New Phenylpropane and Anti-inflammatory Diterpene Derivatives from Amentotaxus formosana

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Biological evaluation
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

One new diterpene, 8(14),15-sandaracopimaradiene-2α,3β,18-triol (1), two new phenylpropane derivatives, i. e., (E)-methyl 2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoate (2) and (E)-2-(3,4-methylene-dioxyphenyl)-3-methoxypropenoic acid (3), and two known diterpenes, ent-8(14),15-sandaracopimaradiene-2α,18-diol (4) and 8(14),15-sandaracopimaradiene-2α,18, 19-triol (5), were isolated from the heartwoods and barks of Amentotaxus formosana, respectively. The anti-inflammatory activity of the diterpenes 1, 4, and 5 was assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, macrophages, and microglial cells. Compounds 1, 4, and 5 showed significant concentration-dependent inhibitory effects on the release of β-glucuronidase from rat neutrophils in response to formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB) with IC₅₀ values of 5.5 ± 1.8, 8.4 ± 2.9 and 19.2 ± 3.3 μM, respectively. Compounds 1 and 5 also showed significant concentration-dependent inhibitory effects on superoxide anion generation in rat neutrophils stimulated with fMLP/CB and phorbol 12-myristate 13-acetate (PMA) with IC₅₀ values of 12.6 ± 1.2 and 9.4 ± 1.7, and 10.7 ± 3.3 and 12.9 ± 0.9 μM, respectively.

Introduction

Amentotaxus formosana Li (Amentotaxaceae) is an endemic tree of southeastern Taiwan. Recently, we have isolated and characterized five lanostanoids, a compound consisting of two diterpenoids substructures, two terpenoids with a novel skeleton, two diterpenoids, and ten known compounds, from the bark and leaves of A. formosana [1], [2], [3]. Previously, we also reported that the terpenoids isolated from this plant showed potent cytotoxicity [3]. A further search for structurally interesting and bioactive compounds from this plant resulted in the isolation of a new diterpenoid 1, two new phenylpropane derivatives 2 and 3 and two known diterpenoids, ent-8(14),15-sandaracopimaradiene-2α,18-diol (4) and 8(14),15-sandaracopimaradiene-2α,18, 19-triol (5) from heartwood and bark, respectively.

It is conceivable that mast cells, neutrophils and macrophages are the important players in inflammatory disorders. Activation of microglial cell also plays a crucial role in inflammatory disease of CNS. Thus, inhibition of the activation of these inflammatory cells appears to be an important therapeutic target for small molecular drugs for the treatment of inflammatory disease. A novel pimarane diterpene, acanthoic acid, has been shown to exhibit an excellent suppression of interleukin-1, tumor necrosis factor-α and cyclooxygenase 2 [4] and as a part of our ongoing search for anti-inflammatory diterpenes from natural sources, we have investigated the anti-inflammatory effects of diterpenes 1, 4 and 5. In the present paper, the structure elucidation of the new constituents, 1 - 3 (Fig. [1]), and the anti-inflammatory activity of 1, 4, and 5 are reported.

Materials and Methods

General experimental procedures

Melting points are reported uncorrected. Optical rotation was obtained on JASCO model DIP-370 digital polarimeter. UV spectra were obtained on JASCO model 7800 UV/VIS spectrophotometer. IR spectra were recorded on a Perkin Elmer 200 FT-IR spectrophotometer. ¹H- (400 MHz) and ¹³C-NMR (100 MHz) spectra were recorded on a Varian Unity-400 spectrometer. Mass spectra were run on a JMSHX 100-mass spectrometer.

Plant material

Whole plants of A. formosana (Amentotaxaceae) were collected at Kaohsiung Hsien, Taiwan, R. O. C., during July 2001. A voucher specimen (9001) has been deposited at the Department of Medicinal Chemistry, School of Pharmacy, Kaohsiung Medical University.

