Design, synthesis and biological evaluation of a hybrid compound of berberine and magnolol for improvement of glucose and lipid metabolism

Article abstract of DOI:10.1039/c6ra15100k

The discovery and structural optimization of lead compounds is the main task in the research and development of new drugs. In order to search for new compounds with greater activities and lower toxicity, the synthesis of two or more basic structures of drugs bonded together has been designed on the basis of combination principles. Here, a hybrid compound consisting of berberine (Ber) and magnolol (Mag) was synthesized and its biological activities were evaluated. We named the hybrid compound Huanghousu (HHS), and its size and structure were confirmed by spectroscopy (¹H NMR, ¹³C NMR and HRMS spectra). The LD₅₀ of HHS was determined in NIH mice by intraperitoneal injection according to the modified Karber's method. Treatment of transgenic aP2-SREBP-1c mice with HHS markedly reduced blood triglycerides (TG) and improved sugar tolerance. In addition, we also evaluated the effects of berberine, magnolol and HHS on the proliferation and differentiation of 3T3-L1 preadipocytes and investigated the underlying mechanism. The adipocyte differentiation-related genes PPARγ and C/EBPα were tested using a real-time quantitative polymerase chain reaction (real-time PCR). In addition, FAS, UCP2 and adiponectin mRNA, which are related to adipocyte adipogenesis, were also measured in mature 3T3-L1 adipocytes induced by differentiation medium. The efficacy of HHS in preventing obesity was somewhat greater than that of magnolol or berberine. Taken together, these results indicated that HHS was effective in improving disorders of glucose and lipid metabolism in vivo and regulating lipid metabolism-related gene expression in vitro.

Full text of DOI:10.1039/c6ra15100k

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Design, synthesis and biological evaluation of a hybrid compound berberine and
magnolol for improvement of glucose and lipid metabolism
a
b
c
a
a
c
b
Yan Li, Xiao Yuan, Xianglu Rong, Ying Gao, Zhibin Qiu, Zhipeng Zhang, Dongbin Zhou, and
Weimin Lia∗
Abstract
The discovery and structure optimization of lead compounds is the main task in new drug
research and development. In order to search new compounds with greater activities and lower
toxicity, the synthesis of two or more drug basic structure stitched together were designed on the
basis of combination principles. Here, a hybrid compound consisting of berberine (Ber) and
magnolol (Mag) was synthesized, and its biological activities were evaluated. We name the hybrid
1
compound Huanghousu (HHS), and its size and structure were confirmed by spectroscopic ( H
13
NMR, C NMR and HRMS spectra). The LD50 of HHS was determined in NIH mice by
intraperitoneal injection according to Modified karber’s method. Treatment of transgenic
aP2-SREBP-1c mice with HHS markedly reduced blood triglycerides (TG) and improved the sugar
tolerance. Additionally, we also evaluated the effects of berberine, magnolol and HHS on the
proliferation and differentiation of 3T3-L1 preadipocytes and to explore the underlying the
mechanism. The adipocyte differentiation-related gene, PPARγ and C/EBPα, were evaluated using
Real-time Quantitative polymerase chain reaction (Real-time PCR). Additionally, FAS, UCP2 and
adiponectin mRNA, which were related to adipocyte adipogenesis, were also measured in mature
3
T3-L1 adipocytes induced by differentiation medium. The efficacy of HHS in preventing obesity
was somewhat more effective than magnolol or berberine. Taken together, these results
indicated that HHS was effectively improving disorders of glucose and lipid metabolism in vivo
and regulating lipid metabolism-related gene expression in vitro.
1
. Introduction
Diabetes mellitus (DM) is a complex endocrine and metabolic disorder characterized by high levels of glucose and
1
,2
lipid in the blood. Recent study finds that the estimated prevalence of diabetes in Chinese adults is 11.6% and
3
the prevalence of prediabetes is 50.1%. It is predicted that approximated 4% of the population worldwide suffer
4
from diabetes and there is expected to increase by 5.4% in 2025. Obesity is a key risk factor for type 2 diabetes as
2
it desensitizes glucose recipient organs to the action of insulin. Growing evidence shows that obesity is
characterized by intraabdominal visceral fat accumulation, which depends on adipocyte proliferation and
5
-7
differentiation of preadipocytes. Lipogenic gene expression in adipocytes is known to be mainly regulated by
8
-10
CCAATꢀenhancer binding proteins (C/EBPs) and peroxisome proliferatorꢀactivated receptorꢀγ(PPARꢀγ).
