The m6 A reader MhYTP2 negatively modulates apple Glomerella leaf spot resistance by binding to and degrading MdRGA2L mRNA.

Glomerella leaf spot (GLS), caused by the fungal pathogen Colletotrichum fructicola, significantly threatens apple production. Some resistances to plant disease are mediated by the accumulation of nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins that are encoded by a major class of plant disease resistance genes (R genes). However, the R genes that confer resistance to GLS in apple remain largely unclear. Malus hupehensis YT521-B homology domain-containing protein 2 (MhYTP2) was identified as an N6 -methyladenosine RNA methylation (m6 A) modified RNA reader in our previous study. However, whether MhYTP2 binds to mRNAs without m6 A RNA modifications remains unknown. In this study, we discovered that MhYTP2 exerts both m6 A-dependent and -independent functions by analysing previously obtained RNA immunoprecipitation sequencing results. The overexpression of MhYTP2 significantly reduced the resistance of apple to GLS and down-regulated the transcript levels of some R genes whose transcripts do not contain m6 A modifications. Further analysis indicated that MhYTP2 binds to and reduces the stability of MdRGA2L mRNA. MdRGA2L positively regulates resistance to GLS by activating salicylic acid signalling. Our findings revealed that MhYTP2 plays an essential role in the regulation of resistance to GLS and identified a promising R gene, MdRGA2L, for use in developing apple cultivars with GLS resistance.

γ-Aminobutyric acid plays a key role in alleviating Glomerella leaf spot in apples.

The fungal disease Glomerella leaf spot (GLS) seriously impacts apple production. As a nonprotein amino acid, γ-aminobutyric acid (GABA) is widely involved in biotic and abiotic stresses. However, it is not clear whether GABA is involved in a plant's response to GLS, nor is its molecular mechanism understood. Here, we found that exogenous GABA could significantly alleviate GLS, reduce lesion lengths, and increase antioxidant capacity. MdGAD1 was identified as a possible key gene for GABA synthesis in apple. Further analysis indicated that MdGAD1 promoted antioxidant capacity to improve apple GLS resistance in transgenic apple calli and leaves. Yeast one-hybrid analysis identified the transcription factor MdWRKY33 upstream of MdGAD1. Electrophoretic mobility shift assay, ß-glucuronidase activity, and luciferase activity further supported that MdWRKY33 bound directly to the promoter of MdGAD1. The content of GABA and the transcription level of MdGAD1 in the MdWRKY33 transgenic calli were higher than that of the wild type. When MdWRKY33 transgenic calli and leaves were inoculated with GLS, MdWKRY33 positively regulated resistance to GLS. These results explained the positive regulatory effects of GABA on apple GLS and provided insight into the metabolic regulatory network of GABA.

Glomerella fusarioides-catalyzed structural transformation of steroidal drugs mesterolone and methasterone, and anti-inflammatory activity of resulting derivatives.

Transformation of steroidal drug mesterolone (1) with Glomerella fusarioides yielded two new (17α-hydroxy-1α-methyl-5α-androstan-3-one-11α-yl acetate (2) and 15α-hydroxy-1-methyl-5α-androstan-1-en-3,17-dione (3)), and four known derivatives (15α,17ß-dihydroxy-1α-methyl-5α-androstan-3-one (4), 15α-hydroxy-1α-methyl-5α-androstan-3,17-dione (5), 1α-methyl-androsta-4-en-3,17-dione (6) and 15α,17ß-dihydroxy-1-methyl-5α-androstan-1-en-3-one (7). Similarly, G. fusarioides-catalyzed transformation of steroidal drug methasterone (8) afforded four new metabolites, 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (9), 3a,11α,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (10), 1ß,3ß,17ß-trihydroxy-2α,17α-dimethyl-5α-androstane (11), and 11α,17ß-dihydroxy-2,17α-dimethylandrosta-1,4-diene-3-one (12). Structures of new derivatives were determined by using 1D-, and 2D-NMR, HREI-MS, and IR spectroscopic data. New derivative 3 was identified as a potent inhibitor of NÈ® production with the IC50 value of 29.9 ± 1.8 µM, in comparison to the standard l-NMMA (IC50 = 128.2 ± 0.8 µM) in vitro. In addition, methasterone (8) (IC50 = 83.6 ± 0.22 µM) also showed a significant activity comparable to new derivative 12 (IC50 = 89.8 ± 1.2 µM). New derivatives 2 (IC50 = 102.7 ± 0.5 µM), 9 (IC50 = 99.6 ± 5.7 µM), 10 (IC50 = 123.5 ± 5.7 µM), and 11 (IC50 = 170.5 ± 5.0 µM) showed a moderate activity. NG-MonomethylL-arginine acetate (IC50 = 128.2 ± 0.8 µM) was used as standared NO⋅- free radicals have an important role in the regulation of immune responses and cellular events. Their overproduction is associated with the pathogenesis of numerous ailments, such as Alzheimer's cardiac disorders, cancer, diabetes, and degenerative diseases. Therefore, inhibition of NÈ® production can help in the treatment of chronic inflammation and associated disorders. All derivatives were found to be non-cytotoxic to human fibroblast (BJ) cell line. The results presented here form the basis of further research for the development of new anti-inflammatory agents with improved efficacy through biotransformation approaches.