Extraction and isolation

The heart wood (1.6 kg) of A. formosana was chipped and extracted with CHCl₃ at room temperature. The CHCl₃ extract (6.5 g) was separated by column chromatography (CC) on silica gel (300 g, 3 × 50 cm, CH₂Cl₂/MeOH, 5 : 2, 1 mL/min, t_R of 1: between 2 and 5 mL; cyclohexane/EtOAc, 3 : 4, 1 mL/min, t_R of 2: between 3 and 5 mL; t_R of 3: between 8 and 11 mL) to yield 1 (3 mg) (R_f: 0.3 - 0.4, UV detection), 2 (3 mg) (R_f: 0.3 - 0.4, UV detection), and 3 (5.8 mg) (R_f: 0.3 - 0.4, UV detection).

The air-dried barks (700 g) were extracted with CH₂Cl₂. The CH₂Cl₂ extract (15 g) was separated by medium pressure liquid chromatography (MPLC) on silica gel (20 × 300 mm, CH₂Cl₂/Me₂CO, 9 : 1, 10 mL/min, t_R of 4: between 50 and 70 mL; t_R of 5: between 100 and 120 mL) to yield 4 (3 mg) (R_f: 0.2 - 0.3, UV detection), [α]_D ²⁵: + 5° (c 0.13, MeOH) and 5 (4 mg) (R_f: 0.2 - 0.3, UV detection), [α]_D ²⁵: -13° (c 0.13, MeOH). The known compounds were characterized by comparing the various spectroscopic data reported in the literature [5], [6], [7].

8(14),15-Sandaracopimardiene-2α,3β,18-triol (1): Colorless powder. [α]_D ²⁸: + 11° (c 0.12, MeOH); IR: ν_max = 3385 cm^-1; ¹H-NMR: see Table [1]; ¹³C-NMR: see Table [1]; EI-MS : m/z (rel. int.) = 320 (M⁺, 100), 305 (65), 287 (14), 271 (34), 269 (22), 239 (8), 187 (28), 135 (45); HR-EI-MS: m/z = 320.2348 (calcd. for C₂₀H₃₀O₃ [M]⁺: 320.2351).

(E)-Methyl 2-(3,4-methylenedioxyphenyl)-3-methoxypropenoate (2): Oily substance. UV (MeOH): λ_max (log ε) = 265 nm (3.5); IR: ν_max = 1712, 1633, 1504 cm^-1; ¹H-NMR [(CD₃)₂CO, 400 MHz]: see Table [2]; ¹³C-NMR [(CD₃)₂CO, 100 MHz]: see Table [2]; EI-MS : m/z (rel. int.) = 236 (M⁺, 50), 205 ([M - OMe]⁺, 4), 191 ([M - OMe - Me + H]⁺, 9), 177 ([M - COOMe]⁺, 2), 162 ([M - COOMe - Me]⁺, 18), 134 (14), 75 (100); HR-EI-MS: m/z = 236.0687 (cacld. for C₁₂H₁₂O₅ [M]⁺: 236.0685).

(E)-2-(3,4-Methylenedioxyphenyl)-3-methoxypropenoic acid (3): Amorphous powder. UV (MeOH): λ_max (log ε) = 265 nm (3.63); IR: ν_max = 3447, 1673, 1618, 1502 cm^-1; ¹H-NMR [(CD₃)₂CO, 400 MHz]: see Table [2]; ¹³C-NMR [(CD₃)₂CO, 100 MHz]: see Table [2]; EI-MS: m/z (rel. int.) = 222 (M⁺, 1) , 178 ([M - COOH + H]⁺, 1), 165 ([M - COOH - CH₂ + 2H]⁺, 50), 149 ([M - COOH - CH₂ - H₂O + 4H]⁺, 40), 121(23), 81(49), 69 (100); HR-EI-MS: m/z = 222.0524 (calcd. for C₁₁H₁₀O₅ [M]⁺: 222.0528).

