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Chemical Constituents and Antiviral Activity from Marine Endophytic Fungi from Red Sea Alga Padina pavonica.

Byline: Usama Wahid Hawas and Radwan Al-Farawati

Summary: Three endophytic fungal strains, Fusarium equiseti, Scopulariopsis fusca and Geotrichum candidum were isolated from the inner tissue of the brown alga Padina pavonica, collected from the Red Sea. The organic extracts of their liquid cultures were evaluated for their inhibition of hepatitis C virus (HCV) NS3-NS4A protease. As a result, F. equiseti showing a high-level inhibition of HCV protease (IC50 27.0 ug/ml) was selected for further investigation on its secondary metabolites. The fungus was identified by its morphology and 18S rDNA. Bioassay-guided fractionation of the EtOAc extract of the fungus culture broth revealed seven known metabolites. The structures of these metabolites were determined by MS and NMR spectral analysis. The isolated compounds were explored for inhibition of HCV NS3-NS4A protease activity.

Key words: Red Sea, Brown alga Padina pavonica, Fusarium equiseti, HCV protease.

Introduction

Hepatitis C virus (HCV) is a growing public health problem, affecting an estimated more than 3% of the world's population [1, 2]. No vaccine right now to prevent HCV infection and HCV protease (HCV PR) inhibition is considered to be one of the important excellent target for selective anti-HCV chemotherapy [3, 4]. Marine endophytic fungi are microorganisms living in intra-cellular healthy tissues without causing any diseases to their marine hosts [5]. Endophytic microbes improve the resistance of the host marine organisms to the outside intimidations by producing bioactive metabolites acting as antimicrobial and insecticidal [6]. Recently, marine endophytic fungi are being considered as untapped microbial reservoir that can be expected to provide a wide variety of structurally unique and biologically potent marine natural products [7, 8].

Fusarium equiseti, a naturally occurring endophytic fungus produced different metabolites such as fusarochromanones that they have been shown to have both stimulatory and inhibitory effects on various mammalian cells [9, 10]. The effects of fusarochromanones on the growth of human melanoma cells both in vitro and in vivo at low concentrations (0.1-1 nmol/l) were found to be cytotoxic to many melanoma cell lines [11]. The naturally occurring Zearalenone produced by F. equiseti is a macrocyclic lactone with high binding affinity to oestrogen responsive proliferation of MCF-7 cells and only low acute toxicity [12].

Motivated by a search for bioactive metabolites as antiviral from the fermentation products of endophytic fungi isolated from the inner tissues of the Red Sea alga Padina pavonica. This paper deals with the isolation and structure elucidation of seven secondary metabolites isolated from the active fungus F. equiseti, and their inhibitory effect on HCV NS3-NS4A protease using a SensoLyte TM 520 HCV protease assay kit.

Experimental

General Experimental Procedures

Silica gel (60-120 mesh; Merck) and Sephadex LH-20 (Pharmacia) were used for Column Chromatography. Culture media of Czapek agar and potato dextrose broth were procured from Lab M (Bury, Lancashire, UK). Fluorescent TLC was used (Merck). All compound spots changed after spraying with anisaldehyde/sulfuric acid followed by heating at 120degC. NMR spectra were measured on a Bruker 600 (1H, 600 MHz; 13C, 150 MHz). ESI-mass spectra were recorded on a Finnigan LCQ ion trap mass spectrometer.

Chemicals and Enzymes

Culture media: Glucose, (Acros Organics), bacteriological peptone, malt extract, yeast extract powder and nutrient agar (Lab M Limited), K2HPO4 (Laboratory Reagent), MgSO4 (Oxford Laboratory Reagent), agar (Sisco Research Laboratories Pvt. Ltd). Hepatitis Virus C NS3 protease inhibitor 2 (cat#AS-25346), SensolyteTM 520 HCV Protease assay kit Fluorimetric (cat#AS-71145), HCV NS3/4A protease and SensolyteTM Green protease Assay Kit Fluorimetric (cat#AS-71124) were purchased from AnaSpec Inc., San Jose, Ca, USA. Chymotrypsin inhibitor (Soybean trypsin) was purchased from Sigma Aldrich Co., and Becton Dickinson Falcon TM Microtest 384-well 120 uL black assay plates, no lid, non-sterile, was purchased from Becton Dickinson Inc, Franklin Lakes, USA.