It
has long been known that transient expression of C/EBPβ and C/EBPδ gene are manifested in the early stage of
a
School of Chinese Material Medical, Guangzhou University of Chinese Medicine, Guangzhou 510006, People’s
Republic of China. Eꢀmail addresses: 13925023915@139.com; Fax: +86 20 39358290.
b
Guangzhou Pi
＆
Pi Technology Inc, Guangzhou 510006, People’s Republic of China
c
Center Laboratory, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China
1
13
†
Electronic supplementary information (ESI) available: The H NMR, C NMR and HRMS spectra for
synthesized compounds; HPLC profile for HHS; The oligonucleotide primer sequence used for realꢀtime PCR. See
DOI: 10.1039/x0xx00000x
1

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cell differentiation, and the C/EBPα is expressed in the late stage. Once C/EBPα is activated, it can trigger the
expression of several genes that influence intracellular lipid droplets deposition, insulin resistance,
metabolism of adipose and balance of energy. It has been reported that activation of PPARꢀγ upꢀregulates FAS and
UCP2 expression, resulting in augmentation of glucose uptake and lipogenesis.
Traditional Chinese medicine (TCM) is based on herbal extracts containing natural active components. In
TCM, the herb couples, as the basic composition units of Chinese herbal formulae, have specific clinical
11,12
significance.
Coptis Magnoliae Decoction is a representative formula composed of Rhizoma coptidis and
Magnolia officinalis in mass prescriptions, which is obtained from The Complete Record of Holy Benevolence, a
traditional Chinese medical classic compiled in the Song dynasty. Berberine and magnolol are as the major active
components, which can be used as lead compounds of chemical drug for the corresponding structure modification.
Berberine is an isoquinoline alkaloid isolated from Chinese herb Rhizoma coptidis, which mainly exists in
ranunculaceae, rutaceae and berberidaceae. The high medicinal value of berberine and the physiological activeties
of berberine derivatives have recently attracted remarkable attention for its extensive pharmacological effects and
1
3,14
15
usefulness in biomedical applications.
Studies have shown that berberine possesses the antiꢀinflammatory,
1
6-18
19,20
21,22
hypotensive, antiꢀplatelet aggregative,
hypoglycemic
and hypolipidemic
activities. Also, derivatization
of berberine core structure has resulted in the development of molecules with positively enhanced biological
2
3,24
effects.
Magnolol is a polyphenolic binaphthalne compound from Chinese medicinal herb Magnolia officinalis,
2
5
26,27
which has been reported to have multiple biological effects, including antiꢀinflammatory, antiꢀoxidant,
2
8
29
antiꢀangiogenic and antidiabetic activities. Moreover, Rhizoma coptidis and Magnolia officinalis are widely
used for the treatment of diabetes and hyperlipidemia in Chinese traditional herbal medicines and prescriptions.
Although the researches on the synthesis and structure modification of berberine and magnolol have been
well done recently, little information has been available on the synthesis of a hybrid compound consisting of
berberine and magnolol. Therefore, this study aims to investigate the structure combination process for berberine
and magnolol and to synthesize a new compound with the pharmacodynamic activity. In this study, the structure of
1
13
the new compound was confirmed by spectroscopic ( H NMR, C NMR and HRMS spectra). We investigated
hypoglycemic and hypolipidemic activities of the new compound using transgenic aP2ꢀSREBPꢀ1c mice in vivo.
Furthermore, we determined the antiꢀobesity effects using 3T3ꢀL1 in vitro, which measured mRNA expression of
adipogenic transcription factors using Realꢀtime PCR.
2
. Rusults and discussion
2
.1 Design of berberine and magnolol hybrid compound (HHS)
The synthesis route of HHS was conveniently undertaken as outlined in Scheme 1. With berberine hydrochloride
as starting material, berberrubine was synthesized by 9ꢀposition demethylation reaction
under microwave irradiation in excellent yields (92%). Afterwards, berberrubine 1 on reaction with 1,
ꢀdibromoethane in acetonitrile as the solvent, followed by the heating reflux reaction gives 9ꢀ(2ꢀethyl bromide)
berberine hydrochloride 2 in good yields (56%). Finally, the hybrid compound 3 (HHS) could be obtained by the
reaction of 9ꢀ(2ꢀethyl bromide) berberine hydrochloride 2 with magnolol and Na CO (1:1.5:6) in acetonitrile
under reflux in moderate yields (24%). After crystallization and purification, the structure characterizations of
a
1
2
2
3
1
13
those products were analyzed by H NMR, C NMR and HRMS spectra, and the profile for HHS was established
†
by HPLC (ESI ).
Previous studies have suggested that berberrubine was usually synthesized by vacuum pyrolysis, with
3
0
low yield and purity due to uneven heating. In the preliminary experiment, we adopted microwave synthesis
31,32
method as described by references,
but no chemical transformation occurred. After that, we used DMF with a
2

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high boiling point and good stability as reaction medium under microwave irradiation to afford berberrubine in 92%
yield. 9ꢀ(2ꢀethyl bromide) berberine hydrochloride was synthesized by a solvent acetonitrile. There have been few
studies on using two natural active molecules stitched together through chemical bond to synthesize a hybrid
compound.
