Synthesis and in vitro photocytotoxicity of 9-/13-lipophilic substituted berberine derivatives as potential anticancer agents

Article abstract of DOI:10.3390/molecules25030677

The objective of this study was to synthesize the 9-/13-position substituted berberine derivatives and evaluate their cytotoxic and photocytotoxic effects against three human cancer cell lines. Among all the synthesized compounds, 9-O-dodecyl- (5e), 13-dodecyl- (6e), and 13-O-dodecyl-berberine (7e) exhibited stronger growth inhibition against three human cancer cell lines, (HepG2, HT-29 and BFTC905), in comparison with structurally related berberine (1). These three compounds also showed the photocytotoxicity in human cancer cells in a concentration-dependent and light dose-dependent manner. Through flow cytometry analysis, we found out a lipophilic group at the 9-/13-position of berberine may have facilitated its penetration into test cells and hence enhanced its photocytotoxicity on the human liver cancer cell HepG2. Further, in cell cycle analysis, 5e, 6e, and 7e induced HepG2 cells to arrest at the S phase and caused apoptosis upon irradiation. In addition, photodynamic treatment of berberine derivatives 5e, 6e, and 7e again showed a significant photocytotoxic effects on HepG2 cells, induced remarkable cell apoptosis, greatly increased intracellular ROS level, and the loss of mitochondrial membrane potential. These results over and again confirmed that berberine derivatives 5e, 6e, and 7e greatly enhanced photocytotoxicity. Taken together, the test data led us to conclude that berberine derivatives with a dodecyl group at the 9-/13-position could be great candidates for the anti-liver cancer medicines developments.

Full text of DOI:10.3390/molecules25030677

molecules
Article
Synthesis and In Vitro Photocytotoxicity of
9-/13-Lipophilic Substituted Berberine Derivatives as
Potential Anticancer Agents
Hong-Jhih Lin 1,†, Jinn-Hsuan Ho 2,†, Li-Chen Tsai 1, Fang-Yu Yang 1, Ling-Ling Yang 3,
Cheng-Deng Kuo 4,5 , Lih-Geeng Chen 1, Yi-Wen Liu 1 and Jin-Yi Wu 1,
*
1
Department of Microbiology, Immunology and Biopharmaceuticals, College of Life Sciences, National Chiayi
University, Chiayi 60004, Taiwan; s1030730@mail.ncyu.edu.tw (H.-J.L.); s1003500@mail.ncyu.edu.tw (L.-C.T.);
s1060645@mail.ncyu.edu.tw (F.-Y.Y.); lgchen@mail.ncyu.edu.tw (L.-G.C.); ywlss@mail.ncyu.edu.tw (Y.-W.L.)
Department of Chemical Engineering, College of Engineering, National Taiwan University of Science and
Technology, Taipei 10607, Taiwan; jhho@mail.ntst.edu.tw
Department of Pharmacognosy, School of Pharmacy, College of Pharmacy, Taipei Medical University,
Taipei 11031, Taiwan; llyang@tmu.edu.tw
Division of Chest Medicine, Department of Internal Medicine, Changhua Christian Hospital,
Changhua 50006, Taiwan; cdkuo23@gmail.com
2
3
4
5
Department of Medical Research, Taipei Veterans General Hospital, Taipei 11217, Taiwan
Correspondence: jywu@mail.ncyu.edu.tw; Tel.: +886-5-271-7925
*
†
These authors made equal contribution to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 January 2020; Accepted: 2 February 2020; Published: 5 February 2020
Abstract: The objective of this study was to synthesize the 9-/13-position substituted berberine
derivatives and evaluate their cytotoxic and photocytotoxic effects against three human cancer
cell lines. Among all the synthesized compounds, 9-O-dodecyl- (5e), 13-dodecyl- (6e), and
13-O-dodecyl-berberine (7e) exhibited stronger growth inhibition against three human cancer cell
lines, (HepG2, HT-29 and BFTC905), in comparison with structurally related berberine (1). These three
compounds also showed the photocytotoxicity in human cancer cells in a concentration-dependent
and light dose-dependent manner. Through flow cytometry analysis, we found out a lipophilic
group at the 9-/13-position of berberine may have facilitated its penetration into test cells and
hence enhanced its photocytotoxicity on the human liver cancer cell HepG2. Further, in cell cycle
analysis, 5e
,
6e, and 7e induced HepG2 cells to arrest at the S phase and caused apoptosis upon
6e, and 7e again
irradiation. In addition, photodynamic treatment of berberine derivatives 5e
,
showed a significant photocytotoxic effects on HepG2 cells, induced remarkable cell apoptosis, greatly
increased intracellular ROS level, and the loss of mitochondrial membrane potential. These results
over and again confirmed that berberine derivatives 5e, 6e, and 7e greatly enhanced photocytotoxicity.
Taken together, the test data led us to conclude that berberine derivatives with a dodecyl group at the
9-/13-position could be great candidates for the anti-liver cancer medicines developments.
Keywords: berberine; lipophilic substituent; anti-cancer activity; photocytotoxicity; reactive
oxygen species
1. Introduction
Although significant advances have been made in preventing and treating cancer, the mortality
rate remains to be unacceptably high. Meanwhile, many of the current chemotherapeutics are limited
by significant side effects, unpredictable efficacies, and/or acquired resistances [1–3].
Molecules 2020, 25, 677; doi:10.3390/molecules25030677
www.mdpi.com/journal/molecules

Molecules 2020, 25, 677
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Human hepatocellular carcinoma (HCC) and colorectal carcinoma are the most common types of
malignancy in sub-Saharan Africa and Southeast Asia, like Taiwan and China. It is also the fifth most
common and lethal cancer worldwide. Most of all, HCC is the most frequent malignant tumor and the
second leading cause of cancer-related death in Taiwan [4]. The hepatoma cells have high proliferative
and metastasis rates. It is usually treated by surgical removal, followed by chemotherapy [5,6
].
Doxorubicin is widely used in HCC chemotherapy. However, the agent failed to significantly improve
the 5-year survival rate of the patients partly for their severe side effects, which, among others, include
hepatic dysfunction and heart failure. As a group, we have been pursuing projects aimed at developing
novel cancer therapeutics that improve the prognosis of the disease with less adverse side effects.
Many natural products have been rich resources for potential drug development. Alkaloids,
among which are a well-known nitrogen-containing group of diverse plant secondary metabolites [
Especially, among them, berberine ( , Figure 1), is an isoquinoline alkaloid isolated from many
medicinal herbs, such as Coptis chinensis, Berberis aristata, and Phellodendron amurense. It is widely used
as traditional Chinese medicine [ 10]. Ayurvedic medicine in China and India [11]. It has a wide
range of biochemical and pharmacological potencies including antibacterial, anti-inflammatory, and
anti-cancer effects [12 13]. Further, berberine has a long history of medical application and was widely
7].
1
8–
,
used as an anti-cancer drug to treat hepatoma [14], breast cancer [15], bladder cancer [16], and colon
cancer [17]. Furthermore, berberine has significant cytotoxicity against human cancer cell lines by
triggering a significant increase in G1 arrest in the cell cycle phase of HepG2 cells [18]. It also induced
cell cycle arrest at the G2/M phase and caused apoptosis of human gastric cancer SNU-5 cells [19].
However, owing to its hydrophilic nature, or lower bioavailability, berberine is absorbed poorly in
intestines. This results in a low efficacy in suppressing cancer cell growth [20]. Besides, the anti-cancer
activity of berberine and its derivatives had recently been shown as an inhibitor of topoisomerase I
and II [21,22].
Figure 1. Chemical structures of berberine (1) and berberrubine (2).
In addition, a recent study reported various analogs of berberine on the 9-/13-position using
different chain lengths and terminal amino groups were synthesized and evaluated on the DNA-binding
affinity or as G-quadruples stabilizing ligands [23
lipophilic substituent into the 9-O-position of the berberine backbone and enhanced the anti-proliferative
activities against human cancer cell lines [27 30]. However, structural modification on the 13-position
–26]. The study also showed the introduction of a
–
of the berberine backbone has rarely been reported [31]. To explore its intestinal absorption and
inhibitory function, in this study, we modified berberine analogs and evaluated its photo-induced
cytotoxicity in vitro on human liver HepG2, bladder BFTC905 and colon HT29 cancer cell lines. The
type of functional groups, n-alkyl derivatives of berberine at the 9- or 13-position, was investigated for
the structure-activity relationship (SAR).
2. Results and Discussion
2.1. Chemistry
In general, the desired berberine derivatives were synthesized with long-chain n-alkyl bromide
through 9-/13-alkylation. As shown in Scheme 1, in specific, synthesis of 5a–e, 6a–e, and 7a–e was

Molecules 2020, 25, 677
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carried out by reacting berberrubine (
2) with selected n-alkyl bromide in dry acetone or acetonitrile
using anhydrous potassium carbonate (K2CO3) as a base under nitrogen for 8-24 h according to
the reported procedure [27]. The reduction of berberine ( ) using sodium borohydride (NaBH4)
gave intermediate in 74% yield. The addition of long-chain n-alkyl group to berberrubine ( ) and
13-hydroxyberberine ( ) gave the products of 9-/13-O-position derivatives in 40–86% yield. The
compound was reacted with n-alkyl aldehyde in an ethanol and acetic acid solvent mixture, and then
1
3
2
4
3
acidified with 2 N HCl to yield the desired 13-position derivatives 6a–e in 29%–76% yield [31].
Scheme 1. Reagents and conditions: (a) 190 ◦C, 20-30 mmHg, 30-60 min; (b) n-alkyl bromide, K2CO3,
CH3CN, reflux, 16-24 h; (c) NaBH4, K2CO3, MeOH, 0 ◦C, 2 h; (d) R-CHO, EtOH, HOAc, 85-95 ◦C, 5
h; then ether, HCl(aq), rt, 2 h; (e) MCPBA, CH2Cl2, -20 ~ -30 ◦C, 2 h; then Na2SO3, rt, 1 h; (f) n-alkyl
bromide, NaI, Et3N, MeOH, reflux, 16-24 h.
Further, the reaction mixture was refluxed for 8–24 h, and the progress of the reaction was
monitored by TLC. The mixture was then cooled to room temperature, and the solvent was removed
under reduced pressure. The crude product was chromatographed on silica gel column and eluted with
CH2Cl2/MeOH (15/1) solvent to afford the desired 9-/13-position berberine derivatives 5a
–e, 6a–e, and
7a in quantitative yields (Scheme 1). All compounds were purified by flash column chromatography
–
e
and were thoroughly characterized by 1H, 13C, 2D NMR spectroscopy, liquid chromatography-mass
spectroscopy, and high-resolution mass spectroscopy. The full signal assignment of 1H and 13C NMR
techniques including DEPT, COSY, HSQC, and HMBC analyses. All of the final compounds showed
>98% purity by HPLC analysis.
2.2. Biological Activity
2.2.1. In Vitro Anti-Proliferative Activity by MTT Assay
The cytotoxic effect of the compounds was tested by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [32] and rated by half-maximal inhibitory
concentration (IC50). IC50 is a measure of the effectiveness of a compound in inhibiting biological or

