Ginkgolide B produced endophytic fungus (Fusarium oxysporum) isolated from Ginkgo biloba

Fitoterapia 83 (2012) 913–920

Contents lists available at SciVerse ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Ginkgolide B produced endophytic fungus (Fusarium oxysporum) isolated from Ginkgo biloba Yuna Cui a, Dawei Yi a, Xiufeng Bai a, Baoshan Sun b, Yuqing Zhao a,⁎, Yixuan Zhang a,⁎⁎ a b

Shenyang Pharmaceutical University, Shenyang, P.R. China INIA Dois Portos, Instituto Nacional de Recursos Biológicos, I.P., 2565–191, Dois Portos, Portugal

a r t i c l e

i n f o

Article history: Received 17 December 2011 Accepted in revised form 4 April 2012 Available online 16 April 2012 Keywords: Ginkgo biloba Endophytic fungus Fermentation Fusarium oxysporum Ginkgolide B

a b s t r a c t To screen the presence of ginkgolide B-producing endophytic fungi from the root bark of Ginkgo biloba, a total of 27 fungal isolates, belonging to 6 different genus, were isolated from the internal root bark of the plant Ginkgo biloba. The fungal isolates were fermented on solid media and their metabolites were analyzed by TLC. The obtained potential ginkgolidesproducing fungus, the isolate SYP0056 which was identified as Fusarium oxysporum, was successively cultured in the liquid fermentation media, and its metabolite was analyzed by HPLC. The ginkgolide B was successfully isolated from the metabolite and identified by HPLC/ ESI-MS and 13C-NMR. The current research provides a new method to produce ginkgolide B by fungal fermentation, which could overcome the natural resource limitation of isolating from the leaves and barks of the plant Ginkgo biloba. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The endophytic fungi live inside the host plants without causing discernible manifestation of disease [1,2]. They play important roles in the process of host plant growth and systematic evolution [3,4]. It has been recognized that many endophytic fungi have the capability to produce important novel structurally-active secondary metabolites with anticancer, antimicrobial or other biological activities [5,6]. Stierle et al. successfully isolated a strain of Taxomyces andreanae from phloem of Taxus brevifolia first, which can produce taxol and related chemicals at the concentration of 24–50 ng/L [7]. From then on, more and more endophytic fungi from pharmaceutical plants, such as Camptotheca acuminata [8], pine [9] and Taxus plants [10–12] were isolated. Recent studies on new biologically-active metabolites from endophytic microorganisms residing in a well-known medicinal plant G. biloba have shown that Colletotrichum sp. could

⁎ Corresponding author. Tel.: + 86 2423986521; fax: + 86 2423986522. ⁎⁎ Corresponding author. Tel.: + 86 2423986576; fax: + 86 2423986401. E-mail addresses: [email protected] (Y. Zhao), [email protected] (Y. Zhang). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.04.009

produce flavones which exhibited potent anti-cancer and antioxidant activities [13,14], Alternaria No. 28 could produce cytotoxic metabolites [15] and Chaetomium globosum ZY-22 could produce two polyhydroxylated steroids [16]. On the other hand, Ginkgo tree is the source of various phytochemicals like flavonoids, terpenoids, and other compounds [17,18], among which, bilobalide and ginkgolides are shown to be beneficial to human health [19]. The ginkgolides are a kind of diterpenoide lactones classified as A, B, C, J, and M [20]. Among different effects, ginkgolide B (GB) in particular, shows potent antagonist effects on platelet activating factors (PAF), involved in the development of a number of cardiovascular, renal, respiratory and central nervous system disorders [21,22]. Hence, the structure–activity relationships of ginkgolides as PAF receptor antagonists hold great appeal to medical chemists. All ginkgolides are 20-carbon cage molecules that incorporate six rings of five carbons each which include a tetrahydrofuran group, a spirononane system, three lactone rings and a tert-butyl group. Structurally, ginkgolides differ only in the number and position of hydroxyl functions. Ginkgolide B is characterized by the presence of two hydroxyl groups on C1 and C3 of spirononane framework. The C7 position of ginkgolides showed critical importance of PAF receptor activity, Ginkgolide C is 25-

