Article pubs.acs.org/JAFC
New Tyrosinase Inhibitors from Paecilomyces gunnii Ruili Lu,† Xiaoxiao Liu,† Sha Gao, Wancun Zhang, Fan Peng, Fenglin Hu,* Bo Huang, Longyun Chen, Guanhu Bao,* Chunru Li, and Zengzhi Li Anhui Agricultural University, Hefei, Anhui 230036, People’s Republic of China S Supporting Information *
ABSTRACT: Through screening 50 strains of entomopathogenic fungi and rescreening of 7 strains of Paecilomyces gunnii, a methanol extract of liquid-cultivated mycelia of P. gunnii was found to have the strongest tyrosinase inhibitory activity. Preparative high-speed counter-current chromatography (HSCCC) guided by high-performance liquid chromatography (HPLC)−electrospray ionization (ESI)−high-resolution mass spectrometry (HRMS) was employed for the isolation and purification of the active components, and three new compounds with half inhibition concentration (IC50) of 0.11, 0.17, and 0.14 mM against diphenolase were obtained from the extract, respectively. Their chemical structures were identified by HRMS, oneand two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy as paecilomycones A, B, and C. Structure and activity studies showed that the tyrosinase inhibition activities are positively related to the number of hydroxyl groups on the paecilomycones. KEYWORDS: Paecilomyces gunnii, phenalenones, tyrosinase inhibition
■
DOPA was from TCI Co., Ltd. (Shanghai, China). L-Tyrosine was from Solarbio Co., Ltd. (Shanghai, China). Tyrosinase was from Worthington Co., Ltd., Lakewood, NJ. Reverse osmosis Milli-Q water (Millipore, Bedford, MA) was used for all solutions and dilutions. Thin-layer chromatography (TLC) plates were bought from Qingdao Haiyang Chemcals Co., Ltd. Instrumentation. The TBE-300B HSCCC with a TBP-50A pump, a coiled column (inner diameter of 2.6 mm, 300 mL), a UVD-200 detector, and a HX 105 temperature regulator was from Tauto Biotech Company (Shanghai, China). The Agilent 6210 time-of-flight HPLC− mass spectrometry (MS) system with a binary high-pressure mixing pump, an auto sampler, a column oven, a photodiode array detector (PAD), an Agilent workstation, and a high-resolution mass spectrometry (HRMS) with an electrospray ionization (ESI) source was from Agilent Technologies (Santa Clara, CA). Analytical separations were carried out on a Waters Atlantis column (5 μm, 150 × 3.9 mm inner diameter). The RVC2-25 centrifugal vacuum concentrator was a product of the Christ Company of Germany. Nuclear magnetic resonance (NMR) experiments were carried out using a Bruker Avance 400 MHz NMR spectrometer (Bruker, Switzerland). A bioassay was performed on a Spectra M2 microplate reader purchased from Molecular Device, Sunnyvale, CA. Fungal Culture and Workup. All tested entomopathogenic fungi strains have been catalogued and deposited in the Research Center on Entomopathogenic Fungi (RCEF, WDCM1031), Anhui Agricultural University. The selected 50 fungal isolates (see Table S1 of the Supporting Information) were cultured on Sabouraud dextrose agar yeast (SDAY) medium (40 g/L glucose, 10 g/L yeast extract, 10 g/L peptone, and 20 g/L agar). The activated fungal culture was transferred to 100 mL of sterile water and vortexed to prepare a homogenized fungal suspension. The suspension was inoculated 500 μL per dish on cellophane membrane precovered plates and cultured at 25 °C for 12 days. The final cultured mycelia were scraped, freezedried, and then pulverized for use.
INTRODUCTION Tyrosinase, also known as polyphenoloxidase, a coppercontaining oxidoreductase widely found in mammals, insects, plants, and microorganisms, is the key and rate-limiting enzyme of melanin synthesis in vivo.1−3 Tyrosinase is mainly involved in two reactions during the process of melanin formation.3−5 On the one hand, tyrosinase catalyzes hydroxylation of L-tyrosine to produce L-3,4-dihydroxyphenylalanine (L-DOPA) as a single phenol enzyme. On the other hand, it oxidizes L-DOPA to produce DOPA quinone as a two-phenol oxidizing enzyme and further formation of melanoma through a series of reactions of DOPA quinine. Tyrosinase is closely related to aging, wound healing, and browning of fruits and vegetables.4−8 It also plays an important role in the metamorphosis and immunity of insects.9 The insecticidal mechanism of entomopathogenic fungi shows that the insect is able to prevent fungal infection through a browning defense response under the action of tyrosinase, while the entomopathogenic fungi can reduce the defensive function of the insect through destroying the role of browning.9−11 Another study showed that tyrosinase is also involved in the tanning of insect to enhance the defense capabilities against entomopathogenic fungi.12 Thus, tyrosinase inhibitors are likely to exist in secondary metabolites of entomopathogenic fungi as important insecticidal components to suppress the defense system of the insects. On the basis of the above hypothesis, tyrosinase inhibitor searching was carried out on our entomopathogenic fungal library.
