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

Abstract

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.

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1. Introduction

Diabetes mellitus (DM) is a complex endocrine and metabolic disorder characterized by high levels of glucose and lipids in the blood.^1,2 A recent study found that the estimated prevalence of diabetes in Chinese adults is 11.6% and the prevalence of prediabetes is 50.1%.³ It is estimated that approximately 4% of the population worldwide suffer from diabetes, which is expected to increase by 5.4% in 2025.⁴ Obesity is a key risk factor for type 2 diabetes as it desensitizes glucose recipient organs to the action of insulin.² Growing evidence shows that obesity is characterized by the intra-abdominal accumulation of visceral fat, which depends on the proliferation of adipocytes and differentiation of preadipocytes.^5–7 The expression of lipogenic genes in adipocytes is known to be mainly regulated by CCAAT-enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPARγ).^8–10 It has long been known that transient expression of the C/EBPβ and C/EBPδ genes is manifested in the early stage of cell differentiation, and C/EBPα is expressed in the late stage. Once C/EBPα is activated, it can trigger the expression of several genes that influence the intracellular deposition of lipid droplets, insulin resistance, the metabolism of adipose and the balance of energy. It has been reported that the activation of PPARγ upregulates the expression of FAS and UCP2, which results in increases in glucose uptake and lipogenesis.

Traditional Chinese medicine (TCM) is based on herbal extracts containing natural active components. In TCM, herb couples, as the basic compositional units of Chinese herbal formulae, have specific clinical significance.^11,12 Coptis Magnolia Decoction is a representative formula composed of Rhizoma coptidis and Magnolia officinalis in mass prescriptions, which is derived from The Complete Record of Holy Benevolence, a traditional Chinese medical classic compiled during the Song dynasty. Berberine and magnolol are its major active components, which can be used as lead compounds for chemical drugs with the corresponding structural modifications. Berberine is an isoquinoline alkaloid isolated from the Chinese herbal extract Rhizoma coptidis, which mainly exists in Ranunculaceae, Rutaceae and Berberidaceae. The high medicinal value of berberine and the physiological activities of berberine derivatives have recently attracted significant attention owing to their extensive pharmacological effects and usefulness in biomedical applications.^13,14 Studies have shown that berberine possesses anti-inflammatory,¹⁵ hypotensive, anti-platelet aggregation,^16–18 hypoglycemic^19,20 and hypolipidemic^21,22 activities. Also, derivatization of the berberine core structure has resulted in the development of molecules with enhanced biological effects.^23,24 Magnolol is a polyphenolic biphenyl compound from the Chinese medicinal herb Magnolia officinalis, which has been reported to have multiple biological effects, including anti-inflammatory,²⁵ antioxidant,^26,27 antiangiogenic²⁸ and antidiabetic²⁹ 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 research into the synthesis and structural modification of berberine and magnolol has been extensively carried out recently, little information has been available on the synthesis of hybrid compounds consisting of berberine and magnolol. Therefore, this study aims to investigate the process of the structural combination of berberine and magnolol and to synthesize a new compound with pharmacodynamic activity. In this study, the structure of the new compound was confirmed by spectroscopy (¹H NMR, ¹³C NMR and HRMS spectra). We investigated the hypoglycemic and hypolipidemic activities of the new compound using transgenic aP2-SREBP-1c mice in vivo. Furthermore, we determined its antiobesity effects using 3T3-L1 cells in vitro, which measured mRNA expression of adipogenic transcription factors using real-time PCR.

2. Results and discussion

2.1 Design of berberine–magnolol hybrid compound (HHS)

The synthesis of HHS was conveniently undertaken, as outlined in Scheme 1. With berberine hydrochloride as a starting material, berberrubine 1 was synthesized by demethylation of the 9-position under microwave irradiation in excellent yield (92%). Afterwards, berberrubine 1 on reaction with 1,2-dibromoethane in acetonitrile as the solvent, followed by heating under reflux, gave 9-(2-bromoethyl)berberine hydrochloride 2 in good yield (56%). Finally, the hybrid compound 3 (HHS) was obtained by the reaction of 9-(2-bromoethyl)berberine hydrochloride 2 with magnolol and Na₂CO₃ (1 : 1.5 : 6) in acetonitrile under reflux in moderate yield (24%). After crystallization and purification, the structural characterization of these products was performed by ¹H NMR, ¹³C NMR and HRMS spectroscopy, and the profile of HHS was established by HPLC (ESI†).

