Synthesis and Preliminary Biologic Activity Evaluation of Nitric Oxide-Releasing Andrographolide Derivatives in RIN-m Cells

Abstract

Pancreatic β-cell dysfunction and death are important feature of diabetes mellitus. Beta-cell protection has demonstrated clinical benefits in the treatment of this disease. In the present study, andrographolide derivatives with nitric oxide (NO)-releasing capability were synthesized and their protective effects against tert-butyl hydroperoxide (t-BHP) induced cell damage were investigated in RIN-m cells. Compound 6b was found to release a moderate amount of NO and was more potent than its natural parent andrographolide in inhibiting cell apoptosis. These findings suggested that andrographolide derivatives with NO-releasing capacity may be a potential therapy for diabetes.

Andrographolide (Fig. 1), the main bioactive component of the medicinal plant Andrographis paniculata, has a variety of biological activities including anti-inflammation,¹⁾ anti-bacteria,^2–4) anti-malaria,⁵⁾ hepatoprotection,^6,7) immunomodulation,⁸⁾ and anti-cancer.^9,10) In addition, previous studies showed that andrographolide lowered blood glucose levels and possessed anti-diabetic properties.¹¹⁾ It was reported that the anti-diabetic effects of andrographolide were attributed to its anti-inflammation and hypoglycemic effects.¹²⁾ It has been documented that andrographolide attenuated inflammation primarily through inhibition of the nuclear factor-kappa B (NF-κB) signalling pathway.^13,14) Jin et al. reported that andrographolide inhibited tumor necrosis factor-alpha (TNF-α) induced insulin resistance and improved insulin sensitivity through inhibition of the NF-κB pathway.¹⁵⁾ In an effort to find more effective therapies for diabetes, we have recently synthesized numerous andrographolide derivatives, investigated their anti-diabetic activities and their potential mechanisms of action.¹⁶⁾

Fig. 1. The Structures of Andrographolide and AL-1

Nitric oxide (NO) is a small, diffusible and highly reactive molecule with many important biological and physiological functions. The role NO plays in insulin secretion has been controversial.¹⁷⁾ Although excessive NO production within β-cells may trigger oxidative/nitrosative stress leading to cell death, NO also served as an important β-cell stimulus-secretion coupling factor.^18–20) Evidence suggested that exogenous NO and endogenous glucose-stimulated β-cell NO augmented insulin release.²¹⁾ In addition, studies had demonstrated that NO stimulated insulin secretion by stimulating calcium release from mitochondria.²²⁾ A recent report also suggested that low levels of NO protected β-cells from thapsigargin-induced apoptosis.²³⁾

Building on our previous discovery that andrographolide and its lipoic acid conjugate AL-1 (Fig. 1) significantly protected β-cells in diabetic rats and the finding that NO offered protection to β-cells, we wanted to know if andrographolide derivatives bearing NO-donating moieties could release NO to offer additional protective effects to β-cells.²⁾ We herein reported synthesis of several novel andrographolide derivatives with NO-releasing capability. The target compounds were evaluated for their in vitro protective effects against tert-butyl hydroperoxide (t-BHP) induced cell damage. The new compounds were expected to release NO to offer additional protection to islet β-cells.

Results and Discussion

Chemistry

The procedures for synthesis of compounds 3b–6b were illustrated in Chart 1. Treatment of andrographolide 1 with 2,2-dimethoxypropane (DMP) produced the key intermediate 2.²⁾ Intermediate 2 was then reacted with nitrooxy-substituted acids (for 3a–5a) or bromo-substituted acids (for 6a) to afford the corresponding esters 3a–6a. The reaction of intermediate 2 with 3-nitrooxypropionic acid produced compound 3a, not the desired 14-(3-nitrooxypropionyl)andrographolide due to β-elimination of 3-nitrooxypropionic acid under alkaline conditions. Compounds 3b–6b were obtained by removing of the protective groups. All target products were purified by column chromatography and their structures were characterized by spectroscopic (MS and ¹H-NMR) methods and confirmed by elemental analysis.