914

Y. Cui et al. / Fitoterapia 83 (2012) 913–920

fold less potent than ginkgolide B as a PAF receptor antagonist, due to the presence of the 7beta-OH [23]. Another important feature contributed to the activity of PAF receptor antagonist is the t-butyl group which is prominent outside the main framework. The QSAR models were also used to account for why ginkgolide B has unusual higher bioactivity than other ginkgolides [24]. The objective of the present study was to explore an endophytic fungus (Fusarium oxysporum) isolated from G. biloba with the capability to produce biologically-active ginkgolide B, which was further to be isolated and purified. 2. Experimental procedures 2.1. Plant material Healthy root barks were collected from G. biloba grown at the forest site located in the Changbai Mountain, P.R. China. The samples were surface-sterilized by washing in 70% ethanol (v/v) for 1 min, followed by 0.1% mercuric chloride (v/v) for 8 min. The root barks were rinsed five times in sterile distilled water then employed to isolate endophytic fungi. 2.2. Mediums The modified potato dextrose agar (PDA) medium was prepared as follows: the components of PDA medium were dissolved in the 20% (v/v) ginkgo bark extract solution instead in water. The ginkgo bark extract solution was prepared by boiling pieces (2 cm×2 cm) of 200 g ginkgo barks in 1 L distilled water and then leaching the ginkgo bark. Solid fermentation medium: 10 g/L glucose, 10 g/L groundnut flour, 10 g/L soybean flour, 40 g/L maize starch, 3 g/L NH4Cl, 3 g/L KH2PO4, 3 g/L NaAc∙3H2O, 4 g/L CaCO3, 3 g/L MgSO4·7H2O, 20 g/L agar, pH 6.0, sterilized at 115 °C for 20 min. Liquid seed medium: 20 g/L glucose, 3 g/L groundnut flour, 3 g/L NH4Cl, 3 g/L KH2PO4, 3 g/L MgSO4·7H2O, 4 g/L CaCO3, 1 g/L NaAc·3H2O, pH 6.0, sterilized at 115 °C for 20 min.

Table 1 HPLC spectral information of authentic compounds. Reference substances (0.2 mg mL− 1)

GA

GB

GC

BB

Retention time (min) HPLC peak area

16.375 50,708

18.492 45,875

8.748 58,342

7.937 34,699

The potential ginkgolides-producing fungi were cultivated in liquid seed medium for 24 h at 28 °C, 200 r/min, 15 mL culture was inoculated into 500 mL Erlenmeyer flasks containing 300 mL of the liquid fermentation medium and cultured for 6 days at 28 °C, 200 r/min. The mycelial pellet was harvested by filtration and frozen at −20 °C over night for molecular taxonomy of the isolates. The fermentation broths and ground mycelia were subjected to ultrasound-assisted extraction three times with acetic ether at room temperature for 5 min. All extracts were combined and dried under vacuum. The residues were redissolved in methanol to the concentration of 7.6 mg/mL. Authentic compounds, Ginkgolide A, B, C and bilobalide (Chinese Biological Product Detection Institute), were dissolved in methanol to the concentration of 0.2 mg/mL separately. The sample (10 μL) was analyzed by reverse-phase HPLC (Shimzdzu LC-10AT pump, Japan) using an RP-C18 Kromasil column (4.6×250 mm, 5 μm) with UV absorbance at 220 nm at column temperature of 40 °C. The separation was performed at a flow rate of 0.6 mL/min with MeOH/H2O (45:55, v/v) as the mobile phase.

2.5. Isolation of GB' The dried acetic ether extract was subjected to silica gel column chromatography (200–300 mesh) in a gradient CHCl3/ MeOH (50:1, 30:1, 20:1, 10:1, v/v) to yield six fractions A–F. Fraction C was chromatographed to prepared HPLC eluted with cyclohexane/acetone (2:1 and 1:1, v/v) to afford the compound GB’.

2.3. Isolation of endophytic fungi The sterilized root barks were cut into pieces of about 1 cm 2. Up to six pieces of root material was plated on the modified PDA medium supplemented with 100 μg/mL streptomycin sulphate (Sigma, St Louis, America) to incubate at 25 °C for 2–15 days. At the first emergence of fungal growth from the root pieces, the fungus was subcultured onto another modified PDA plate and incubated for 3–5 days.