Scheme 1 Synthesis of HHS. Reagents and conditions: (1) DMF, microwave power (400 W), reflux, 15 min, 92%; (2) acetonitrile, 1,2-dibromoethane, reflux, 3 h, 56%; (3) acetonitrile, anhydrous sodium carbonate, reflux, 8 h, 24%.

Previous studies have suggested that berberrubine was usually synthesized by vacuum pyrolysis, with low yield and purity owing to uneven heating.³⁰ In a preliminary experiment, we adopted a microwave synthesis method as described in the literature,^31,32 but no chemical transformation occurred. After that, we used DMF, which has a high boiling point and high stability, as the reaction medium under microwave irradiation to afford berberrubine in 92% yield. 9-(2-Bromoethyl)berberine hydrochloride was synthesized using acetonitrile as the solvent. There have been few studies on the use of two natural active molecules bonded together through a chemical bond to synthesize a hybrid compound.

2.2 Acute toxicity study

After intraperitoneal injection of HHS, the various behaviors of mice were observed closely for the first 4 h. Mice administered with HHS at a dose of 132.1 mg kg⁻¹ displayed no significant changes in behavior or breathing or postural abnormalities. However, significant changes occurred in mice in other groups, including unsteady walking, difficult breathing, and death, even the existence of moderate organ damage. Moreover, the mortality of mice treated with doses of 188.7 and 385.0 mg kg⁻¹ was 0.2 and 0.8, respectively, in a dose-dependent manner. The LD₅₀ value (lethal dose 50) was 279 mg kg⁻¹, and the 95% confidence interval was 234.6–332.5 mg kg⁻¹ (Table 1). The results obtained from this study demonstrate that after a single intraperitoneal injection the acute toxicity of HHS was lower than that of berberine, whereas the toxicity of berberine in previous reports was shown by LD₅₀ values ranging between 38.8 and 85.5 mg kg⁻¹ (ref. 33) and an LD₅₀ value of 50 mg kg⁻¹.³⁴

Table 1 Results of acute toxicity testing of HHS administered by intraperitoneal injection to NIH mice within 14 d^a

Group	Dose (mg kg^−1)	Mortality	LD50 (mg kg^−1)	95% confidence interval (mg kg^−1)
a These data were calculated according to the modified Karber's method.
1	550.0	10/10	279.3	234.6–332.5
2	385.0	8/10
3	269.5	4/10
4	188.7	2/10
5	132.1	0/10
Control	0	0
		∑p = 2.4

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2.3 Effect of HHS on glucose and lipid metabolism in aP2-SREBP-1c mice

It has been known that an excessive amount of white tissue (obesity) is a crucial factor in insulin resistance.^35–37 In contrast to all other mouse models of insulin resistance,^38,39 aP2-SREBP-1c mice are not obese, but exhibit a distinct syndrome that includes lipodystrophy, insulin resistance, diabetes mellitus and fatty liver.^40,41 In the present study, we examined the effect of HHS on glucose and lipid metabolism disorders. An OGTT was performed at the end of 9 weeks. As shown in Fig. 1A, the blood glucose level in the model group was much higher than that in the control group. However, the mice treated with HHS displayed a significant decrease in blood glucose level compared with the 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 weeks, the plasma TG level in the model group was significantly higher than that in the control group, whereas the HHS group exhibited a significantly decreased TG level compared with the model group. According to an analysis of the TG level, compared with 0 weeks (0.85 ± 0.43) the TG levels at 8 and 13 weeks in the control group displayed no significant change, and the level in the model group increased significantly. Conversely, after treatment with HHS for 8 and 13 weeks, the plasma TG level decreased significantly compared with 0 weeks. These data indicated that HHS improved glucose and lipid metabolism in aP2-SREBP-1c mice.