Candidate Effector Proteins from the Maize Tar Spot Pathogen Phyllachora maydis Localize to Diverse Plant Cell Compartments.

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins, of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmEC) proteins remain unknown, and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super yellow fluorescent protein-tagged PmEC proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating that the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed that most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane, whereas an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest that effector candidate proteins from P. maydis target diverse cellular organelles and could thus provide valuable insights into their putative functions, as well as host processes potentially manipulated by this fungal pathogen.

A TIR-NBS-LRR Gene MdTNL1 Regulates Resistance to Glomerella Leaf Spot in Apple.

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is one of the most devastating apple diseases. Our previous study reported that the GLS resistance locus was defined on the chromosome 15 region. Here, we further found a single-nucleotide polymorphism (SNP) site (SNP7309212) in the GLS resistance that was able to distinguish resistant cultivars (lines) from susceptible ones. On the basis of the SNP site, we cloned a TNL gene from the GLS resistant locus and named it MdTNL1 (NCBI Accession Number: ON402514). This gene contains a toll/interleukin-1 receptor transmembrane domain (TIR), nucleotide-binding sites (NBS), and leucine-rich repeat (LRR) domain. Subcellular location indicated that MdTNL1 was expressed in the nucleus and cell membrane. Ectopic overexpression of MdTNL1 in Nicotiana benthamiana caused cell death. We further demonstrated allelic polymorphisms in MdTNL1. It is noteworthy that NBS and LRR domains of the MdTNL1 protein serve as the repository for generating allelic diversity. Quantitative real-time PCR (qRT-PCR) assay revealed that MdTNL1 was highly expressed in resistant apple cultivar 'Fuji' after inoculation with C. fructicola, whereas susceptible cultivar 'Golden Delicious' exhibited low expression after inoculation. Over-expression of MdTNL1-1 in susceptible apple fruits and leaves improved disease resistance, while in 'Orin' calli, silencing the MdTNL1-1 gene conversely decreased GLS resistance. In conclusion, we identified a GLS associated with SNP7309212 and demonstrated that a TIR-NBS-LRR gene MdTNL1-1 positively regulates GLS resistance in apple.

Fungal mediated biotransformation of melengestrol acetate, and T-cell proliferation inhibitory activity of biotransformed compounds.

Glomerella fusaroide, and Rhizopus stolonifer were effectively able to transform the steroidal hormone melengestrol acetate (MGA) (1) into four (4) new metabolites, 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (2), 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-1,4,6-triene-3,20-dione (3), 17α-acetoxy-6,7α-epoxy-6ß-methyl-16-methylenepregna-4,6-diene-3,20-dione (4), and 17α-acetoxy-11ß,15ß-dihydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (5). All these compounds were structurally characterized by different spectroscopic techniques. The objective of the current study was to assess the anti-inflammatory potential of melengestrol acetate (1), and its metabolites 2-5. The metabolites and the substrate were assessed for their inhibitory effects on proliferation of T-cells in vitro. The substrate (IC50 = 2.77 ± 0.08 µM) and its metabolites 2 (IC50 = 2.78 ± 0.07 µM), 4 (IC50 = 2.74 ± 0.1 µM), and 5 (IC50 = < 2 µM) exhibited potent T- cell proliferation inhibitory activities, while compound 3 (IC50 = 29.9 ± 0.09 µM) showed a moderate activity in comparison to the standard prednisolone (IC50 = 9.73 ± 0.08 µM). All the metabolites were found to be non-toxic against 3T3 normal cell line. This study thus identifies some potent compounds active against T-cell proliferation. Their anti-inflammatory potential, therefore, deserves to be further investigated.

A new enantioselective CE method for determination of oxcarbazepine and licarbazepine after fungal biotransformation.