■
MATERIALS AND METHODS
Chemicals and Reagents. All solvents used for high-speed counter-current chromatography (HSCCC) were of analytical grade (Sinopharm Chemical Reagent Co., Ltd.). High-performance liquid chromatography (HPLC)-grade methanol and formic acid were from Tedia Company, Fairfield, OH. Arbutin was from Hubei Artec Carbohydrate Chemistry Co., Ltd. Kojic acid was from Shanxi Sciphar Hi-Tech Industry Co., Ltd. Ninhydrin was from Sigma Corporation. L© 2014 American Chemical Society
Received: Revised: Accepted: Published: 11917
August 31, 2014 November 7, 2014 November 10, 2014 November 10, 2014 dx.doi.org/10.1021/jf504128c | J. Agric. Food Chem. 2014, 62, 11917−11923
Journal of Agricultural and Food Chemistry
Article
HPLC−PAD−HRMS Analysis. A total of 10 μL of methanol solution of the sample (1.0 mg/mL) was injected into the analytical column. The flow rate was 0.8 mL/min. The column was eluted with a water/methanol (both contain 0.05% formic ammonium) gradient from 90:10 to 55:45 for 10 min, from 55:45 to 40:60 for a further 15 min, from 40:60 to 0:100 (%) for another 5 min, and pure methanol for the final 15 min. The eluates were monitored with a PAD at full length scan from 200 to 600 nm and a HRMS detector under negative and positive modes. Drying gas of the ESI ion source was 12 L/min at 325 °C. The nebulizer pressure was 35 psig. The capillary voltage was 3500 V for negative mode and 4000 V for positive mode. The fragmentor voltage was 175 V for negative mode and 215 V for positive mode. The scan range was 50−1200 amu. HPLC−MS−Bioactivity Combined Analysis. A total of 10 μL of methanol solution of the sample (3.0 mg/mL) was injected into the analytical column and eluted with the above conditions. A total of 20% of the eluates was monitored with the MS detector through a distribution valve. The other 80% of the eluates was automatically collected every 5 min. The injection was repeated 9 times. Every three collections of the same time were combined as one sample and condensed to dry with the centrifugal concentrator. The dried eluates were dissolved with 15 μL of DMSO, and 10 μL of the solutions were transferred to 96 well microtiter plates for tyrosinase inhibitory activity detection. Selection of the Two-Phase Solvent System for HSCCC. The two-phase solvent system was selected according to the partition coefficient (K) of each target component. The concentration of the target component in the upper phase (CA) and lower phase (CB) was analyzed by HPLC−MS. K = CA/CB. According to the fact that the target components are methanolsoluble and possibly have moderate polarity, we selected a two-phase solvent system (n-hexane/ethyl acetate/methanol/water), which was suitable for the separation of compounds with medium polarity.13 To obtain a better separation effect by HSCCC, a partition coefficient (K) between 0.5 and 2 is highly needed and the retention rate of the stationary phase has to be kept more than 50%.14 On the basis of the selected basic solvent system and HSCCC separation pretests, we created a series of solvent systems (Table 1), and system 5 with 72% retention rate of the stationary phase was selected as the optimal solvent system for target compound preperation. HSCCC Separation. The sample solution was prepared by dissolving the crude sample in a solvent mixture consisting of equal volumes of both upper and lower phases (4 mL for each phase). In each separation, the coiled column of HSCCC was first entirely filled with the upper phase as the stationary phase at a flow rate of 9.99 mL/ min. Then, the lower phase (mobile phase) was pumped into the column at a flow rate of 2.0 mL/min, while the apparatus was run at a speed of 850 rpm. After the mobile phase front emerged and hydrodynamic equilibrium was established in the column, the sample solution (8 mL) containing 100 mg of crude extract was injected. All through the experiment, the separation temperature was controlled at 25 °C. The effluent of the column was monitored at 254 nm and automatically collected in a 10 mL test tube per 2 min. Structural Identification and Statistical Analysis. The structural identification of each HSCCC peak fraction was carried out by the analysis of HPLC−PAD−ESI−HRMS, 1H and 13C NMR data, and two-dimensional (2D) NMR. Student’s paired t test for comparison of means was performed with the obtained data.