Molecules 2020, 25, 677
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biochemical function. The cytotoxic effects of the 15 9-O-, 13- or 13-O-position substituted berberine
derivatives were tested against three human cancer cell lines and one normal murine embryonic liver
cell line. The cancer cells under study were HepG2 (liver carcinoma), HT29 (colon carcinoma), and
BFTC905 (bladder carcinoma). As shown in Table 1, we found firstly, as a trend, the anti-cancer activity
of these compounds increased with the alkyl chain length, or lipophilic characteristics, of the substitutes.
Thus, the remarkable anti-cancer activity of the berberine derivatives with 9-/13-position substitution of
the dodecyl moiety can be attributed to their enhanced bioavailability and cell membrane permeability
through the increased lipophilicity [33].
Table 1. Cytotoxicity of berberine and its derivatives against three human cancer cell lines for 24 h.
IC50 (µM) b
Compound
clogP a
HepG2 c
HT29 e
BFTC905 d
Berberine (1)
Berberrubine (2)
–0.77
–0.71
1.20
2.26
3.32
4.37
6.49
1.31
2.37
3.81
4.87
5.54
1.37
2.43
3.49
4.54
5.60
–1.68
> 40
> 40
26.11 ± 6.07
> 40
> 40
36.93 ± 9.60
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8.27 ± 1.69
2.83 ± 0.84
0.59 ± 0.21
0.61 ± 0.18
0.32 ± 0.08
8.11 ± 0.15
2.19 ± 0.42
1.19 ± 0.32
0.67 ± 0.19
0.77 ± 0.18
8.21 ± 1.47
2.64 ± 0.31
0.98 ± 0.26
0.91 ± 0.40
0.83 ± 0.30
82.12 ± 2.31
6.40 ± 2.20
1.74 ± 0.22
1.18 ± 0.31
0.89 ± 0.11
0.76 ± 0.02
> 20
9.76 ± 1.02
1.08 ± 0.23
0.75 ± 0.19
0.87 ± 0.15
9.94 ± 1.70
2.09 ± 0.47
1.00 ± 0.24
0.96 ± 0.29
0.78 ± 0.13
42.58 ± 1.16
b
> 20
14.70 ± 4.41
3.88 ± 1.06
2.79 ± 0.44
1.34 ± 0.32
> 20
8.45 ± 0.16
2.77 ± 0.03
1.76 ± 0.64
1.39 ± 0.08
> 20
5.69 ± 0.66
2.21 ± 0.21
1.39 ± 0.19
1.23 ± 0.06
53.28 ± 2.64
Cisplatin
a
Calculated log value of partition coefficient by ChemDraw Ultra 11.0. Compound concentration (µM) required
to inhibit tumor cell proliferation rate by 50%. Data are expressed as the mean
of 3–5 independent experiments. Human hepatocarcinoma cell lines. Human bladder cancer cell lines. Human
colon adenocarcinoma cell lines.
±
SD from the dose-response curves
c
d
e
As was shown in Figure 2, the cytotoxicity effect of three bioactive derivatives; compounds 5e
(9-O-dodecylberberine), 6e (13-dodecylberberine), and 7e (13-O-decylberberine). In essence, all showed
significant cell growth inhibition with the IC50 values of 0.32
respectively, against human liver carcinoma HepG2 cells. Compared to berberine (
>40 M, these three derivatives evinced 48- to 125-fold stronger cytotoxicity potency. In other words,
±
0.08, 0.77
±
0.18 and 0.83
±
0.30 µM,
1)’s IC50 value of
µ
through a simple structure-activity relationship (SAR) analysis, we have shown that the cell growth
inhibitory potency closely related to the chain length of n-alkyl groups. Meanwhile, to check potential
side effects, we treated normal Madin–Darby Canine Kidney (MDCK) cell lines with berberine (
compounds 5e, yet detected no significant cell death (data not shown). Furthermore, only a marked
effect on cell death (under 20%) was observed at the maximum concentration (4 M) of 5e and 6e after
48 h treatment, except for berberine ( ), which exhibited slight toxic. It appeared that compounds
5e 6e and 7e are cytotoxic to human colon cancer cells with no significant adverse effects on normal
MDCK cells.
1) and
µ
1
,

Molecules 2020, 25, 677
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Figure 2. In vitro cytotoxicity of berberine (1), 5e, 6e, and 7e in (A) HepG2, (B) BFTC905, and (C) HT29
cells after incubation with different compounds at various concentrations for 24 h. The error bars
denote the standard derivation of three independent experiments.
To confirm the findings above, we calculated partition coefficient (clogP) values of 9-/13-substituted
berberine derivatives and compared it with the cytotoxic activity. As shown in Table 1, clogP values
correlated well with tested anti-cancer activity for 9-/13-substituted berberine derivatives. These
results just once again verified that the lipophilicity or the chain length of the n-alkyl moieties of
9-/13-substituted berberine derivatives played a very important role in their anti-cancer activity.
Further, the positive clogP values of berberine derivatives 5e, 6e, and 7e showed that the
introduction of the long-chain n-alkyl groups to the berberine scaffold at the 9-O-, 13-, and 13-O-position
will moderately augment the lipophilicity of berberine, while n-alkyl substituents at the 9-O-, 13-, and
13-O-positions of berberine remarkably increased the lipophilicity of berberine (Table 1). It is also worth
noting, berberine derivatives with a moderate carbon chain length (5e, 6e, and 7e) would significantly
enhance its lipophilicity and yield much more favorable transmembrane permeability. In general, the
clogP value is a good index in estimating the distribution of drugs within the cells. Thus, hydrophobic
drugs with high 1-octanol/water partition coefficients are preferentially distributed to hydrophobic
compartments such as the lipid bilayers of cells while hydrophilic drugs (low 1-octanol/water partition
coefficients) preferentially are found in aqueous compartments such as blood serum.

Molecules 2020, 25, 677
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2.2.2. Absorption and Emission Spectra of Berberine (1) and its Derivatives (5e, 6e, and 7e)
The absorption and fluorescence spectra of berberine (1) and its derivatives (5e, 6e, and 7e) in
MeOH were measured (Figure 3). The absorption spectrum of the derivative 6e, with the dodecyl group
at the 13-position of berberine, exhibited a moderate bathochromic shift (ca. 8.3 nm) than the other
compounds. Its absorptivity was obviously significantly decreased compared with that of berberine
(1) and other derivatives. Figure 3B showed the emission spectra of berberine (1) and its derivatives
(
5e 6e, and 7e) in MeOH (10 M). All gave the excitation wavelength at 420 nm with the emission
,
µ
maxima of 529, 531, 516, and 524 nm, respectively. The data suggested that compounds 6e and 7e have
higher energy of the emission spectra in MeOH, which, in turn, may enhance its photocytotoxicity in
photodynamic therapy.
Figure 3.
) Emission spectral traces of the berberine (
wavelength (λex) are 420 nm].
(
A
) Electronic absorption spectra of berberine (
1
) and its derivatives (5e
, 6e, and 7e) in MeOH.
(B
1) and its derivatives (5e,
6e, and 7e) in MeOH [excitation
2.2.3. Intracellular Uptake of Berberine (1) and Its Derivatives (5e, 6e, and 7e)
Intracellular uptake of an anti-cancer agent is to check if the medicine would be able to target
key organelles of the cell. Using time-dependent HPLC analysis, we studied the cellular uptake
of berberine (
derivatives at different time points, viz. 0.25, 1, 2, 4, and 8 h. The emission of the berberine (
its derivatives inside the cells was monitored (Figure 4). The percentage incorporation of berberine
) and its derivatives (5e 6e, and 7e) into the cells is shown in Figure 4. The data suggested that
both the berberine ( ) and its derivatives internalized into the cancer cells to the same extent with
1
) and its derivatives. First, cells were treated with 10
µM of berberine (1) and its
1
) and
(1
,
1
similar rates of internalization. A nearly 100% uptake was observed when the cells were incubated
with the compounds for 2-4 h. As shown in Figure 4, we found that the intracellular concentrations of
berberine (
the plateau for another 6 h. The uptake amounts of compounds 5e
than those of berberine ( ). This observation confirmed our calculated higher partition coefficients
(clogP) of compounds 5e 6e, and 7e. Thus, berberine derivatives (5e 6e, and 7e) with moderate carbon
1
) and its three derivatives increased rapidly in 1 h, reached a plateau at 2 h, then stayed at
,
6e, and 7e were significantly higher
1
,
,
chain length showed the most significant enhancement in lipophilicity, implying more preferable
trans-membrane permeability than berberine.