Fig. 1 In vivo effects of HHS on glucose and lipid metabolism in aP2-SREBP-1c mice. Mice at 12 weeks of age were divided into 3 groups. HHS was suspended in a 5% aqueous solution of CMC-Na and administered orally for 13 weeks. (A) 9 weeks after final dosing, following an overnight fast, an oral gavage of 50% glucose (2 g kg⁻¹ body weight) was performed on each animal. Blood glucose levels were estimated from blood samples just prior to glucose administration (0 min) and 20, 60 and 120 min after glucose administration. (B) By an oral glucose tolerance test (OGTT), the area under a blood glucose concentration curve (AUC) was calculated from the formula: AUC = [1/4 × 0 min (mmol L⁻¹) + 1/2 × 20 min (mmol L⁻¹) + 3/4 × 60 min (mmol L⁻¹) + 120 min (mmol L⁻¹)]. (C) At the end of 8 and 13 weeks, mice were anesthetized with diethyl ether after fasting for 12 h and plasma triglyceride levels were measured. Each group was composed of 8 mice. Data are presented as means ± SD. *p < 0.05, **p < 0.01 (one-way ANOVA) compared with the model group.

2.4 Cytotoxicity of HHS to 3T3-L1 preadipocytes

In the present study, the cytotoxicity of berberine, magnolol and HHS to 3T3-L1 preadipocytes was investigated using CCK-8. The results are shown in Fig. 2. Within the range of 0–80 μM, the cell viability of the magnolol group decreased with an increase in concentration in a dose-dependent manner (Fig. 2A). When the concentration was greater than 20 μM, berberine had an obvious inhibitory effect on 3T3-L1 preadipocytes (Fig. 2B). However, the result showed that HHS could significantly reduce cell viability when the concentration reached 10 μM (Fig. 2C). Moreover, the IC₅₀ values of berberine, magnolol and HHS were 19.43 ± 0.56, 27.93 ± 0.63 and 11.54 ± 0.52 μM, respectively. These observations suggested that HHS exerted a stronger effect than berberine or magnolol on the viability of 3T3-L1 preadipocytes.

Fig. 2 Cytotoxic activity of magnolol (Mag) (A), berberine (Ber) (B) and HHS (C). Cells were seeded in a 96-well plate at a density of 5000–8000 cells per well and incubated for 24 h. Then, various concentrations of berberine, magnolol and HHS (1.25, 2.5, 5, 10, 20, 40 and 80 μM) were added to the medium, which was incubated for 48 h. Cells treated with 0.1% DMSO served as controls (con). Values are means ± SD (n = 3 in each group).

2.5 Differentiation of 3T3-L1 preadipocytes and Oil Red O staining

The cultured 3T3-L1 preadipocytes were in a fibroblast-like form with a long fusiform or polygonal shape (Fig. 3A). The cells could successfully be trans-differentiated into adipocytes with an appreciable amount of accumulated lipid droplets after induction for 10 days, which was confirmed by staining with Oil Red O (Fig. 3B and C).⁴² Lipids and Oil Red O were extracted using isopropanol, and the absorbance was measured at 520 nm. The result of staining with Oil Red O is shown in Fig. 3D. The OD₅₂₀ value was significantly increased in the model group compared with the control group.

Fig. 3 3T3-L1 preadipocytes were successfully differentiated into adipocytes. Differentiated 3T3-L1 cells were identified using staining with Oil Red O. (A) Controls (preadipocytes), (B) differentiated adipocytes, (C) model (differentiated adipocytes, using staining with Oil Red O), (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 the mean ± SD. **p < 0.01 compared with the model group.