The purity (> 95%) of compounds 1, 4, and 5, used for anti-inflammatory tests, was determined by HPLC.

Biological evaluation

Compound stock solutions (30 mM in DMSO) were prepared and stored at -25 °C, and were diluted with DMSO to a 1 - 20 mM range at room temperature before experiments. The final percentage of DMSO in the reaction mixture was less than 0.5 % (v/v).

Rat (Sprague-Dawley) peritoneal mast cells [8] and peripheral blood neutrophils [9] were isolated and incubated with test compounds for 5 min at 37 °C before stimulation with 10 μg/mL of compound 48/80 for another 15 min or with 1 μM formyl-Met-Leu-Phe (fMLP) plus 5 μg/mL of cytochalasin B (CB) for another 45 min, respectively. The degranulation of mast cells and neutrophils was assessed by the determination of histamine [10] and β-glucuronidase [11], respectively, in the supernatant. The total content of histamine and β-glucuronidase was measured from the Triton X-100-treated cells.

In the superoxide anion generation experiments, neutrophils were stimulated with fMLP/CB or 3 nM phorbol 12-myristate 13-acetate (PMA) for 30 min in the presence of cytochrome c, and the superoxide anion generation was measured in terms of superoxide dismutase-inhibitable cytochrome c reduction [12].

Murine macrophage-like RAW 264.7 cells and microglial cell line N9 cells [13] were plated in 96-well plates, and incubated with test compounds for 1 h at 37 °C before stimulation with 1 μg/mL of lipopolysaccharide (LPS) or 10 ng/mL of LPS plus 10 U/mL of interferon-γ, respectively, for 24 h. Nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in the cell medium was determined by the Griess reaction [14] and ELISA, respectively.

Statistical analysis

Data are presented as the mean ± s. e.m from 4 - 6 separate experiments. Statistical analyses were performed using the Bonferroni t-test method after ANOVA for multigroup comparison and the Student’s t-test method for two groups comparison. P < 0.05 was considered significant. Analysis of linear regression (at least three data within 20 - 80 % inhibition) was used to calculate IC₅₀ values.

Results and Discussion

The molecular formula of 1 was determined to be C₂₀H₃₂O₃ by HR-EI-MS (m/z = 320.2348 [M]⁺), which was consistent with the ¹H- and ¹³C-NMR data. The IR absorption of 1 implied the presence of an OH group (3385 cm^-1). The structure of 1 was deduced from extensive analysis of 1D and 2D data, including those from COSY 90, HMQC, HMBC and NOESY experiments in CD₃OD. The ¹H-NMR spectra of 1 (Table [1]) revealed the presence of three Me groups at δ = 0.73, 0.92 and 1.04 (each s), two protons geminal to two secondary OH group at δ = 3.67 (ddd, J = 12.4, 9.6, 4.4 Hz) and 3.39 (d, J = 9.6 Hz), an AB system [δ = 3.24 and 3.48 (each d, J = 11.2 Hz, 2H)], characteristic of a CH₂OH group, and four olefinic protons. Three of the olefinic protons, attributed to a monosubstituted olefin, constituted an ABX system [δ = 4.86 (H_A), 4.89 (H_B), 5.76 (X) (J _AB = 1.6, J _AX = 10.8, J _BX = 17.6 Hz)], while one olefinic proton was isolated [δ = 5.25 (s)]. In the ¹³C-NMR spectrum of 1 (Table [1]), the chemical shift values of 1 were almost identical to the corresponding data of 8(14),15-sandaracopimaradiene-2α-18-diol except for C-1 to C-4, and C-18 to C-20 [4]. The above information suggested that 1 was a sandaracopimaradiene diterpenetriol. The COSY cross-peaks of H_β-1/H-2 and H-2/H-3, and the HMBC correlations of H_β-18/C-5 and C-3, Me-19/C-18, C-4, and C-3, H-3/C-19, H-2/C-3, Me-20/C-1, C-5, C-10, and C-9, and the NOESY cross-peaks of H_β-1/H-2, H-2/Me-19 and Me-20, H_α-1/H-3, Me-17/H_β-12, H-5/H_α-18, and H-9/H_α-1 established the structure of 1. The relative configurations at OH-2, CH₂OH-4, H-5, and H-9 are on the α-side and OH-3, Me-4, Me-10 and Me-17 on the β-side of 1. Thus the new diterpene, 1 was characterized as 8(14),15-sandaracopimaradiene-2α,3β,18-triol (1). The EI-MS of 1 showed significant peaks at m/z = 305 ([M - CH₃]⁺), 287 ([M - Me - H₂O]⁺), 269 ([M - Me - 2 H₂O]⁺), and 239 ([M - Me - 2 H₂O - CH₂OH + H]⁺), which also supported the structure of 1.

