeable PI dye in small amount
of death cells with broken cell wall. After treating with
diferent concentrations (0.5, 2.5, 5, and 10 μM) of deriva-
tive 9, there was an obvious red fuorescence enhancement
accompanied with a blue fuorescence decrease, due to
the apoptosis of normal HeLa cells. The percentage of
cell apoptosis was 46% when the concentration of 9 was
2.5 μM and reached 95% with the concentration of 10 μM
(Fig. 4), which revealed the high cytotoxicity efect of 9 on
HeLa cell lines. Based on the fact that apoptosis has been
proved to play important role in the depression of tumor
cells and keeping tissue homeostasis [33], drugs that can
efectively induce tumor cell apoptosis are urgently desir-
able owing to their low toxic side-efects during cancer
treatment [34]. Consequently, compound 9 might be a
promising anticancer agent which can induce cell apop-
tosis at low concentrations.
Fluorescence photomicrographs and percentage
of apoptosis
Efect of berberine derivative 9 on cell cycle
Fluorescence photomicrographs of HeLa cells treated
with different concentrations of berberine derivative
9 (0.5, 2.5, 5, and 10 μM) were measured by a Hoechst
33342/PI staining strategy (Fig. 3). Hoechst 33342 is a
cell membrane penetrating dye which can bind with DNA
in normal cells and gives blue fuorescence [31]. In con-
trast, the cell-impermeable propidium iodide (PI) can bind
with DNA in apoptosis cells with broken cell membrane,
therefore producing red fuorescence [31]. Hence, dual
staining of these two dyes can be applied to distinguish
apoptosis and normal cells on a fuorescence microscope
Some anticancer agents inhibit cell proliferation by inter-
fering the cell cycle processes [35], while others result in
cell apoptosis [36]. To confrm whether the decrease in cell
proliferation was infuenced by the cell cycle arrest, we
measured the DNA content of nuclei in HeLa cells by fow
cytometry (Fig. 5). Treatment of HeLa cells with diferent
concentrations of 9 (5 and 10 μM) resulted in signifcant
increase in cell populations (41.59% and 37.02%) at S phase,
but the cell populations at G1 (54.44% and 60.51%) and
G2 (3.97% and 2.47%) phases showed no obvious changes.
Scheme 2 Synthesis of carbohydrate-modifed berberine derivatives 17–22
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Journal of the Iranian Chemical Society
These results indicated that berberine derivative 9 might
inhibit cell proliferation by G2 phase arrest.
of Caspase-3, Parp, and Bax and reduced the production
of P53, thus proving the apoptosis of tumor cells. Further-
more, the over-expression of PCNA was slightly inhibited
after treatment of HeLa cells with 9, which indicated that
derivative 9 might have some efect on the S phase of cell
cycle. Interestingly, there was no efect on the expression
levels of BCL-2.
Expression of cell apoptosis proteins
PCNA is a well-kwon proliferating cell nuclear antigen
which is associated with the DNA replication in the S phase
of cell cycle [37], and over-expression of PCNA can lead to
the proliferation of many human cancer cells [38]. Caspase-3
has been proved an executioner of cell apoptosis, which can
result in cell morphological changes and apoptosis in the
presence of many substrates [39]. One of the most important
substrates is Parp, which is also a widely used diagnostic
agent for confrming tumor cell apoptosis [40]. Furthermore,
P53 tumor suppressor gene is an important regulated tran-
scription factor which can induce the expression of Bax and
reduce the production of BCL-2 and then cause tumor cell
apoptosis [38]. In our experiments, western blot analysis
was utilized to investigate the expression of the above pro-
teins related to tumor cell proliferation and apoptosis. As
depicted in Fig. 6, addition of increasing concentrations of
berberine derivative 9 signifcantly stimulated the expression
Conclusions
In summary, this paper synthesized some hydrophobic
9-O-alkylated berberine derivatives with diferent length of
lipid chain for investigating their antitumor activities. As a
comparison, some hydrophilic carbohydrate-modifed ber-
berine derivatives were also synthesized by a published pro-
cedure [28]. All these berberine derivatives were subjected
to antitumor investigation on various cancer cell lines, and
the results indicated that hydrophobic modifcation of ber-
berine on its 9-O-position with long lipid chain could give
derivatives with high antitumor activity. Berberine deriva-
tive 9 with an octanyl group on its 9-O-position showed
Fig. 2 Cytotoxic efects of berberine derivatives on HeLa cell lines after 24 h of incubation. Data are presented as mean SD. *P<0.05,
**P<0.01 versus control
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Journal of the Iranian Chemical Society
Table 1 IC50 values (μM)
for berberine, cisplatin, and
berberine derivatives 8 and 9.
Cells were treated with diferent
concentrations (40, 20, 10, 5,
2.5, 1.25, and 0.625 μM) of
drugs for 24 h
Compound
IC50 values (μM)
A549
HepG2
HeLa
MCF7
PC12
8
9
8.56 1.34
5.54 0.22
>40
8.34 1.45
5.54 0.52
>40
9.58 1.89
5.02 0.12
>40
7.65 2.22
4.87 0.24
>40
8.52 2.12
6.54 1.31
>40
Berberine
Cisplatin
25.03 0.85
24.91 0.41
20.32 1.52
19.70 1.01
21.69 1.88
Fig. 4 The percentage of apoptosis HeLa cells after treatment with
diferent concentrations (0.5, 2.5, 5, and 10 μM) of berberine deriva-
tive 9. Data are presented as mean SD. *P<0.05, **P<0.01 versus
control
cell populations (41.59% and 37.02%) at S phase, which
indicated that compound 9 might inhibit cell proliferation
by G2 phase arrest. Western blot analysis revealed that com-
pound 9 could induce the down-regulation or up-regulation
of various cell apoptosis proteins, such as Caspase-3, Parp,
Bax, and P53. All these results proved that hydrophobic
modifcation of berberine with long lipid chain can pro-
vide derivatives with high antitumor activity, for example,
9-O-octanylated berberine 9 should be a potential target for
further investigation.
Fig. 3 The efect of diferent concentrations (0.5, 2.5, 5, and 10 μM)
of berberine derivative 9 on fuorescence photomicrographs of HeLa
cells detected by dual staining of Hoechst 33342/PI after 24 h of incu-
bation. Con: Non-treated cells
excellent in vitro antitumor activity and could induce 84% of
cell apoptosis at very low concentration (5 μM). More inter-
estingly, compound 9 could result in an increase in HeLa
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Journal of the Iranian Chemical Society
Fig. 5 Efect of berberine
derivative 9 on cell cycle distri-
bution (a) and S phase variation
(b). HeLa cells were treated
with diferent concentrations
(2.5, 5, and 10 μM) of 9 for 24 h
and untreated HeLa cells were
used as a control
Fig. 6 Western blot analysis of
the efect of berberine deriva-
tive 9 (0.5, 2.5, 5, and 10 μM)
on cell apoptosis proteins of
HeLa cells. GAPDH served as
the internal control
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Journal of the Iranian Chemical Society
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Acknowledgments This research was supported by grants from the
Young Science Foundation of Shandong Academy of Sciences (No.
2020QN0018), the Young Doctoral Cooperation Foundation of Qilu
University of Technology (Shandong Academy of Sciences) (No.
201713SH2024), the Natural Science Foundation of Shandong Prov-
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