PMFs (the main component was nobiletin, with 98% purity) were extracted from Pericarpium Citri Reticulatae in our laboratory. The ELISA kit for insulin detection was purchased from Gibco BRL (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories, Inc. (Logan, UT, USA). The reverse transcriptase-polymerase chain reaction (RT-PCR) kit and DNaseI (RNase-free) were purchased from Life Technologies (Gaithersburg, MD, USA). MTT, diethylpyrocarbonate, RPMI-1640 and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). HEPES, kanamycin and ampicillin were purchased from Amersham (Piscataway, NJ, USA). Trypsin and standard PMF samples were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Pericarpium Citri Reticulatae were obtained from Zaolutang (Xian, China). High-performance liquid chromatography (HPLC) grade methanol and other chemicals (analytical grade) were obtained from Merck (Darmstadt, Germany).

PMFs were extracted from dried Pericarpium Citri Reticulatae as previously described (19,20). The measurement of nobiletin, the main component of PMFs, was performed on a Thermo Finnigan HLPC system (Thermo-Finnigan, San Jose, CA, USA) as previously described (16,21).

The rat INS-1 pancreatic β-cell line was cultured in RPMI-1640 medium supplemented with 10% FBS, 10 mmol/l HEPES, 11.1 mmol/l glucose, 50 μg/ml ampicillin, 50 μg/ml kanamycin, 2.0 mmol/l glutamine, 1.0 mmol/l sodium pyruvate and 50 μmol/l β-mercaptoethanol. The cells were maintained at 37°C and in 5% CO₂.

For the measurement of cell doubling time, the INS-1 cells were harvested by trypsinization and seeded into normal 96-well dishes (2.5×10⁵/well). The optical density at 490 nm was detected at the indicated times by the VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

The INS-1 cells (2.5×10⁴/well) were seeded into 96-well plates. The medium was changed after 40–50% confluence was reached. The cells were incubated with fresh medium containing different concentrations of PMFs (0, 12.5, 25, 50, 60, 70, 80 and 90 μg/ml) for 72 h and the medium was changed every 24 h. The control cells were treated with DMSO for the same time. After 72 h, cell viability was assessed using the MTT method as previously described (21). Briefly, the cells were pulsed with 20 μl of a 5-mg/ml MTT stock in PBS and incubated for 4 h, after which time 150 μl of DMSO was added. The plates were then placed on a shaker for 10 min and absorption was read on a VICTOR 3 multilabel plate reader (Perkin-Elmer, Turku, Finland) using a test wavelength of 490 nm. Test reagents (DMSO) alone were added to the medium to provide a blank.

The INS-1 cells (2.5×10⁴/well) were seeded into 24-well plates. After cell confluence reached 60–70%, the medium was removed and the cells were treated with Krebs-Ringer bicarbonate buffer (KRB; 129 mmol/l NaCl, 5 mmol/l NaHCO₃, 4.8 mmol/l KCl, 1.2 mmol/l KH₂PO₄, 1.2 mmol/l MgSO₄·7H₂O and 2.5 mmol/l CaCl₂) without glucose for 60 min to ensure that the cells were in a non-glucose metabolic state. Subsequently, KRB was removed and the cells were incubated with fresh KRB containing different concentrations of glucose (3, 11 and 20 mmol/l) and PMFs (12.5, 25, 37.5 and 50 μg/ml) for 60 min. After the 60-min incubation, the supernatants were collected for insulin measurement by ELISA, according to the manufacturer’s instructions.

The cells were lysed with TRIzol and RNA was extracted with the RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The RNA samples were treated with DNase and used for RT-PCR according to the manufacturer’s instructions. The sequences of the primers used were as follows: β-actin: sense, 5′-AGAAGGATTCCTATGTGGGCG-3′ and antisense, 5′-CATGTCGTCCCAGTTGGTGAC-3′; insulin: sense, 5′-CAGCTCCACACTCCAGGTAC-3′ and antisense, 5′-CTTTCGCTGGGCTCTGAAGG-3′; pancreatic and duodenal homeobox 1 (PDX1): sense, 5′-ATGAATAGTGAG GAGCAGTACTACG-3′ and antisense, 5′-CCGGGGTTC CTGCGGTC-3′. For relative quantification, we calculated the n-fold differential expression with the ΔCt method (Ct, threshold cycle of PCR amplification at which the product is first detected by fluorescence), which compares the amount of target gene amplified normalized to the β-actin endogenous reference. The sizes of the amplified products were measured by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide staining (22).

The results are expressed as the means ± standard deviation. One-way analysis of variance (ANOVA) and the Student’s t-test were performed using SPSS software for Windows, version 12.0, 2003 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. All the assays were performed in triplicate.

In this study, PMFs were extracted from dried tangerine peel with ethyl acetate at room temperature and the extract was analyzed by HPLC (Fig. 1). PMF crystals (1.05 g) were extracted from 1 kg dried tangerine peel and the content of PMFs was 83.9%, according to the peak area regression method. These compounds were used for the subsequent experiments.

To ensure that the INS-1 cells were in an optimal state for the subsequent experiments, the cell doubling time was determined using a test wavelength of 490 nm. As shown in Fig. 2, the growth of the cells followed an exponential trend between 48 and 144 h. After culture for 120 h, the cell proliferation rate decreased. The confluence reached 70–80% after 6 h in culture and did not increase any further. These preliminary tests formed the basis for the subsequent experiments.

To investigate the cytotoxic effect of PMFs on INS-1 cells, the cell viability under treatment with different concentrations of PMFs was measured with the MTT assay. The cells were incubated with different concentrations of PMFs for 72 h. As shown in Table I, following a 72-h incubation, no cytotoxicity was observed in the 0–70 μg/ml concentration range compared to the DMSO-treated group (P>0.05). However, when INS-1 cells were treated with PMF concentrations of ≥80 μg/ml, a cytotoxic effect was observed after 12 h of incubation. Morphological changes, such as cell shrinkage and condensation, were observed. After 24 h of incubation, there were more cells in suspension and the number of suspended cells increased at later times. Significant differences were observed (P<0.05).

To achieve a preliminary understanding of the molecular mechanism of PMF function in diabetes, the INS-1 cells were treated with different concentrations of glucose, mimicking a hyperglycemic environment. The insulin secretion by INS-1 cells treated with different concentrations of glucose and PMFs was assessed using ELISA. As shown in Fig. 3, following incubation with the same concentration of glucose and different concentrations of PMFs, no significant difference was observed among groups (P>0.05). At the same concentration of PMFs, no significant difference was observed in insulin secretion at different concentrations of glucose (P>0.05).

Subsequently, we investigated whether PMFs affect the expression of insulin mRNA using RT-PCR. The INS-1 cells were treated with 25 μg/ml PMFs and were collected at indicated times for RNA isolation and RT-PCR. As shown in Fig. 4A and B, the incubation with PMFs exerted no significant effect on insulin mRNA expression in the INS-1 cells compared to that in the control cells (P>0.05).

We also analyzed the mRNA levels of PDX1, which is an insulin secretion-stimulating factor. Following incubation with 25 μg/ml PMFs for different time periods (Fig. 4C and D), the PDX1 mRNA levels were not found to be significantly altered compared to those in the control group (P>0.05).