Neurite Outgrowth Activity of Cyathane Diterpenes from Sarcodon cyrneus, Cyrneines A and B

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Cell culture and evaluation of neurite outgrowth
• Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)
• Measurement of lactate dehydrogenase activity (LDH)
• Results and Discussion
• Acknowledgements
• References

Abstract

Two new cyathane diterpenes, cyrneines A (1) and B (2), were isolated from the mushroom Sarcodon cyrneus. The structures of the novel diterpenoids were determined by analysis of their spectroscopic data. Neither cyrneine A nor cyrneine B at 100 μM showed cytotoxicity as determined by LDH analysis. The stimulating activity on neurite outgrowth of cyrneines was evaluated. Rat pheochromocytoma cells (PC12), used as a model system of neuronal differentiation, were cultured with cyrneine A (100 μM), cyrneine B (100 μM) or NGF (100 ng/ml) for 24 h. Interestingly, cyrneines A and B significantly promoted neurite outgrowth in addition to NGF as a positive control.

Introduction

The cyathane diterpenes represent an interesting group of complex molecules isolated for the first time in 1971 from the mushroom Cyathus helenae [1]. After this first report several other cyathane diterpenes have been isolated from Cyathus sp. [2] and different mushrooms such as Hericium erinaceum [3] and Sarcodon scabrosus [4], [5]. All the isolated compounds showed interesting biological activities such as antibacterial [6] and anti-inflammatory [7], [8]. In our ongoing search for natural bioactive agents [9], we studied the mushroom Sarcodon glaucopus. From the methanolic extract we isolated two new cyathane diterpenes, glaucopines A and B [10], and from the hexane extract glaucopine C [11]. All the three compounds (Fig. [1]) were tested for their topical anti-inflammatory activity.

Furthermore, more recently cyathanes have attracted attention for their action as stimulators of neurotrophic factors in vitro [3], [4], [12], [13].

Glial cells produce neurotrophic factors to support neurons and among them NGF (nerve growth factor) has pleiotropic effects on the promotion of neuronal differentiation and survival in various neurons [14]. Neurotrophic factors and inducers of neurotrophic factor biosynthesis have great therapeutic interest for the treatment of neurodegenerative diseases such Alzheimer’s and Parkinson’s. Since neurotrophic factors are polypeptides they cannot cross the blood-brain barrier and are easily proteolysed peripherally. For this reason in the last years there has been an increasing interest towards small organic molecules which could enhance the production of neurotrophic factors such as NGF. Some NGF inducers have been already reported, such as catecholamines [15], active metabolite vitamin D [16], benzoquinones [17], hericenones [18] and erinacines [3].

The interesting biological activities shown by Sarcodon scabrosus metabolites and particularly by scabronine G [12], [13] prompted us to examine Sarcodon cyrneus Maas Geest (Basidiomycetes). The phytochemical screening of this mushroom led to the isolation and to the structure elucidation of two new compounds, cyrneine A (1) and B (2). Their biological activity on the production of neurotrophic factors and as stimulators of neurite outgrowth was evaluated.

Materials and Methods

General experimental procedures

IR spectra were obtained from neat samples on a Jasco FT/IR 410 infrared spectrophotometer. ¹H- and ¹³C-NMR spectra were recorded in CDCl₃ on a Bruker Avance-DRX 400 spectrometer at 400 and 100.62 MHz, respectively, using TMS as the internal standard. Optical rotations were measured with a Jasco DIP-1000 digital polarimeter. GC-MS analysis were performed on an HP-6890 instrument equipped with a mass selective detector HP-5973. Elemental analyses were carried out on a Carlo Erba Model 1106 Elemental Analyzer. Column chromatography was performed using Merck silica gel 60 (70 - 120 mesh). Thin layer chromatography was performed on Merck silica gel 60 F₂₅₄ .

Plant material

Sarcodon cyrneus was collected in October 2004 near Perugia (Italy) and identified by Prof. Rita Pagiotti. A voucher specimen (RP # 62) is deposited at the Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia.