The molecular formula of 2 was determined to be C₁₂H₁₂O₅ by HR-EI-MS (m/z = 236.0687 [M]⁺), which was consistent with the ¹H- and ¹³C-NMR data. The IR absorption of 2 implied the presence of a carboxylate (1712 cm^-1), an exocyclic C = C (1633 cm^-1), which were confirmed by ¹³C-NMR data (off-resonances at δ = 127.7, 161.3), and an aromatic ring (1504 cm^-1) functionality. The proposed structure for 2 (Fig. [1]) was deduced from extensive analysis of 1D and 2D NMR data, including those from COSY, HMQC, HMBC and NOESY experiments in CDCl₃ (Table [2]). The ¹H-NMR spectrum of 2 revealed the presence of a methylenedioxy group, two methoxy groups [δ = 3.66 (s, 3H), 3.89 (s, 3H)], and three aromatic protons of ABX coupling [δ = 6.79 (d, J = 1.2 Hz), 6.80 (d, J = 7.2 Hz), and 6.80 (dd, J = 7.2, 1.2 Hz)], and one olefinic proton [(δ = 7.52 (s)). The HMBC correlations of MeO-1/C-1, H-3/C-1, and H-3/MeO-3 confirmed the C-1 was linked through C-2 to C-3. The NOESY showed cross-peaks between MeO-3/H-2′, and MeO-1/H-6′. The above information, and the HMBC correlations between H-2′/H-6′ and C-2 supported that compound 2 was characterized as (E)-methyl 2-(3,4-methylenedioxyphenyl)-3-methoxypropenoate. The EI-MS (see Extraction and Isolation) data also supported the characterization of 2.

The molecular formula of 3 was determined to be C₁₁H₁₀O₅ by HR-EI-MS (m/z = 222.0524 [M]⁺), which was consistent with the ¹H- and ¹³C-NMR data. The IR absorption of 3 was indicative of a hydroxy group (3447 cm^-1), a carboxylic acid (1673 cm^-1), exocyclic C = C (1618 cm^-1) and an aromatic ring (1502 cm^-1) functionality. The UV spectrum resembled that of 2. The ¹H-NMR data of 3 were very similar to those of 2, except for the absence of a proton signal of the methyl ester moiety and the presence of a proton signal of a carboxylic acid. The ¹³C-NMR spectra of 3 was assigned by ¹H-decoupled and DEPT spectra and comparison with those of corresponding data of 2. The EI-MS (see Extraction and Isolation) and ¹³C-NMR data also supported the characterization of 3. In the ¹³C-NMR spectra of 3 (Table [2]), the chemical shift values of C-1 to C-7′ were almost identical to the corresponding data of 3 (Table [2]) except for C-1 and C-6′. Based on these results, the carboxylic acid moiety was linked at C-1. Thus compound 3 was characterized as (E)-2-(3,4-methylenedioxyphenyl)-3-methoxypropenoic acid (3).