The present work describes, for the first time, the simultaneous separation of oxcarbazepine (OXC) and its active metabolite 10-hydroxy-10,11-dihydrocarbamazepine (licarbazepine, Lic) by chiral CE. The developed method was employed to monitor the enantioselective biotransformation of OXC into its active metabolite by fungi. The electrophoretic separations were performed using 10 mmol/L of a Tris-phosphate buffer solution (pH 2.5) containing 1% w/v of ß-CD phosphate sodium salt (P-ß-CD) as running electrolyte, -20 kV of applied voltage and a 15°C capillary temperature. The method was linear over the concentration range of 1000-30 000 ng/mL for OXC and 75-900 ng/mL for each Lic enantiomer (r ≥ 0.9952). Within-day precision and accuracy evaluated by RSD and relative errors, respectively, were lower than 15% for all analytes. The validated method was used to evaluate the enantioselective biotransformation of OXC, mediated by fungi, into its active metabolite Lic. This study showed that the fungi Glomerella cingulata (VA1) and Beuveria bassiana were able to enantioselectively metabolize the OXC into Lic after 360 h of incubation. Biotransformation by the fungus Beuveria bassiana showed 79% enantiomeric excess for (S)-(+)-Lic, while VA1 gave an enantiomeric excess of 100% for (S)-(+)-Lic. This study opens a new route to the drug (S)-(+)-licarbazepine.

Microbial transformation of cycloastragenol.

The microbial transformation of cycloastragenol by the fungi Cunninghamella blakesleeana NRRL 1369 and Glomerella fusarioides ATCC 9552, and the bacterium Mycobacterium sp. NRRL 3805 were investigated. Both fungi mainly provided hydroxylated metabolites together with products formed by cyclization, dehydrogenation and Baeyer-Villiger oxidation resulting in a ring cleavage. The bacteria yielded only a single oxidation product, namely, 3-oxo-cycloastragenol. Structures of the metabolites were elucidated by 1-D ((1)H,(13)C), 2-D NMR (COSY, HMBC, HMQC) and HRMS analyses.

Microbial transformation of Isopimpinellin by Glomerella cingulata.

Microbial transformation studies conducted on isopimpinellin (1) by the fungus Glomerella cingulata have revealed that 1 was metabolized to give the corresponding reduced acid, 5,8-dimethoxy-6,7-furano-hydrocoumaric acid (2). The structure of metabolite 2 was elucidated by high-resolution mass spectrometry (HR-MS), extensive NMR techniques, including (1)H NMR, (13)C NMR, (1)H-(1)H correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC) and heteonuclear multiple bond coherence (HMBC). The biotransformed product 2 showed weak a in vitro ß-secretase (BACE1) inhibitory effect.

Biotransformation of bergapten and xanthotoxin by Glomerella cingulata.

The biotransformation of bergapten (1) by the fungus Glomerella cingulata gave the corresponding reduced acid, 6,7-furano-5-methoxy hydrocoumaric acid (2), a new compound. Xanthotoxin (3) was also converted to the corresponding reduced acid cnidiol b (4) and demethylated metabolite xanthotoxol (5) by G. cingulata. The structure of the new compound 2 was elucidated by high-resolution mass spectrometry, extensive NMR techniques, including (1)H NMR and (13)C NMR, (1)H-(1)H correlation spectroscopy, heteronuclear multiple quantum coherence, and heteonuclear multiple bond coherence. The methyl ester or methyl ether or methyl ester and ether derivatives of 2 and 4 were synthesized. All compounds were tested for the beta-secretase (BACE1) inhibitory activity in vitro. The methyl ester and ether derivative 8 was shown to possess BACE1 inhibitory activity, and a IC(50) value was 0.64 +/- 0.04 mM.

Microbial transformation of (-)-isolongifolol by plant pathogenic fungus Glomerella cingulata.

The biotransformation of terpenoids using the plant pathogenic fungus as a biocatalyst to produce useful novel organic compounds was investigated. The biotransformation of sesquiterpen alcohol, (-)-isolongifolol (1) was investigated using plant pathogenic fungus Glomerella cingulata as a biocatalyst. Compound 1 was converted to (-)-(3R)-3-hydroxy-isolongifolol and (-)-(9R)-9-hydroxy-isolongifolol by G. cingulata.

Suppression of SOS-inducing activity of chemical mutagens by metabolites from microbial transformation of (-)-isolongifolene.