Seven strains of Paecilomyces gunnii were selected for rescreening (Figure 1). The strains were cultured with the same medium and
Figure 1. Inhibibition rates of mycelia extracts and fermentation broth extracts from different strains against diphenolase of tyrosinase.
condition as that of the 50 fungal isolates or cultured in liquid medium (40 g/L glucose, 10 g/L yeast extract, and 10 g/L peptone) under shaking at 130 rpm and 25 °C for 10 days. The mycelia of the liquid cultures were separated from fermentation media. All of the mycelia were freeze-dried and then pulverized to save for use. The fermentation broths were condensed to 1/4 of their original volume under reduced pressure and then freeze-dried. The freeze-dried samples were sonicated in methanol by a solid/ liquid ratio of 1 g/30 mL at 25 °C for 30 min and then statically extracted at 4 °C for 6 h. After centrifugation, the supernatants of the extracts were concentrated under reduced pressure and lyophilized to give the crude methanol extracts. Tyrosinase Inhibition Assay. Extracts and isolated compounds (initial concentration of 5.0 mg/mL) were first dissolved in dimethyl sulfoxide (DMSO) and then diluted to different concentrations using DMSO. L-DOPA and tyrosinase were dissolved in 50 mmol/L phosphate buffer (pH 6.8). A total of 150 μL of substrate solution (1.0 mmol/L) and 10 μL of sample solution were added to 96 well microtiter plates and incubated at 30 °C for 5 min, and then 40 μL of tyrosinase solution (100 μg/mL) was quickly added and immediately mixed. After thorough mixing by vortex, initial absorbance at 475 nm was measured. The enzyme activity was obtained by calculation of the slope of the time line through tracking and measuring the optical density (OD475) at a wavelength of 475 nm. The molar extinction coefficient of the product at 475 nm was calculated as 3700 mol L−1 cm−1, and the relative inhibition rate was calculated as follows: relative inhibition rate (%) = (1 − ΔA m /ΔAc) × 100% where ΔAm is the enzyme activity of the sample and ΔAc is the enzyme activity of the control (sample was replaced by an equal volume of solvent). Triplicate experiments were performed, and half inhibition concentration (IC50) of the extract on tyrosinase was determined for each compound. The inhibition type was assayed by the Lineweaver−Burk plot, and the inhibition constant was determined by the second plot of the apparent Km/Vm or 1/Vm versus the concentration of the inhibitor.
Table 1. Distribution Coefficient (K) of the Target Compounds in Different Solvent Systems K 1 2 3 4 5
n-hexane/ethyl acetate/ methanol/acetic acid/water
compound 1
compound 2
compound 3
5:5:5:0:5 2:5:2:0:5 2.5:5:2.5:0:5 2.5:5:2.5:0.15:5 3.5:5:3.5:0.15:5
0.45 7.35 1.55 5.32 1.56
0.18 2.25 0.90 1.45 0.86
0.14 1.80 0.68 0.91 0.53
11918
dx.doi.org/10.1021/jf504128c | J. Agric. Food Chem. 2014, 62, 11917−11923
Journal of Agricultural and Food Chemistry
Article
Figure 2. (A) HPLC chromatograms of the methanol extract from liquid mycelia of P. gunnii. (B) Inhibition rate of HPLC fractions on diphenolase activity.
Figure 3. HPLC−PAD−HRMS analysis of compounds 1−3: (A) HPLC and full wavelength scan spectrum (PAD), (B) HRMS of the compounds in positive mode, and (C) HRMS of the compounds in negative mode.