Scheme 1 Synthesis of HHS. Reagents and conditions: ① DMF, microwave power (400 W), reflux, 15 min,
9
2%; ② acetonitrile, 1,2ꢀdibromoethane, reflux, 3 h, 56%; ③ acetonitrile, anhydrous sodium carbonate, reflux, 8
h, 24%.
2
.2 Acute toxicity study
After intraperitoneal injection of HHS, the various situations of mice were observed strictly in the first 4 h. The
mice administered with HHS at a dose of 132.1 mg/kg showed no significant changes in behavior, breathing and
postural abnormalities. However, the significant changes occurred in the mice of other groups, including unsteady
walking, difficult breathing, and death, even with exsistence of moderate organ damages. Moreover, the mortality
of treated mice at dose of 188.7 and 385.0 mg/kg was 0.2 and 0.8 in a doseꢀdependent manner. The LD50 value
(
lethal dose 50) was 279 mg/kg, and 95% confidence limit was 234.6~332.5 mg/kg (Table 1). The results obtained
from this study demonstrate that, after single intraperitoneal injection, the Acute toxicity of HHS was lower than
the berberine, while the toxicity of berberine in the previous reports was of the LD50s ranging between 38.8 and
3
3
34
8
5.5 mg/kg and LD50 being 50 mg/kg.
Table 1 The results of acute toxicity testing of HHS in NIH mice administered by intraperitoneal injection within
4 d.
1
Dose
mg/kg)
550.0
385.0
269.5
188.7
132.1
0
LD50
95% confidence limit
(mg/kg)
Group
Mortality
(
(mg/kg)
1
10/10
8/10
4/10
2/10
0/10
0
2
3
2
79.3
234.6～332.5
4
5
control
∑
p=2.4
These data were calculated according to Modified karber’s method.
2
.3 Effect of HHS on glucose and lipid metabolism in aP2-SREBP-1c mice
3
5-37
It has been known for excessive amounts of white tissue (obesity) are the crucial factors in insulin resistance.
38,39
In contrast to all other mouse models of insulin resistance,
aP2ꢀSREBPꢀ1c mice were not obese, which exhibit
4
0,41
a distinct syndrome that includes lipodystrophy, insulin resistance, diabetes mellitus and fatty liver.
In the
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present study, we examined the effect of HHS on glucose and lipid metabolism disorder. OGTT was performed at
end of 9 weeks. As shown in Fig. 1A, the blood glucose level in model group was much higher than that in control
group. However, the mice treated with HHS showed a significant decrease in blood glucose level compared with
model group at 20 and 60 min. Moreover, the AUC was significantly lower in the HHS group than that in the
model group (Fig. 1B). As shown in Fig. 1C, at the end of 8 and 13 week, the plasma TG level in model group was
significantly higher than that in control group, while HHS group showed significantly decreased TG level
compared to model group. According analysis of TG level, compared with 0 week (0.85±0.43), the TG level at 8
and 13 weeks in control group was no significant change, and the level in model group was increased significantly.
Conversely, after HHS treatment for 8 and 13 weeks, plasma TG level significantly decreased compared with 0
week. These data indicated that HHS improved glucose and lipid metabolism in aP2ꢀSREBPꢀ1c mice.
Fig. 1 In vivo effects of HHS on the glucose and lipid metabolism in aP2ꢀSREBPꢀ1c mice. Mice at 12 weeks of
age were divided into 3 groups. HHS was suspended in 5% CMCꢀNa aqueous solution and administered orally for
1
3 weeks. (A) After 9 weeks after final dosing, following an overnight fast, an oral gavage of 50% glucose (2 g/kg
body weight) was performed on each animal. Blood glucose levels were estimated from the blood samples just
prior to glucose administration (0 min) and 20, 60 and 120 min postꢀglucose administration. (B) By oral glucose
tolerance test (OGTT), area under blood glucose concentration curve (AUC) was calculated from the formula:
AUC=[1/4 × 0 min (mmol/L) + 1/2 × 20 min (mmol/L) + 3/4 × 60 min (mmol/L) + 120 min (mmol/L)]. (C) At the
end of 8 and 13 weeks, mice were anesthetized with diethyl ether after fasting for 12 h, plasma triglyceride levels
were measured. Each group was composed of 8 mice. Data are presented as means ± SD. P<0.05, ∗∗P<0.01
∗
(
oneꢀway ANOVA) compared with model group.