Fungal Isolation and Culture Conditions

The healthy exterior sample of brown alga, Padina pavonica was collected from the Egyptian Red Sea site at a depth of 3-5 m from the coast of South Hurgada in March 2010. In the laboratory, specimens were washed by sterile water and processed for identification by the Coral Reef Ecology and Biology group, National Institute of Oceanography and Fisheries, Suez, Egypt. Fungi F. equiseti, S. fusca and G. candidum were isolated as an epiphyte using Potatoes dextrose agar (PDA) medium containing (g/l) potato (200), glucose (10), and agar (15) at pH 7.5 prepared in 50% sea water supplemented with penicillin benzyl sodium salt (0.02 g/l) to avoid any bacterial growth. After 7-10 days a white, velvety colonies were observed.

Morphological and Molecular Identification of the Endophytic Isolates

The pure isolated fungal strains of P. pavonica were identified mainly by morphological methods by scrutinizing the culture, the mechanism of spore production, and the characteristics of the spores by macroscopic (colony morphology, texture, color, shape and size) and microscopic examination. According to the HCV protease bioassays results, F. equiseti was subjected for DNA extraction and 18S rDNA sequence comparison.

The DNA of the active fungus F. equiseti was extracted using GenElute(tm) Genomic DNA Kit, sigma Aldrich. The 18s rDNA was amplified by polymerase chain reaction (PCR) using pair of primers ITS1 (5'-TTTTTTGGCGCATTCGTGTA-3') and ITS4 (5'-CCCCATGCCCTTCAC-TGGGC-3'), and the intervening 5.8S rDNA region. Multiple sequence alignment and molecular phylogeny was performed using BioEdit [13]. Molecular identification of this isolate based on 18S rDNA sequencing was carried out. According to sequencing similarities and multiple alignments the fungus was found to be in a close relation to Fusarium equiseti (AF141949.1) with a 99% identity. DNA sequencing was carried out by Sequencer Scientific Bourg El-Arab, Alexandria, Egypt.

Extraction and Isolation of Metabolites

The isolated fungus F. equiseti was cultivated on Czapek's peptone liquid medium containing (g/l) glucose (30), yeast (2), peptone (10), NaNO3 (3), KH2PO4 (0.5), KCl (0.5), in 50% sea water at 30degC. Two weeks old fermentation broth (12 L) was separated from the fungal mat. The medium and fungal mycelia were diluted with distilled water to enable them to be easily blended using the Ultra Turrax model 25 at 8000 min-1 for further extraction by ethyl acetate (EtOAc). The resultant extracts were combined and dried using a rotavapour with heating water bath up to 40degC. The resultant crude extract was chromatographed over silica gel column using n-hexane as starting non polar eluent and gradually increasing the polarity by EtOAc as polar solvent in mixture eluent (10%, 20%, until 100% EtOAc/hexane), followed by 10% and 50% MeOH/EtOAc regarding to the TLC of the crude extract as a reference during the fractionation.

A total of 57 fractions were collected (100 ml each), which were combined in 7 fractions according to TLC analysis. Fraction I was chromatographed on a silica gel column using CH2Cl2/MeOH (9.5:0.5, v/v) yielded compounds 1 and 2, while fraction II was purified on Sephadex LH-20 using CH2Cl2/MeOH (3:2, v/v) afforded compounds 3 and 4. Fraction III was further separated using preparative TLC with CH2Cl2/MeOH (9:1, v/v) followed by Sephadex LH-20 chromatography using MeOH/CH2Cl2/n-hexane (1:1:1, v:v:v) as eluent to give compounds 5 and 6.