Chart 1. Synthesis of Compounds 3b–6b

Reagents and conditions: (a) 2,2-Dimethoxypropane, PPTS, toluene, 81°C, 1 h; (b) (1) RCOOH, HBTU, DIEA, CH₂Cl₂, rt, 1 h; (2) DBU, NaHCO₃, H₂O, CH₂Cl₂, rt, 2 h, 29–80.9%; (c) AcOH, H₂O, rt, 30 min, 79.1–88.7%; (d) (1) AgNO₃, CH₃CN, rt, 8 h; (2) AcOH, H₂O, rt, 30 min, 60.4%.

Effects on t-BHP Injured RIN-m Cells

In order to assess the protective effect of the target compounds, a tert-butyl hydroperoxide (t-BHP)-induced cell damage model was established.²⁴⁾ In RIN-m cells, t-BHP decreased cell viability dose-dependently. Approximately 50% of the cells survived after a 4 h exposure to 100 µm t-BHP. Most of the new compounds exhibited moderate to strong protective effects for t-BHP injured cells. As shown in Fig. 2, andrographolide improved cell viability to 71.1% at a concentration of 5 µm. Compounds 3b, 4b, 5b, 6b displayed strong protective effects in a dose-dependent manner, especially at 1–5 µm. At 1 µm, their protective effects were equal to that of andrographolide at 5 µm or even better. Notably, compound 6b, the nitrooxy-substituted crotonate, showed the strongest protective effect (76.1% at 0.2 µm), which was 25-fold stronger than that of andrographolide.

Fig. 2. Protective Effects against t-BHP-Induced Damage in RIN-m Cells

The results were expressed as the percentage of that of the untreated cells. Data were processed statistically by a single-tail Student’s t-test. * p<0.05, ^# p<0.01 compared to t-BHP group.

NO Assay

In order to further explore the relationship between NO released by the andrographolide-NO donor derivatives and their protective effects, the levels of NO produced by compounds 4b, 5b and 6b were measured by the Griess assay. As shown in Fig. 3, compounds 4b and 5b released lower levels of NO (2.6, 2.3 µm, respectively). By contrast, compound 6b produced a higher amount of NO (7.3 µm). As shown in Fig. 2, compound 6b was much more potent than compound 3b in protecting cells. Both compounds 3b and 6b had α,β-unsaturated group, but 6b was armed with a NO-donating group while 3b was not. From these results, we concluded that the enhanced protective effects of compound 6b may derive from its additional NO releasing effects.

Fig. 3. Compounds 4b–6b Release NO in Vitro

NO₂⁻ concentrations which represent the quantity of NO were determined by the Griess assay. Griess reagent can combine with NO₂⁻ and form chromospheres after 10 min at 30°C. The absorbance then was measured at 540 nm. The levels of NO produced by each compound were calculated, according to the standards of different concentrations of nitrate. Data were expressed as means of each compound tested at each time point and intra-group variations were less than 10%.

Analysis of structure–activity relationships (SAR) of the new compounds revealed some important information: (a) esterification at the C-14 of the natural andrographolide enhanced protective effect to t-BHP induced RIN-m cell damage; (b) enhanced NO-releasing capability increased the compound’s protective effects.

Conclusion

In conclusion, we synthesized several NO-releasing andrographolide derivatives, and evaluated their protective effects against t-BHP-induced cell damage. All the target compounds exhibited protective effects against t-BHP-induced cell damage. Compound 6b was at least 25-fold more potent than its parent andrographolide. These results demonstrated that NO-releasing andrographolide derivatives were more potent than the parent andrographolide in protecting cells from oxidative damage. Our findings suggest that compound 6b merits further investigation as potential anti-diabetes agent.

Experimental

Chemistry

¹H-NMR spectra and ¹³C-NMR spectra were recorded on a Bruker AV 300 spectrometer at 300 MHz. High resolution-electrospray ionization-mass spectra (HR-ESI-MS) was performed on an Agilent 6210 ESI/time-of-flight (TOF) mass spectrometer. ESI-MS was obtained on a Finnigan LCQ Advantage MAX mass spectrometer (ABI Company, 4000 Q TRAP). Elemental analysis was performed on a Vario EL instrument and all values were within ±0.4% of the theoretical values unless otherwise noted.