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Figure 4. Intracellular accumulation of berberine (
HepG2 cells (1 M of berberine and its derivatives (5e
106) were treated with 10
then incubated for 0 h, 0.25 h, 1 h, 2 h, 4 h, or 8 h. Intracellular concentrations of berberine (
derivatives (5e 6e, and 7e) were detected using HPLC analysis. Data are shown as the mean
1
) and its derivatives (5e
,
6e, and 7e) in HepG2 cells.
6e, and 7e) and
) and its
SD (n =
×
µ
,
1
±
,
3) of two independent experiments.
2.2.4. Photocytotoxicity of Berberine (1) and Its Derivatives (5e, 6e, and 7e)
To determine the photo-induced cytotoxicity of berberine (1) and its derivatives (5e, 6e, and 7e) in
HepG2 cells, proliferation assay was performed. Exponentially growing HepG2 cells were exposed for
10 min to visible light (420 nm, 5.9 mW/cm2) in the presence of increasing concentrations of berberine
(1) and its derivatives. Subsequently, the effect of the treatments on cell proliferation was determined
by the cell counts right after damage and after 20 h of incubation in fresh media, free of berberine
derivatives. It is notable that, as shown in Figure 5, the berberine derivatives caused a quite similar
dose-dependent reduction of cell proliferation under visible light irradiation. On the contrary, no
growth inhibition was seen by the irradiation alone (see data point at 0
µM of berberine derivatives)
and in non-irradiated cells pre-incubated with 0.5
Figure 5).
µM of berberine derivatives (see open symbols in
In light of the dark cytotoxicity findings described above, we extended our study further on
the anti-proliferation efficacy of berberine ( ), compounds 5e 6e, and 7e against HepG2 cells over
a 24 h period with data on berberine ( ) as the base case. As shown in Table 2, first of all, the dark
cytotoxicity of compounds 5e 6e, and 7e demonstrated, once again, an impressive 125-, 52-, and 48-fold,
respectively, stronger bioactivity than that of berberine ( in vitro. In addition, the growth inhibition
of HepG2 cells induced by these three samples was a concentration- and light dose-dependent manner.
1
,
1
,
1
)
Table 2. In vitro dark cytotoxicity and photocytotoxicity of berberine (
7e) on three human cancer cell lines for 24 h.
1
) and its derivatives (5e
,
6e, and
IC50 (µM) a
BFTC905 c
Compd.
HepG2 b
HT29 d
Dark
Dark
Light
Dark
Light
Light
1
>40
3.48 ± 0.69
0.28 ± 0.15
0.15 ± 0.06
0.14 ± 0.06
>40
15.20 ± 1.74
0.29 ± 0.11
0.20 ± 0.09
0.17 ± 0.05
>40
>40
5e
6e
7e
0.52 ± 0.17
1.42 ± 0.38
1.66 ± 0.48
2.74 ± 0.66
2.58 ± 0.87
2.64 ± 0.33
3.42 ± 0.79
3.04 ± 0.32
2.64 ± 0.44
0.78 ± 0.25
0.28 ± 0.02
0.23 ± 0.02
a
Compound concentration (
mean
Human bladder cancer cell lines. Human colon adenocarcinoma cell lines.
µ
M) required to inhibit tumor cell proliferation rate by 50%. Data are expressed as the
b
±
SD from the dose-response curves of at 3–5 independent experiments. Human hepatocarcinoma cell lines.
c
d

Molecules 2020, 25, 677
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Figure 5. In vitro photocytotoxicity of HepG2 cells incubated with (A) berberine (1), (B) 5e, (C) 6e,
and ( 7e at various concentration for 4 h and then illuminated under a visible light (420 nm)
D)
for 5, 10, 20 min, or no light (dark series). The error bars denote the standard derivation of three
independent experiments.
The cytotoxicity of berberine (
with HepG2 cells in the dark as well as after exposure to visible light (420 nm) for 10 min. Further, the
photo-induced cytotoxic effects of berberine ( ) and its derivatives on three human cancer cell lines
1) and its derivatives (5e, 6e, and 7e) evaluated after incubation
1
was assessed using a light dose of 3.6 J/cm2 at 420 nm wavelength, respectively. The resulting cells
were irradiated with visible light (420 nm) for 10 min irradiation. After photo-irradiation, the cell
viability was measured by MTT assay, of which these results are shown in Figure 5 along with the IC50
values in Table 2. The control experiment without light irradiation showed slight cytotoxicity (85% ~
95% of the cell viability). The dark cytotoxicity of these berberine derivatives was evaluated in HepG2
cells at the concentration of 0.5
6e and 7e showed no significant cytotoxicity in the dark (Figure 5), except over 1
Figure 5 showed the cell survival ratio as a function of the concentrations of berberine derivatives. The
µM without photo-irradiation. We found that the berberine derivatives
µM. Furthermore,
IC50 value of 6e was 0.15
of 5e (0.28 µM).
µM, which was almost the same as that of 7e (0.14 µM) and lower than that
2.2.5. Cell Morphological Assessment
The apoptotic cells change in morphology, such as chromatin condensation, nuclear shrinking,
and DNA fragmentation. It can be characterized through Hoechst 33,258 stained cell nucleus [34
under a fluorescent microscope. Thus, to further study the role of apoptosis played in the superior
cytotoxicity of compounds 5e 6e, and 7e, we incubated HepG2 cells in 0.5 M of berberine ( ), or
its three derivatives separately. As shown in Figure 6A, while the nuclei of the cells were round in
shape homogenously in the control without the testing compound, those treated with berberine ( ),
compound 5e 6e, or 7e showed typical morphological features of apoptosis such as cell blebbing and
nuclear shrinkage. Figure 6B shows that HepG2 cells treated with 0.5 M of compound 7e stained with
]
,
µ
1
1
,
µ
Hoechst 33,258 in dark or light, and examined under fluorescence microscopy for topical morphological
changes, such as chromatin condensation and DNA fragmentation [34]. Evidently, the proliferation of

Molecules 2020, 25, 677
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the cancer cells was inhibited by the testing compounds in vitro. These results showed that compounds
5e, 6e, or 7e induce apoptosis in HepG2 cells through irradiation.
Figure 6. Morphological changes in HepG2 cells treated with berberine (
at the dark or light (420 nm, 10 min) for 4 h (magnification, 200 ). ( ) Upper panels show the cell
morphology under phase-contrast microscopy, red arrows indicate the apoptotic cells. ( ) HepG2 cells
were treated with 7e (0.5 M) for 4 h by irradiation and stained with Hoechst 33258 staining nuclear
1), 5e, 6e, or 7e at 0.5 µM
×
A
B
µ
patterns detected through fluorescence microscopy (magnification, 200
the apoptotic cells by irradiation.
×). The yellow arrows indicate
2.2.6. Cell Cycle Distribution Analysis Using Flow Cytometry
To probe the apoptotic effects of berberine (
1) and its derivatives on cell cycle progression of
HepG2 cells were treated once again with these compounds, separately, at 0.5
µM concentrations for 2
h after irradiation (420 nm, 10 min) for 24 h. Then, the samples were further treated with propidium
iodide (PI) staining [35], and of which the sub-G1 phase was analyzed by flow cytometry. Besides, the
cells with no test compound were the control of the experiment. Berberine (1) and its derivatives (5e,
6e, and 7e) showed significant photocytotoxic property in visible light at 420 nm wavelength. To find
out whether the said property plays a role in the apoptosis of the cells, flow cytometric analysis of
the HepG2 cells treated with berberine (1) and its derivatives (5e, 6e, and 7e) was carried out at 0.5
µM concentrations. Cells initially treated with the compounds in the dark and tested before and after
being exposed to visible light (420 nm) and then stained with propidium iodide (PI) to observe any
fragmented DNA or an increase in the sub-G1 population. As illustrated in Figure 7, the percentage of
S phase increased by 32.71% and 37.58% in the dark and light for the control, respectively, in the sample
with 5e increased to 41.14%, 49.71% for 6e, and 44.36% for 7e compared to the 37.58% for the control in
the light. The increased S phase percentage and the sub-G1 population in compounds 5e
treatment suggested that 5e 6e, and 7e induced S phase arrest and caused apoptosis in HepG2 liver
cancer cells. At first, berberine ( ) and its derivatives (5e 6e and 7e) being non-cytotoxic in the dark at
0.5 M concentration, but it induced significant apoptosis in the cells at a concentration of 0.5 M upon
visible light irradiation for 24 h. In addition, the percentage of apoptotic cell populations increased
, 6e, and 7e
,
1
,
µ
µ

Molecules 2020, 25, 677
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with increasing compounds concentration (Figure 7). Lastly, a significant induction of apoptosis in the
cells occurred even in the dark with an increasing concentration of derivatives.
Figure 7. Effect of berberine (
visible light exposure. ( ) Cell cycle distribution after treatment with berberine (
M concentration for 2 h after irradiation (420 nm, 10 min) in HepG2 cells for 24 h. (
difference of cell cycle distribution changed after treatment with berberine ( ), 5e 6e, and 7e at 0.5
SD of three independent experiments
1), 5e
,
6e, or 7e on HepG2 cell cycle distribution using without or with
), 5e 6e, or 7e at 0.5
) The quantitative
A
1
,
µ
B
1
,
µM
concentration in HepG2 for 24 h. Data are shown as the mean
and *** p < 0.001 compared with the dark group.
±
2.2.7. Measurement of Intracellular ROS Production by Irradiation
Several alkaloids induce apoptosis by generating singlet oxygen (1O2) in mitochondria were
reported [36]. Using dichlorofluorescein diacetate (DCFH-DA) assay, we checked if berberine (
compounds 5e 6e, or 7e could stimulate the generation of reactive oxygen species (ROS) after irradiation
at visible light (420 nm) in HepG2 cells. We found that the fluorescence intensity of DCF in the cells
was right-shifted after the cells were treated with all four compounds in a 0.5 M concentration upon
irradiation. Shown in Figure 8A, berberine ( ), compounds 5e 6e, or 7e all stimulated to release
intracellular 1O2 from HepG2 cells and exhibited a more profound effect than on the 1O2 generation
in HepG2 cells after 24 h of treatment (P < 0.05, Figure 8B). These data showed that berberine ( ),
compounds 6e and 7e induced apoptosis by increasing the intracellular ROS generation of HepG2 cells
1),
,
µ
1
,
1

