一株分離自黑果枸杞的內(nèi)生真菌米曲霉代謝產(chǎn)物

楊秀芳，王鵬飛，馬養(yǎng)民，賈強強

陜西科技大學化學與化工學院 教育部輕化工助劑化學與技術(shù)重點實驗室，西安 710021

Introduction

An endophyte is a bacterial (including actinomycete)or fungal microorganism，which spends the whole or part of its life cycle colonizing inter-and/or intra-cellularly inside the healthy tissues of the host plant，typically causing no apparent symptoms of disease［1］.Endophytic fungi of medicinal plants are a hotspot in recent research.Lycium ruthenicum Murr.is a traditional Chinese medicine plant，which only grew in Tibet region.The research on L.ruthenicum is limited to the chemical composition of its fruit and plant，and the chemical constituents from its endophytes were only reported in our group.The endophytic Aspergillus oryzae RER4 was isolated from its root，and its broth culture exhibited antimicrobial activity with zones of inhibition towards Escherichia coli，Pseudomonas aeruginosa，Staphylococcus aureus and Bacillus subtilis at 22，18，11 and 17 mm，respectively，for concentration at 10 mg/mL in the antimicrobial agar diffusion assay.［2］

We investigated the metabolites of A.oryzae RER4 residing in the root of L.ruthenicum，which leading to the isolation of nine compounds 1-9.In this study，we report the isolation，antioxidant and antimicrobial activities of these compounds.

Material and Methods

General

Column chromatography (CC):silica gel (SiO2:200-300 mesh，Qingdao Marine Chemical Group Co.);Sephadex LH-20 (Pharmacia Co.).1H-，13C NMR spectra:Bruker Avance III-400.

Fungal material and cultivation

The fungus RER4 was isolated from the healthy root of L.ruthenicum collected at the Dingbian，Gansu province，China，in July 2011.This fungus was inculcated into the medium (rice 50 g，water 60 mL，steamed under 120 ℃，0.1 MPa)incubated for 28 days at 25 ℃，in total 13 kg rice.

Extraction，isolation and purification

The fermentation was subjected to extraction with EtOAc three times and were concentrated under reduced pressure to give an EtOAc extract (70 g).This crude extract was fractionated by silica gel CC using petroleum ether-EtOAc gradient elution to provide fractions(Fr.1-Fr.5).Fr.2 (11.7 g)was subjected to repeated CC (silica gel)to yield compounds 1(32 mg)and 2(28 mg).Fr.3 (13.8 g)was separated into five fractions，followed by Sephadex LH-20 (MeOH)，CC (silica gel)to afford compounds 3(19.1 mg)，4(22 mg)，5(33.6 mg)，F(xiàn)r.4 (7.2 g)was subjected to repeated CC (silica gel)to yield compounds 6 (30 mg)and 7(15 mg).Fr.5 (8.5 g)was subjected to repeated CC(silica gel)and Sephadex LH-20 (MeOH)to yield compounds 8 (10.5 mg)and 9 (38.2 mg).

Antioxidant and antibacterial activity assays

Free radical scavenging activity was assayed by DPPH method［3］.Antibacterial activity assay of compounds were performed by the method of minimum inhibitory concentrations (MICs)［4］.

Results and Discussion

Identification of fungus

This fungus was identified on the base of the analysis of morphology (Fig.1) and molecular biology techniques.Compared the DNA (analyzed by Sangon Biotech)with the data base in Genbank (http://ncbi.nlm.nih.gov/blast)，finally the fungus RER 4 was identified with Aspergillus oryzae (Genbank accession number KF198066)through a system phylogenetic tree based on rDNA-ITS sequence (Fig.2，by the software of MEGA 5.0).