■
RESULTS AND DISCUSSION Preliminary Screening of Tyrosinase Inhibitory Activity of Fungal Extracts. Screening was conducted on the methanol extracts of solid-cultured mycelia of the selected 50 strains by testing their inhibition rates against tyrosinase diphenolase in Table S1 of the Supporting Information. An obvious difference was found in the tyrosinase inhibition activity among different species and strains of the same species. Among the measured strains, P. gunnii showed relative higher inhibitory activity; therefore, further rescreening experiment was carried out on multiple strains of P. gunnii. Rescreening of Seven Strains of P. gunnii. To better understand the tyrosinase inhibitory activities of the active strains of P. gunnii, the methanol extracts of the solid-cultured mycelia, liquid-cultured mycelia, and broth were tested (Figure 1 and Table S2 of the Supporting Information). The inhibitory activities of the liquid-cultured mycelia were significantly (p < 0.05) higher than that of the others, and the differences between mycelia and broth were very significant (p < 0.01; see Table S2 of the Supporting Information), indicating that the
active ingredients mainly exist in the mycelia. Of all of the tested samples, the methanol extract of the liquid-cultured mycelia of the strain RCEF0199 showed the highest activity; therefore, it was selected for further bioassays and follow-up searching for the active compounds. IC50 of the Extract on Tyrosinase. With L-DOPA as a substrate, inhibitory rates of different concentrations of the methanol extract of the strain RCEF0199 on tyrosinase were measured. The amount of product formation is proportional to the reaction time, and the response slope of the line is the enzyme activity. The effect curve was obtained through plotting the concentrations versus enzyme activities. The results showed that the enzyme activity decreased with an increased concentration of the extract, and the IC50 of the extract on tyrosinase was calculated as 48 μg/mL (see Figure S1A of the Supporting Information). Tyrosinase Inhibitory Mechanism of the Extract. The inhibition mechanism on the enzyme by the methanol extract for oxidation of L-DOPA was investigated. In the experiment, the concentration of the substrate L-DOPA was constant while changing the concentration of the extract and the enzyme. The 11919
dx.doi.org/10.1021/jf504128c | J. Agric. Food Chem. 2014, 62, 11917−11923
Journal of Agricultural and Food Chemistry
Article
Table 2. 1H and 13C NMR Spectral Data (δ in ppm) of Compounds 1−3 in DMSO-d6a 1
13
H NMR
position 1 2 3 3a 3b 4 5 6 6a 7 8 9 9a OCH3 CH3 OH NH2 a
1
2
3
6.51 s
6.53 s
6.41 s
6.82 s
6.83 s
6.67 s
4.00 s 2.74 s
4.01 s 2.76 s 12.85 s, 13.53 s
3.96 s 2.75 s
C NMR
1
2
3
172.3 97.4 166.8 111.6 127.8 144.9 117.3 162.2 105.4 172.0 131.7 166.8 103.3 56.7 25.8
169.2 97.4 166.8 111.5 130.6 145.1 117.3 171.2 105.7 162.7 196.6 166.8 100.0 56.7 25.8
174.3 96.3 166.6 110.7 129.6 144.8 117.2 165.0 106.5 169.9 131.0 174.0 102.3 56.4 25.8
8.95 br s
s, singlet; br s, broad signal.
Information). The collected fractions were analyzed by HPLC, and fractions with the same component were combined. After solvent evaporation under vacuum at 50 °C, compound 1 (20.4 mg), compound 2 (5.2 mg), and compound 3 (12.5 mg) were obtained with the purities of 96.8, 95.8, and 95.0%, respectively, based on HPLC analysis (panels 1A−3A of Figure 3). Structural Identification. Full wavelength scan of active ingredient absorption spectra are shown in panels 1A−3A of Figure 3. Identification of peak fractions was performed with ESI−HRMS (panels 1B−3C of Figure 3), 1H and 13C NMR, and 2D NMR (see Figure S3 of the Supporting Information). The 1H and 13C NMR data are given in Table 2. Compound 1, peak 1 in Figure 2A, is a yellow powder. The ultraviolet (UV) spectrum (panel 1A of Figure 3) shows the existence of a long conjugated system. UV (MeOH) λmax (log ε): 218 (5.0), 256 (sh, 2.6), and 380 (2.0). ESI−HRMS (m/z) showed the pseudo-molecular peak at 289.07083 (M + H, calcd. 289.07066) in positive mode and 287.05662 (M − H, calcd. 287.05611) in negative mode (panels 1B and 1C of Figure 3). Thus, its molecular formula was calculated as C15H12O6, which suggested that it has a carbon skeleton with 10 degrees of unsaturation. The 1H and 13C NMR (Table 2) spectra of compound 1 indicated the presence of one naphthalene ring system, which has 10 aromatic carbons, including 1 methine carbon (δH 6.82, δC 117.23) and 9 quaternary carbons (δC 111.6, 144.9, 162.2, 105.4, 170.0, 131.07, 173.0, and 103.3), as well as 1 methoxy group (δH 4.00, δC 56.7), 1 methyl group (δH 2.74, δC 25.8), and 1 conjugated carbonyl system (δCO 172.3, δC 97.3 and 166.8). The above NMR and HRMS data suggested that it has a very high degree of unsaturation, consistent with a tricyclic phenalenone framework. A literature search showed that it is very similar to the known phenalenone compound myeloconone A2 (Figure 4), which was found from the lichen Myeloconis erumpens,15 except that compound 1 has a hydroxyl group instead of a methoxyl group at position C-8. Further evidence for the structure of compound 1 was provided by heteronuclear multiple-bond correlation (HMBC) long-range C−H connectivities, as illustrated in Figure 4. The