Extraction and isolation

48 g of the fruiting bodies of lyophilized S. cyrneus were extracted with hexane (1 L) at r. t. for 24 h. After vacuum filtration the residue was extracted with MeOH (1 L) at r. t. for 24 h. The extract was evaporated giving 11.3 g of residue. A solution of this crude extract in EtOAc (100 mL) was washed with water (5 times, 20 mL). The organic phase was dried over Na₂SO₄ and evaporated leading to 2.27 g of a brown syrup that was fractioned by silica gel column chromatography (4 × 45 cm) using CH₂Cl₂/EtOAc (9 : 1, 200 mL; 5 : 1, 150 mL; 4 : 1, 100 mL; 7 : 3, 300 mL; 3 : 2, 100 mL) and MeOH (100 mL). The eluents were combined to 18 fractions (1 - 18) on the basis of TLC analysis. Fr. 14, eluted with CH₂Cl₂-EtOAc 7 : 3, gave cyrneine A (1) (0.056 g) as a white solid. Fr. 15 (0.120 mg, eluted with CH₂Cl₂/EtOAc 3 : 2) was further purified by silica gel CC (1.5 × 20 cm, CH₂Cl₂/EtOAc 7 : 3) to give cyrneine B (2) as yellowish oil (0.052 g).

1,14-Dihydroxycyatha-3,5(10),11-triene-12-carbaldehyde (1): white powder (Et₂O); m. p. 206 °C (Et₂O); [α]_D ^25.4: + 470.7° (c 0.44, CH₂Cl₂); IR (neat): ν_max = 3411, 2924, 1672 and 1567 cm^-1; ¹H- and ¹³C-NMR see Table [1]; MS: m/z = 316 [M⁺] (40), 301 (31), 283 (20), 206 (100), 191 (48); anal. calcd. for C₂₀H₂₈O₃: C 75.91 %, H 8.92 %; found: C 75.82 %, H 8.97 %.

4,14-Dihydroxy-1-oxocyatha-2,5(10),11-triene-12-carbaldehyde (2): yellowish oil; [α]_D ^28.6: -99.9° (c 0.37, CH₂Cl₂); IR (neat): ν_max = 3428, 2925 and 1693 cm^-1; ¹H- and ¹³C-NMR see Table [1]; MS: m/z = 330 (70), 312 (20), 297 (5), 283 (5), 269 (7), 251 (5), 153 (100); anal. calcd. for C₂₀H₂₆O₄: C 72.70 %, H 7.93 %; found: C 72.76 %, H 7.85 %.

Cell culture and evaluation of neurite outgrowth

PC12 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated 10 % fetal calf serum (FCS) (Cell Culture Laboratory; Cleveland, OH, USA) and 5 % horse serum (HS) (Invitrogen; Grand Island, NY, USA), penicillin (50 units/mL) and streptomycin (50 μg/mL) in a 5 % CO₂ incubator. 1321N1 human astrocytoma cells were grown in DMEM supplemented with heat-inactivated 5 % FCS and the above antibiotics. The neurite outgrowth assay was performed as described previously [19]. In brief, PC12 cells were seeded onto 24-well plates (1 × 10⁵ cells/well) and cultured for 24 h. Media were replaced with DMEM containing 1 % FCS, 0.5 % HS, antibiotics and drugs (cyrneines or NGF (Sigma; St. Louis, MO, USA)), then the cells were cultured for additional 24 h. The cell morphology was assessed under the microscope. Neurite extension from PC12 cells was regarded as an index of neuronal differentiation, processes with a length equivalent to one or more diameters of the cell body were regarded as neuritis. The differentiation of PC12 cells was evaluated by examining the proportion of neurite-positive cells to total cells in randomly selected fields (a field/well). The mean differentiation score was obtained for more than 100 cells in each well. Data were expressed as the means ± standard error of mean (SEM) of three different wells, and the significant differences were analyzed with Dunnett’s test.

Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA from 1321N1 cells was extracted by using a total RNA extraction kit (Nippon Gene; Toyama, Japan), and semi-quantitative RT-PCR was carried out by using a RT-PCR kit (Toyobo; Osaka, Japan). NGF mRNA expression was examined as described previously [13] except for the use of different primers. For analysis of human NGF mRNA, the sense primer (5′-CCA AGG GAG CAG CTT TCT ATC CTG G-3′) and the antisense primer (5′-GGC AGT GTC AAG GGA ATG CTG AAG T-3′), were used. The human NGF cDNA fragment (189 base pairs) was amplified during 30 cycles (94 °C for 60 s, 56 °C for 60 s, 72 °C for 120 s). β-Actin transcripts amplified during 17 cycles with the sense primer (5′-AGG GAA ATC GTG CGT GAC AT-3′) and antisense primer (5′-TCC TGC TTG CTG ATC CAC AT-3′) were used as an internal control.

Measurement of lactate dehydrogenase activity (LDH)

The cytotoxicity of cyrneines was determined by measuring lactate dehydrogenase (LDH) activity. PC12 cells were incubated with cyrneines or A23187 (Sigma) in DMEM containing 1 % FCS, 0.5 % HS and above antibiotics for 24 h in 48 well plates (1.25 × 10⁵ cells/well). After drug incubation, the culture media containing LDH released from dead cells were collected and centrifuged to remove contaminating cells (1,900 g, 2 min, 4 °C). To measure the total LDH contents in alive cells, Triton X-100 (0.1 %) was added to the culture as well. The supernatants (5 μL) were incubated for 10 min at 37 °C with assay solution (100 μL) [Tris-HCl (100 mM), sodium lactate (50 mM), β-NAD⁺ (2.6 mM), NO₂-TB (nitrotetrazolium blue) (0.49 mM), 1-methoxy-PMS (1-methoxy-5-methylphenazinium methyl sulfate) (0.04 mM), pH 8.4]. The reaction was terminated with 100 μL of 1.0 M HCl. The absorbance of the produced formazan at 595 nm was measured with a plate reader.

Results and Discussion

The fruiting bodies of S. cyrneus, collected near Perugia (Italy), were frozen, lyophilized, and sequentially extracted with n-hexane and methanol. The methanolic extract was evaporated and partitioned between EtOAc and water. The methanol extract was subjected to silica gel column chromatography, affording a series of fractions, which led to two pure compounds (1 and 2) (Fig. [1]).

The molecular formula of compound 1, C₂₀H₂₈O₃, was deduced from MS, ¹H-NMR and JMODXH (J-modulated spin echo; C, H) experiments. The IR spectrum showed absorptions at 3411, 2924, 1672 and 1567 cm^-1, implying hydroxy and carbonyl functions. The carbon resonances at δ_C = 194.5 (CH), 138.5 (C), 144.7 (CH), 120.3 (CH), 153.9 (C) in the JMODXH spectrum showed the presence of an α,β:γ,δ-unsaturated aldehyde (Table [1]). The olefinic signals at δ_C = 143.9 and 140.9 ppm indicated the presence of a tetrasubstituted double bond. Furthermore, the presence of two sp ³ oxygenated carbons at δ = 81.3 (CH) and δ = 74.6 (CH), four methyl signals at δ = 16.6, 21.4, 21.9 and 26.7, four methylene signals at δ = 29.7, 33.7, 34.9 and 37.6 ppm, one methine at δ = 26.9 ppm and finally two quaternary carbons signals at δ = 48.0 and 50.3 ppm, were deduced from the JMODXH spectrum.

The ¹H-NMR spectrum confirmed the presence of the unsaturated aldehyde by signals at δ = 9.48, 6.75 and 5.93 ppm. In addition, two oxygenated methines at δ = 3.98 (t, J = 8.7 Hz) and 3.79 (bd, J = 4.9 Hz) were observed. The four methyls appeared as two doublets [δ = 0.97 (d, J = 6.9 Hz) and 0.89 (d, J = 6.9 Hz)] and two singlets (δ = 0.89 and 0.87).

The combined use of H-H COSY and HMQC evidenced the presence of four spin systems (see a - d in Fig. [2]). These substructures were connected by HMBC correlations between the protons H-2 (δ_H = 2.72 and 2.27) and C-4 (δ_C = 140.9) and C-9 (δ_C = 50.3), between H-18 (δ_H = 2.91) and C-3 (δ_C = 143.9), between H-17 (δ_H = 0.96) and C-1 (δ_C = 81.3), between H-14 (δ_H = 3.79) and C-5 (δ_C = 153.9) and C-12 (δ_C = 138.5), between H-7 (δ_H = 2.44 and 1.34) and C-6 (δ_C = 48.0) and C-14 (δ_C = 74.6). These relationships are represented in Fig. [2].