2.6 Effects of HHS on expression of differentiation-related genes in 3T3-L1 adipocytes

Research shows that PPARγ and C/EBPα play major roles in the process of adipocyte differentiation, and are also critical in the regulation of lipid metabolism and glucose homeostasis.^43–46 To quantitatively describe the effects 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 B, the levels of PPARγ2 and C/EBPα mRNA were significantly increased in the MD group compared with the NC group, and HHS was effective in reducing their levels. Ten days after initiating treatment, the groups treated with Mag and Ber at 5 μM experienced a significant effect on PPARγ2 and C/EBPα mRNA levels compared with the MD group, which was consistent with previous studies.^47–49 The group treated with Mag at 2.5 μM did not undergo substantial change in PPARγ2 and C/EBPα mRNA levels. Treatment with HHS significantly decreased the PPARγ2 mRNA level in a dose-dependent manner. However, surprisingly, the group treated with HHS at 5 μM underwent a less effective decrease in the C/EBPα mRNA level than the group treated with Ber at 5 μM. Although the group treated with HHS experienced a significant decrease in the C/EBPα mRNA level, this was not in a dose-dependent manner. By a comparison of the effects of both doses of HHS on PPARγ2 and C/EBPα, we found that HHS at 2.5 μM led to a greater reduction in mRNA expression than berberine or magnolol. Thus, we concluded that HHS to some extent displayed a 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 3T3-L1 cells. Three independent experiments were performed and data are shown as means ± SD. *p < 0.05, **p < 0.01 compared with the DM group. NC = control group (cells treated with 0.1% DMSO). MD = model group (mature 3T3-L1 adipocytes).

2.7 Effects of HHS on expression of adipogenesis-related genes in 3T3-L1 adipocytes

Adipocyte differentiation is a complex process that is controlled and regulated by various adipocyte-specific genes such as FAS, UCP2, adiponectin and others, which participate in creating the adipocyte phenotype.⁵⁰ Here, we focused more on downstream genes of PPARγ2 and C/EBPα and studied how their mRNA expression is influenced by HHS. When 3T3-L1 preadipocytes differentiated into adipocytes, we tested the mRNA levels of FAS, UCP2 and adiponectin in all groups. As shown in Fig. 5A–C, significant increases in the mRNA expression of FAS and UCP2 were observed in the MD group compared with the NC group, which confirmed the presence of lipid accumulation. After treatment with berberine, magnolol and HHS, it was found that cells treated with 5 μM HHS experienced greater reductions in the mRNA expression of FAS and UCP2 than those treated with berberine and magnolol, whereas the mRNA expression of adiponectin increased more obviously, which demonstrated a good antiobesity effect.⁵¹

Fig. 5 Effects of HHS on mRNA expression levels of (A) FAS, (B) UCP2 and (C) adiponectin in 3T3-L1 cells. Three independent experiments were performed and data are shown as means ± SD. *p < 0.05, **p < 0.01 compared with the MD group. NC = control group (cells treated with 0.1% DMSO). MD = model group (mature 3T3-L1 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 modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). Insulin (INS), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX) and Oil Red O were purchased from Sigma Aldrich (St Louis, MO). RNAiso Plus, PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time), and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) were purchased from Takara (Tokyo, Japan). All other chemicals and reagents were of analytical grade and were purchased from Aldrich Chemical Co. (Beijing, China). ¹H and ¹³C NMR spectra were recorded using a Bruker Avance 400 at room temperature with tetramethylsilane (TMS) as an internal standard and chloroform-d (CDCl₃) as solvent. Mass spectra were recorded using a DSQ mass spectrometer (Thermo, USA).

3.2 Synthesis of HHS

3.2.1 General procedure for the synthesis of berberrubine (1). Berberine (1 g) was weighed and dissolved in DMF (25 mL), and then several zeolites were added. The reaction mixture was refluxed for 15 min at a microwave power of 400 W. After completion, the reaction mixture was diluted with 40 mL water and refrigerated until crystallization was complete. The combined extracts were washed with petroleum ether and evaporated in a vacuum. The crude product obtained was purified by PIPO-02 macroporous resin to obtain pure compound 1 (berberrubine) in 92% yield.
Berberrubine 1. Red needle crystals; mp 281.6–282.4 °C. ¹H NMR (CDCl₃, 300 MHz) δ: 7.35 (1H, s, H-1), 6.76 (1H, s, H-4), 3.04 (1H, t, J = 5.8, H-5), 4.53 (1H, t, J = 5.8, H-6), 9.20 (1H, s, H-8), 6.83 (1H, d, J = 9.1, H-11), 7.46 (1H, d, J = 9.1, H-12), 7.97 (1H, s, H-13), 5.95 (1H, s, H-15), 3.81 (1H, s, H-16); ¹³C NMR (CDCl₃, 300 MHz) δ: 106.27 (C-1), 121.81 (C-1a), 151.42 (C-2), 149.97 (C-3), 109.14 (C-4), 131.16 (C-4a), 57.04 (C-6), 143.72 (C-8), 134.21 (C-8a), 147.84 (C-9), 147.84 (C-10), 120.21 (C-11), 123.21 (C-12), 131.16 (C-12a), 124.22 (C-13), 136.23 (C-14), 103.68 (C-15), 55.94 (C-16); LC-MS (ESI⁺, m/z) [C₁₉H₁₆NO₄]⁺: 322.2 [M − Cl]⁺.