In this study, biotransformation of (-)-isolongifolene (1) by Glomerella cingulata and suppressive effect on umuC gene expression by chemical mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) and aflatoxin B(1) (AFB(1)) of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Initially, 1 was carried out the microbial transformation by G. cingulata. The result found that 1 was converted into (-)-isolongifolen-9-one (2), (-)-(2S)-13-hydroxy-isolongifolen-9-one (3), and (-)-(4R)-4-hydroxy-isolongifolen-9-one (4) by G. cingulata, and their conversion rates were 60, 25, and 15%, respectively. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB(1) in the umu test. Comound 2 showed gene expression by chemical mutagens furylfuramide and AFB(1) was suppressed 54 and 50% at <0.5 mM, respectively. Compound 2 is the most effective compound in this experiment.

Biotransformation of isoimperatorin and imperatorin by Glomerella cingulata and beta-secretase inhibitory activity.

Biotransformation studies conducted on the furanocoumarins isoimperatorin (1) and imperatorin (3) have revealed that 1 was metabolized by Glomerella cingulata to give the corresponding reduced acid, 6,7-furano-5-prenyloxy hydrocoumaric acid (2), and 3 was transformed by G. cingulata to give the dealkylated metabolite, xanthotoxol (4) in high yields (83% and 81%), respectively. The structures of the new compound 2 have been established on the basis of spectral data. The metabolites 2 and 4 were tested for the beta-secretase (BACE1) inhibitory activity in vitro, and metabolite 2 slightly inhibited the beta-secretase activity with an IC(50) value of 185.6+/-6.8 microM. The metabolite 4 was less potent activity than compounds 1-3. In addition, methyl ester (2Me), methyl ether (2a) and methyl ester and ether (2aMe) of 2 were synthesized, and investigated for the ability to inhibit beta-secretase. Compound 2aMe exhibited the best beta-secretase inhibitory activity at the IC(50) value 16.2+/-1.2 microM and found to be the 2aMe showed competitive mode of inhibition against beta-secretase with K(i) value 11.3+/-2.8 microM.

Enantioselective analysis of propranolol and 4-hydroxypropranolol by CE with application to biotransformation studies employing endophytic fungi.

A CE method is described for the enantioselective analysis of propranolol (Prop) and 4-hydroxypropranolol (4-OH-Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused-silica capillary, 4% w/v carboxymethyl-beta-CD in 25 mmol/L triethylamine/phosphoric acid (H(3)PO(4)) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid-liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25-10.0 microg/mL for each 4-OH-Prop enantiomer and 0.10-10.0 microg/mL for each Prop enantiomer (r>or=0.995). Within-day and between-day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4-OH-Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically, Glomerella cingulata (VA1) biotransformed 47.8% of (-)-(S)-Prop to (-)-(S)-4-OH-Prop with no formation of (+)-(R)-4-OH-Prop in 72 h of incubation.

Use of solid phase microextraction (SPME) for profiling the volatile metabolites produced by Glomerella cingulata.

The profile of volatile organic compounds (VOCs) released from Glomerella cingulata using solid phase microextraction (SPME) with different fibers, Polydimethylsiloxane (PDMS), Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), Carboxen/Polydimethylsiloxane (CAR/PDMS) and Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), was investigated. C4-C6 aliphatic alcohols were the predominant fraction of VOCs isolated by CAR/PDMS fiber. Sesquiterpene hydrocarbons represented 20.3% of VOCs isolated by PDMS fiber. During the growth phase, Ochracin was produced in the large majority of VOCs. 3-Methylbutanol and phenylethyl alcohol were found in the log phase of it. Alcohols were found in cultures of higher age, while sesquiterpenes were found to be characteristic of initial growth stage of G. cingulata.

Stereoselective analysis of thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: an investigation of rac-thioridazine biotransformation by some endophytic fungi.

The purpose of this study was to develop a method for the stereoselective analysis of thioridazine-2-sulfoxide (THD-2-SO) and thioridazine-5-sulfoxide (THD-5-SO) in culture medium and to study the biotransformation of rac-thioridazine (THD) by some endophytic fungi. The simultaneous resolution of THD-2-SO and THD-5-SO diastereoisomers was performed on a CHIRALPAK AS column using a mobile phase of hexane:ethanol:methanol (92:6:2, v/v/v)+0.5% diethylamine; UV detection was carried out at 262 nm. Diethyl ether was used as extractor solvent. The validated method was used to evaluate the biotransformation of THD by 12 endophytic fungi isolated from Tithonia diversifolia, Viguiera arenaria and Viguiera robusta. Among the 12 fungi evaluated, 4 of them deserve prominence for presenting an evidenced stereoselective biotransformation potential: Phomopsis sp. (TD2) presented greater mono-2-sulfoxidation to the form (S)-(SE) (12.1%); Glomerella cingulata (VA1) presented greater mono-5-sulfoxidation to the forms (S)-(SE)+(R)-(FE) (10.5%); Diaporthe phaseolorum (VR4) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(FE) (84.4% and 82.5%, respectively) and Aspergillus fumigatus (VR12) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(SE) (31.5% and 34.4%, respectively).