Molecules 2020, 25, 677
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Figure 8. ROS generation in HepG2 cells treated with berberine (
The effects of HepG2 cells treated with berberine ( ), 5e 6e, and 7e at 0.5
after irradiation (420 nm, 10 min) for 24 h on percentage distribution of ROS generation by DCFH-DA
staining. ( ) Average intensity of DCFH-DA fluorescence in HepG2 cells treated with berberine ( ) and
its derivatives 5e SD. *** p <
1), 5e
,
6e, and 7e after irradiation. (
A)
1
,
µM concentration for 2 h
B
1
,
6e, and 7e at 0.5
µ
M concentration. The results are presented as mean
±
0.001 compared with control, ## p < 0.01 and ### p < 0.001 compared with the dark group.
2.2.8. Measurements of Mitochondrial Membrane Potential Changes
The depletion of mitochondrial membrane potential (MMP) is an early marker of the apoptotic
process. To determine whether an early loss of MMP occurred upon the treatment of berberine (
and its derivatives in HepG2 cells, we performed MMP measurement with rhodamine 123 staining.
Delineated in Figure 9, the MMP decreased remarkably at the concentrations of 0.5 M of berberine ( ),
0.25 M of compound 5e 6e, or 7e. The percentage of cells with membrane potential loss increased from
20.53% to 32.90% at 24 h. Thus berberine ( ), compound 5e 6e, or 7e severely reduced the mitochondrial
1
)
µ
1
µ
,
1
,
membrane potential of HepG2 cells. It was documented that P53 could also down-regulate Bcl-2
and up-regulate Bax, which are related to mitochondria-mediated apoptosis [37]. Further, several
studies have also reported that apoptosis involves a disruption of the mitochondrial membrane
integrity, a critical cell death process. Moreover, the depolarization of the mitochondrial membrane
potential is a characteristic feature of apoptosis [38,39]. The report also indicated that the reduction
of the membrane potential, the release of mitochondrial cytochrome c, a critical step, is followed
by the formation of apoptosomes [39]. Thus, we found the loss of MMP upon the treatments of
berberine (
finding of the loss of mitochondrial potential induced by berberine (
concentration of 0.25 M. These observations suggested that berberine derivatives 6e and 7e induced
mitochondrial-dependent apoptosis in HepG2 by irradiation.
1
), compound 5e
,
6e, or 7e were consistent with the published results. Thus, does the
1
), compounds 5e 6e, and 7e at a
,
µ

Molecules 2020, 25, 677
12 of 22
Figure 9. Measurement of the mitochondrial membrane potential. (
A
) HepG2 cells (1
×
105 cells/mL)
were incubated with 0.5 M concentration of berberine ( ) and its derivative 5e
µ
1
,
6e and 7e at 0.25 µM
concentration for 2 h upon visible light irradiation (420 nm, 10 min) for 24 h and then stained with 2
mg/mL of Rh123 for 20 min under gentle shaking. After washing with PBS, cells were immediately
analyzed by flow cytometry and data were analyzed using the FCS Express software. (
intensity of RH123 fluorescence in HepG2 cells treated with 0.5 M of berberine ( ) and its derivatives
SD. *** p < 0.001 compared
B) Average
µ
1
5e
,
6e and 7e at 0.25
µ
M concentration. The results are presented as mean
±
with control, ### p < 0.001 compared with dark group.
3. Materials and Methods
3.1. General Information
All chemical reagents and solvents in commercial quality were purchased from Sigma-Aldrich
(St. Louis, MO, USA), Fluka (Roma, Italy), TCI (Tokyo, Japan), and Alpha Aesar without further
purification. All chemical reagents in commercial quality were used as received (Sigma-Aldrich, St.
Louis, MO, USA) and were used without further purification. Solvents were dried and the synthesized
compounds were purified using standard techniques. Reactions-progression was monitored by TLC
on aluminum plates coated with silica gel with a fluorescent indicator (Merck 60 F254). Melting points
of the synthesized compounds were determined by Fargo MP-2D apparatus and were uncorrected.
The electronic absorption spectra were measured on a Shimadzu UV-1601 UV-vis spectrophotometer.
Fluorescence spectra were taken on a Shimadzu RP-5301 spectrofluorometer. NMR spectra were
recorded using TMS as an internal standard in CDCl3 at 500 MHz for 1H and at 125 MHz for 13
(Bruker Biospin GmbH AVANCE III 500 MHz, Rheinstetten, Germany) (see Supplementary Materials).
Chemical shift ( ) were reported in parts per million (ppm) measured relative to the internal standards
(TMS), and the coupling constant (J) were expressed in Hertz (Hz). Column chromatography was
performed with silica gel SiliaFlash® G60 (60–200
m) purchased from SiliCycle Inc. (Quebec City, QC,
Canada). In general, the reactions were carried out under anhydrous conditions in dry solvent and
C
δ
µ

Molecules 2020, 25, 677
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nitrogen atmosphere. The purity of these compounds was based on the analysis of HPLC (Hitachi
High-Technologies, Tokyo, Japan) equipped with a 280 nm detector and LiChroCART RP-C18 column
(4.6 mm i.d.
×
250 mm, 5 µm, Merck, Darmstadt, Germany). The mobile phase was composed of
MeOH-H2O (0.05% TFA) (90:10) and the flow rate was 1.0 mL/min. The purity of all compounds
was more than 98%. The mass spectra were acquired using a Thermo Finnigan model LXQ (Thermo
Electron Co., Waltham, MA, USA) ion trap mass spectrometer equipped with ESI source interference
and controlled by Xcalibur 2.06. The mass spectra were acquired in a positive ion mode or a negative
ion mode. Berberrubine (2), derivatives 3, 4, 5a, 5c, and 5e were synthesized according to previous
methods [27].
3.2. Synthesis of 9-O-Sunstituted Berberine Derivatives (5a–e)
To a solution of berberrubine (1 mmol) in dry acetonitrile (10 mL) was added various n-alkyl
2
bromide (1.2 mmol) and the reaction mixture was refluxed for 4–8 h. The progress of the reaction was
monitored by TLC. Then, the reaction mixture was cooled to room temperature and the solvent was
removed under reduced pressure. The crude product was chromatographed on a silica gel column
and eluted with ethyl acetate/methanol (2/1) solvent to afford the desired berberine derivatives 5a–e.
9-O-Butylberberine bromide (5a). Berberrubine (
the general procedure to give the desired bromide salt (5a) as a yellowish brown solid, yield: 86%; UV
(MeOH) (log
): 431 (3.77), 350 (4.41), 267 (4.43), 231 (4.45), 205 (4.41) nm; 1H NMR (500 MHz,
9.75 (s, 1H, H-8), 8.94 (s, 1H, H-13), 8.20 (d, J = 9.1 Hz, H-12), 7.99 (d, J = 9.1 Hz, H-11),
2) was treated with n-butyl bromide according to
λ
max
ε
DMSO-d6):
δ
7.80 (s, 1H, H-1), 7.09 (s, 1H, H-4), 6.17 (s, 2H, -OCH2O-), 4.95 (t, J = 6.3 Hz, 2H, H-6), 4.29 (t, J = 6.8
Hz, 2H, H-1’), 4.05 (s, 3H, -OCH3), 3.21 (t, J = 6.3 Hz, 2H, H-5), 1.86 (p, J = 7.2 Hz, 2H, H-2’), 1.52 (sex,
J = 7.4 Hz, 2H, H-3’), 0.99 (t, J = 7.4 Hz, 3H, H-4’); 13C NMR (125 MHz, DMSO-d6):
δ 150.4, 149.8,
147.7, 145.3, 142.9, 137.5, 133.0, 130.7, 126.7, 123.3, 121.7, 120.5, 120.2, 108.4, 105.4, 102.1, 73.9, 57.0, 55.3,
31.5, 26.3, 18.6, 13.8; LC-MS (ESI+, m/z) C23H24NO4+: 378.33 [M–Br]+; HRMS-ESI: m/z calculated for
C23H22NO4+: 378.1705 [M − Br]+, found for 378.1708.
9-O-Hexylberberine bromide (5b). Berberrubine (2) was treated with n-hexyl bromide according to
the general procedure to give the desired bromide salt (5b) as a yellowish brown solid, yield: 69%;
UV (MeOH) (log
): 431.6 (3.92), 350.2 (4.54), 266.5 (4.60), 229.1 (4.67) nm; 1H NMR (500 MHz,
9.75 (s, 1H, H-8), 8.94 (s, 1H, H-13), 8.20 (d, J = 9.1 Hz, H-12), 7.99 (d, J = 9.1 Hz, H-11),
λ
max
ε
DMSO-d6):
δ
7.80 (s, 1H, H-1), 7.09 (s, 1H, H-4), 6.17 (s, 2H, -OCH2O-), 4.95 (t, J = 6.3 Hz, 2H, H-6), 4.28 (t, J = 6.8
Hz, 2H, H-1’), 4.05 (s, 3H, -OCH3), 3.21 (t, J = 6.3 Hz, 2H, H-5), 1.87 (p, J = 7.2 Hz, 2H, H-2’), 1.52 (p, J
= 7.4 Hz, 2H, H-3’),1.37-1.33 (m, 4H, H-4’, H-5’), 0.90 (t, J = 7.0 Hz, 3H, H-6’); 13C-NMR (125 MHz,
DMSO-d6):
δ 150.5, 149.8, 147.7, 145.3, 142.9, 137.5, 133.1, 130.7, 126.7, 123.4, 121.7, 120.5, 120.3, 108.5,
105.5, 102.1, 74.3, 57.1, 55.3, 31.1, 29.5, 26.4, 25.0, 22.1, 14.0; LC-MS (ESI+, m/z) C25H26NO4+: 406.33
[M–Br]+; HRMS-ESI: m/z calculated for C25H26NO4+: 406.2018 [M − Br]+, found for 406.2021.
9-O-Octylberberine bromide (5c). Berberrubine (2) was treated with n-octyl bromide according to
the general procedure to give the desired bromide salt (5c) as a bright yellow solid, yield: 74%; UV
(MeOH) (log
): 432 (3.79), 350 (4.42), 268 (4.44), 230 (4.47), 204 (4.48) nm; 1H NMR (500 MHz,
9.75 (s, 1H, H-8), 8.95 (s, 1H, H-13), 8.19 (d, J = 9.1 Hz, H-12), 7.99 (d, J = 9.1 Hz, H-11),
λ
max
ε
DMSO-d6):
δ
7.80 (s, 1H, H-1), 7.09 (s, 1H, H-4), 6.17 (s, 2H, -OCH2O-), 4.95 (t, J = 6.2 Hz, 2H, H-6), 4.28 (t, J = 6.8 Hz,
2H, H-1’), 4.06 (s, 3H, -OCH3), 3.21 (t, J = 6.2 Hz, 2H, H-5), 1.87 (p, J = 7.2 Hz, 2H, H-2’), 1.48 (p, J = 7.3
Hz, 2H, H-3’), 1.33 (m, 8H, H-4’–H-7’), 0.88 (t, J = 6.8 Hz, 3H, H-8’); 13C NMR (125 MHz, DMSO-d6):
δ
150.4, 149.8, 147.7, 145.3, 142.9, 137.5, 133.0, 126.7, 123.3, 121.7, 120.5, 120.2, 108.4, 105.4, 102.1, 74.2,
57.0, 55.3, 31.2, 29.5, 28.8, 28.7, 26.3, 25.3, 22.1, 14.0; LC-MS (ESI+, m/z) C27H32NO4+: 434.41 [M–Br]+;
HRMS-ESI: m/z calculated for C27H30NO4+: 434.2331 [M − Br]+, found for 434.2332.
9-O-Decylberberine bromide (5d). Berberrubine (2) was treated with n-decyl bromide according
to the general procedure to give the desired bromide salt (5d) as a bright yellow solid, yield: 71%;
UV (MeOH) max (log
): 431.0 (3.68), 350.2 (4.31), 266.4 (4.35), 229.8 (4.38) nm; 1H-NMR (500 MHz,
DMSO-d6): 9.75 (s, 1H, H-8), 8.95 (s, 1H, H-13), 8.19 (d, J = 9.1 Hz, H-12), 7.99 (d, J = 9.1 Hz, H-11),
λ
ε
δ