Fig.1 Morphologic diagram of endophytic fungi RER4

Fig.2 System phylogenetic tree of strain RER4 based on rDNA-ITS sequence

Compound identification

Compound 1 White crystal1H NMR (400 MHz，CDCl3)δ:3.66(1H，m，H-3)，5.59(1H，d，J=3.2 Hz，H-6)，5.40(1H，t，J=2.0 Hz，H-7)，0.65(3H，s，H-18)，0.95(3H，s，H-19)，1.05(3H，d，J=6.5 Hz，H-21)，5.21(2H，m，H-22，23)，0.85(6H，t，J=6.4 Hz，H-26，27)，0.93(3H，d，J=6.9 Hz，H-28);13C NMR (100 MHz，CDCl3)δ:38.3(C-1)，31.9(C-2)，70.4(C-3)，40.7(C-4)，139.7(C-5)，119.5(C-6)，116.2(C-7)，141.4(C-8)，46.2(C-9)，37.0(C-10)，21.1(C-11)，39.0(C-12)，42.8(C-13)，54.5(C-14)，23.0(C-15)，28.3(C-16)，55.7(C-17)，12.0(C-18)，17.6(C-19)，40.4(C-20)，21.1(C-21)，135.5(C-22)，131.9(C-23)，42.8(C-24)，33.0(C-25)，19.6(C-26)，19.9(C-27)，16.2(C-28).Comparing the NMR data with reference［5］，compound 1 was determined to be ergosterol.

Compound 2 Yellow crystal1H NMR(400 MHz，DMSO-d6)δ:3.85(2H，s，H-3)，6.76(1H，d，J=3.2 Hz，H-6)，5.40(1H，t，J=2.0 Hz，H-7)，0.65(3H，s，H-18)，0.95(3H，d，J=2.4 Hz，H-5)，9.35(1H，s，6-OH)，6.66(1H，dd，J=8.6，2.4 Hz，H-7)，6.97(1H，d，J=8.6 Hz，H-8);13C NMR(100 MHz，DMSO-d6)δ:33.7(C-2)，112.2(C-3)，147.1(C-4)，114.6(C-5)，110.9(C-6)，154.3(C-7)，125.5(C-8).Compound 2 was identified as 5-hydroxy-benzofuran-2 (3H)-one by comparing the NMR data with references［6］.

Compound 3 White crystal1H NMR(400 MHz，DMSO-d6)δ:8.65(1H，s，2-OH)，6.58(1H，d，J=8.4 Hz，H-3)，6.47(1H，dd，J=8.4，2.4 Hz，H-4)，8.76(1H，s，5-OH)，6.52(1H，d，J=2.4 Hz，H-6)，3.46(2H，s，H-7)，3.57(3H，s，H-9);13C NMR(100 MHz，DMSO-d6)δ:122.0(C-1)，148.2(C-2)，115.8(C-3)，114.7(C-4)，149.9(C-5)，117.9(C-6)，35.5(C-7)，172.2(C-8)，51.9(C-9).The above spectral data with were agree with the literature valuas of 3，4-di-hydroxyphenyl acetate［7］.

Compound 4 Yellow crystal1H NMR(400 MHz，DMSO-d6)δ:8.04(1H，s，H-2)，6.34(1H，s，H-4)，4.29(2H，s，H-7);13C NMR(100 MHz，DMSO-d6)δ:139.7(C-2)，146.1(C-3)，110.2(C-4)，168.5(C-5)，174.3(C-6)，59.8(C-7).The NMR data of compound 4 were identical to the 5-hydroxymethylfuran-3-carboxylic acid［8］.

Compound 5 Yellow crystal1H NMR(400 MHz，DMSO-d6)δ:8.63(1H，s，1-NH)，5.72(1H，d，J=9.2 Hz，H-2)，6.78(1H，d，J=9.2 Hz，2-OH)，9.71(1H，s，6-OH)，6.56(1H，d，J=15.9 Hz，H-8)，6.46(1H，m，H-9)，1.93(3H，t，0.78 Hz，H-10);13C NMR(100 MHz，DMSO-d6)δ:75.5(C-2)，174.5(C-3)，165.3(C-3a)，146.3(C-5)，142.5(C-6)，169.4(C-7)，112.0(C-7a)，119.3(C-8)，131.9(C-9)，19.2(C-10).Compound 5 was identified as pyranonigrins A by physicochemical date and spectral data with those in the literature［9］.

Compound 6 White crystal1H NMR(400 MHz，DMSO-d6)δ:5.57(1H，d，J=1.8 Hz，H-3)，6.10(1H，d，J=1.8 Hz，H-5)，4.39(1H，t，J=8.4 Hz，H-7)，1.79(1H，m，H-8)，1.44，1.12(2H，m，H-9)，0.83(3H，t，J=7.2 Hz，H-10)，0.78(3H，d，J=6.4 Hz，H-11)，1.87(3H，s，H-2')，3.80(3H，s，-OCH3)，8.31(1H，d，J=8.4 Hz，-NH);13C NMR (100 MHz，DMSO-d6)δ:163.8(C-2)，87.8(C-3)，170.6(C-4)，99.9(C-5)，163.2(C-6)，54.9(C-7)，35.9(C-8)，24.5(C-9)，10.7(C-10)，15.4(C-11)，169.2(C-1')，22.3(C-2')，56.3 (-OCH3).The above spectral data with were in accord with the literature valuas of campyrones A［10］.