2
.4 Cytotoxicity of HHS in 3T3-L1 preadipocyte
In the present study, the cytotoxicity of berberine, magnolol and HHS towards 3T3ꢀL1 preadipocyte was
investigated using CCKꢀ8. The results are shown in Fig. 2. Within the range of 0ꢀ80 ꢀM, the cell viability of Mag
group decreased with increasing concentration, which had a doseꢀdependent manner (Fig. 2A). When the
concentration is more than 20 ꢀM, Berberine had an obvious inhibitory effect toward 3T3ꢀL1 preadipocyte (Fig.
2
B). However, the result showed that HHS could significantly reduced cell viability when the concentration
reached 10 ꢀM (Fig. 2C). Meanwhile, IC50 values of berberine, magnolol and HHS were 19.43±0.56, 27.93±0.63
and 11.54±0.52 ꢀM. These observations suggested that HHS exerted stronger effect than berberine, magnolol on
the viability of 3T3ꢀL1 preadipocyte.
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Fig. 2 The cytotoxic activity of Mag (A), Ber (B) and HHS (C). Cells were seeded in 96ꢀwellꢀplate at the density
of 5000ꢀ8000 cells per well and incubated for 24h. Then various concentrations of berberine, magnolol and HHS
(
1.25, 2.5, 5, 10, 20, 40 and 80 ꢀM) were added to the medium and incubated for 48 h. The cells treated with 0.1%
DMSO served as control (con). Values are means ± SD (n=3 in each group).
2
.5 Differentiation of 3T3-L1 preadipocytes and Oil Red O staining
The cultured 3T3ꢀL1 preadipocytes were in a fibroblastꢀlike from with long fusiform or polygon shape (Fig. 3A).
And the cells could successfully be transꢀdifferentiated into adipocytes with an appreciable amount of accumulated
4
2
lipid droplets after 10 days of induction, which were confirmed by using Oil RedꢀOstaining (Fig. 3B and 3C).
Lipid and Oil Red O were extracted using isopropanol, and absorbance was measured at 520nm. the result of oil
red O staining shown in Fig. 3D. The OD520 value was significantly increased in model group compared with the
control group.
A
B
C
D
Fig. 3 3T3ꢀL1 preadipocytes were successfully differentiated into adipocytes. Differentiated 3T3ꢀL1 cells were
identified using Oil Red O staining. (A) Control (preadipocytes), (B) differentiated adipocytes, (C) Model
(
differentiated adipocytes, using Oil Red O staining), (D) Oil Red O was extracted from the cells using isopropyl
alcohol, and the absorbance was measured at 520 nm. Three independent experiments were performed and data are
shown as mean ± SD. ∗∗P<0.01 compared with Model group.
2
.6 Effects of HHS on expression of differentiation-related gene in 3T3-L1 adipocytes
Research shows that PPARγ and C/EBPα play major roles in the process of adipocyte differentiation, which are
4
3-46
also critical in the regulation of lipid metabolism and glucose homeostasis.
To quantitatively describe the effect
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of berberine, magnolol and HHS at different concentrations on adipocyte differentiation, we examined the
expression of PPARγ2 and C/EBPα using Real time PCR. As shown in Fig. 4A and 4B, the PPARγ2 and C/EBPα
mRNA levels were significantly increased in MD group compared to the NC group, and HHS was effective in
reducing their levels. Ten days after initiating treatment, the Mag and Ber treated group at 5 ꢀM had a significant
effect on PPARγ2 and C/EBPα mRNA levels compared with MD group, which were consistent with previous
47-49
studies.
And the Mag treated group at 2.5 ꢀM did not induce efficient change in PPARγ2 and C/EBPα mRNA
levels. HHS treatment significantly decreased PPARγ2 mRNA level in a doseꢀdependent manner. However,
surprisingly, the HHS treated group at 5 ꢀM was less effective in decreasing C/EBPα mRNA level than the Ber
group at 5 ꢀM. Although the HHS treated group had a significant decrease in C/EBPα mRNA level, it showed no
doseꢀdependent manner. Through the comparison of the effects of both doses HHS on PPARγ2 and C/EBPα, we
found that the HHS at 2.5 ꢀM had a higher reduction in the mRNA expression than berberine or magnolol. Thus,
we concluded that HHS to some extent showed stronger inhibitory effect on PPARγ2 and C/EBPα mRNA levels
than berberine or magnolol.
Fig. 4 Effects of HHS on mRNA expression levels of PPARγ2 and C/EBPα in 3T3ꢀL1 cells. Three independent
experiments were performed and data are shown as mean ± SD. P<0.05, ∗∗P<0.01 compared with DM group. NC
as control group: the cells treated with 0.1% DMSO, MD as model group: 3T3ꢀL1 mature adipocytes.