Table 2 HPLC spectral information of fermentation products of isolate SY0056. Fermentation metabolites of isolate SY0056 (7.6 mg mL− 1)

GA'

GB'

GC'

BB'

Retention time (min) HPLC peak area

16.668 3426

18.150 30,169

8.542 11,906

8.005 66,403

2.4. Screening of ginkgolides-producing fungi Each fungal endophyte was cultivated on the sterile solid fermentation medium for 7 days at 28 °C, in 60% humidity. Then the medium was collected and crushed followed by immersed into 0.5 mL acetic ether to extract the fermental metabolites. The mixture of the medium and acetic ether was centrifuged at 10000 r/min for 5 min. 4 μL of supernatant was applied for TLC detection. Blank controls were composed of sterile solid fermentation medium incubated without strains under the identical conditions.

Table 3 HPLC spectral information of the mixture of isolate SY0056 products and authentic compounds. Fermentation sample (7.6 mg mL− 1) + reference substances (0.2 mg mL− 1)

GA + GA'

GB + GB'

GC + GC'

BB + BB'

Retention time (min) HPLC peak area

16.59 53,995

18.775 76,291

8.757 65,164

8.173 96,603

Y. Cui et al. / Fitoterapia 83 (2012) 913–920

2.6. Identification of GB' The LCQ liquid chromatograph-mass spectrograph (Finnigan Ltd., American) was taken together with the electronic spraying ionization (ESI) technique to identify the GB'. Mass spectrograph condition was as follows: negative ion source spraying voltage 4.25 kV, capillary voltage 6.25 kV, capillary temperature 180 °C, sheath airflow velocity 70 flux unit (a. u.), accessorial airflow velocity 10 a.u. Chromatographic analyzing condition: chromatographic column: Hypersil C18 (250×6.0 mm, 5 μm), flowing phase MeOH/H2O (45/55, v/v), flow rate 0.5 mL/min, column temperature 40 °C, injection volume 10 μL. 13 C-NMR (150 MHz) analysis was also used for structural identification of GB’. The NMR equipment was a Bruker APX600 (Bruker Corporation, Germany). 13C-NMR spectral data of the analyzed compound were obtained in CD3OCCD3 with TMS as internal standard.

2.7. Morphological taxonomy The isolate SY0056 was cultivated on PDA plates for 5 days at 25 °C in darkness to measure the colonies' diameters, and was

915

continuously incubated for 3 days under natural illumination to observe the colonies' morphology. A square piece of culture medium was cut together with mycelia, put on a sterile glass slide, and covered with a coverslip. The slide was incubated in the Petri dish for a week. The coverslip was transferred to a new slide to make a usual glass slide specimen for observation [25].

2.8. Molecular taxonomy 0.5 g mycelia of endophytic fungi was ground with a sterile mortar in liquid nitrogen. DNA was extracted by the CTAB method [26]. The fungal ITS fragments were amplified using the universal primers ITS1 and ITS4 [27]. The PCR reaction mixtures (20 μL) were composed of 1 μL genomic DNA (100 ng), 2 μL 10× PCR reaction buffer, 2 μL 2 μM MgCl2, 0.5 μL 10 μM forward and reverse primers, 0.5 μL each 2.5 μM deoxyribonucleotide triphosphate, 0.2 μL 5 U of Ex Taq DNA polymerase (TaKaRa, DaLian, P.R. China), and 13.3 μL PCR aquae sterilisata. The PCR reaction programs were pre-heating at 94 °C for 5 min, 25 cycles of 94 °C for 1 min, 55 °C for 40 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR products were separated on 1% (w/v) agarose gel and purified

Fig. 1. ESI-MS/MS spectra of fermentation product GB’. Full scan of MS (A), MS/MS scan of [M-H]− (m/z 423.0) (B).

916

Y. Cui et al. / Fitoterapia 83 (2012) 913–920

using a DNA Gel Exaction Kit (TaKaRa, DaLian, P.R. China). The resulting DNA was sequenced directly using the same primers (Sangon Biological Engineering Technology & Services Co., Ltd, Shanghai, P.R. China). The ITS sequence of the endophytic fungi was compared with the data available in NCBI using BLAST searches to estimate its phylogenetic relationships.

3. Results and discussion 3.1. Isolation of culturable endophytic fungi from the root bark of ginkgo A total of 27 fungal isolates, belonging to 6 different genus, were acquired from the internal root bark of G. biloba. In this study, these isolates were morphologically identified as Alternaria, Aspergillus, Colletotrichum, Fusarium, Myrothecium and Penicillium [28], which reveals the amount of endophytic fungi living in the internal part of Ginkgo tree.