All these data allowed us to identify compound 1 as 1-hydroxyallocyathin B₂ [20]. With the gross structure of 1 in hand, the relative stereochemistry of cyrneine A was deduced from NOESY correlations. As indicated in Fig. [3], NOESY correlations between H-1 and H-2a and H-8a and between H-8a and H-16 indicated a β-orientation of the C-1 hydroxy group. Correlations between H-14 and H-13, H-7b and H-16 led us to attribute a β-orientation to the C-14 hydroxy group. Furthermore, correlations between H-16 and H-13b and H-8b and between H-7a and H-17 showed that the 6-membered ring exists in a chair conformation and H-8b, H-13b and C-6 methyl are α-axial, while H-7a and C-9 methyl are β-axial.

The ¹H-NMR spectrum of compound 2 in CDCl₃ was unclear due to the overlapping of several signals between 3.0 and 1.5 ppm. For this reason we tested several deuterated solvents to register the NMR spectra, and best results were obtained using C₆D₆.

Compound 2 differed from the already reported glaucopine C (3) in the replacement of the methine at C-4 (δ_C = 63.5) with an oxygenated tetrasubstituted carbon (δ_C = 85.7) and showed the same basic pattern of HMBC and NOESY correlations as in 3 [11]. The major differences between the two compounds resided in the chemical shifts of the methyl groups at C-9 and C-6. Due to the γ-effect exerted by the hydroxy group at C-4, C-17 in compound 2 resonated at higher fields (20.0 ppm vs. 22.8 ppm) while the C-6 methyl was upshifted by 2.2 ppm (26.8 ppm vs. 28.7). All these data allowed us to identify compound 2 as 4-hydroxyglaucopine C [11].

Finally, the ability of the cyrneine A (1) to stimulate the production of neurotrophic factors was investigated. When cyrneine A at 100 μM was tested according to the reported procedure [13] to evaluate its ability to induce the expression of NGF in 1321N1 human astrocytoma cells, the effect of the cyrneine A was marginal compared with scabronine G-methyl ester (data not shown). Next, rat pheochromocytoma cells (PC12), used as a model system of neuronal differentiation, were cultured with cyrneine A (50 or 100 μM), cyrneine B (50 or 100 μM) or NGF (100 ng/mL) for 24 h. Interestingly, cyrneines A and B significantly promoted neurite outgrowth in concentration-dependent manners, and the effects of both compounds at 100 μM were comparable to that of NGF as a positive control (Fig. [4]). The cytotoxicity of cyrneines was determined by the LDH assay. PC12 cells were cultured in the presence of cyrneine A (100 μM), cyrneine B (100 μM) and A23187 (10 μM) as a positive control for 24 h in the same media as above, then the LDH assay was performed. As a result, although A23187 caused cytotoxicity (3.5-fold LDH release of control), neither cyrneine A nor cyrneine B was cytotoxic (0.59-fold and 0.90-fold by cyrneines A and B, respectively).

Previously, we had shown that scabronines, similar diterpenes, enhanced the biosynthesis of neurotrophic factors including NGF in 1321N1 human astrocytoma cells [13]. Furthermore, these compounds themselves failed to induce neurite outgrowth in PC12 cells. Interestingly, although the structures of cyrneines are really similar to those of scabronines, cyrneines showed distinct pharmacological effects in this study, i. e., these compounds promoted neurite outgrowth in PC12 cells, whereas the effect of cyrneine A (100 μM) on NGF expression in 1321N1 cells was marginal compared with scabronine G-methyl ester. Also, it has been reported that other similar diterpenes such as erinacines enhanced NGF biosynthesis in rat astroglial cells. [3] Although their effects on neurite outgrowth were not examined in the study, it would be of interest to know whether erinacines have an additional effect on neurite genesis. Judging from these facts, minor differences in functional groups on cyathane structure can influence the cellular responses, thus further study is essential to investigate the structure-activity relationship in addition to the mechanism of these diterpenes.