∗
2
.7 Effects of HHS on expression of adipogenesis-related gene in 3T3-L1 adipocytes
Adipocyte differentiation is a complex process that is controlled and regulated by various adipocyteꢀspecific genes,
50
such as FAS, UCP2, adiponectin and others, which participate in creating the adipocyte phenotype. Here we
focused more on downstream genes of PPARγ2, C/EBPα and studied how their mRNA expression is influenced by
HHS. When 3T3ꢀL1 preadipocyte cells differentiated into adipocytes, we tested mRNA levels of FAS, UCP2 and
adiponectin in all groups. And as shown in Fig. 5A, 5B and 5C, a significant increase in the mRNA expression of
FAS and UCP2 were observed in the MD group compared to the NC group, which confirmed the presence of lipid
accumulation. After treatment with the berberine, magnolol and HHS, it was found that 5 ꢀM HHSꢀtreated cells
showed greater reductions in the mRNA expression of FAS and UCP2 than those of berberine, magnololꢀtreated;
while, it increased the mRNA expression of adiponectin more obviously which demonstrated the good antiꢀobesity
51
effect.
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Fig. 5 Effects of HHS on mRNA expression levels of FAS, UCP2 and adiponectin in 3T3ꢀL1 cells. Three
independent experiments were performed and data are shown as mean ± SD. P<0.05, ∗∗P<0.01 compared with
MD group. NC as control group: the cells treated with 0.1% DMSO, MD as model group: 3T3ꢀL1 mature
adipocytes.
∗
3
. Experimental
3
.1 Reagents and chemicals
Berberine and Magnolol (purity ≥ 98%) were purchased from the China Drugs and Biological Products
Inspection Institute (Beijing, China). Dulbecco's Eagle's Medium (DMEM) and Fetal Bovine Serum (FBS) were
purchased from Gibco BRL (Grand Island, NY). Insulin (INS), 3ꢀIsobutylꢀ1ꢀmethylxanthine (IBMX),
dexamethasone (DEX) and Oil Red O were purchased from Sigma Aldrich (St Louis, MO). RNAiso Plus,
TM
®
PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time), SYBR Premix Ex Taq™ II (Tli RNaseH
Plus) were purchased from Takara (Tokyo, Japan). All other chemicals and reagents were of analytical grade and
1
13
purchased from Aldrich Chemical Co. (Beijing, China). Hꢀ and CꢀNMR spectra were recorded on Bruker
AVANCE 400 at room temperature with tetramethylsilane (TMS) as an internal standard and chloroformꢀd
CDCl ) as solvents. Mass spectra were recorded with a DSQ mass spectrometer (Thermo, USA).
3
(
3
3
.2 Synthesis of HHS
3
.2.1 General procedure for the synthesis of berberrubine (1)
Berberine (1g) was weighted and dissolved in DMF (25ml), and then added several zeolites. The reaction
mixture was refluxed for 15 min at the 400 W microwave power. After completion, the reaction mixture was
diluted with 40 ml of water and refrigerated until well crystallization. The combined extracts were washed with
petroleum ether and evaporated in vacuum. The crude product obtained was purified by PiPoꢀ02 macroporousresin
resin to get pure compound 1 (berberrubine) in yields (92%).
Berberrubine 1
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1
Red needle crystal; Mp 281.6～282.4
℃
. H NMR (CDCl
3
, 300 MHz) δ: 7.35 (1H, s, Hꢀ1), 6.76 (1H, s, Hꢀ4),
3
9
.04 (1H, t, J = 5.8, Hꢀ5), 4.53 (1H, t, J = 5.8, Hꢀ6), 9.20 (1H, s, Hꢀ8), 6.83 (1H, d, J = 9.1, Hꢀ11), 7.46 (1H, d, J =
1
3
3
.1, Hꢀ12), 7.97 (1H, s, Hꢀ13), 5.95 (1H, s, Hꢀ15), 3.81 (1H, s, Hꢀ16); C NMR (CDCl , 300 MHz) δ: 106.27
(
Cꢀ1), 121.81 (Cꢀ1a), 151.42 (Cꢀ2), 149.97 (Cꢀ3), 109.14 (Cꢀ4), 131.16 (Cꢀ4a), 57.04 (Cꢀ6), 143.72 (Cꢀ8), 134.21
(
Cꢀ8a), 147.84 (Cꢀ9), 147.84 (Cꢀ10), 120.21 (Cꢀ11), 123.21 (Cꢀ12), 131.16 (Cꢀ12a), 124.22 (Cꢀ13), 136.23 (Cꢀ14),
+
+
+
1
4
03.68 (Cꢀ15), 55.94 (Cꢀ16); LCꢀMS (ESI , m/z) [C19H16NO ] : 322. 2 [MꢀCl] .