Of all the plants in the world, only a few grass species were thoroughly studied for their endophytes. Thus, it is of significance to find interesting strains among a variety of plants. Recently statistical analyses showed that 51% of the biologically active metabolites obtained from endophytes are previously unknown, compared with only 38% of novel substances from soil microflora, so endophytes could be regarded as a prolific source of structurally novel secondary metabolites with biological activities [4,29]. Recent studies on endophytic microorganisms residing in a well-known medicinal plant G. biloba, have shown some new biologically active metabolites with potential anti-cancer, antioxidant activities et al. For example, Chaetomium globosum, isolated in the leaves of G. biloba, produced four compounds, chaetomugilin D, together with chaetomugilin A, chaetoglobosins A and chaetoglobosins C, which all displayed significant growth inhibitory activity against the brine shrimp (Artemia salina) and Mucor miehei [30]. Extract of another endophyte of G. biloba, Alternaria No.28, were elucidated to contain eight compounds (alterperylenol, altertoxin I, alternariol, alternariol

Fig. 2. ESI-MS/MS spectra of authentic compound, ginkgolide B. Full scan of MS (A), MS/MS scan of [M-H]− (m/z 423.0) (B).

Y. Cui et al. / Fitoterapia 83 (2012) 913–920

monomethyl ether, tenuazonic acid and its derivative, together with ergosterol and ergosta-4, 6, 8, 22-tetraen-3-one) by means of spectroscopic analysis. Among them, tenuazonic acid and its derivative showed significant cytotoxic effects in vitro against brine shrimp (Artemia salina), with mortality rates of 73.6% and 68.9%, respectively, at a concentration of 10 μg/mL, and they were first isolated from endophytic fungi [15]. Chaetomium globosum ZY-22, an endophytic fungus, also separated from the leaves of G. biloba, which produced a novel C29-polyhydroxysterol, named globosterol, along with a tetrahydroxylated ergosterol. The C29-polyhydroxysterol is a sterol possesses an unprecedented 25-methyl Δ22-C10-side chain and Δ7-3β, 6β, 9α-hydroxy-steroid nucleus, which represents the first example for C29-steroids of the group [16]. 3.2. Screening of ginkgolide B-producing fungi TLC detection of solid fermentation products showed that only one isolate, SY0056, produced some metabolites with the same Rf values to those of ginkgolide B (GB), ginkgolide C (GC), bilobalide (BB), the Rf values were 0.75, 0.69 and 0.55 respectively. Therefore the isolate SY0056 was further incubated in liquid fermentation medium. Under the same HPLC conditions, the liquid fermentation extracts of SY0056 and authentic compounds were analyzed separately to detect fungal ginkgolides and bilobalide. The results showed retention time and peak shapes of extracts of strain SY0056 (7.6 mg/mL) were identical to those of the authentic compounds (each of 0.2 mg/mL), GA, GB, GC, BB (Tables 1 and 2). Some compounds in the extract of strain SY0056 have the identical retention time (tR) to the authentic compounds at 8.005 min (close to BB), 8.542 min (close to GC), 16.668 min (close to GA) and 18.150 min (close to GB) respectively, therefore these components were named as BB', GC', GA', and GB', separately. Moreover, when the mixture of fermentation extracts (7.6 mg/mL) and four authentic compounds (each of 0.2 mg/mL) was analyzed, there were still four peaks appeared in the HPLC chromatogram with the identical shape and retention time to BB, GC, GA and GB (Table 3), also, the quantity of peaks’ areas of the mixture increased distinctly, corresponding to the summation area of BB + BB’, GC + GC’, GA + GA’ and GB + GB’ separately (Table 3). The components of GA’, GB’, GC’ and BB’ might be structurally identical to ginkgolide A, B, C and bilobalide.

917

Table 5 13 C-NMR spectral data of GB'. Number

GB

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 t-Bu

73.84 91.87 82.98 98.51 71.75 78.65 36.66 48.61 67.46 69.11 174.01 109.67 170.30 41.57 176.42 7.90 32.08 28.91