Acknowledgements

We are grateful to MIUR (Italy), to University of Perugia, and to Hokuto Life Sciences Foundation (Japan) for financial support.

References

• 1 Allbutt A D, Ayer W A, Brodie H J, Johri B N, Taube H. Cyathin, a new antibiotic complex produced by Cyathus helenae .  Can J Microbiol. 1971;  17 1401-7
  PubMedSearch in Google Scholar
• 2 Hecht H J, Höfle G, Steglich W, Anke T, Oberwinkler F. Striatin A, B, and C: novel diterpenoid antibiotics from Cyathus striatus . J Chem Soc, Chem. Commun 1978: 665-6
  Search in Google Scholar
• 3 Kawagishi H, Shimada A, Shirai R, Okamoto K, Ojima F, Sakamoto H. et al . Erinacines A, B. and C, strong stimulators of nerve growth factor (NGF)-synthesis from the mycelia of Hercium erinaceum .  Tetrahedron Lett. 1994;  35 1569-72
  CrossrefPubMedSearch in Google Scholar
• 4 Ohta T, Takako K, Norihiro K, Obara Y, Norimichi N, Yasushi O, Yoshiaki T, Yoshiteru O. Scabronine A, a novel diterpenoid having potent inductive activity of the nerve growth factor synthesis, isolated from the mushroom Sarcodon scabrosus .  Tetrahedron Lett. 1998;  39 6 229-32
  PubMedSearch in Google Scholar
• 5 Kita T, Takaya Y, Oshima Y, Ohta T, Aizawa K, Hirano T. et al . Scabronins B, C, D, E and F, novel diterpenoids showing stimulating activity of nerve growth factor-synthesis, from mushroom Sarcodon scabrosus .  Tetrahedron. 1998;  54 11 877-86
  PubMedSearch in Google Scholar
• 6 Anke T, Oberwinkler F, Steglich W, Höfle G. Antibiotics from Basidiomycetes, 1. The striatins - new antibiotics from the Basidiomycete Cyathus striatus (Huds. Ex Pers.) Willd.  J Antibiot. 1977;  30 221-5
  PubMedSearch in Google Scholar
• 7 Hirota M, Moritura K, Shibata H. Anti-inflammatory compounds from the bitter mushroom, Sarcodon scabrosus .  Biosci Biotechnol Biochem. 2002;  66 179-84
  PubMedSearch in Google Scholar
• 8 Kamo T, Imura Y, Hagio T, Makabe H, Shibata H, Hirota M. Anti-inflammatory cyathane diterpenoids from Sarcodon scabrosus .  Biosci Biotechnol Biochem. 2004;  68 1362-5
  CrossrefPubMedSearch in Google Scholar
• 9 Rosas-Romero A, Martinez Manchado C, Crescente O, Acosta M, Curini M, Epifano F. et al . Anti-inflammatory sesquiterpene lactones from Lourteigia ballotaefolia .  Planta Med. 2002;  68 841-3
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Curini M, Maltese F, Marcotullio M C, Menghini L, Pagiotti R, Rosati O. et al . Glaucopines A and B, new cyathane diterpenes from the fruiting bodies of Sarcodon glaucopus .  Planta Med. 2005;  71 194-6
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Marcotullio M C, Pagiotti R, Campagna V, Maltese F, Fardella G, Altinier G. et al .Glaucopine C, a new diterpene from the fruiting bodies of Sarcodon glaucopus . Nat Prod Res, in press
• 12 Obara Y, Nakahata N, Kita T, Takaya Y, Kobayashi H, Hosoi S. et al . Stimulation of neurotrophic factors secretion from 1321N1 human astrocytoma cells by novel diterpenoids, scabronines A and G.  Eur J Pharmacol. 1999;  370 79-84
  CrossrefPubMedSearch in Google Scholar
• 13 Obara Y, Kobayashi H, Ohta T, Ohizumi Y, Nakahata N. Scabronine G-methyl ester enhances secretion of neurotrophic factors mediated by an activation of protein kinase C-ξ.  Mol Pharmacol. 2001;  59 1287-97
  PubMedSearch in Google Scholar
• 14 Levi Montalcini R. The nerve growth factor: 35 years later.  Science. 1987;  237 1154-62
  CrossrefPubMedSearch in Google Scholar
• 15 Furukawa Y, Furukawa S. Catecholamines induce an increase in nerve growth factor content in the medium of mouse L-M cells.  J Biol Chem. 1986;  261 6039-47
  PubMedSearch in Google Scholar
• 16 Veenstra T D, Fahnestock M, Kumar R. AP-1 site in the nerve growth factor expression in osteoblast.  Biochemistry. 1998;  37 5988-94
  CrossrefPubMedSearch in Google Scholar
• 17 Takeuchi R, Murase K, Furukawa Y, Furukawa S, Hayashi K. Stimulation of nerve growth factor synthesis/secretion by 1,4-benzoquinone and its derivative in cultured mouse astroglial cells.  FEBS Lett. 1990;  261 63-6
  CrossrefPubMedSearch in Google Scholar
• 18 Kawagishi H, Ando M, Shinba K, Sakamoto H, Yoshida S, Ojima F. et al . Chromans, hericenones F, G and H from the Mushroom Hericium erinaceum .  Phytochemistry. 1993;  32 175-8
  PubMedSearch in Google Scholar
• 19 Obara Y, Aoki T, Kusano M, Ohizumi Y. β-Eudesmol induces neurite outgrowth in rat pheochromocytoma cells accompanied by an activation of mitogen-activated protein kinase.  J Pharmacol Exp Ther. 2002;  301 803-11
  CrossrefPubMedSearch in Google Scholar
• 20 Tori M, Toyoda N, Sono M. Total synthesis of allocyathin B₂, a metabolite of bird's nest fungi.  J Org Chem. 1998;  63 306-13
  CrossrefPubMedSearch in Google Scholar