Compound 7 was obtained from fraction IV using MeOH (100%) on a Sephadex LH-20 column. The isolated compounds were subjected to NMR and MS spectroscopic analysis.

Cyclo-L-Ala-L-Leu (1)

White powder (4.6 mg); TLC: Rf = 0.34(CH3OH/CHCl3, 5:95); [a]D20 -78.3 (c, 1.0, MeOH); 1H NMR (CDCl3) d: 4.03 (1H, m, H-6), 3.92 (1H, m, H-3), 1.85 (2H, dd, H-8), 1.72/1.62 (2H, dd, 7-CH2), 1.43 (3H, d, J = 6 Hz, 6-CH3), 0.96 (3H, d, J = 6 Hz, 9-CH3), 0.94 (3H, d, J = 6 Hz 10-CH3); 13C NMR (CDCl3) d: 170.5 (C-5), 170.0 (C-2), 53.9 (C-3), 51.3 (C-6), 44.4 (C-8), 24.6 (C-7), 23.3 (C-10), 21.6 (C-9), 20.8 (6-CH3); (+)ESI-MS: m/z 207 [M+Na]+, 391 [M+2Na]+, HRESI-MS: m/z 207.1104 [M+Na]+, calcd. for C9H16N2O2, m/z 184.1094

Cyclo(L-Tyr-L-Pro) (2)

White amorphous powder (3 mg); TLC: Rf = 0.23 (CH3OH/CHCl3, 5:95); [a]D20 -69.3 (c, 0.5, MeOH); 1H NMR (CD3OD) d: 7.01 (2H, d, J = 8 Hz, H-2'/6'), 6.67 (2H, d, J = 8 Hz, H-3'/5'), 3.99 (1H, m, H-3), 3.51 (1H, m, H-6), 3.02 (2H, d, J = 6 Hz, H-10), 2.41/2.05 (2H, m, H-9), 1.74 (2H, m, H-7), 1.21 (2H, m, H-8); 13C NMR (CD3OD, 150 MHz) d: 170.8 (C-5), 166.9 (C-2), 157.6 (C-4'), 132 (C-2'/6'), 127.7 (C-1'), 116.2 (C-3'/5'), 60.0 (C-3), 57.7 (C-6), 49.8 (C-9), 37.5 (C-10), 29.3 (C-7), 22.7 (C-8). HRESI-MS (m/z 261.1237 [M+H]), calcd. for C14H17N2O3, m/z 261.2960.

Cordycepi (3'-Deoxyadenosine; 9-cordyceposidoadenosine; Adenine cordyceposide) (3)

White powder (21 mg); TLC: Rf = 0.39 (CH3OH/CHCl3, 1:9); 1H NMR (DMSO-d6): d 8.32 (1H, s, H-8), 8.11 (1H, s, H-2), 7.23 (2H, s, 6-NH2), 5.79 (1H, d, J = 2.4 Hz, H-1'), 5.61 (1H, d, J = 4.0 Hz, 2'-OH), 4.54 (1H, m, H-2'), 4.31 (1H, m, H-4'), 3.61/3.43 (2H, m, 5'-CH2), 2.21/1.89 (2H, m, 3'-CH2); 13C NMR (DMSO-d6): d 156.3 (C-4), 152.4 (C-2), 148.9 (C-6), 139.1 (C-8), 119.2 (C-5), 90.6 (C-1'), 80.4 (C-4'), 74.3 (C-2'), 62.7 (C-5'), 34.2 (C-3'); (+) ESI-MS: m/z 252 [M+H]+ and 274 [M+Na]+; (+) HRESI-MS m/z 252.10912 [M+H]+.