[21,22]; therefore this study was focused on whether the isolate SY0056 could produce ginkgolide B. Because the content of GB’ in fermentation extract was too low to acquire, it was purified by silica gel column chromatography and preparative HPLC to isolate a slight amount of compound GB'. The ESI-MS/MS spectra of the fungal GB' were shown in Fig. 1, while the spectra of the authentic compound GB were shown in Fig. 2, and their ESI-MS/MS information was summarized in Table 4. The molecular weight of GB is 424. GB yielded a single HPLC peak at retention time of 22.53 min, [M-H]− at m/z 423.0, [2M-H]− at m/z 847.0, [M-2CO-H]− at m/z 367.3, [M-CO-H]− at m/z 394.7 and [M-2CO-H]− at m/z 367.0 (Fig. 2). Comparatively, the fungal GB' also yielded a single HPLC peak at 22.98 min, which was close to the retention time of GB, and formed similar ion fragment peaks to GB, such as m/z 423.0 (presumed to be [M-H]−), m/z 367.0 (presumed to be [M-2COH]−) and m/z 394.8 (presumed to be [M-CO-H]−), m/z 366.9 (presumed to be [M-2CO-H]−) (Fig. 1). On the basis of HPLC/ESIMS/MS, the fungus SY0056 did generate ginkgolide B in vitro. 13 C-NMR (CD3OCCD3, 150 MHz) data and its structure were shown in Table 5 and Fig. 3. The carbon signals of the

3.3. Identification of GB' Among the well-known ginkgolides, ginkgolide B has the strongest functional activity against platelet activating factors

Table 4 HPLC/ESI-MS spectral information of GB and GB'. HPLC/ESI-MSn

GB (reference substance)

GB' (fermentation product)

Ion

HPLC tR(min) Full scan MS (m/z)-

22.53 423.0 847.0 367.3 394.7 367.0

22.98 423.0

– [M-H]− [2 M-H]− [M-2CO-H]− [M-CO-H]− [M-2CO-H]−

MS2 423.0 (m/z)

367.0 394.8 366.9

Fig. 3. Chemical structure of ginkgolide B.

918

Y. Cui et al. / Fitoterapia 83 (2012) 913–920

fermentation product GB' in the 13C-NMR spectrum were similar as those of GB [28]. Ginkgolides are specific inhibitor of human platelet aggregation and inhibit the binding of (3H)-PAF-acether to its membrane platelet receptor. The cis-fused cyclopentanoid F, A, D and C (Fig. 5(A)) are folded in such a way which a semispherical cavity is sufficiently large to receive most atoms such as the trimethylammonium group. The two paralleled side of the cavity are defined by the C11 and C15 lactone carbonyls induced in the F and C ring, respectively. The tetrahydrofuran ring D occupies a central position in the cage, its etheral oxygen, along with F and C ring ester oxygen and C10 hydroxyl oxygen constitute a polydentate system similar to that observed in the “Crown ether series”. This electron-rich cavity is ideally suited to the charged bingding of cationic or positive polarized molecules. Another important feature contributed to the activity of PAF receptor antagonist is the t-butyl group which is prominent outside the main framework. Due to the shape of the cavity, if binding does occur, it may well be stereospecific. Active center of ginkgolide as a PAF receptor antagonist may be E, F rings and t-butyl group [31,32]. Presently, the main ginkgolides isolated were ginkgolides A, B, C, J, M, among which, the most powerful PAF receptor antagonist was ginkgolide B. Ginkgolide A and B are

A

characterized by the presence of two hydroxyl groups on C1 and/ or C3. Ginkgolide C, J and M with a hydroxyl group on C7 in an αposition of the lipophilic t-butyl moiety are less active. Regarding to ginkgolide J and M, the loss of the hydroxyl group on C1 or C3 further decreases the activity. As the ginkgolide becomes less polar, its PAF receptor antagonistic activity increases. Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. However, bilobalide is not with the characterized structural properties as ginkgolides (Fig. 5(B)), which may be account for why the bilobalide has no favorable properties equal to ginkgolide B as PAF receptor antagonist which has strong neuroprotective properties. 3.4. Identification of fungus isolate SY0056 As for ITS-5.8S rDNA fragment of isolate SY0056, its ITS BLASTN result only showed 100% identity with a lot of isolates of Fusarium oxysporum (such as Genbank: FJ618521, FJ154076, FJ605243, EU839398). The molecular taxonomy was also supported by the following morphology investigation. After 5 days in darkness and 3 days under natural illumination of single cultivation on PDA plate at 25 °C, the colonies of SY0056

B

C

10mm

10mm

D

E

Fig. 4. Image of isolate SY0056. Image of Conidiophores and conidiogenous cells (A), image of macroconidia and microconidia (B and C), colonies of isolate SY0056 on PDA at 25 °C for 5 days in darkness (D), reverse colonies of isolate SY0056 on PDA at 25 °C for 5 days in darkness (E).