Prof. Maria Carla Marcotullio

Dipartimento di Chimica e Tecnologia del Farmaco

Sez. Chimica Organica

Università di Perugia

Via del Liceo 1

06123 Perugia

Italy

Phone: +39-075-585-5107

Fax: +39-075-585-5116

Email: marcotu@unipg.it

References

• 1 Allbutt A D, Ayer W A, Brodie H J, Johri B N, Taube H. Cyathin, a new antibiotic complex produced by Cyathus helenae .  Can J Microbiol. 1971;  17 1401-7
  PubMedSearch in Google Scholar
• 2 Hecht H J, Höfle G, Steglich W, Anke T, Oberwinkler F. Striatin A, B, and C: novel diterpenoid antibiotics from Cyathus striatus . J Chem Soc, Chem. Commun 1978: 665-6
  Search in Google Scholar
• 3 Kawagishi H, Shimada A, Shirai R, Okamoto K, Ojima F, Sakamoto H. et al . Erinacines A, B. and C, strong stimulators of nerve growth factor (NGF)-synthesis from the mycelia of Hercium erinaceum .  Tetrahedron Lett. 1994;  35 1569-72
  CrossrefPubMedSearch in Google Scholar
• 4 Ohta T, Takako K, Norihiro K, Obara Y, Norimichi N, Yasushi O, Yoshiaki T, Yoshiteru O. Scabronine A, a novel diterpenoid having potent inductive activity of the nerve growth factor synthesis, isolated from the mushroom Sarcodon scabrosus .  Tetrahedron Lett. 1998;  39 6 229-32
  PubMedSearch in Google Scholar
• 5 Kita T, Takaya Y, Oshima Y, Ohta T, Aizawa K, Hirano T. et al . Scabronins B, C, D, E and F, novel diterpenoids showing stimulating activity of nerve growth factor-synthesis, from mushroom Sarcodon scabrosus .  Tetrahedron. 1998;  54 11 877-86
  PubMedSearch in Google Scholar
• 6 Anke T, Oberwinkler F, Steglich W, Höfle G. Antibiotics from Basidiomycetes, 1. The striatins - new antibiotics from the Basidiomycete Cyathus striatus (Huds. Ex Pers.) Willd.  J Antibiot. 1977;  30 221-5
  PubMedSearch in Google Scholar
• 7 Hirota M, Moritura K, Shibata H. Anti-inflammatory compounds from the bitter mushroom, Sarcodon scabrosus .  Biosci Biotechnol Biochem. 2002;  66 179-84
  PubMedSearch in Google Scholar
• 8 Kamo T, Imura Y, Hagio T, Makabe H, Shibata H, Hirota M. Anti-inflammatory cyathane diterpenoids from Sarcodon scabrosus .  Biosci Biotechnol Biochem. 2004;  68 1362-5
  CrossrefPubMedSearch in Google Scholar
• 9 Rosas-Romero A, Martinez Manchado C, Crescente O, Acosta M, Curini M, Epifano F. et al . Anti-inflammatory sesquiterpene lactones from Lourteigia ballotaefolia .  Planta Med. 2002;  68 841-3
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Curini M, Maltese F, Marcotullio M C, Menghini L, Pagiotti R, Rosati O. et al . Glaucopines A and B, new cyathane diterpenes from the fruiting bodies of Sarcodon glaucopus .  Planta Med. 2005;  71 194-6