Molecules 2020, 25, 677
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7.80 (s, 1H, H-1), 7.09 (s, 1H, H-4), 6.17 (s, 2H, -OCH2O-), 4.95 (t, J = 6.2 Hz, 2H, H-6), 4.27 (t, J = 6.8 Hz,
2H, H-1’), 4.05 (s, 3H, -OCH3), 3.21 (t, J = 6.2 Hz, 2H, H-5), 1.87 (p, J = 7.2 Hz, 2H, H-2’), 1.48 (p, J =
7.3 Hz, 2H, H-3’), 1.40-1.26 (m, 12H, H-4’–H-9’), 0.85 (t, J = 6.8 Hz, 3H, H-10’); 13C-NMR (125 MHz,
DMSO-d6):
δ 150.4, 149.8, 147.7, 145.3, 142.9, 137.5, 133.0, 130.7, 126.7, 123.3, 121.7, 120.5, 120.2, 108.4,
105.4, 102.1, 74.2, 57.0, 55.3, 31.3, 29.5, 29.0, 28.9, 28.8, 28.7, 26.3, 25.3, 22.1, 14.0; LC-MS (ESI+, m/z)
C29H34NO4+: 462.43 [M–Br]+; HRMS-ESI: m/z calculated for C29H34NO4+: 462.2644 [M
for 462.2646.
−
Br]+, found
9-O-Dodecylberberine bromide (5e). Berberrubine (2) was treated with n-dodecyl bromide according
to the general procedure to give the desired bromide salt (5e) as a dark yellowish brown solid, yield:
61%; UV (MeOH)
MHz, DMSO-d6):
λ
max
(log
ε
): 431 (3.42), 351 (4.03), 267 (4.07), 230 (4.10), 202 (4.19) nm; 1H NMR (500
δ 9.77 (s, 1H, H-8), 8.98 (s, 1H, H-13), 8.21 (d, J = 9.1 Hz, H-12), 8.00 (d, J = 9.1 Hz,
H-11), 7.82 (s, 1H, H-1), 7.10 (s, 1H, H-4), 6.18 (s, 2H, -OCH2O-), 4.96 (t, J = 6.1 Hz, 2H, H-6), 4.28 (t, J =
6.8 Hz, 2H, H-1’), 4.05 (s, 3H, -OCH3), 3.21 (t, J = 6.1 Hz, 2H, H-5), 1.88 (d, J = 7.4 Hz, H-2’), 1.47 (q, J
= 7.5 Hz, 2H, H-3’), 1.29 (m, 16 H, H-4’–H-11’), 0.86 (t, J = 6.8 Hz, 3H, H-12’); 13C NMR (125 MHz,
DMSO-d6):
δ 150.4, 149.8, 147.7, 145.3, 142.9, 137.5, 133.0, 130.7, 126.7, 123.3, 121.7, 120.5, 120.3, 108.4,
105.5, 102.1, 74.2, 57.0, 55.3, 48.6, 31.3, 29.5, 29.08, 29.05, 28.9, 28.7, 26.3, 25.3, 22.1, 14.0; LC-MS (ESI+,
m/z) C31H38NO4+: 490.50 [M
found for 490.2958.
−
Br]+; HRMS-ESI: m/z calculated for C31H38NO4+: 490.2957 [M
−
Br]+,
3.3. Synthesis of 13-Sunstituted Berberine Derivatives (6a–e)
To a stirred solution of berberine ( ) (3.71 g, 10 mmol) and K2CO3 (3.6 g, 30 mmol) in methanol
1
(125 mL), 5% NaOH (5 mL) solution containing NaBH4 (0.30 g, 7.5 mmol) was added dropwise. The
reaction mixture was stirred at room temperature for 1 h and the precipitated product was filtered,
washed with 30% ethanol (20 mL) and 80% ethanol (20 mL), and then recrystallized from absolute
ethanol to provide dihydroberberine (
3
) (2.5 g, 74%) as a brown like solid, yield: 74%; UV (MeOH)
λ
max
(log δ
ε
): 353.9 (3.96), 268 (4.01), 228.6 (4.09) nm;1H-NMR (500 MHz, DMSO-d6)
7.29 (s, 1H, H-13),
6.82 (d, J = 8.4 Hz, H-12), 6.76 (s, 1H, H-1), 6.69 (d, J = 8.4 Hz, 1H, H-11), 6.05 (s,1H, H-1), 6.00 (s, 2H,
-OCH2-), 4.20 (s, 2H, H-8), 3.76 (s, 3H, -OCH3), 3.71 (s, 3H, -OCH3), 3.05 (t, J = 5.8 Hz, 2H, H-6), 2.79 (t,
J = 5.8 Hz, 2H, H-5); 13C-NMR (125 MHz, DMSO-d6)
δ
149.9, 146.9, 146.4, 143.9, 141.0, 128.6, 128.3,
123.9, 121.6, 118.4, 111.7, 107.9, 103.5, 101.0, 96.0, 60.2, 55.7, 48.8, 48.3, 28.9.
To a stirred solution of dihydroberberine ( ) (170 mg, 5.0 mmol) in 80% ethanol (8 mL) and HOAc
3
(2 mL), n-alkyl aldehyde (2 mL) was added. The reaction mixture was heated to 85–95 ◦C for 5 h. The
solvent was removed by evaporation, and the residue was acidified by 2 N HCl (5 mL), and then
stirred at room temperature for 1 h. The solid was collected by filtration and then purified by flash
chromatography over silica gel, affording the title compounds 6a–e.
13-Butylberberine bromide (6a). Dihydroberberine (3) was treated with butanal according to the
general procedure to give the desired chloride salt (6a) as a yellowish brown solid, yield: 68%; UV
1
(MeOH)
λ
max
(log
ε
): 420.3 (3.93), 341.9 (4.49), 265.1 (4.61), 231.9 (4.58) nm; H-NMR (500 MHz,
DMSO-d6):
δ
9.88 (s, 1H, H-8), 8.21 (d, J = 9.5 Hz, 1H, H-12), 8.18 (d, J = 9.5 Hz, 1H, H-11), 7.28 (s, 1H,
H-1), 7.16 (s, 1H, H-4), 6.18 (s, 2H, -OCH2O-), 4.78 (t, J = 8.0 Hz, 2H, H-6), 4.09 (s, 3H, -OCH3), 4.08 (s,
3H, -OCH3), 3.33 (t, J = 8.0 Hz, 2H, H-5), 3.07 (t, J = 6.2 Hz, 2H, H-1’), 1.75 (p, J = 6.6 Hz, 2H, H-2’), 1.41
(hex, J = 7.2 Hz, 2H, H-3’), 0.91 (t, J = 6.9 Hz, 3H, H-4’); 13C-NMR (125 MHz, DMSO-d6):
δ 150.3, 149.1,
146.6, 144.4, 144.3, 135.8, 134.2, 134.1, 132.3, 125.9, 121.5, 121.3, 109.2, 108.4, 102.2, 62.1, 57.1, 57.0, 32.6,
28.8, 27.4, 22.0, 13.5; LC-MS (ESI+, m/z) C24H26NO4+: 392.28 [M − Cl]+; HRMS-ESI: m/z calculated for
C24H26NO4+: 392.1862 [M − Cl]+, found for 392.1861.
13-Hexylberberine bromide (6b). Dihydroberberine (3) was treated with hexanal according to the
general procedure to give the desired chloride salt (6b) as a bright yellow solid, yield: 76%; UV (MeOH)
λ
max
(log
ε
): 420.3 (3.73), 341.9 (4.29), 265.4 (4.40), 232 (4.37) nm; 1H-NMR (500 MHz, DMSO-d6):
δ
9.91 (s, 1H, H-8), 8.22 (d, J = 9.7 Hz, 1H, H-12), 8.20 (d, J = 9.7 Hz, 1H, H-11), 7.30 (s, 1H, H-1), 7.18 (s,
1H, H-4), 6.20 (s, 2H, -OCH2O-), 4.80 (s, 2H, H-6), 4.10 (s, 3H, -OCH3), 4.10 (s, 3H, -OCH3), 3.33 (s,2H,