The anti-inflammatory activity of the diterpenes 1, 4, and 5 was studied in vitro for their inhibitory effects on chemical mediators released from mast cells, neutrophils, macrophages, and microglial cells. Compounds 1, 4, and 5 (each at 30 μM) did not show significant inhibition of mast cells degranulation stimulated with compound 48/80 (data not shown), while they had potent and concentration-dependent inhibitory effects on fMLP-induced neutrophil degranulation with IC₅₀ values of 5.5 ± 1.8, 8.4 ± 2.9 and 19.2 ± 3.3 μM, respectively. The IC₅₀ value of compound 5 was significantly greater than the IC₅₀ values of compounds 1 and 4 (both P < 0.05), while no differences between the potencies of 1 and 4 in inhibiting neutrophils degranulation were noticed. It is likely that the introduction of an OH group at C-19 of 8(14),15-sandaracopimaradiene-2α,18-diol to yield compound 5 decreased the inhibitory activity. Trifluoperazine (IC₅₀ = 12.2 ± 0.3 μM) was used as a positive control. Compound 1 (P < 0.05) but not 4 was more potent than trifluoperazine in terms of the IC₅₀ values.

FMLP and PMA stimulate superoxide anion generation in neutrophils through different mechanisms, in which the former stimulates neutrophils via the activation of G protein-coupled receptors, while the later bypasses the membrane receptor and directly activates protein kinase C [15]. In these experiments, both compounds 1 and 5 inhibited the fMLP-induced superoxide anion generation (IC₅₀ values of 12.6 ± 1.2 and 10.7 ± 3.3 μM, respectively) and the PMA-induced response (IC₅₀ values of 9.4 ± 1.7 and 12.9 ± 0.9 μM, respectively) with similar potencies, while compound 4 (30 μM) inhibited fMLP- (34.4 ± 4.8 % inhibition, P < 0.05) but not the PMA-induced response. The positive control, trifluoperazine inhibited the fMLP- and PMA-induced superoxide anion generation with IC₅₀ values of 7.3 ± 1.5 and 2.9 ± 0.6 μM, respectively. These results indicated that the introduction of an OH group at C-3 or C-19 of 8(14),15-sandaracopimaradiene-2α,18-diol improves the inhibitory potency.

Compounds 1, 4, and 5 (each at 30 μM) failed to significantly inhibit the NO production from both RAW 264.7 and N9 cell lines (data not shown). Compound 1 inhibited TNF-α production from N9 cells (44.0 ± 7.5 % inhibition at 30 μΜ, P < 0.05), while 4 and 5 had no significant inhibitory activity. Moreover, compounds 1, 4, and 5 failed to affect the TNF-α production from RAW 264.7 cells induced by LPS (data not shown).

The present study suggests that the inhibitory effect on the release of chemical mediators by 1, 4, and 5, and on the superoxide anion generation by 1 and 5 in neutrophils, and the slight inhibitory effect of compound 1 on the production of TNF-α from microglial-like cell line N9 cells may have value in the therapeutic treatment or prevention of certain central as well as peripheral inflammatory diseases associated with excess production of chemical mediators.

Acknowledgements

This work was supported by a grant from the National Science Council of Republic of China. (NSC 90-2323-B037-002).

References

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Prof. Chun-Nan Lin

School of Pharmacy

Kaohsiung Medical University

Kaohsiung 807

Taiwan

Republic of China

Phone: +886-7-312-1101∼9 ext 2163.