Ara-A (Adenosine; spongouridine; Adenine 9-N-b-arabinofuranoside) (4)

White powder (17 mg); TLC: Rf = 0.56 (CH3OH/CHCl3, 1:9); 1H NMR (DMSO-d6): d 8.35 (1H, s, H-8), 8.14 (1H, s, H-2), 7.35 (2H, s, 6-NH2), 5.87 (1H, d, J = 6.6 Hz, H-1'), 4.62 (1H, t, J = 5.4 Hz, H-3'), 4.14 (1H, dd, J = 3 Hz, H-2'), 3.97 (1H, dd, J = 3 Hz, H-4'), 3.66/3.56 (2H, dd, J = 10.2and3 Hz,5'-CH2); 13C NMR (DMSO-d6): d 156.6 (C-4), 152.8 (C-2), 149.5 (C-6), 140.4 (C-8), 119.8 (C-5), 88.3 (C-1'), 86.3(C-4'), 73.9 (C-2'), 71.1(C-3'), 62.1 (C-5'); (+) ESI-MS: m/z 268 [M+H]+.

Chrysophanol (1,8-Trihydroxy-3-methyl-anthraquinone) (5)

Orange powder (6 mg); TLC: Rf = 0.69 (CH3OH/CHCl3, 5:95); 1H NMR (DMSO-d6): d = 12.02 (2H, br, OH-1/8), 7.81 (1H, t, J = 7.6, H-6), 7.72 (1H, d, J = 7.6 Hz, H-5), 7.56 (1H, br, H-4), 7.39 (1H, d, J = 7.6 Hz, H-7), 7.23 (1H, br, H-2), 2.44 (3H, s, 3-CH3); 13C NMR (DMSO-d6): d = 192.0 (C-9), 182.0 (C-10), 162.0 (C-1), 161.8 (C-8), 149.5 (C-3), 137.7 (C-6), 133.7 (C-10a), 133.4 (C-4a), 124.9 (C-7), 124.5 (C-2), 120.9 (C-4), 119.7 (C-5), 116.3 (C-9a), 114.2 (C-8a), 22.0 (3-CH3); (-) ESI-MS: m/z = 253[M - H]-.

o-Hydroxyemodin (1,3,8-Trihydroxy-6-hydroxymethylanthraquinone) (6)

Red powder (3.8 mg); TLC: Rf = 0.64 (CH OH/CHCl , 5:95); 1H NMR (DMSO-d ): d = 12.17 (2H, br, 1-and 8-OH), 7.65 (1H, d, J = 1.3 Hz, H-4), 7.25 (1H, d, J = 1.3 Hz, H-2), 7.09 (1H, d, J = 2.3 Hz, H-5), 6.53 (1H, d, J = 2.3 Hz, H-7), 4.61 (2H, s, 6-CH2); 13C NMR (DMSO-d6): d = 189.4 (C-9), 182.1 (C-10), 167.3 (C-6), 165.1 (C-8), 161.8 (C-1), 153.0 (C-3), 135.5 (C-10a), 133.4 (C-4a), 121.2 (C-2), 117.4 (C-4), 114.7 (C-9a), 110.3 (C-5), 108.5 (C-8a), 108.7 (C-7), 62.5 (3-CH2); (-) ESI-MS: m/z = 285 [M - H]-.

Communiol D (7)

White powder (4.3 mg); TLC: Rf = 0.43 (CH3OH/CHCl3, 1:9); [a]D +3.2 (c0.5, CHCl3); 1H NMR (CDCl3/CD3OD): d = 5.21 (1H, br, H-11), 4.62 (1H, m, H-3), 3.98 (1H, dd, J = 7and3.5 Hz, H-7), 3.74 (1H, dd, J = 7and3.5 Hz, H-8), 3.72 (2H, t, J = 6 Hz, H-1), 3.22 (1H, m, H-5), 2.23 (2H, m, H-6), 1.87 (2H, m, H-4), 1.79 (2H, m, H-2), 1.42 (2H, m, H-9), 1.02 (3H, t, J = 6.5 Hz, 10-CH3); 13C NMR (CDCl3/CD3OD): d = 99.8 (C-11), 79.5 (C-7), 78.3 (C-3), 73.6 (C-8), 61.3 (C-1), 45.3 (C-5), 41.2 (C-4), 32.4 (C-6), 26.7 (C-9), 13.4 (10-CH3); (-) ESI-MS: m/z = 215 [M - H]-.