Procedure for Synthesis of 3,19-Isopropylidene Andrographolide (2)

Compound 2 was prepared according to the method previously reported by our laboratory.²⁾

General Procedure for Synthesis of Compounds 3a–5a

A mixture of the corresponding nitrooxy-substituted acid, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (1.85 mmol) and ethyldiisopropylamine (DIEA) in CH₂Cl₂ (5 mL) was stirred at room temperature for 1 h, and the reaction mixture was then washed with brine (3×5 mL). The organic layer was separated. Aqueous NaHCO₃ (9 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (6.15 mmol) and the organic layer were sequentially added to an ice-cold solution of intermediate 2 (1.54 mmol) in CH₂Cl₂ (5 mL). The resulting reaction mixture was stirred for 2 h, and was then diluted with water and extracted with CH₂Cl₂. The organic layer was neutralized with 1% aqueous acetic acid (10 mL), followed by extraction with CH₂Cl₂ (3×5 mL). The combined organic phases were dried with anhydrous Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography eluting with ethyl acetate–petroleum ether (1 : 2) to produce the title compounds.

14-Acryl-3,19-isopropylidene Andrographolide (3a): White solid; Yield 52.7%; ¹H-NMR (CDCl₃) δ: 7.05 (dt, J=1.8, 6.9 Hz, 1H), 6.47 (dd, J=1.2, 17.1 Hz, 1H), 6.14 (dd, J=10.5, 17.4 Hz, 1H), 6.02 (d, J=6.0 Hz, 1H), 5.95 (dd, J=1.2, 10.5 Hz, 1H), 4.88 (s, 1H), 4.59 (dd, J=6.0, 11.1 Hz, 1H), 4.52 (s, 1H), 4.27 (dd, J=2.1, 11.4 Hz, 1H), 3.95 (d, J=11.7 Hz, 1H), 3.49 (m, 1H), 3.17 (d, J=11.4 Hz, 1H), 2.45 (m, 3H), 2.03–1.65 (m, 6H), 1.40 (s, 3H), 1.36 (s, 3H), 1.25 (m, 4H), 1.19 (s, 3H), 0.91 (s, 3H); ¹³C-NMR (CDCl₃) δ: 16.13, 23.12, 24.90, 25.27, 25.44, 26.11, 34.49, 37.57, 37.91, 38.34, 52.15, 55.87, 63.92, 67.92, 71.57, 76.16, 99.16, 108.93, 123.74, 127.22, 132.79, 147.02, 150.95, 165.55, 169.13; HR-ESI-MS: [M+Na]⁺ m/z 467.2404 (Calcd for C₂₆H₃₆O₆Na: 467.2404); ESI-MS: [M+Na]⁺ m/z 467.4.

14-(5-Nitrooxyvaleryl)-3,19-isopropylidene Andrographolide (4a): White solid; Yield 35.5%; ¹H-NMR (CDCl₃) δ: 7.03 (dt, J=1.5, 6.9 Hz, 1H), 5.95 (d, J=6.0 Hz, 1H), 4.89 (s, 1H), 4.61–4.42 (m, 4H), 4.23 (dd, J=1.8, 11.1 Hz, 1H), 3.95 (d, J=11.7 Hz, 1H), 3.50 (m, 1H), 3.18 (d, J=11.7 Hz, 1H), 2.53–2.32 (m, 5H), 2.10–1.91 (m, 2H), 1.90–1.65 (m, 9H), 1.41 (s, 3H), 1.37 (s, 3H), 1.32–1.25 (m, 3H), 1.20 (s, 3H), 0.94 (s, 3H); ¹³C-NMR (CDCl₃) δ: 16.19, 21.11, 24.86, 25.53, 26.23, 33.31, 34.47, 37.57, 37.93, 38.32, 52.07, 55.89, 63.93, 67.93, 71.61, 76.05, 99.22, 108.85, 123.73, 147.19, 150.82, 169.03, 172.44; HR-ESI-MS: [M+Na]⁺ m/z 558.2673 (Calcd for C₂₈H₄₁NO₉Na: 558.2673); ESI-MS: [M+Na]⁺ m/z 558.3.