Endophytic fungi as models for the stereoselective biotransformation of thioridazine.

The stereoselective kinetic biotransformation of thioridazine, a phenothiazine neuroleptic drug, by endophytic fungi was investigated. In general, the sulfur of lateral chain (position 2) or the sulfur of phenothiazinic ring (position 5) were oxidated yielding the major human metabolites thioridazine-2-sulfoxide and thioridazine-5-sulfoxide. The quantity of metabolites biosynthesized varied among the 12 endophytic fungi evaluated. However, mono-2-sulfoxidation occurred in higher ratio and frequency. Among the 12 fungi evaluated, 4 of them deserve prominence for presenting an evidenced stereoselective biotransformation: Phomopsis sp. (TD2), Glomerella cingulata (VA1), Diaporthe phaseolorum (VR4), and Aspergillus fumigatus (VR12). Both enantiomers of thioridazine were consumed by the fungi; however, the 2-sulfoxidation yielded preferentially the R configuration at the sulfur atom.

GcSTUA, an APSES transcription factor, is required for generation of appressorial turgor pressure and full pathogenicity of Glomerella cingulata.

Glomerella cingulata, which infects a number of different hosts, gains entry to the plant tissue by means of an appressorium. Turgor pressure generated within the appressorium forces a penetration peg through the plant cuticle. A visible lesion forms as the fungus continues to grow within the host. A G. cingulata homolog (GcSTUA) of the genes encoding Asm1, Phd1, Sok2, Efg1, and StuA transcription factors in Magnaporthe grisea and other fungi was cloned and shown to be required for infection of intact apple fruit and penetration of onion epidermal cells. Mobilization of glycogen and triacylglycerol during formation of appressoria by the GcSTUA deletion mutant appeared normal and melanization of the maturing appressoria was also indistinguishable from that of the wild type. However, GcSTUA was essential for the generation of normal turgor pressure within the appressorium. As is the case for its homologs in other fungi, GcSTUA also was required for the formation of aerial hyphae, efficient conidiation, and the formation of perithecia (sexual reproductive structures).

Biotransformation of (+)-cycloisolongifolol by plant pathogenic fungus Glomerella cingulata.

The biotransformation of terpenoids using the plant pathogenic fungus as a biocatalyst to produce useful novel organic compounds was investigated. The biotransformation of sesquiterpen alcohol, (+)-cycloisolongifolol (1) was investigated using plant pathogenic fungus Glomerella cingulata as a biocatalyst. Compound 1 gave one major metabolic product and a number of minor metabolic products. Major product was dehydration at the C-8 position to (+)-dehydrocycloisolongifolene (2). The structure of the product was determined by their spectroscopic data. Glomerella cingulata gave dehydration in the specifically and over 70% conversion.

Biotransformation of cycloartane-type triterpenes by the fungus Glomerella fusarioides.

Biotransformation of three cycloartane-type triterpenes, cycloartenol (1), 24-methylenecycloartanol (2), and cycloartenone (3), by the fungus Glomerella fusarioides was studied. Compound 1 was converted to 3, cycloart-25-ene-3beta,24-diol (4), and cycloartane-3beta,24,25-triol (5). Compound 2 was metabolized to cycloeucalenol (6) and two new compounds, 24-methylcycloartane-3beta,24,24(1)-triol (7) and 24(1)-methoxy-24-methylcycloartane-3beta,24-diol (8). Compound 3 was converted into two new metabolites, 4alpha,4beta,14alpha-trimethyl-9beta,19-cyclopregnane-3,20-dione (9) and 25-hydroxy-24-methoxycycloartan-3-one (14), and four known compounds, viz., cycloartane-3,24-dione (10), 24-hydroxycycloart-25-en-3-one (11), (23E)-25-hydroxycycloart-23-en-3-one (12), and 24,25-dihydroxycycloartan-3-one (13). The structures of four new metabolites, 7, 8, 9, and 14, were established by spectroscopic methods.