Assay for determination of HCV protease inhibitory activity

Samples of 2 uL of culture broth fungi extracts and isolated compounds dissolved in dimethyl sulfoxide (DMSO) were placed in each well of a Samples of 2 uL of culture broth fungi extracts dissolved in dimethyl sulfoxide (DMSO) were placed in each well of a 384-well microplate, then 8 iLof recHCV PR (0.5 ug/mL) were added, and the plate was briefly agitated. Finally, 10 iL of the freshly prepared substrate [Ac-Asp-Glu-Dap (QXLTM520)-Glu-Glu-Abu-COO-Ala-Ser-Cys(5-FAMsp)-NH2] were added with sequential rotational shaking. The reaction mixture was incubated at 37degC for 30 min. The fluorimetric analyses were performed on an automated TECAN GENios plate reader (Mannedorf, Switzerland) with excitation and emission wavelengths at 485 and 520 nm, respectively. Each sample was tested in triplicate. HCV PR inhibition (%) was calculated using the following equation:

% Inhibition = (Fsubstrate - Ftest sample) x 100/Fsubstrate

Where Fsubstrate is the fluorescence of the enzyme and substrate only, and Ftest sample is the fluorescence of the assay mixture with the added sample.

Green Protease Assay

The active HCV-PR sample of extracts and isolated compounds were dissolved in DMSO (2.5 ul; final content, 10% w/v) and placed in the wells of a 384-well microplate. Then 17.5 ul of assay buffer and 2.5 ul of trypsin (0.1 U/ul) were added and the plate was briefly agitated. Finally, 2.5 ul of the freshly diluted protease substrate HiLyte Fuor TM 488-labeled casein were added under sequential rotary shaking and the mixture incubated at 37degC for 30 min. The positive control was the soybean trypsin-chymotrypsin inhibitor. Inhibition was calculated as above for HCV PR assay.

Results and Discussion

Characterization of Isolated Compounds

Three fungal strains Fusarium equiseti, Scopulariopsis fusca and Geotrichum candidum were isolated from the brown alga Padina pavonica. The organic extracts of their liquid cultures were evaluated for their inhibition of HCV NS3-NS4A protease. As a result, F. equiseti showed a high-level inhibition of HCV protease was selected for further chemical investigation on its secondary metabolites.

The active fungus F. equiseti was grown on a static Czapek's peptone liquid medium. The culture broth extract was submitted to a combination of silica gel column, preparative TLC, and Sephadex LH-20 column chromatography to afford seven known metabolites. Compounds 1-7 were detected by TLC on silica gel as yellow and dark spots under UV light. These UV absorbing spots were tentatively identified as anthraquinones, adenosines and diketopiprazines due to their color reaction with KOH and Ehrlich's reagents, respectively. The isolated compounds (Fig. 1) were identified based on the spectral analyses and comparison with literature data.

Table-1: Inhibition of HCV NS3-NS4A protease by compounds 1-7.

###HCV Protease inhibitory activity (%)###Trypsin inhibitory activity (%)

Sample###IC50 (ig/ml)###100 (ig/ml)###IC50 (ig/ml)