results are shown in Figure S1B of the Supporting Information. A group of straight lines through the origin was obtained for the extract, and the slop of the line decreased with the increased concentration of the sample, which indicated that the inhibitory effect of the extract on tyrosinase was a reversible process. Therefore, the extract was a reversible inhibitor of tyrosinase. Competitive Inhibitory Type of the Extract on Tyrosinase. The effects on the enzyme activity of different concentrations of the extract were measured by changing the concentration of the substrate (L-DOPA) while maintaining the amount of the enzyme. Using the Lineweaver−Burk doublereciprocal plot method, a set of straight lines intersecting the y linear axis was obtained, as shown in Figure S1C of the Supporting Information, indicating that the methanol extract was a competitive inhibitor on the tyrosinase. The Michaelis constant (Km) increased with the ascended concentration of the extract. The affinity of the enzyme with the substrate was reduced with the increase of the concentration of the extract. The extract impeded the binding of the enzyme and substrate and led to a lower catalytic activity. The Michaelis constant (Km) was calculated as 1.02 mM without adding extract in the reaction system. The maximum reaction velocity (Vmax) was 186.44 units/min based on the linear slope and Km values. The equilibrium constant (KI) for the extract binding to the free enzyme was obtained from the secondary plot of the line slope versus the concentration of the extract (see Figure S1D of the Supporting Information). The value of KI was 555.56 μg/mL. Bioactive Fractions of the Extract. Combined analysis of HPLC−MS and tyrosinase inhibitory activities shows that the HPLC fraction collected from 10 to 15 min has the strongest bioactivity (Figure 2B). The HPLC chromatogram shows that the main bioactive fraction contains three components, where the purities of the target compounds 1, 2, and 3 were 23.9, 6.6, and 15.7%, respectively, based on the HPLC peak area percentage (Figure 2A). The three compounds will be used as targets for HSCCC separation. HSCCC Preparation for the Target Compounds. The target components were isolated by the optimal solvent system as compounds 3, 2, and 1 with retention times at 70, 110, and 210 min, respectively (see Figure S2 of the Supporting 11920
dx.doi.org/10.1021/jf504128c | J. Agric. Food Chem. 2014, 62, 11917−11923
Journal of Agricultural and Food Chemistry
Article
lene-1,2,3-trione, as shown in Figure 4, and named as paecilomycone B. Compound 3, peak 3 in Figure 2A, is an olive powder. The UV spectrum (panel 3A of Figure 3) shows the existence of a long conjugated system. UV (MeOH) λmax (log ε): 208 (2.2), 260 (sh, 1.3), and 380 (1.3). ESI−HRMS (m/z) showed the pseudo-molecular peak at 288.08624 (M + H, calcd. 288.08665) in positive mode and 286.07217 (M − H, calcd. 286.07210) in negative mode (panels 3B and 3C of Figure 3). Thus, its molecular formula was decided as C15H13NO5. The 1 H and 13C NMR (Table 2) spectra of compound 3 indicated the presence of one naphthalene ring system, which has 10 aromatic carbons, including 1 methine carbon (δH 6.67, δC 117.2) and 9 quaternary carbons (δC 110.7, 144.8, 165.0, 106.5, 169.9, 131.0, 174.0, and 102.3), as well as 1 methoxy group (δH 3.96, δC 56.4), 1 methyl group (δH 2.75, δC 25.8), and 1 conjugated carbonyl system (δCO 174.3, δC 96.3 and 166.6). The above NMR data are very similar to those of compound 1. In comparison of its HRMS and NMR data to those of compound 1, one NH2 group (δH 8.95, br s, 2H) substituted one OH group in the structure of compound 3, which was assigned to NH2-9 because the NH2 group is hydrogen-bonded with the carbonyl group of C-1, which led to the lower field signal (δH 8.95). In combination with the above HRMS and NMR information, compound 1 can be deduced as 9-amino6,7,8-trihydroxy-3-methoxy-4-methyl-1H-phenalen-1-one and named as paecilomycone C (Figure 4).15,16 For further confirmation of the presence of a NH2 group in the deduced structure, compound 3 was tried with the ninhydrin reaction and showed a positive purple reaction in the TLC plate (see Figure S6 of the Supporting Information).17 Inhibition Capacity of Paecilomycones A−C on Tyrosinase. According to the above assay method, initial solutions of paecilomycones were diltuted to 3−9 fold and the inhibitory rates at each dilution were measured. IC50 of the three new phenalenones paecilomycones A−C were 0.11, 0.17, and 0.14 mM, respectively (Table 3), which are almost as
Figure 4. Chemical structures of phenanelones from methanol extracts of liquid-cultivated mycelia of P. gunnii and HMBC correlations (arrows with arrowhead) around the protons in the structure: H-5, CH3, H-2, and OCH3 of compound 1.