Y. Cui et al. / Fitoterapia 83 (2012) 913–920

A HO R1

O

by fungal fermentation. Further study will focus on how to increase the yield of ginkgolides and ginkgolide B.

O 11

O 10

O

References

C t-Bu

1

F

D O B

A

3

Me

7

E

R3

R2

O

O Compounds

R1

R2

R3

Glinkgolide A Glinkgolide B Glinkgolide C Glinkgolide J Glinkgolide M

H

H

OH

OH

H

OH

OH

OH

OH

H

OH

OH

OH

OH

H

B

O O

OH

t-Bu O

OH

O O

919

O

Fig. 5. Chemical structures of glinkgolides (A) and bilobalide (B).

were floccose, white to purplish, and grew fast to attain a diameter of 30–46 mm. On the reverse side, the colonies were grey purple. The microconidia were oval-ellipsoid, straight or a little curved, generally abundantly, mostly non-septate, forming false heads on conidiogenous cells, 5–12.6×2.5–3.6 μm. Macroconidia were sparse, falcate, 3-(5)-septate, 32.2–40×3.5– 4.5 μm, with gradually tapering and curving ends. Basal cells are foot shaped, 1–6 septate, 10–60×2.5–6 μm, mostly 3 septate. Conidiogenous cells are short, monophialidic, 4.4– 15×2.5–4.4 μm, sparse on aerial mycelia, usually abundant and branching in sporodochia. From these cultivation characters, the isolate SY0056 was appraised as Fusarium oxysporum (Fig. 4). According to the above morphological characteristics and the ITS information, the isolate SY0056 was identified as Fusarium oxysporum [33]. 4. Conclusion An endophytic fungus F. oxysporum, originally from the root bark of G. biloba, was first found to produce Ginkgolide B. It provided a new method to overcome the natural resource limitation of ginkgolides from the leaves and barks of G. biloba