Scopulariopsis fusca###55.0+-6.8###17.74+-6.07###>100

Geotrichum candidum###39.0+-4.5###30.22+-2.61###>100

Fusarium equiseti###23.0+-2.5###9.89+-1.96###>100

Cyclo-L-Ala-L-Leu (1)###>1000###Nt###-

Cyclo(L-Tyr-L-Pro) (2)###8.20+-1.7###42.3+-2.15###37.9+-3.8

Cordycepin(3)###25.4+-2.3###64.71+-3.16###>100

Ara-A (4)###21.3+-4.12###39.24+-4.27###>100

Chrysophanol (5)###>1000###Nt###-

u-Hydroxyemodin (6)###10.7+-2.3###56.2+-3.24###48.5+-1.9

Communiol D (7)###>1000###Nt###-

HCV-I2###1.5 +- 0.5

T-I###0.01 +- 0.4

Alkaloid metabolites 1 - 4 with substituted nitrogen atoms appeared as dark spots on TLC under UV light, changed to red with Ehrlich's reagent. These compounds showed both aliphatic (compound 1) and aromatic (compounds 2 - 4) proton characters in their 1H NMR spectra. The compounds were characterized as two diketopiperazines, cyclo-L-Ala-L-Leu (1) [14] and cyclo(L-Tyr-L-Pro) (2) [15]; two nucleosides, cordycepin (3) and Ara-A(4) [16]. The presence of peri-hydroxy anthraquinones in compounds 5 and 6 was detected by their red colour with 5% KOH solution on TLC and with 1.0% vanillin/H2SO4, respectively. The NMR spectra of these compounds revealed tricyclic anthraquinones with chelated hydroxy groups. These compounds were identified as chrysophanol (5) [17] and o-hydroxyemodin (6) [18]. Bis-tetrahydrofurane derivative, communiol D (7) was reported as the fungal metabolite from Podospora communis [19]. However, these compounds were isolated for the first time from Red Sea fungus F. equiseti.

HCV NS3-NS4A protease inhibition

The anti-HCV protease activity was carried out for the crude extracts of the isolated endophytic fungal strains in 100 ug/ml concentration using the hepatitis virus C NS3 protease inhibitor 2 as a positive control. Moreover, the selectivity of the active extracts toward HCV NS3/4A protease (viral protease) and not human serine proteases such as trypsin and chymotrypsin has been confirmed through investigating the inhibitory activity of these extracts and/or their isolated chemical constituents on Human recombinant Trypsin.

The isolated compounds 1-7 from the EtOAc extract were tested for their inhibitory activity against HCV PR using HCV NS3 protease inhibitor 2 as a positive control [20]. Some of these isolated compounds showed potent activity against HCV NS3-NS4A protease with IC50 values from 10.7 to 58.3 ug/ml, compared to their EtOAc extract with IC50 value 27 ug/ml. Compounds 2 and 6 showed the most potent inhibitory effect with IC50 values of 18.20 and 10.7 ug/ml, respectively, while adenosine compounds 3 and 4 exhibited mild inhibitory with an IC50 value of 24.5 and 22.3 ug/ml, respectively. Compounds 1, 5 and 7 were inactive as inhibitor of HCV PR as compared to other constituents.

The selectivity of the aforementioned active extracts against HCV PR was tested via their ability to inhibit the human protease trypsin [21]. At concentrations up to 100 ig/ml Fusarium equiseti extract showed the most potent inhibitory activity against human trypsin with 9.89%. Ara-A (2) and o-Hydroxyemodin (6) were approximately 4 times more selective as inhibitor of human trypsin than HCV PR (Table-1). These findings suggested that these active compounds 2 and 6 selectively inhibited HCV PR and have mild interfere with human physiological processes requiring trypsin activity.

Conclusion

In conclusion, searching for marine-derived bioactive compounds led to the investigation of seven metabolites from the fungus F. equiseti. This showed potent HCV protease activity compared to the fungi isolated from the Red sea alga Padina pavonica. Cyclo(L-Tyr-L-Pro) (diketopiperazine) and o-hydroxyemodin (anthraquinone) exhibited potent inhibitory effect on HCV NS3-NS4A protease with mild interfere with human physiological processes requiring trypsin activity, which warrants for further investigation of other members of these widely distributed class of diketopiperazines and anthraquinones in marine and terrestrial metabolites.

Acknowledgement

This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah under grant No (G-1436-150-443). The authors, therefore, acknowledge with thank DSR technical and financial support.

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