3
.2.2 General procedure for the synthesis of 9ꢀ(2ꢀethyl bromide) berberine hydrochloride (2)
Compound 1 (3.23 mmol ) was refluxed in acetonitrile (200 ml), with stirring and heating to 85
℃. Then, 1,
2ꢀDibromoethane (20 ml) was added and the reaction mixture was refluxed for 3 h. The combined extract
continued to refrigerate until well crystallization. After completion, the crude product obtained was diluted with
acetonitrile and petroleum ether and evaporated in vacuum to get pure compound 2 [9-(2-ethyl bromide)
berberine hydrochloride] in yields (56%).
9-(2-ethyl bromide) berberine hydrochloride 2
1
Yellow crystal; Mp 273.6
～
274.4
℃
. H NMR (CDCl
3
, 300 MHz) δ: 7.68 (1H, s, Hꢀ1), 6.97 (1H, s, Hꢀ4),
3
9
5
2
1
.87 (2H, t, J = 5.8, Hꢀ5), 4.74 (2H, t, J = 5.8, Hꢀ6), 9.87 (1H, s, Hꢀ8), 8.04 (1H, d, J = 9.1, Hꢀ11), 8.15(1H, d, J =
.1, Hꢀ12), 8.74 (1H, s, Hꢀ13), 6.11 (2H, s, Hꢀ15), 3.30 (3H, s, Hꢀ16), 4.65 (2H, t, J = 5.6, Hꢀ17), 4.12 (2H, t, J =
13
3
.6, Hꢀ18); C NMR (CDCl , 300 MHz) δ: 106.43 (Cꢀ1), 151.64 (Cꢀ2), 149.39 (Cꢀ3), 109.29 (Cꢀ4), 131.77 (Cꢀ4a),
7.59 (Cꢀ5), 57.53 (Cꢀ6), 143.27 (Cꢀ8), 134.68 (Cꢀ8a), 146.24 (Cꢀ9), 146.24 (Cꢀ10), 121.57 (Cꢀ11), 124.94 (Cꢀ12),
31.77 (Cꢀ12a), 127.53 (Cꢀ13), 139.26 (Cꢀ14), 121.34 (Cꢀ14a), 103.31 (Cꢀ15), 56.88 (Cꢀ16), 57.53 (Cꢀ17), 74.49
+
+
+
(
Cꢀ18); LCꢀMS (ESI , m/z) [C H BrNO ] : 429.1 [Mꢀ Cl] .
21 19 4
3
.2.3 General procedure for the synthesis of HHS (3)
To a solution of compound 2 (1.21mmol) in acetonitrile (150ml), anhydrous sodium carbonate (10.85mmol)
and mgnolol (1.81mmol) were added, stirred and heated to 85℃. The reaction mixture was refluxed for 8 h. The
reaction mixture was immediatly filtered and then dissolved in dimethyl sulfoxide (DMSO). The solution mixture
obtained was purified by C18 column and washed with 30%, 40%, 50%, 60% methanol. The concentrated solution
obtained was purified by a gel silica column and washed with petroleum etheꢀethyl acetate (1:1). The crude
product obtained was evaporated in vacuum to get pure compound 3 (HHS) in yields (24%).
HHS (3)
{
9ꢀ(2'ꢀ((5',5''ꢀdiallylꢀ2''ꢀhydroxyꢀ[1',1"ꢀbiphenyl]ꢀ2'ꢀyl)oxy)ethoxy)ꢀ10ꢀmethoxyꢀ5,6ꢀdihydroꢀ[l,3]dioxolo[4,5ꢀg]iso
quinolino[3,2ꢀa]isoquinolinꢀ7ꢀium}.