[1] Strobel GA. Rainforest endophytes and bioactive products. Crit Rev Biotechnol 2002;22:315–33. [2] Strobel GA. Endophytes as sources of bioactive products. Microbes Infect 2003;5:535–44. [3] Tan RX, Zou WX. Endophytes: a rich source of functional metabolites. Nat Prod Rep 2001;18:448–59. [4] Saikkonen K, Wali P, Helander M, Faeth SH. Evolution of endophyteplant symbioses. Trends Plant Sci 2004;9:275–80. [5] Caruso M, Colombo AL, Fedeli L, Pavesi A, Quaroni S, Saracchi M. Isolation of endophytic fungi and actinomycetes taxane producers. Ann Microbiol 2000;50:3–13. [6] Zhang Y, Mu J, Feng Y, Kang Y, Zhang J, Gu PJ, et al. Broad-spectrum antimicrobial epiphytic and endophytic fungi from marine organisms: isolation, bioassay and taxonomy. Mar Drugs 2009;7:97–112. [7] Stierle A, Strobel G, Stierle D. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of pacific yew. Science 1993;260:214–6. [8] Lin X, Lu CH, Huang YJ, Zheng ZH, Su WJ, Shen YM. Endophytic fungi from a pharmaceutical plant, Camptotheca acuminata: isolation, identification and bioactivity. World J Microbiol Biotechnol 2007;23:1037–40. [9] Ganley RJ, Brunsfeld SJ, Newcombe G. A community of unknown, endophytic fungi in western white pine. Proc Natl Acad Sci 2004;101: 10107–12. [10] Deng BW, Liu KH, Chen WQ, Ding XW, Xie XC. Fusarium solani, Tax-3, a new endophytic taxol-producing fungus from Taxus chinensis. World J Microbiol Biotechnol 2009;25:139–43. [11] Zhang CL, Liu SP, Lin FC, Kubicek CP, Druzhinina IS. Trichoderma taxi sp. nov., an endophytic fungus from Chinese yew Taxus mairei. FEMS Microbiol Lett 2007;270:90–6. [12] Liu KH, Ding XW, Deng BW, Chen WQ. Isolation and characterization of endophytic taxol-producing fungi from Taxus chinensis. J Ind Microbiol Biotechnol 2009;36:1171–7. [13] Wang MX, Chen SL, Yan SZ, Huo J. A preliminary study on the isolation, identification and media of an endophytic fungus producing flavones. J Nanjing Nor Univ (Nat Sci) 2003;26:106–10. [14] Zhang YJ, Wang JF, Huang YJ. Screening for antitumor activities of endophytic fungi isolated from four species of gymnosperm. J Xiamen Univ (Nat Sci) 2002;41:804–9. [15] Qin JC, Zhang YM, Hua L, Ma YT, Gao JM. Cytotoxic metabolites produced by Alternaria No.28, an endophytic fungus isolated from Ginkgo biloba. Nat Prod Commun 2009;4:1473–6. [16] Qin JC, Gao JM, Zhang YM, Yang SX, Bai MS, Ma YT, et al. Polyhydroxylated steroids from an endophytic fungus, Chaetomium globosum ZY-22 isolated from Ginkgo biloba. Steroids 2009;74:786–90. [17] Huh H, Staba EJ, Singh J. Supercritical fluid chromatographic analysis of polyphenols in Ginkgo biloba. J Chromatogr 1992;600:364–9. [18] Kang SS, Kim JS, Kwak WJ, Kim KH. Flavonoids from the leaves of Ginkgo biloba. Korean J Pharmacogn 1990;21:111–20. [19] Laurain D, Guiller JT, Chenieux JC, van Beek TA. Production of ginkgolide and bilobalide in transformed and gametophyte derived cell cultures of Ginkgo biloba. Phytochemistry 1997;46:127–30. [20] Carrier DJ, van Beek TA, van der Heijden R, Veroorte R. Distribution of ginkgolides and terpenoids biosynthetic activity in Ginkgo biloba. Phytochemistry 1998;48:89–92. [21] Smith PF, Maclennan K, Darlington CL. The neuroprotective properties of the Ginkgo biloba leaf: a review of the possible relationship to platelet-activating factor (PAF). J Ethnopharmacol 1996;50:131–9. [22] Negro AJM, Miralles LJC, Ortiz MJL, Abellan AA, Rubio BR. Plateletactivating factor antagonists. Allergol Immunopathol 1997;25:249–58. [23] Vogensen SB, Strømgaard K, Shindou H, Jaracz S, Suehiro M, Ishii S, et al. Preparation of 7-substituted ginkgolide derivatives: potent platelet activating factor (PAF) receptor antagonists. J Med Chem 2003;46:601–8. [24] Zhu W, Chen G, Hu L, Luo X, Gui C, Luo C, et al. QSAR analyses on ginkgolides and their analogues using CoMFA, CoMSIA, and HQSAR. Bioorg Med Chem 2005;13:313–22. [25] Nelson PE, Toussoun TA, Marasas W. Fusarium species—an illustrated manual for identification. London, U.K.: The Pennsylvania State University Press; 1983. p. 142–5. [26] Zhang D, Yang Y, Castlebury LA, Cerniglia CE. A method for the large scale isolation of high transformation efficiency fungal genomic DNA. FEMS Microbiol Lett 1996;145:261–5. [27] Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and application. San Diego: Academic Press Inc.; 1990. p. 315–22.

920

Y. Cui et al. / Fitoterapia 83 (2012) 913–920

[28] Roumestand C, Perly B, Hosford D, Braquet P. Proton and carbon-13 NMR of ginkgolides. Tetrahedron 1989;45:1975–83. [29] Strobel G, Daisy B, Castillo U, Harper J. Natural products from endophytic microorganisms. J Nat Prod 2004;67:257–68. [30] Qin JC, Zhang YM, Gao JM, Bai MS, Yang SX, Laatsch H, et al. Bioactive metabolites produced by Chaetomium globosum, an endophytic fungus isolated from Ginkgo biloba. Bioorg Med Chem Lett 2009;19:1572–4. [31] Le Solleu H, Langlois MH, Kummer E, Dubost JP. Determination of a PAF antagonist pharmacophore using combined molecular electrostatic

potential and molecular lipophilicity potential. Drug Des Discov 1994;12(2):149–67. [32] Ostermann G, Hofmann B, kertscher HP, Till U. PAF-agonistric and -antagonistic behaviour of new synthetic ether phospholipids. II. Relationship between chemical structure and inhibition of PAF-induced human platelet activation. J Lipid Mediat 1991;3(2):225–37. [33] Zhou YL, Xing LJ. Mycology. Beijing: High Education Press. 1989, p128-131, p194-197, p448.