Molecules 2020, 25, 677
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H-5), 3.09 (t, J = 5.7 Hz, 2H, H-1’), 1.76 (s, 2H, H-2’), 1.40 (m, 2H, H-3’), 1.27-1.26 (m, 4H, H-4’, H-5’),
0.86 (t, J = 6.9 Hz, 3H, H-6’); 13C-NMR (125 MHz, DMSO-d6):
150.2, 149.1, 146.6, 144.4, 144.2, 135.8,
δ
134.2, 134.1, 132.2, 125.9, 121.5, 121.3, 120.3, 109.1, 108.4, 102.2, 62.0, 57.0, 30.7, 30.4, 28.9, 28.3, 27.4, 22.1,
+
13.9; LC-MS (ESI+, m/z) C26H30NO4+: 420.26 [M
−
Cl]+; HRMS-ESI: m/z calculated for C26H30NO4
:
420.2175 [M-Cl]+, found for 420.2176.
13-Octylberberine chloride (6c). Dihydroberberine (
general procedure to give the desired chloride salt (6c) as a bright yellow solid, yield: 65%; UV (MeOH)
(log
): 420.1 (3.80), 341.4 (4.35), 265.3 (4.47), 232 (4.44) nm; 1H-NMR (500 MHz, DMSO-d6):
3) was treated with octanal according to the
λ
max
ε
δ
9.91 (s, 1H, H-8), 8.22 (d, J = 9.7 Hz, 1H, H-12), 8.20 (d, J = 9.7 Hz, 1H, H-11), 7.30 (s, 1H, H-1), 7.17
(s, 1H, H-4), 6.20 (s, 2H, -OCH2O-), 4.80 (s, 2H, H-6), 4.10 (s, 3H, -OCH3), 4.10 (s, 3H, -OCH3), 3.33
(s,2H, H-5), 3.09 (t, J = 5.7 Hz, 2H, H-1’), 1.76 (s, 2H, H-2’), 1.38-1.37 (m, 2H, H-3’), 1.28-1.23 (m, 8H,
H-4’-H-7’), 0.86 (t, J = 6.9 Hz, 3H, H-8’); 13C NMR (125 MHz, DMSO-d6):
δ 150.2, 149.0, 146.6, 144.3,
144.2, 135.8, 134.2, 134.0, 132.2, 125.9, 121.4, 121.2, 120.3, 109.1, 108.3, 102.1, 62.0, 57.0, 31.2, 30.4, 28.8,
28.6, 28.4, 27.4, 22.0, 13.9; LC-MS (ESI+, m/z) C28H34NO4+: 448.28 [M–Cl]+; HRMS-ESI: m/z calculated
for C28H34NO4+: 448.2488 [M − Cl]+, found for 448.2487.
13-Decylberberine chloride (6d). Dihydroberberine (3) was treated with decanal according to the
general procedure to give the desired chloride salt (6d) as a bright yellow solid, yield: 43%; UV (MeOH)
λmax (log
ε
): 420.3 (3.73), 341.9 (4.29), 265.3 (4.40), 231.7 (4.39) nm; 1H-NMR (500 MHz, DMSO-d6):
δ
9.90 (s, 1H, H-8), 8.22 (d, J = 9.7 Hz, 1H, H-12), 8.20 (d, J = 9.7 Hz, 1H, H-11), 7.29 (s, 1H, H-1), 7.16
(s, 1H, H-4), 6.19 (s, 2H, -OCH2O-), 4.79 (s, 2H, H-6), 4.09 (s, 3H, -OCH3), 4.09 (s, 3H, -OCH3), 3.33 (s,
2H, H-5), 3.08 (t, J = 5.7 Hz, 2H, H-1’), 1.74 (s, 2H, H-2’), 1.36-1.35 (m, 2H, H-3’), 1.27-1.22 (m, 12H,
H-4’-H-9’), 0.86 (t, J = 6.9 Hz, 3H, H-10’); 13C-NMR (125 MHz, DMSO-d6):
δ 150.2, 149.0, 146.6, 144.3,
144.2, 135.8, 134.2, 134.0, 132.2, 125.9, 121.4, 121.2, 120.3, 109.1, 108.3, 102.1, 62.0, 57.0, 31.3, 30.4, 28.9,
28.8, 28.6, 28.8, 28.4, 27.4, 22.1, 13.9; LC-MS (ESI+, m/z) C30H38NO4+: 476.40 [M–Cl]+; HRMS-ESI: m/z
calculated for C30H38NO4+: 476.2801 [M − Cl]+, found for 476.2801.
13-Dodecylberberine chloride (6e). Dihydroberberine (3) was treated with dodecanal according
to the general procedure to give the desired chloride salt (6e) as a bright yellow solid, yield: 29%;
UV (MeOH) max (log
): 420.6 (3.71), 341.8 (4.26), 265.3 (4.38), 232.0 (4.37) nm; 1H-NMR (500 MHz,
DMSO-d6): 9.90 (s, 1H, H-8), 8.21 (d, J = 9.6 Hz, 1H, H-12), 8.19 (d, J = 9.6 Hz, 1H, H-11), 7.29 (s, 1H,
λ
ε
δ
H-1), 7.16 (s, 1H, H-4), 6.18 (s, 2H, -OCH2O-), 4.79 (s, 2H, H-6), 4.09 (s, 3H, -OCH3), 4.09 (s, 3H, -OCH3),
3.33 (s,2H, H-5), 3.08 (t, J = 5.7 Hz, 2H, H-1’), 1.74 (s, 2H, H-2’), 1.36-1.35 (m, 2H, H-3’), 1.28-1.22 (m,
16H, H-4’-H-11’), 0.85 (t, J = 6.9 Hz, 3H, H-12’); 13C-NMR (125 MHz, DMSO-d6):
δ 150.2, 149.0, 146.5,
144.3, 144.2, 135.8, 134.2, 134.0, 132.2, 125.9, 121.4, 121.2, 120.3, 109.1, 108.3, 102.1, 62.0, 57.0, 31.3, 30.4,
30.0, 28.9, 28.9, 28.8, 28.7, 28.6, 28.4, 27.4, 22.1, 13.9; LC-MS (ESI+, m/z) C32H42NO4+: 504.43 [M
HRMS-ESI: m/z calculated for C32H42NO4+: 504.3114 [M − Cl]+, found for 504.3115.
−
Cl]+;
3.4. Synthesis of 13-O-Sunstituted Berberine Derivatives (7a–e)
Compound
3 (337 mg) was dissolved in 35 mL of CH2Cl2. Under nitrogen atmosphere, the
temperature of the system was kept at -25 ◦C ~ -30 ◦C, and 8 mL of CH2Cl2 solution containing 258 mg
MCPBA (1.5 mmol) was added slowly dropwise. After the addition, the temperature was kept and the
reaction was carried out under agitation for 1 h. Then, the temperature was raised to 0 ◦C and 250 mg
(2.0 mmol) of sodium sulfite was added therein, followed by being stirred at room temperature for
1 h. The reaction was stopped and the reaction mixture was filtered. The filtrate was evaporated to
remove the solvent under reduced pressure and the residue was purified through silica gel column
chromatography (CH2Cl2/CH3OH = 10:1) to obtain compound
4 (280 mg, 80 percent), yield: 80%;UV
(MeOH)
λ
max
(log
ε
): 444.5 (4.08), 367.9 (3.93), 305.3 (4.04), 235.0 (4.44), 212.1 (4.42) nm; 1H-NMR (500
MHz, DMSO-d6):
δ
8.89 (s, 1H, H-8), 8.08 (d, J = 9.3 Hz, 1H, H-12), 8.06 (s, 1H, H-1), 7.52 (d, J = 9.3 Hz,
1H, H-11), 6.88 (s, 1H, H-4), 6.02 (s, 2H, -OCH2O-), 4.61 (t, J = 6.2 Hz, 2H, H-6), 3.95 (s, 3H, -OCH3),
3.93 (s, 3H, -OCH3), 3.01 (t, J = 6.2 Hz, 2H, H-5); 13C-NMR (125 MHz, DMSO-d6):
165.1, 150.2, 145.3,
145.2, 142.2, 130.2, 127.4, 123.9, 123.4, 122.3, 121.4, 117.6, 117.3, 107.5, 107.2, 100.8, 61.1, 56.4, 56.3, 27.6.
δ