Compound 7 White crystal1H NMR(400 MHz，DMSO-d6)δ:12.03(1H，brs，-COOH)，2.20(2H，t，J=7.2 Hz，H-2)，1.59(2H，m，H-3)，3.01(2H，q，J=6.4 Hz，H-4)，1.77 (3H，s，H-6)，7.82 (1H，s，-NH);13C NMR (100 MHz，DMSO-d6)δ:174.1(C-1)，31.0(C-2)，24.5(C-3)，37.8(C-4)，169.0(C-5)，22.5(C-6).The NMR data of compound 7 were identical to the 4-acetamidobutanoic acid［11］.

Compound 8 Yellow crystal1H NMR(400 MHz，CDCl3)δ:2.38(3H，s，2-CH3)，6.01(1H，s，H-3)，14.97(1H，s，5-OH)，4.00(3H，s，6-CH3)，6.40(1H，d，J=2.1 Hz，H-7)，3.93(3H，s，8-OCH3)，6.59(1H，d，J=2.1 Hz，H-9)，6.98(1H，s，H-10);13C NMR (100 MHz，CDCl3)δ:167.0(C-2)，20.2(2-CH3)，106.8(C-3)，183.8(C-4)，103.9 (C-4a)，162.0(C-5)，106.8(C-5a)，160.1(C-6)，55.6(6-OCH3)，96.3(C-7)，161.0(C-8)，54.9(8-OCH3)，97.7(C-9)，140.6(C-9a)，100.6(C-10)，152.7(C-10a).Comparing the NMR data with references［12］，compound 8 was determined to be rubrofusarin B.

Compound 9 Yellow powder1H NMR(400 MHz，DMSO-d6)δ:6.52(1H，s，H-3)，6.99(1H，s，H-6)，7.08(1H，s，H-7)，2.47(3H，s，2-CH3)，12.92(1H，s，5-OH)，10.20(1H，s，8-OH)，3.40(3H，s，10-OCH3)，6.22(1H，s，H-3')，6.56(1H，d，J=2.2 Hz，H-7')，6.14(1H，d，J=2.2 Hz，H-9')，2.14(3H，s，2'-CH3)，15.12 (1H，s，5'-OH)，3.94 (3H，s，6'-OCH3)，3.58(3H，s，8'-OCH3);13C NMR (100 MHz，DMSO-d6)δ:167.9(C-2)，110.0(C-3)，182.3(C-4)，108.0(C-4a)，155.5(C-5)，104.4(C-6)，140.0(C-6a)，105.1(C-7)，158.4(C-8)，116.2(C-9)，156.8(C-10)，106.5(C-10a)，154.7(C-10b)，19.8(2-CH3)，61.0(10-OCH3)，168.5(C-2')，106.7(C-3')，183.9 (C-4')，103.3 (C-4a')，162.0 (C-5)，107.5(C-5a')，160.8(C-6')，96.3(C-7')，161.1(C-8')，96.8 (C-9')，140.0 (C-9a')，105.1 (C-10')，150.2(C-10a')，20.1(2'-CH3)，56.0(6'-OCH3)，55.0(8'-OCH3).These dada were identical to those of recorded for an authentic specimen of asperpyrones A［13］.

Results of antioxidant and antibacterial activity assays

The antioxidant experiment showed that compound 2 had higher DPPH-scavenging activity，with IC50value of 4.98 μg/mL，than the positive control VC(Vitamin C)(IC506.93 μg/mL)，while compound 3 with IC50values of 7.00 μg/mL，similar to that of VC(Table 1).On the other hand，antimicrobial activity tests showed compound 2 and 3 had a strong activity against four bacteria and four fungi，which is similar to the positive control sodium penicillin，streptomycin sulfate and ketoconazole (anti-fungal)，other compounds exhibited different activities (Table 2).

Table 1 The antioxidant activity of all compounds

Table 2 The antimicrobial activity of all compounds

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