aromatic proton singlet at δH 6.82 (H-5) yielded three strong C−H long-range correlations, assigned as 3JCH couplings, to quaternary carbon atoms at δC 111.6 (C-3a) and 105.4 (C-6a) and the methyl carbon atom at δC 25.8. In addition, a weak connectivity to an oxysubstituted carbon atom (δ 162.9) was assigned as a 2JCH correlation to C-6. Similarly, three strong correlations involving the methyl protons at δH 2.74 can be found at δC 111.6 (C-3a), 117.3 (C-5), and 144.9 (C-4), and a weak correlation at δC 166.8 (C-3) also existed. The strong correlations involving δH 6.51 (H-2) are at δC 103.3 (C-9a), 111.6 (C-3a), 166.8 (C-3), and 172.3 (C-1), with a weak correlation at δC 144.9 (C-4). The strong correlation involving the methoxy group δH 4.00 (OCH3-3) is at δC 166.8 (C-3), which confirmed that the methoxy group is at position C-3. Besides, a weak correlation of OCH3-3/C-2 (δC 97.4) was also found. Thus, the 1H and 13C NMR spectra of compound 1 were consistent with a highly oxygenated phenalenone and very similar to those of the known phenalenone myeloconone A2. Finally, in combination with the above HRMS and NMR information, compound 1 can be deduced as 6,7,8,9tetrahydroxy-3-methoxy-4-methyl-1H-phenalen-1-one and named as paecilomycone A (Figure 4).15,16 Compound 2, peak 2 in Figure 2A, is an orange powder. The UV spectrum (panel 2A of Figure 3) shows the existence of a long conjugated system. UV (MeOH) λmax (log ε): 220 (2.6), 255 (2.7), and 332 (1.5). ESI−HRMS (m/z) showed the pseudo-molecular peak at 287.05498 (M + H, calcd. 287.05501) in positive mode and 285.04035 (M − H, calcd. 285.04046) in negative mode (panels 2B and 2C of Figure 3). Thus, its molecular formula was calculated as C15H10O6. The 1 H NMR spectra of compound 2 showed similarity to those of compound 1 (Table 2). However, its molecular formula has one more oxygen atom while short of two hydrogen atoms, which suggests 1 more degree of unsaturaton in the structure. Thus, it should be a more oxygenated phenalenone-type compound compared to compound 1. In the 1H NMR experiment, two lower field single peaks were found at δH 12.85 and 13.53, which are two typically strongly hydrogenbonded hydroxyl groups ascribed to the positions OH-1 and OH-6 (Figure 4). In comparison of the above-mentioned HRMS and NMR data to those of compound 1, its structure was decided as 4,9-dihydroxy-6-methoxy-7-methyl-1H-phena-
Table 3. IC50 of Compounds 1−3 on Tyrosinase compound paecilomycone A (1) paecilomycone B (2) paecilomycone C (3) kojic acid arbutin
IC50 (mM) ± SD 0.11 0.17 0.14 0.10 0.20
± ± ± ± ±
0.010 0.009 0.004 0.003 0.005
strong as that of kojic acid (0.10 mM) and stronger than that of arbutin (0.20 mM). Both kojic acid and arbutin are well-known tyrosinase inhibitors, which are usually used as the positive control of the tyrosinase inhibitor and commercially used in whitening cosmetics and food to prevent browning.18 The three new phenalenones are the main tyrosinase inhibitors in the methanol extract of P. gunnii. The competitive inhibitory mechanism of the extract means that the three new compounds combine with tyrosinase in a manner that competes for the same binding site on the enzyme as the substrate L-DOPA or L-tyrosine. The three phenalenones are natural polyphenols with several phenolic hydroxyl groups in their structure. The tyrosinase inhibition test showed that the number of hydroxyl groups plays an important role in its activity: paecilomycone A has four hydroxyl groups with the highest activity (IC50 = 0.11 mM); paecilomycone B has one 11921
dx.doi.org/10.1021/jf504128c | J. Agric. Food Chem. 2014, 62, 11917−11923
Journal of Agricultural and Food Chemistry
Article
*Telephone: +86-551- 65786401. Fax: +86-551-65786765. Email:
[email protected].