Molecules 2020, 25, 677
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Compound
4
(1.0 g) and potassium carbonate (200 mg) were dissolved in 20 mL of acetone
followed by adding 0.25 mL of alkyl bromide. The obtained reaction solution was heated and refluxed
for 3 h. Then, the reaction mixture was filtered and the filtrate was evaporated under reduced pressure.
The residual was purified through silica gel column (CH2Cl2/CH3OH = 20: 1) to obtain compounds
7a–e.
13-O-Butylberberine bromide (7a). Compound (
the general procedure to give the desired bromide salt (7a) as a yellowish brown solid, yield: 66%;
UV (MeOH) (log
): 427.8 (3.46), 349.7 (3.97), 265.5 (4.07), 232.6 (4.17) nm;1H-NMR (500 MHz,
9.82 (s, 1H, H-8), 8.21 (d, J = 9.3 Hz, 1H, H-12), 8.09 (d, J = 9.3 Hz, 1H, H-11), 7.90 (s, 1H,
4) was treated with n-butyl bromide according to
λ
max
ε
DMSO-d6):
δ
H-1), 7.13 (s, 1H, H-4), 6.17 (s, 2H, -OCH2O-), 4.88 (t, J = 5.8 Hz, 2H, H-6), 4.10 (s, 3H, -OCH3), 4.09 (s,
3H, -OCH3), 3.84 (t, J = 6.4 Hz, 2H, H-1’), 3.17 (t, J = 4.7 Hz, 2H,H-5), 1.80-1.74 (m, 2H, H-2’), 1.53-1.45
(m, 2H, H-3’), 0.91 (t, J = 7.4 Hz, 3H, H-4’); 13C-NMR (125 MHz, DMSO-d6):
δ 150.9, 150.0, 149.2, 146.8,
144.2, 142.0, 132.5, 130.8, 128.8, 126.1, 122.1, 118.4, 108.4, 107.9, 102.1, 75.0, 62.0, 57.1, 56.4, 31.5, 26.9,
18.6, 13.6; HRMS-ESI: m/z calculated for C24H26NO5+: 408.1811 [M − Br]+, found for 408.1812.
13-O-Hexylberberine bromide (7b). Compound (4) was treated with n-hexyl bromide according
to the general procedure to give the desired bromide salt (7b) as a bright yellow solid, yield: 76%;
UV (MeOH) max (log
): 428.0 (3.75), 350.3 (4.26), 266.5 (4.32), 229.0 (4.45) nm; 1H-NMR (500 MHz,
DMSO-d6): 9.81 (s, 1H, H-8), 8.21 (d, J = 9.4 Hz, 1H, H-12), 8.09 (d, J = 9.4 Hz, 1H, H-11), 7.90 (s, 1H,
λ
ε
δ
H-1), 7.13 (s, 1H, H-4), 6.17 (s, 2H, -OCH2O-), 4.87 (t, J = 5.9 Hz, 2H, H-6), 4.10 (s, 3H, -OCH3), 4.09 (s,
3H, -OCH3), 3.84 (t, J = 6.4 Hz, 2H, H-1’), 3.16 (t, J = 5.9 Hz, 2H, H-5), 1.78 (p, J = 7.0 Hz, 2H, H-2’),
1.45 (p, J = 7.5 Hz,2H, H-3’), 1.33-1.23 (m, 4H, H-4’, H-5’), 0.86 (t, J = 6.9 Hz, 3H, H-6’); 13C-NMR (125
MHz, DMSO-d6):
δ 150.9, 150.1, 149.2, 146.8, 144.2, 141.9, 132.5, 130.8, 128.8, 126.1, 122.1, 118.5, 118.4,
+
108.3, 108.0, 102.1, 75.4, 62.0, 57.1, 30.9, 29.4, 25.1, 22.0, 13.9; HRMS-ESI: m/z calculated for C26H30NO5
:
436.2124 [M − Br]+, found for 436.2122.
13-O-Octylberberine chloride (7c). Compound (4) was treated with n-octyl bromide according to
the general procedure to give the desired bromide salt (7c) as a bright yellow solid, yield: 61%; UV
(MeOH) max (log
): 430.6 (3.73), 349.2 (4.23), 265.7 (4.32), 234.0 (4.42) nm; 1H-NMR (500 MHz, CDCl3):
10.33 (s, 1H, H-8), 7.98 (d, J = 9.2 Hz, 1H, H-12), 7.94 (s, 1H, H-1), 7.83 (d, J = 9.2 Hz, 1H, H-11), 6.85 (s,
λ
ε
δ
1H, H-4), 6.09 (s, 2H, -OCH2O-), 5.24 (d, J = 5.8 Hz, 1H, H-6), 4.34 (s, 3H, -OCH3), 4.08 (s, 3H, -OCH3),
3.83 (d, J = 6.5 Hz, 2H, H-1’), 3.29 (t, J = 5.8 Hz, 2H, H-5), 1.83 (p, J = 7.1 Hz, 2H, H-2’), 1.50 (p, J = 7.2
Hz, 2H, H-3’), 1.33-1.26 (m, 8H, H-4’-H-7’), 0.90 (t, J = 6.8 Hz, 3H, H-8’); 13C NMR (125 MHz, CDCl3):
δ
152.3, 150.7, 149.9, 147.1, 146.2, 143.4, 132.1, 130.9, 129.9, 125.4, 123.0, 118.5, 117.9, 108.5, 108.4, 102.0,
75.9, 63.2, 57.1, 31.8, 30.2, 29.3,29.2, 28.2, 26.0, 22.6, 14.1; LC-MS (ESI+, m/z) C28H34NO5+: 464.58 [M
−
Br]+; HRMS-ESI: m/z calculated for C28H34NO5+: 464.2437 [M-Br]+, found for 464.2436.
13-O-Decylberberine chloride (7d). Compound (
the general procedure to give the desired bromide salt (7d) as a bright yellow solid, yield: 58%; UV
(MeOH) max (log
): 428.3 (3.70), 349.6 (4.19), 265.4 (4.28), 223.8 (4.47) nm; 1H-NMR (500 MHz, CDCl3):
10.50 (s, 1H, H-8), 7.97 (d, J = 9.2 Hz, 1H, H-12), 7.94 (s, 1H, H-1), 7.81 (d, J = 9.2 Hz, 1H, H-11), 6.86 (s,
4) was treated with n-decyl bromide according to
λ
ε
δ
1H, H-4), 6.09 (s, 2H, -OCH2O-), 5.31 (d, J = 5.8 Hz, 1H, H-6), 4.34 (s, 3H, -OCH3), 4.09 (s, 3H, -OCH3),
3.82 (d, J = 6.5 Hz, 2H, H-1’), 3.28 (t, J = 5.8 Hz, 2H, H-5), 1.83 (p, J = 7.2 Hz, 2H, H-2’), 1.50 (d, J = 7.2
Hz, 2H, H-3’), 1.32-1.23 (m, 12H, H-4’-H-9’), 0.90 (t, J = 6.9 Hz, 3H, H-10’); 13C-NMR (125 MHz, CDCl3):
δ
151.3, 150.6, 149.9, 147.5, 146.4, 143.9, 132.2, 130.8, 129.8, 125.3, 123.1, 118.5, 117.8, 108.4, 102.0, 75.8,
63.2, 57.0, 56.7, 31.9, 30.1,29.5, 29.33, 29.29, 28.2, 26.0, 22.7, 14.1; LC-MS (ESI+, m/z) C30H38NO5+: 492.62
[M − Br]+; HRMS-ESI: m/z calculated for C30H38NO5+: 492.2750 [M − Br]+, found for 492.2751.
13-O-Dodecylberberine chloride (7e). Compound (4) was treated with n-dodecyl bromide according
to the general procedure to give the desired bromide salt (7e) as a bright yellow solid, yield: 40%;
1
UV (MeOH)
λ
max
(log
ε
): 427.5 (3.63), 350 (4.13), 266.4 (4.20), 225.4 (4.37) nm; H-NMR (500 MHz,
DMSO-d6):
δ 9.80 (s, 1H, H-8), 8.20 (d, J = 9.4 Hz, 1H, H-12), 8.09 (d, J = 9.4 Hz, 1H, H-11), 7.89 (s,
1H, H-1), 7.13 (s, 1H, H-4), 6.16 (s, 2H, -OCH2O-), 4.86 (t, J = 5.9 Hz, 2H, H-6), 4.09 (s, 3H, -OCH3),
4.08 (s, 3H, -OCH3), 3.84 (t, J = 6.4 Hz, 2H, H-1’), 3.16 (t, J = 5.9 Hz, 2H, H-5),1.77 (p, J = 7.0 Hz, 2H,

Molecules 2020, 25, 677
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H-2’), 1.43(m, 2H, H-3’), 1.24 (m, 16H, H-4’-H-11’), 0.85 (t, J = 6.9 Hz, 3H, H-12’); 13C-NMR (125 MHz,
DMSO-d6): 150.9, 150.1, 149.2, 146.8, 144.2, 142.0, 132.5, 130.9, 128.8, 126.1, 122.2, 118.5, 118.4, 108.3,
δ
108.0, 102.1, 75.4, 62.0, 57.1, 56.4, 31.3, 29.4, 29.04, 29.02, 28.96, 28.89, 28.75, 28.7, 26.9, 25.4, 22.1, 14.0;
LC-MS (ESI+, m/z) C32H42NO5+: 520.68 [M-Br]+; HRMS-ESI: m/z calculated for C32H42NO5+: 520.3063
[M − Br]+, found for 520.3066.
3.5. Biological Activity
3.5.1. Cell Lines and Cell Culture Conditions
All cells were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan).
Human colon cancer cells (HT-29) was cultured in RPMI 1640 (HyClone, South Logan, UT, USA)
medium, supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, Carlsbad, CA,
USA) and antibiotics (100 mg/mL streptomycin and 100 U/mL penicillin, Gibco, Life Technologies,
USA). Human bladder cancer cells (BFTC 905) and human liver carcinoma (HepG2) cell lines were
cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone, USA) medium, supplemented with
10% fetal bovine serum (FBS) and antibiotics. Human cancer cells were maintained in humidified
atmosphere with 5% CO2 and 95% air at 37 ◦C in a carbon dioxide incubator (SANYO, CO2 incubator,
Osaka, Japan).
3.5.2. Cell Viability Inhibition Assay
Cell viability was assessed by staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT, Bionovas Biotechnology Co., Ltd., Toronto, Ontario, Canada) [32]. Briefly, the cancer
cells were plated at 5
incubator for 24 h. After medium removal, 100
×
103 – 1
×
104 cells into 96-well plates incubated at 37 ◦C in a humidified 5% CO2
µL of culture medium with 0.1% DMSO containing
the test compounds at different concentrations was added to each well and incubated at 37 ◦C for
another 24 h. At the end of treatment, final concentration of 0.5 mg/mL MTT was added, and cells were
incubated for a further 1.5 h. The absorbance was recorded on an ELISA plate reader (SpectraMax
340PC384, Molecular Devices, Orleans, CA, USA) at a 540 nm wavelength. Inhibition ratio (%) was
calculated using the following equation:
Inhibition ratio (%) = [(Acontrol − Atreated)/Acontrol] × 100%,
(1)
Atreated and Acontrol are the average absorbance of three independent experiments from treated and
control groups, respectively.
Cell viability was determined the test compound concentration required to inhibit tumor cell
proliferation by 50% (IC50) from the dose-response curves. All data average values from triplicate
samples and the experiments were repeated at least three times.
3.5.3. Measurement of Absorption and Fluorescence Spectra
Absorption spectrum (250–500 nm) of berberine and its derivatives (5e
on a UV–vis spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan) with a quartz cuvette (path
length 1 cm, accuracy 0.2 nm). Fluorescence emission spectrum were recorded on a fluorescence
, 6e, and 7e) were recorded
±
spectrophotometer (RF-5301PC, Shimadzu, Kyoto, Japan) with excitation at 420 nm and a measure of
emission from 450 to 700 nm (slit 3/3 nm) in a 1 cm quartz cuvette. A final concentration of berberine and
its derivatives 5e, 6e, and 7e were 10 µM diluted using methanol. Appropriate blanks corresponding
to the methanol were subtracted to correct the background.
3.5.4. Light Source
The UVA light box consisting of 8 UVA lamps (Blacklight blue, F8T5BLB, Sankyo Denki Co. Ltd.,
Osaka, Japan) was custom made [37]. The light spectral irradiance of the light box was determined