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Marcotullio M C, Pagiotti R, Campagna V, Maltese F, Fardella G, Altinier G. et al .Glaucopine C, a new diterpene from the fruiting bodies of Sarcodon glaucopus . Nat Prod Res, in press
• 12 Obara Y, Nakahata N, Kita T, Takaya Y, Kobayashi H, Hosoi S. et al . Stimulation of neurotrophic factors secretion from 1321N1 human astrocytoma cells by novel diterpenoids, scabronines A and G.  Eur J Pharmacol. 1999;  370 79-84
  CrossrefPubMedSearch in Google Scholar
• 13 Obara Y, Kobayashi H, Ohta T, Ohizumi Y, Nakahata N. Scabronine G-methyl ester enhances secretion of neurotrophic factors mediated by an activation of protein kinase C-ξ.  Mol Pharmacol. 2001;  59 1287-97
  PubMedSearch in Google Scholar
• 14 Levi Montalcini R. The nerve growth factor: 35 years later.  Science. 1987;  237 1154-62
  CrossrefPubMedSearch in Google Scholar
• 15 Furukawa Y, Furukawa S. Catecholamines induce an increase in nerve growth factor content in the medium of mouse L-M cells.  J Biol Chem. 1986;  261 6039-47
  PubMedSearch in Google Scholar
• 16 Veenstra T D, Fahnestock M, Kumar R. AP-1 site in the nerve growth factor expression in osteoblast.  Biochemistry. 1998;  37 5988-94
  CrossrefPubMedSearch in Google Scholar
• 17 Takeuchi R, Murase K, Furukawa Y, Furukawa S, Hayashi K. Stimulation of nerve growth factor synthesis/secretion by 1,4-benzoquinone and its derivative in cultured mouse astroglial cells.  FEBS Lett. 1990;  261 63-6
  CrossrefPubMedSearch in Google Scholar
• 18 Kawagishi H, Ando M, Shinba K, Sakamoto H, Yoshida S, Ojima F. et al . Chromans, hericenones F, G and H from the Mushroom Hericium erinaceum .  Phytochemistry. 1993;  32 175-8
  PubMedSearch in Google Scholar
• 19 Obara Y, Aoki T, Kusano M, Ohizumi Y. β-Eudesmol induces neurite outgrowth in rat pheochromocytoma cells accompanied by an activation of mitogen-activated protein kinase.  J Pharmacol Exp Ther. 2002;  301 803-11
  CrossrefPubMedSearch in Google Scholar
• 20 Tori M, Toyoda N, Sono M. Total synthesis of allocyathin B₂, a metabolite of bird's nest fungi.  J Org Chem. 1998;  63 306-13
  CrossrefPubMedSearch in Google Scholar

Prof. Maria Carla Marcotullio

Dipartimento di Chimica e Tecnologia del Farmaco

Sez. Chimica Organica

Università di Perugia

Via del Liceo 1

06123 Perugia

Italy

Phone: +39-075-585-5107

Fax: +39-075-585-5116

Email: marcotu@unipg.it