Fax: +886-7-556-2365

Email: lincna@cc.kmu.edu.tw

References

• 1 Su H J, Day S H, Yang S Z, Chiang M Y, Lin C N. Lanostanoids of Amentotaxus formosana .  J Nat Prod. 2002;  65 79-81
  CrossrefPubMedSearch in Google Scholar
• 2 Day S H, Su H J, Lin C N, Yang S Z. Constituents with a novel skeleton isolated from Amentotaxus formosana .  Helv Chem Acta. 2002;  85 2377-82
  CrossrefPubMedSearch in Google Scholar
• 3 Su H J, Wang L W, Lin C N, Day S H, Wei B L, Yang S Z, Won S J. A diterpenoid with a new skeleton and cytotoxic terpenoids isolated from Amentotaxus formosana .  Helv Chem Acta. 2003;  86 2645-52
  CrossrefPubMedSearch in Google Scholar
• 4 Suh Y G, Kim Y H, Park M H, Choi Y H, Lee H Y, Moon J Y, Min K H, Shin D Y, Jung J K, Park O H, Jeon R K, Park H S, Kang S A. Pimarane cyclooxygenase 2 (COX-2) inhibitor and its structure-activity relationship.  Bioorg Med Chem Lett. 2001;  11 559-62
  CrossrefPubMedSearch in Google Scholar
• 5 Del Rayo Camacho M, Phillipson J D, Croft S L, Kirby G C, Warhurst D C, Solis P N. Terpenoids from Guarea rhophalocarpa .  Phytochemistry. 2001;  56 203-10
  CrossrefPubMedSearch in Google Scholar
• 6 Carman R M, Corbett R E, Grant P K, McGrath M JA, Munro M HG. The diterpenes of Dacrydium colensoi . Tetrahedron Lett 1966: 3173-79
  Search in Google Scholar
• 7 Cambie R C, Burfitt I R, Goodwin T E, Wenkert E. The structure of hallol.  J Org Chem. 1975;  40 3789-3791
  PubMedSearch in Google Scholar
• 8 Wang J P, Hsu M F, Ouyang C, Teng C M. Edematous response caused by [Thi^5,8,D-Phe⁷]bradykinin, a B₂ receptor antagonist, is due to mast cell degranulation.  Eur J Pharmacol. 1989;  161 143-9
  CrossrefPubMedSearch in Google Scholar
• 9 Wang J P, Raung S L, Kuo Y H, Teng C M. Daphnoretin-induced respiratory burst in rat neutrophils is, probably, mainly through protein kinase C activation.  Eur J Pharmacol. 1995;  288 341-8
  CrossrefPubMedSearch in Google Scholar
• 10 Håkanson R, Rönnbert A L. Improved fluorometric assay of histamine.  Analyt Biochem. 1974;  60 560-7
  PubMedSearch in Google Scholar
• 11 Barrett A J. in: Lysosomes: A Laboratory Handbook. Dingle JT. (ed) Amsterdam; Elsevier 1977: pp 118-20
  Search in Google Scholar
• 12 Markert M, Andrews P C, Babior B M. Measurement of O₂.^- production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils.  Methods Enzymol. 1984;  105 358-65
  PubMedSearch in Google Scholar
• 13 Corradin S B, Manuel J, Donini S D, Quattrocchi E, Ricciardi-Castagnoli P. Inducible nitric oxide synthase activity of cloned murine microglial cells.  Glia. 1993;  7 255-62
  CrossrefPubMedSearch in Google Scholar
• 14 Minghetti L, Nicolini A, Polazzi E, Créminon C, Maclouf J, Levi G. Inducible nitric oxide synthase expression in activated rat microglial cultures is down-regulated by exogenous prostaglandin E₂ and by cyclooxygenase inhibitor.  Glia. 1997;  19 152-60
  CrossrefPubMedSearch in Google Scholar
• 15 Segal A W, Abo A. The biochemical basis of the NADPH oxidase of phagocytes.  Trends Biochem Sci. 1993;  18 43-7
  CrossrefPubMedSearch in Google Scholar

Prof. Chun-Nan Lin

School of Pharmacy

Kaohsiung Medical University

Kaohsiung 807

Taiwan

Republic of China

Phone: +886-7-312-1101∼9 ext 2163.

Fax: +886-7-556-2365

Email: lincna@cc.kmu.edu.tw