Molecules 2020, 25, 677
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using a NIST-Traceable radiometer (ILT1400 Portable radiometer, International Light Technologies Inc.,
MA, USA). The light dose of the UVA light box, with the maximum emission at 420 nm, was routinely
measured using a photochemical reactor (PR-1000, Panchum Scientific Corp., Kaohsiung, Taiwan). In
the study, the light-irradiation doses were determined at 1.8, 3.6, 7.2 J/cm2 for 420 nm (dose rate 5.9
mW/cm2), which were obtained by approximately 5, 10, and 20 minutes of exposure, respectively. To
determine visible light dose using the following equation:
E (J/cm2) = t (s) ×P (W/cm2),
(2)
where E stands for visible light energy, t represents time expressed in second and, finally, P is
lamp potency.
3.5.5. Photocytotoxic Assay
The photocytotoxicity of berberine (
the MTT colorimetric assay with quantification of formazan formed by cleavage of the tetrazolium
rings of MTT in DMSO by spectral measurement [32]. Various concentrations of the berberine ( ) and
berberine derivatives (5e 6e, and 7e) dissolved in 1% DMSO were added to the cells and incubation
1) and its derivatives (5e, 6e, and 7e) were studied by using
1
,
was continued for 4 h in the dark, followed by photo-irradiation with visible light of 420 nm (1.8,
3.6 and 7.2 J/cm2 for 5, 10 and 20 min of irradiation, respectively) by using a photochemical reactor
(PR-1000, Panchum Scientific Corp., Kaohsiung, Taiwan) filled with 8 fluorescent black tubes lamps
(Blacklight blue, F8T5BLB, Sankyo Denki Co. Ltd., Osaka, Japan). For these experiments, the cells
were seeded in 96-well microliter plates (5000 cells/well) and grown for at least 24 h in 100
µL DMEM
containing 10% FBS. The growth medium was replaced with fresh medium containing 3% FBS and
berberine (5, 10, 20, and 40 µM) and its derivatives (0.05, 0.25, 0.5, and 1.0 µM) and the cells were
incubated for 4 h at 37 ◦C. At the end of the incubation, the drug-containing medium was removed,
and the cells were washed with fresh medium and irradiated for 5, 10, and 20 min with visible light
(420 nm). After the incubation period for 20 h, 10
incubated for an additional 1.5 h at 37 ◦C, 100
L DMSO was added to dissolve the formazan crystals.
The absorbance of the samples was measured using an ELISA microplate reader (SpectraMax 340PC384
µL MTT (5 mg/mL) was added to each well and
µ
,
Molecular Devices, Orleans, CA, USA) and selecting 540 nm as the lest wavelengths. The survival of
the treated cells was calculated as the percentage of their absorbance referred to the absorbance of the
untreated (no drug, no light) cells taken as 100% viability. The IC50 values were obtained by nonlinear
regression analysis (Sigma 11.0 software).
3.5.6. Intracellular Uptake Profiling of Berberine and its Derivatives in HepG2 Cells
In order to establish a time profile for intracellular accumulation of these berberine derivatives, an
HPLC analysis was carried out. The HepG2 cells treated with different berberine (
1) and its derivatives
(
5e 6e, and 7e) were allowed to grow in 5 mL liquid culture. The cultures were incubated in a shaking
,
incubator in the dark. After 0 h, 0.25 h, 1 h, 2 h, 4h, and 8 h, aliquots of cells were harvested by
centrifugation at 6000 rpm for 5 min. The supernatant liquid was discarded and the cell pellet was
washed thrice with 1 mL of 2
After this, the cells were gently resuspended in 0.2 mL methanol and then were filtered with a 0.22
×
PBS in order to remove the uninternalized berberine and its derivatives.
µm
filter into HPLC analysis. The HPLC analysis was carried out by exciting the sample at 350 nm and
emission at 530 nm.
3.5.7. Cell Morphological Assessment
To detect morphological evidence of apoptosis, cells were visualized following DNA staining
with the fluorescent dye Hoechst 33258 [34]. The HepG2 cells were plated in a 6 cm dish at a density
of 7
×
105 cells/well and were incubated overnight. The cells were treated with 0.5
µM concentrations
berberine (1), compounds 5e, 6e, and 7e for 4 h in the dark and treated with visible light (420 nm) for

Molecules 2020, 25, 677
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10 min. Additionally, the HepG2 cells of compound 7e treatment were stained with 10
µM Hoechst
33258 (Sigma-Aldrich, St. Louis, MO, USA). After Cells were incubated in a dark room for 5 min, the
cells were washed with PBS and then were observed by a fluorescent microscope (Zeiss, Axio Observer
A1, Japan).
3.5.8. Cell Cycle Distribution Analysis
Flow cytometry was used to obtain the cell cycle distribution and the apoptotic rate [35]. The HepG2
cells were plated at 1
×
106 cells per 6 cm dish and cultured overnight. Then, various concentrations of
0.5 M berberine ( ), compounds 5e, 6e, and 7e were added for 2 h. At the end of the incubation, the
µ
1
drug-containing medium was removed, and the cells were washed with fresh medium and irradiated
for 10 min with visible light (420 nm). The cells of each dish were harvested, washed once with PBS,
and fixed with methanol at 4 ◦C. After 24 h, the cells of each dish were harvested and washed once
with PBS. The cells were suspended in 473
µL of PBS containing 40 µg/mL propidium iodide (PI) and
40 g/mL RNase. Cells were incubated in a dark room for 30 min at room temperature then subjected
µ
to cell cycle analysis using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) and
analyzed using the ModFit 3.0 software (Verity Software House, ME, USA).
3.5.9. Measurement of Intracellular Singlet Oxygen Production
Intracellular oxidant stress was monitored by measuring changes in fluorescence resulting from
intracellular probe oxidation. Intracellular production of ROS, namely singlet oxygen (1O2), was
measured using the DCFH-DA (Molecular Probes, USA) probe, respectively [36]. The level of
intracellular singlet oxygen was detected using fluorescent dye DCF sensitivity. Briefly, the HepG2
(4 µM of berberine (1) and 0.25 µM of its derivative (5e,
104 cells/ml) cells were pretreated with 0.5
×
6e and 7e) for 2 h. At the end of the incubation, the drug-containing medium was removed, and the
cells were washed with fresh medium and irradiated for 10 min with visible light (420 nm). After the
incubation period for 24 h, intracellular peroxide level was detected by DCFH-DA as an intracellular
fluorescence probe. After incubation with 20 µM DCFH-DA for 1 h, the cells were harvested and
washed with PBS twice. A flow cytometer (Becton Dickinson, San Jose, CA, USA) was used to detect
dichlorofluorescein (DCF). Data were collected and analyzed by ModFit 3.0 software (Verity Software
House, ME, USA).
3.5.10. Measurement of the Mitochondrial Membrane Potential
The changes in the mitochondrial membrane potential (MMP) were determined by using the
fluorescent probe rhodamine 123 (Sigma, St Louis, USA). The dye Rh123 selectively enters mitochondria
without influencing the membrane potential and is retained inside the mitochondria [37]. Once the
MMP is lost, rhodamine 123 is subsequently washed out of the cells, leading to the decline of the
fluorescence signal. HepG2 cells (1
×
105 cells/mL) were incubated with concentrations of 0.5
6e, and 7e) for 2 h. At the end of the incubation, the
µM
berberine ( ) and 0.25 M of its derivative (5e,
1
µ
drug-containing medium was removed, and the cells were washed with fresh medium and irradiated
for 10 min with visible light (420 nm) for 24 h. Then, stained with 2 mg/mL of rhodamine 123 for 20 min
under gentle shaking. After washing with PBS, cells were immediately analyzed by flow cytometer
(Becton Dickinson, San Jose, CA, USA) and data were analyzed using the ModFit 3.0 software (Verity
Software House, ME, USA).
3.5.11. Statistical Analysis
The values shown are mean
evaluated by Student’s t-test of SigmaPlot 11.0 and shown significantly different in * p < 0.05, ** p <
0.01, and *** p < 0.001.
±
SD of three independent experiments. Data are statistically

Molecules 2020, 25, 677
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4. Conclusions
In summary, berberine was mortified with n-alkyl groups of different chain lengths and studied
for their photo-induced cytotoxicity against three human cancer cell lines. Out of the 15 synthesized
derivatives, we found 3; compounds 5e
The increasing chain length of n-alkyl groups enhanced lipophilicity and the cellular permeability of
the derivatives. Besides, its derivatives, compounds 5e 6e, and 7e had exhibited low dark toxicity and
higher photocytotoxicity toward the cancer cell lines under testing. Thus derivatives 5e 6e, and 7e
, 6e, and 7e, worked the best in vitro against the cancer cell lines.
,
,
with a dodecyl group on the 9-, 13- or 13-O-position of berberine, respectively, showed high cellular
uptake, photo-induced cytotoxicity in vitro, ROS generation, and induced cell apoptosis after 420 nm
visible light irradiation. In the cell cycle distribution and apoptotic analysis, compound 5e, 6e, or 7e
induced S phase arrested and apoptosis in HepG2 cells after visible light irradiation. In Hoechst 33,258
staining analysis, these three compounds markedly induce apoptosis, which was further confirmed by
the Sub-G1 rate of cell cycle analysis. Taken together, the presented results showed that compounds 5e
6e, and 7e increased cellular accumulation and enhanced the anti-cancer activity by photo-induced
therapy against the cancer cells. We thus believe they could be novel anti-cancer drug candidates.
,
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/3/677/s1.
Author Contributions: H.-J.L. and J.-H.H. suggested the research work and discussed the experimental data;
H.-J.L., L.-C.T., and F.-Y.Y. finalized the experimental work, interpreted the results, and prepared the figures; L.-G.C.
and Y.-W.L. conceived and designed the biological experiments; L.-L.Y., C.-D.K., and J.-Y.W. wrote the paper,
edited, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This study is supported by grants V103E2-002 and V104E2-002 from the Taipei Veterans General
Hospital, Taipei, Taiwan, ROC.
Acknowledgments: Gratitude is intended to Christopher Liu for his valuable editorial revision.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2–4, 5a–e, 6a–e and 7a–e are available from the authors.
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