14-(7-Nitrooxyheptanoyl)-3,19-isopropylidene Andrographolide (5a): Colorless liquid; Yield 80.9%; ¹H-NMR (CDCl₃) δ: 7.02 (dt, J=1.5, 6.9 Hz, 1H), 5.97 (d, J=6.0 Hz, 1H), 4.89 (s, 1H), 4.58 (dd, J=6.2, 11.3 Hz, 1H), 4.54 (s, 1H), 4.46 (t, J=6.6 Hz, 2H), 4.22 (dd, J=1.8, 11.1 Hz, 1H), 3.95 (d, J=11.7 Hz, 1H), 3.49 (m, 1H), 3.17 (d, J=11.4 Hz, 1H), 2.50–2.33 (m, 5H), 2.04–1.90 (m, 2H), 1.90–1.60 (m, 8H), 1.45–1.38 (m, 6H), 1.36 (s, 3H), 1.20 (s, 3H), 0.93 (s, 3H); ¹³C-NMR (CDCl₃) δ: 14.21, 16.22, 23.11, 24.85, 25.38, 26.10, 26.96, 33.85, 34.45, 37.56, 37.84, 38.30, 52.03, 55.88, 63.94, 67.74, 71.12, 76.04, 99.21, 108.90, 123.83, 147.15, 150.71,169.14 173.09; HR-ESI-MS: [M+Na]⁺ m/z 586.2986 (Calcd for C₃₀H₄₅NO₉Na: 586.2986); ESI-MS: [M+H]⁺ m/z 564.6.

General Procedure for Synthesis of Compounds 3b–5b

The corresponding intermediate (3a–5a) was added to a solution (5 mL) of acetic acid–water (7 : 3, v/v), and the solution was stirred at room temperature for 30 min. Water was added, and the solution was neutralized with NaHCO₃. The solution was extracted with CH₂Cl₂ (3×5 mL). The combined organic phases were dried with anhydrous Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography eluting with ethyl acetate–petroleum ether (2 : 1) to afford the title compounds.

14-Acrylandrographolide (3b): White solid; Yield 79.1%; mp: 134–136°C; ¹H-NMR (CDCl₃) δ: 7.04 (dt, J=1.8, 6.9 Hz, 1H), 6.48 (dd, J=1.5, 17.3 Hz, 1H), 6.14 (dd, J=10.5, 17.2 Hz, 1H), 6.01 (d, J=6.0 Hz, 1H), 5.96 (dd, J=1.5, 10.5 Hz, 1H), 4.86 (s, 1H), 4.58 (dd, J=6.1, 11.2 Hz, 1H), 4.49 (s, 1H), 4.27 (dd, J=2.0, 11.2 Hz, 1H), 4.17 (dd, J=2.1, 11.1 Hz, 1H), 3.48 (m, 1H), 3.32 (t, J=9.9 Hz, 1H), 2.77 (dd, J=2.4, 9.0 Hz, 1H), 2.60–2.30 (m, 3H), 2.03–1.70 (m, 6H), 1.30–1.20 (m, 7H), 0.65 (s, 3H); ¹³C-NMR (CDCl₃) δ: 15.09, 22.70, 23.65, 25.28, 28.16, 36.94, 37.66, 38.80, 42.88, 55.14, 55.77, 64.10, 67.87, 71.57, 80.43, 108.88, 123.77, 127.19, 132.88, 146.62, 150.84, 165.57, 169.13; MS (ESI): [M+Na]⁺ m/z 427.1; Anal. Calcd for C₂₃H₃₂O₆: C, 68.29; H, 7.97. Found: C, 68.02; H, 8.09.

14-(5-Nitrooxyvaleryl)andrographolide (4b): White solid; Yield 88.7%; mp: 63–65°C; ¹H-NMR (CDCl₃) δ: 7.03 (dt, J=1.5, 6.9 Hz, 1H), 5.96 (d, J=6.0 Hz, 1H), 4.89 (s, 1H), 4.57 (dd, J=6.0, 11.1 Hz, 1H), 4.53–4.42 (m, 2H), 4.24 (dd, J=1.8, 11.1 Hz, 1H), 4.20 (d, J=11.7 Hz, 1H), 3.51 (m, 1H), 3.35 (m, 1H), 2.80 (m, 1H), 2.58–2.26 (m, 6H), 2.05–1.92 (m, 1H), 1.91–1.70 (m, 9H), 1.38–1.18 (m, 10H), 0.68 (s, 3H); ¹³C-NMR (CDCl₃) δ: 15.09, 21.04, 22.76, 23.62, 25.35, 26.16, 28.01, 33.25, 36.93, 37.65, 38.76, 42.66, 55.06, 55.74, 64.08, 67.86, 71.63, 72.55, 80.14, 108.66, 123.73, 146.91, 150.76, 169.15, 172.47; MS (ESI): [M+Na]⁺ m/z 518.4; Anal. Calcd for C₂₅H₃₇NO₉: C, 60.59; H, 7.53; N, 2.83. Found: C, 60.38; H, 7.51; N, 2.83.