3.2.2 General procedure for the synthesis of 9-(2-bromoethyl)berberine hydrochloride (2). Compound 1 (3.23 mmol) was refluxed in acetonitrile (200 mL) with stirring and heating to 85 °C. Then, 1,2-dibromoethane (20 mL) was added and the reaction mixture was refluxed for 3 h. The combined extract continued to be refrigerated until crystallization was complete. After completion, the crude product obtained was diluted with acetonitrile and petroleum ether and evaporated in a vacuum to obtain pure compound 2 [9-(2-bromoethyl)berberine hydrochloride] in 56% yield.
9-(2-Bromoethyl)berberine hydrochloride 2. Yellow crystals; mp 273.6–274.4 °C. ¹H NMR (CDCl₃, 300 MHz) δ: 7.68 (1H, s, H-1), 6.97 (1H, s, H-4), 3.87 (2H, t, J = 5.8, H-5), 4.74 (2H, t, J = 5.8, H-6), 9.87 (1H, s, H-8), 8.04 (1H, d, J = 9.1, H-11), 8.15 (1H, d, J = 9.1, H-12), 8.74 (1H, s, H-13), 6.11 (2H, s, H-15), 3.30 (3H, s, H-16), 4.65 (2H, t, J = 5.6, H-17), 4.12 (2H, t, J = 5.6, H-18); ¹³C NMR (CDCl₃, 300 MHz) δ: 106.43 (C-1), 151.64 (C-2), 149.39 (C-3), 109.29 (C-4), 131.77 (C-4a), 27.59 (C-5), 57.53 (C-6), 143.27 (C-8), 134.68 (C-8a), 146.24 (C-9), 146.24 (C-10), 121.57 (C-11), 124.94 (C-12), 131.77 (C-12a), 127.53 (C-13), 139.26 (C-14), 121.34 (C-14a), 103.31 (C-15), 56.88 (C-16), 57.53 (C-17), 74.49 (C-18); LC-MS (ESI⁺, m/z) [C₂₁H₁₉BrNO₄]⁺: 429.1 [M − Cl]⁺.

3.2.3 General procedure for the synthesis of HHS (3). To a solution of compound 2 (1.21 mmol) in acetonitrile (150 mL), anhydrous sodium carbonate (10.85 mmol) and magnolol (1.81 mmol) were added and the solution was stirred and heated to 85 °C. The reaction mixture was refluxed for 8 h. The reaction mixture was immediately filtered and then dissolved in dimethyl sulfoxide (DMSO). The solution mixture obtained was purified by a C18 column and washed with 30%, 40%, 50%, and 60% methanol. The concentrated solution obtained was purified by a silica gel column and washed with petroleum ether–ethyl acetate (1 : 1). The crude product obtained was evaporated in a vacuum to obtain pure compound 3 (HHS) in 24% yield.
HHS (3). {9-(2′-((5′,5′-Diallyl-2′-hydroxy-[1′,1′′-biphenyl]-2′-yl)oxy)ethoxy)-10-methoxy-5,6-dihydro-[l,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium}.