hydroxyl group with the lowest activity (IC50 = 0.17 mM); and paecilomycone C has three hydroxyl groups with mid-activity (IC50 = 0.14 mM) (Figure 4). Phenalenone originated from very restricted natural resources, including Fungi Imperfecti.19 P. gunnii is one species of Fungi Imperfecti, which suggested that its presence of related phenalenones is reasonable and it should share a similar biosynthetic pathway to produce the phenalenones through folding of a linear C14-polyketide.19 Phenalenones were reported to have antibacterial, antitumor, anti-HIV activity through inhibition of HIV-1 integrase, and antiprotozoal activities.19−21 Recently, two phenalenone compounds were found to exhibit considerable effects against the malaria parasite, one of which maintained the same level of activity in a chloroquine-resistant strain, which is the first report of the antimalarial activity of this type of scaffold compound.22 The structure of the newly isolated compound paecilomycone A is very similar to the anti-HIV target funalenone (anti-HIV, IC50 = 1.7 μM),20 while there is a NH2 group at C-9 of paecilomycone C instead of the normal OH group, which could be a hint that the two new compounds could be potential effective anti-HIV compounds and highly valuable for continuing studies. Entomogenous fungi are a treasury for searching for bioactive compounds, and many interesting results have been found from them.23−30 However, few tyrosinase inhibitors were reported from these species. This study revealed that tyrosinase inhibitors truly existed in the secondary metabolites of entomopathogenic fungi. Tyrosinase is one of the main enzymes involved in immunity and defense response of insects;9−11,31 therefore, tyrosinase inhibitors from metabolites of entomopathogenic fungi, such as paecilomycones A−C, must play an important role in virulence of the fungus and can possibly be used as virulence factors for high-virulent strain screening and used in novel pesticide production. Most of the currently used pesticides kill insects by targeting their nervous system and have a neurotoxic effect in mammals as well;9,32 however, tyrosinase inhibitors targeting the immune and defensive systems of insects may have wider application prospects. Additionaly, similar to some popularly used tyrosinases in the food industry and cosmetics, paecilomycones A−C also have the possibility to be used in these areas.
■
Author Contributions †
Ruili Lu and Xiaoxiao Liu contributed equally to this work.
Funding
This work was financially supported by the China National Science Foundation (30871676), the Foundation of the Ministry of Science and Technology of Anhui Province (1408085MC46 and KJ2012A107), the Anhui Agricultural University Talents Foundation (YJ2011-06), and the Anhui Outstanding Youth Science Foundation (1108085J04). Notes
The authors declare no competing financial interest.
■
(1) Parvez, S.; Kang, M.; Chung, H. S.; Bae, H. Naturally occurring tyrosinase inhibitors: Mechanism and applications in skin health, cosmetics and agriculture industries. Phytother. Res. 2007, 21, 805− 816. (2) Chang, T. S. An updated review of tyrosinase inhibitors. Int. J. Mol. Sci. 2009, 10, 2440−2475. (3) Chang, T. M. Tyrosinase and tyrosinase inhibitors. J. Biocatal. Biotransform. 2012, 1, 1−2. (4) Kumar, C. M.; Sathisha, U. V.; Dharmesh, S.; Rao, A. G.; Singh, S. A. Interaction of sesamol (3,4-methylenedioxyphenol) with tyrosinase and its effect on melanin synthesis. Biochimie 2011, 93, 562−569. (5) Demirkiran, O.; Sabudak, T.; Ozturk, M.; Topcu, G. Antioxidant and tyrosinase inhibitory activities of flavonoids from Trifolium nigrescens subsp. petrisavi. J. Agric. Food Chem. 2013, 61, 12598− 12603. (6) Lin, Y. F.; Hu, Y. H.; Lin, H. T.; Liu, X.; Chen, Y. H.; Zhang, S.; Chen, Q. X. Inhibitory effects of propyl gallate on tyrosinase and its application in controlling pericarp browning of harvested longan fruits. J. Agric. Food Chem. 2013, 61, 2889−2895. (7) Espin, J.; Wichers, H. Effect of captopril on mushroom tyrosinase activity in vitro. Biochim. Biophys. Acta 2001, 1544, 289−300. (8) Jimbow, K.; Luo, D.; Chen, H.; Hara, H.; Lee, M. H. Coordinated messenger-RNA and