Yellow solid; Mp 145.2 146.1
2H, t, J = 5.8, Hꢀ5), 4.74 (2H, t, J = 5.8, Hꢀ6), 9.38 (1H, s, Hꢀ8), 7.90 (1H, d, J = 9. 1, Hꢀ11), 8.04 (1H, d, J = 9.1,
1
～
3
℃. H NMR (CDCl , 500 MHz) δ: 7.64 (1H, s, Hꢀ1), 6.91 (1H, s, Hꢀ4), 3.87
(
Hꢀ12), 8.56 (1H, s, Hꢀ13), 6.11 (2H, s, Hꢀ15), 3.30 (3H, s, Hꢀ16), 4.65 (2H, t, J = 5. 6, Hꢀ17), 4.12 (2H, t, J = 5.6,
Hꢀ18), 7.00 (1H, d, Hꢀ21), 7.11 (1H, d, J = 8.45, Hꢀ23), 6.69 (1H, d, J = 8.45, Hꢀ24), 3.00 (1H, d, Hꢀ25), 6.39 (1H,
1
3
d, Hꢀ30), 5.99ꢀ5.86 (1H, m, J = 9.6, 16.9, Hꢀ26), 5.81ꢀ5.67 (1H, m, J = 9.6, 16.9, Hꢀ27), 7.04 (1H, s ); C NMR
(
(
CDCl
Cꢀ6), 143.27 (Cꢀ8), 134.13 (Cꢀ8a), 146.70 (Cꢀ9), 152.06 (Cꢀ10), 123.88 (Cꢀ12), 145.30 (Cꢀ12a), 127.65 (Cꢀ13),
21.34 (Cꢀ14a , 121.75 (Cꢀ11), 103.97 (Cꢀ15), 56.88 (Cꢀ16), 69.31 (Cꢀ17), 75.19 (Cꢀ18), 155.69 (Cꢀ19), 125.04
Cꢀ20), 132.70 (Cꢀ21), 129.66 (Cꢀ22), 129.25 (Cꢀ23), 113.69 (Cꢀ24), 40.49 (Cꢀ25), 131.76 (Cꢀ26), 116.09 (Cꢀ27),
53.83 (Cꢀ28), 126.51 (Cꢀ29), 129.92 (Cꢀ30), 133.57 (Cꢀ31), 129.25 (Cꢀ32), 115.86 (Cꢀ33), 40.65 (Cꢀ34), 131.93
3
, 300 MHz) δ: 106.81 (Cꢀ1), 152.38 (Cꢀ2), 150.21 (Cꢀ3), 109.65 (Cꢀ4), 131.76 (Cꢀ4a), 28.39 (Cꢀ5), 57.83
1
(
1
+
+
+
(
6
Cꢀ35), 116.59 (Cꢀ36); LCꢀMS (ESI , m/z) [C39H36NO ] : 615.3 [Mꢀ Cl] .
3
.3 Acute toxicity study
The mice strain used in this study was NIH, which was bred originally by the National Institutes of Health
8

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DOI: 10.1039/C6RA15100K
(
NIH). Two sexes of SPF (Specific Pathogen Free) NIH mice were investigated by regular methods for
toxicological evaluation. NIH mice weighing 18ꢀ22g [No. SCXK (Yue) 2008ꢀ0002], with half male and female,
were purchased from The Medical Laboratory Animal Center of Guangdong (Guangdong, China). Mice were
housed in temperatureꢀcontrolled animal facility with 12ꢀhour light and dark cycle and free to access water and
food. Procedures for animal care and handling were in strict accordance with the Chinese Legislation on the Care
and Use of Laboratory Animals. The Animal Experiment Center of Guangdong Pharmaceutical University
approved the experimental procedures and protocols conducted during this study. After acclimating for 1 week, ten
mice were treated with each dose level of HHS (132.1, 188.7, 269.5, 385.0 and 550.0 mg/kg). After
intraperitoneal injection of HHS according to Modified karber’s method, mice were observed for 14 days,
recording signs of toxicity and mortality.
3
.4 In vivo pharmacologic test
Transgenic aP2ꢀSREBPꢀ1c mice were introduced from Jackson Lab (USA) at 6 weeks of age, and
commissioned Model Animal Research Center of Nanjing University to purification [NO. J003393
SCXK(Su)2010ꢀ0001]. Mice were housed in temperatureꢀcontrolled animal facility with 12ꢀhour light and dark
cycle and free to access water and food. Procedures for animal care and handling were in strict accordance with the
Chinese Legislation on the Care and Use of Laboratory Animals. The Animal Experiment Center of Guangdong
Pharmaceutical University approved the experimental procedures and protocols conducted during this study. After
the processes of animal reproduction and genotype identification, transgenic mice at 12 weeks of age were divided
into model group and HHS group, and the wildꢀtype littermates as control group (8 mice in each group). Then,
animals were treated with 40 mg/kg HHS, or 0.5% CMCꢀNa aqueous solution orally once daily by gavage for 13
weeks. At the end of 8 and 13 weeks, the animals were anesthetized with diethyl ether after fasting for 12 h, and
blood samples were collected by retroꢀorbital venous plexus puncture. Serum triglyceride levels were determined
using enzymatic kits. At the end of 9 weeks, following an overnight fast, an oral gavage of 50% glucose (2 g/kg
body weight) was performed on each animal. Blood glucose levels were estimated from the blood samples just
prior to glucose administration (0 min) and 20, 60 and 120 min postꢀglucose administration. By oral glucose
tolerance test (OGTT), area under blood glucose concentration curve (AUC) was calculated from the formula:
AUC= [1/4
× 0 min (mmol/L) + 1/2 × 20 min (mmol/L) + 3/4 × 60 min (mmol/L) + 120 min (mmol/L)]. All
samples were stored in –80°C until further analysis.
3
.5 In vitro cell culture and biological studies
3
.5.1 Cell culture
3
T3ꢀL1 preadipocyte was obtained from the Cell bank of The Chinese Academy of Sciences (Shanghai, China)
and maintained in DMEM and supplemented with 10% FBS and 1% penicillin–streptomycin. Cells were cultured
at 37 °C and under a humidified atmosphere of 5% CO in a cell culture incubator.