14-(7-Nitrooxyheptanoyl)andrographolide (5b): Colorless liquid; Yield 85.0%; ¹H-NMR (CDCl₃) δ: 6.99 (dt, J=1.5, 6.9 Hz, 1H), 5.92 (d, J=6.0 Hz, 1H), 4.86 (s, 1H), 4.55 (dd, J=6.0, 11.1 Hz, 1H), 4.47 (s, 1H), 4.44 (t, J=6.6 Hz, 2H), 4.21 (dd, J=1.8, 11.1 Hz, 1H), 4.16 (d, J=11.1 Hz, 1H), 3.47 (m, 1H), 3.32 (m, 1H), 3.02–2.78 (m, 2H), 2.52–2.25 (m, 5H), 2.05–1.90 (m, 1H), 1.90–1.55 (m, 10H), 1.48–1.29 (m, 5H), 1.29–1.14 (m, 6H), 0.65 (s, 3H); ¹³C-NMR (CDCl₃) δ: 15.12, 22.75, 23.64, 24.56, 25.32, 26.53, 28.06, 28.56, 33.82, 36.96, 37.66, 38.77, 42.74, 55.09, 55.77, 64.09, 67.09, 71.70, 73.12, 80.22, 108.75, 123.85, 146.85, 150.60, 169.17, 173.09; MS (ESI): [M+Na]⁺ m/z 546.4; Anal. Calcd for C₂₇H₄₁NO₉: C, 61.93; H, 7.89; N, 2.67. Found: C, 61.70; H, 7.99; N, 2.58.

Procedure for Synthesis of Compounds 6a

To a stirred solution of the corresponding bromo-substituted acid (2.0 mmol) and HBTU (2.2 mmol) in CH₂Cl₂ (5 mL) was added DIEA (2.2 mmol). The mixture was stirred at room temperature for 1 h. The reaction mixture was then washed with brine (3×5 mL). The organic layer was separated. Aqueous NaHCO₃ (4 mL), DBU (2.7 mmol) and the organic layer were sequentially added to an ice-cold solution of intermediate 2 (1.0 mmol) in CH₂Cl₂ (5 mL). The resulting mixture was stirred for 40 min, and diluted with water and extracted with CH₂Cl₂. The organic layer was neutralized with 1% aqueous acetic acid (10 mL), followed by extraction with CH₂Cl₂ (3×5 mL). The combined organic layers were dried with anhydrous Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography eluting with ethyl acetate–petroleum ether (1 : 2) to afford the title compound.

14-(E-4-Nitrooxycrotonyl)-3,19-isopropylidene Andrographolide (6a): White solid; Yield 55.8%; ¹H-NMR (CDCl₃) δ: 7.08 (m, 2H), 6.04 (m, 2H), 4.89 (s, 1H), 4.58 (dd, J=6.0, 11.4 Hz, 1H), 4.52 (s, 1H), 4.27 (dd, J=1.8, 11.1 Hz, 1H), 4.03 (dd, J=1.2, 7.2 Hz, 2H), 3.95 (d, J=11.7 Hz, 1H), 3.49 (m, 1H), 3.17 (d, J=11.4 Hz, 1H), 2.45 (m, 3H), 2.05–1.90 (m, 2H), 1.90–1.65 (m, 5H), 1.40 (s, 3H), 1.36 (s, 3H), 1.33–1.21 (m, 5H), 1.20 (s, 3H), 0.93 (s, 3H); ¹³C-NMR (CDCl₃) δ: 14.21, 16.18, 23.12, 24.91, 25.28, 26.12, 27.07, 28.67, 34.50, 37.56, 37.91, 38.37, 52.15, 55.87, 63.92, 68.14, 71.52, 76.16, 99.17, 109.13, 123.04, 123.65, 144.00, 147.00, 151.08, 164.90, 169.06; HR-ESI-MS: [M+Na]⁺ m/z 559.1678 (Calcd for C₃₀H₄₅NO₉Na: 559.1665); ESI-MS: [M+Na]⁺ m/z 559.5 (⁷⁹Br), 561.5 (⁸¹Br) (1 : 1).