Yellow solid; mp 145.2–146.1 °C. ¹H NMR (CDCl₃, 500 MHz) δ: 7.64 (1H, s, H-1), 6.91 (1H, s, H-4), 3.87 (2H, t, J = 5.8, H-5), 4.74 (2H, t, J = 5.8, H-6), 9.38 (1H, s, H-8), 7.90 (1H, d, J = 9.1, H-11), 8.04 (1H, d, J = 9.1, H-12), 8.56 (1H, s, H-13), 6.11 (2H, s, H-15), 3.30 (3H, s, H-16), 4.65 (2H, t, J = 5.6, H-17), 4.12 (2H, t, J = 5.6, H-18), 7.00 (1H, d, H-21), 7.11 (1H, d, J = 8.45, H-23), 6.69 (1H, d, J = 8.45, H-24), 3.00 (1H, d, H-25), 6.39 (1H, d, H-30), 5.99–5.86 (1H, m, J = 9.6, 16.9, H-26), 5.81–5.67 (1H, m, J = 9.6, 16.9, H-27); ¹³C NMR (CDCl₃, 300 MHz) δ: 106.81 (C-1), 152.38 (C-2), 150.21 (C-3), 109.65 (C-4), 131.76 (C-4a), 28.39 (C-5), 57.83 (C-6), 143.27 (C-8), 134.13 (C-8a), 146.70 (C-9), 152.06 (C-10), 123.88 (C-12), 145.30 (C-12a), 127.65 (C-13), 121.34 (C-14a), 121.75 (C-11), 103.97 (C-15), 56.88 (C-16), 69.31 (C-17), 75.19 (C-18), 155.69 (C-19), 125.04 (C-20), 132.70 (C-21), 129.66 (C-22), 129.25 (C-23), 113.69 (C-24), 40.49 (C-25), 131.76 (C-26), 116.09 (C-27), 153.83 (C-28), 126.51 (C-29), 129.92 (C-30), 133.57 (C-31), 129.25 (C-32), 115.86 (C-33), 40.65 (C-34), 131.93 (C-35), 116.59 (C-36); LC-MS (ESI⁺, m/z) [C₃₉H₃₆NO₆]⁺: 615.3 [M − Cl]⁺.

3.3 Acute toxicity study

The strain of mice used in this study was NIH, which was originally bred by the National Institutes of Health (NIH). Both sexes of SPF (specific pathogen-free) NIH mice were investigated by regular methods for toxicological evaluation. NIH mice weighing 18–22 g [no. SCXK (Yue) 2008-0002], half male and half female, were purchased from the Medical Laboratory Animal Center of Guangdong (Guangdong, China). Mice were housed in a temperature-controlled animal facility with a 12 hour light and dark cycle and were 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⁻¹). After intraperitoneal injection of HHS according to the modified Karber's method, mice were observed for 14 days and signs of toxicity and mortality were recorded.

3.4 In vivo pharmacologic test

Transgenic aP2-SREBP-1c mice were introduced from Jackson Lab (USA) at 6 weeks of age and the Model Animal Research Center of Nanjing University was commissioned for purification [no. J003393 SCXK (Su) 2010-0001]. Mice were housed in a temperature-controlled animal facility with a 12 hour light and dark cycle and were 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 a model group and an HHS group, and the wild-type littermates served as a control group (8 mice in each group). Then, the animals were treated with 40 mg kg⁻¹ HHS or a 0.5% aqueous solution of CMC-Na 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 retro-orbital 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⁻¹ body weight) was performed on each animal. Blood glucose levels were estimated from blood samples just prior to glucose administration (0 min) and 20, 60 and 120 min after glucose administration. By an oral glucose tolerance test (OGTT), the area under a blood glucose concentration curve (AUC) was calculated from the formula: AUC = [1/4 × 0 min (mmol L⁻¹) + 1/2 × 20 min (mmol L⁻¹) + 3/4 × 60 min (mmol L⁻¹) + 120 min (mmol L⁻¹)]. All samples were stored at −80 °C until further analysis.

3.5 In vitro cell culture and biological studies

3.5.1 Cell culture. 3T3-L1 preadipocytes were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China), maintained in DMEM and supplemented with 10% FBS and 1% penicillin–streptomycin. Cells were cultured at 37 °C under a humidified atmosphere of 5% CO₂ in a cell culture incubator.