2
3
.5.2 CCKꢀ8 assay
Cell viability was assessed using a Cell Counting kit (CCKꢀ8). Cells were seeded in 96ꢀwellꢀplate at the
density of 5000ꢀ8000 cells per well and incubated for 24 h. Then various concentrations of berberine, magnolol
and HHS were added to the medium and incubated for 48 h. Berberine, magnolol and HHS was respectively
dissolved in DMSO with a concentration of 100 mM and stored at ꢀ20℃. The final concentrations of berberine,
magnolol and HHS were 1.25, 2.5, 5, 10, 20, 40 and 80 M, respectively. The subsequently diluted solution was
ꢀ
prepared in the medium with a final DMSO concentration of 0.1%. The control group always treated with the same
amount of DMSO as used in the corresponding experiments. CCKꢀ8 solution was added to each well and the plate
was incubated in an incubator for 2ꢀ4 h, and then the absorbance was measured at 450 nm with a microplate reader
9

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DOI: 10.1039/C6RA15100K
(
Thermo Fisher, USA). IC50 was taken as the concentration that caused 50% inhibition of cell viabilities and
calculated by the Logit method.
3
.5.3 Differentiation of 3T3ꢀL1 adipocytes
For 3T3ꢀL1 adipocytes differentiation, the cells were grown in 6ꢀwell plates to full confluence for two days.
Then cells were induced in differentiation DMEM containing 10% FBS, 0.5 mmol/L IBMX, 1.0 ꢁmol/L DEX and
0 ꢁg/ml insulin. After two days of incubation, the cell culture differentiation medium was changed to DMEM
1
containing 10% FBS and 10 ꢁg/ml insulin. After two days, the cell were grown in DMEM containing 10% FBS for
an additional ten days. When 80–90% of cells exhibited adipocyte morphology, 3T3ꢀL1 adipocytes were used in
5
2
experiments. The methods of differentiation of 3T3ꢀL1 adipocytes was performed as previously described.
.5.4 Oil red O staining and Determination of Lipid Content
The cells were washed twice with phosphateꢀbuffered saline (PBS), and then fixed for 30 min with 10%
3
formaldehyde at room temperature. Then the cells were washed twice with PBS and stained for 1 h in freshly
filtered Oil Red O solution (0.5% in 60% isopropanol). After washing three times with distilled water, the cells
were imaged using a Microscope. Lipid and Oil Red O were extracted using isopropanol, and absorbance was
measured at 520nm using a spectrophotometer.
3
.5.5 Quantitative realꢀtime PCR
Total RNA was extracted from 3T3ꢀL1 adipocytes using RNAiso Plus according to the manufacturer’s
instructions. Then 1ug total RNA from each sample was reverseꢀtranscribed to cDNA according to the protocol of
the reverse transcript system. RNA was treated with gDNA Eraser at 42℃ for 2 min to remove genomic DNA
contamination. Then the cDNA was highly efficient synthesized at 37℃ for 15 min. After cDNA synthesis, the
TM
gene expression levels were analyzed by Quantitative realꢀtime PCR conducted using the CFX96 RealꢀTime
PCR Detection system (BioꢀRad). The relative amount of each gene was calculated using the 2 ꢁꢁCT. All results
ꢀ
were obtained from at least three independent experiments. The mRNA levels of all genes were normalized using
β
ꢀactin as internal control. The oligonucleotide primers used in the experiments are shown in the ESI.
.6 Statistical analysis
3
Data were presented as means ± SD. Statistical analysis was done using SPSS 20.0 statistical software by
oneꢀway analysis of variance (ANOVA) followed by Fisher's LSD tests. Graphical representations were performed
using GraphPad Prism 5. Differences were considered significant when P < 0.05.
4
. Conclusion
In the present study, based on association principle, we designed and synthesized a hybrid compound berberine
and magnolol, as a mutual drug, possessing potential biological activities as well as safety and low toxicity. The
method of piecing the two molecules together is carried out to obtain the target product based on chemical bonding.
The results of in vivo study demonstrated that, administration with 40 mg/kg HHS orally to aP2ꢀSREBPꢀ1c mice
reduced plasma glucose and lipid levels. Additionally, HHS could obviously downꢀregulate the expression of
PPARγ2, C/EBPα, FAS and UCP2 mRNA and upꢀregulate the expression of adiponectin mRNA in 3T3ꢀL1
adipocytes. Although the underlying action mechanism for HHS is not currently well understood, the HHS induced
reduction of TG in mouse model is remarkable. For further studies, as the 1, 9, 10, 13ꢀposition of berberine are
used to structural modification, the association of these positions with magnolol can be considered.
1
0

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[
1
2