Procedure for Synthesis of Compounds 6b

Compound 6a (0.61 mmol) was dissolved in dry acetonitrile (5 mL), followed by the addition of silver nitrate (0.73 mmol). The mixture was stirred in dark for 8 h at room temperature. The mixture was then filtered and concentrated in vacuo. Dichloromethane was added. The solution was washed with brine, dried with Na₂SO₄. Solvent was removed, resulting in a white solid. Without further purification, the white solid was added to a solution of acetic acid–water (7 : 3, v/v) (5 mL), and the solution was stirred at room temperature for 30 min. After addition of water, the solution was neutralized with NaHCO₃ and then extracted with CH₂Cl₂. The organic phase was dried with Na₂SO₄ and concentrated. The crude product was purified by column chromatography eluting with ethyl acetate–petroleum ether (2 : 1) to produce compound 6b.

14-(E-Nitrooxycrotonyl)andrographolide (6b): White solid; Yield 60.4%; mp: 49–50°C; ¹H-NMR (CDCl₃) δ: 7.04 (dt, J=1.8, 6.9 Hz, 1H), 6.97 (dt, J=5.1, 15.9 Hz, 1H), 6.12 (dt, J=1.8, 15.9 Hz, 1H), 6.01 (d, J=6.0 Hz, 1H), 5.10 (dd, J=1.8, 5.1 Hz, 2H), 4.86 (s, 1H), 4.57 (dd, J=6.0, 11.4 Hz, 1H), 4.46 (s, 1H), 4.27 (dd, J=1.8, 11.1 Hz, 1H), 4.17 (d, J=11.1 Hz, 1H), 3.48 (m, 1H), 3.33 (d, J=10.8 Hz, 1H), 2.52–2.12 (m, 6H), 2.02–1.67 (m, 6H), 1.32–1.15 (m, 7H), 0.65 (s, 3H); ¹³C-NMR (CDCl₃) δ: 15.10, 22.71, 23.64, 25.39, 28.11, 36.94, 37.65, 38.83, 42.82, 55.13, 55.75, 64.10, 68.26, 69.95, 71.43, 80.37, 108.77, 123.19, 123.60, 139.70, 146.67, 151.08, 164.50, 169.01; MS (EI): [M+H]⁺ m/z 480.4; Anal. Calcd for C₂₄H₃₃NO₉: C, 60.11; H, 6.94; N, 2.92. Found: C, 59.77; H, 7.22; N, 2.65.

Biological Study

Effects on t-BHP Injured RIN-m Cells

Cell viability was measured by the MTT assay. RIN-m cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 units/mL penicillin–streptomycin (Gibco). Cells were placed into 96-well cell culture plates (10000/well, 100 µL) and incubated for 20 h at 37°C under 5% CO₂. Compounds at different concentrations were added, and the cells were incubated at 37°C for 3 h. After incubation, cells were treated with or without (control) t-BHP (100 µm). Cells were then incubated for another 4 h. A solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added, and the cells were incubated for another 4 h before dimethyl sulfoxide was added. After the crystals were completely dissolved (15 min), the absorbance was read at 490 nm with a spectrophotometer (BioTek Synergy HP, U.S.A.). The results were expressed as the percentage of the control.

NO Assay

The NO formation was determined by using Griess reagent kit according to the manufacturer’s instruction. Briefly, 0.2 mm of each compound in phosphate buffer solution (PBS) containing 5% dimethyl sulfoxide and 5.0 mm l-cysteine at pH 7.4 was incubated at 37°C for 15–300 min, and was then sampled every 15 min for 120 min and then every 30 min for the remaining time. The collected samples (50 µL) were mixed with the Griess reagent І (50 µL) and ІІ (50 µL), and then incubated at 37°C for 10 min, followed by measuring at 540 nm. Different concentrations of nitrite were used as standards to calculate the concentrations of NO formed by each compound.

Acknowledgment

This work was supported in part by Grants from the China Natural Science Fund (30772642 and 30973618 to YW; 81001683 and U1032007 to LX), The Guangdong Province (2008A030101007 to YW) and Guangzhou City (2008Z1-E691 to PY) Science and Technology Funds as well as the 211 Project of Jinan University.

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