3.5.2 CCK-8 assay. Cell viability was assessed using a cell counting kit (CCK-8). Cells were seeded in a 96-well plate at a density of 5000–8000 cells per well and incubated for 24 h. Then, various concentrations of berberine, magnolol and HHS were added to the medium, which was incubated for 48 h. Berberine, magnolol and HHS were dissolved in DMSO with a concentration of 100 mM and stored at −20 °C. The final concentrations of berberine, magnolol and HHS were 1.25, 2.5, 5, 10, 20, 40 and 80 μM. Subsequently, a diluted solution was prepared of the medium with a final DMSO concentration of 0.1%. The control group was always treated with the same amount of DMSO as was used in the corresponding experiments. CCK-8 solution was added to each well and the plate was incubated in an incubator for 2–4 h, and then the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher, USA). The IC₅₀ was taken to be the concentration that caused 50% inhibition of cell viability and was calculated by the logit method.

3.5.3 Differentiation of 3T3-L1 adipocytes. For the differentiation of 3T3-L1 adipocytes, cells were grown in 6-well plates to full confluence for two days. Then, cells were induced in differentiation DMEM containing 10% FBS, 0.5 mmol L⁻¹ IBMX, 1.0 μmol L⁻¹ DEX and 10 μg mL⁻¹ insulin. After incubation for two days, the cell culture differentiation medium was changed to DMEM containing 10% FBS and 10 μg mL⁻¹ insulin. After two days, the cells were grown in DMEM containing 10% FBS for an additional ten days. When 80–90% of the cells exhibited adipocyte morphology, 3T3-L1 adipocytes were used in experiments. The method of differentiation of 3T3-L1 adipocytes was performed as previously described.⁵²

3.5.4 Oil Red O staining and determination of lipid content. Cells were washed twice with phosphate-buffered saline (PBS) and then fixed for 30 min with 10% formaldehyde at room temperature. Then, the cells were washed twice with PBS and stained for 1 h in a freshly filtered solution of Oil Red O (0.5% in 60% isopropanol). After washing three times with distilled water, the cells were imaged using a microscope. Lipids and Oil Red O were extracted using isopropanol, and the absorbance was measured at 520 nm using a spectrophotometer.

3.5.5 Quantitative real-time PCR. Total RNA was extracted from 3T3-L1 adipocytes using RNAiso Plus according to the manufacturer's instructions. Then, 1 μg total RNA from each sample was reverse-transcribed into cDNA according to the protocol of the reverse transcription system. RNA was treated with gDNA Eraser at 42 °C for 2 min to remove genomic DNA contamination. Then, cDNA was synthesized with high efficiency at 37 °C for 15 min. After synthesis of cDNA, gene expression levels were analyzed by quantitative real-time PCR conducted using the CFX96™ real-time PCR detection system (Bio-Rad). The relative amount of each gene was calculated using the 2^−ΔΔC_T formula. All results were obtained from at least three independent experiments. The mRNA levels of all genes were normalized using β-actin as an internal control. The oligonucleotide primers used in the experiments are shown in the ESI.†

3.6 Statistical analysis

Data are presented as means ± SD. Statistical analysis was performed using SPSS 20.0 statistical software by one-way analysis of variance (ANOVA) followed by Fisher's LSD tests. Graphical representations were performed using GraphPad Prism 5. Differences were considered to be significant when p < 0.05.

4. Conclusion

In the present study, based on the association principle, we designed and synthesized a hybrid compound of berberine and magnolol as a combined drug, which possessed potential biological activities, as well as safety and low toxicity. The method of linking the two molecules together was carried out to obtain the target product based on chemical bonding. The results of an in vivo study demonstrated that the administration of 40 mg kg⁻¹ HHS orally to aP2-SREBP-1c mice reduced plasma glucose and lipid levels. In addition, HHS could significantly downregulate the expression of PPARγ2, C/EBPα, FAS and UCP2 mRNA and upregulate the expression of adiponectin mRNA in 3T3-L1 adipocytes. Although the underlying mechanism of action of HHS is not currently well understood, the reduction in TG levels induced by HHS in a mouse model is remarkable. For further studies, as the 1, 9, 10, and 13-positions of berberine are open to structural modification, the binding of these positions to magnolol can be considered.

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Footnote

† Electronic supplementary information (ESI) available: The ¹H NMR, ¹³C NMR and HRMS spectra of the synthesized compounds; HPLC profile of HHS; the oligonucleotide primer sequence used for real-time PCR. See DOI: 10.1039/c6ra15100k

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