protein expression of human lamp-1 in induction of melanogenesis after UV-B exposure and contransfection of human tyrosinase and TRP-1 CDNAs. Pigm. Cell Res. 1994, 7, 311−319. (9) Guerrero, A.; Rosell, G. Biorational approaches for insect control by enzymatic inhibition. Curr. Med. Chem. 2005, 12, 461−469. (10) Ortiz-Urquiza, A.; Keyhani, N. O. Action on the surface: Entomopathogenic fungi versus the insect cuticle. Insects 2013, 4, 357−374. (11) Hussein, K. A.; Abdel-Rahman, M. A. A.; Abdel-Mallek, A. Y.; El-Maraghy, S. S.; Joo, J. H. Pathogenicity of Beauveria bassiana and Metarhizium anisopliae against Galleria mellonella. Phytoparasitica 2012, 40, 117−126. (12) Kramer, K. J.; Hopkins, T. L. Tyrosine metabolism for insect cuticle tanning. Arch. Insect Biochem. Physiol. 1987, 4, 279−301. (13) Ito, Y. Golden rules and pitfalls in selecting optimum conditions for high-speed count-current chromatography. J. Chromatogr. A 2005, 1065, 145−168. (14) Black, B.; Sun, C.; Zhao, Y. Y.; Gänzle, M. G.; Curtis, J. M. Antifungal lipids produced by lactobacilli and their structural identification by normal phase LC/atmospheric pressure photoionization−MS/MS. J. Agric. Food Chem. 2013, 22, 5338−5346. (15) Ernst-Russell, M. A.; Chai, C. L. L.; Elix, J.; McCarthyC, P. M. Myeloconone A2, a new phenalenone from the lichen Myeloconis erumpens. Aust. J. Chem. 2000, 53, 1011−1013. (16) Hölscher, D.; Schneider, B. Phenalenones from Strelitzia reginae. J. Nat. Prod. 2000, 63, 1027−1028. (17) Harding, V. J.; Warneford, F. H. S. The ninhydrin reaction with amino acids and ammonium salts. J. Biol. Chem. 1916, 25, 319−335.
ASSOCIATED CONTENT
S Supporting Information *
Inhibiton of the methanol extracts of the strain RCEF0199 on tyrosinase diphenolase (Figure S1), preparative HSCCC separation of the crude sample from P. gunnii using two-step elution with solvent systems (Figure S2), 1H and 13C NMR and HMBC of compound 1 (Figure S3), 1H and 13C NMR of compound 2 (Figure S4), 1H and 13C NMR of compound 3 (Figure S5), ninhydrin reaction with the three compounds shown only in compound 3 reacted positively with a purple color (Figure 6), inhibition rates of mycelia extracts from different strains against diphenolase of tyrosinase (Table S1), and paired t tests on inhibibition rates of mycelia extracts and fermentation broth extracts (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.
■
REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*Telephone/Fax: +86-551-5786887. E-mail: hufenglin@ hotmail.com. 11922
dx.doi.org/10.1021/jf504128c | J. Agric. Food Chem. 2014, 62, 11917−11923
Journal of Agricultural and Food Chemistry
Article
(18) Kim, Y. J.; Uyama, H. Tyrosinase inhibitors from natural and synthetic sources: Structure, inhibition mechanism and perspective for the future. Cell. Mol. Life Sci. 2005, 62, 1707−1723. (19) Cooke, R. G.; Edwards, J. M. Naturally occurring phenalenones and related compounds. Fortschr. Chem. Org. Naturst. 1981, 40, 153− 190. (20) Shiomi, K.; Matsui, R.; Isozaki, M.; Chiba, H.; Sugai, T.; Yamaguchi, Y.; Masuma, R.; Tomoda, H.; Chiba, T.; Yan, H.; Kitamura, Y.; Sugiura, W.; Omura, S.; Tanaka, H. Fungal phenalenones inhibit HIV-1 integrase. J. Antibiot. 2005, 5, 65−68. (21) Rosquete, L. I.; Cabrera-Serra, M. G.; Piñero, J. E.; MartínRodríguez, P.; Fernández-Pérez, L.; Luis, J. G.; McNaughton-Smith, G.; Abad-Grillo, T. Synthesis and in vitro antiprotozoal evaluation of substituted phenalenone analogues. Bioorg. Med. Chem. 2010, 18, 4530−4534. (22) Gutiérrez, D.; Flores, N.; Abad-Grillo, T.; McNaughton-Smith, G. Evaluation of substituted phenalenone analogues as antiplasmodial agents. Exp. Parasitol. 2013, 135, 456−458. (23) Molnar, I.; Gibson, D. M.; Krasnoff, S. B. Secondary metabolites from entomopathogenic Hypocrealean fungi. Nat. Prod. Rep. 2010, 27, 1241−1275. (24) Isaka, M.; Srisanoh, U.; Sappan, M.; Supothina, S.; Boonpratuang, T.; Sterostreins, F.-O. Illudalanes and norilludalanes from cultures of the basidiomycete Stereum ostrea BCC 22955. Phytochemistry 2012, 79, 116−120. (25) Hu, F. L.; He, Y. Q.; Huang, B.; Li, Z. Z. Secondary metabolites in a soybean fermentation broth of Paecilomyces militaris. Food Chem. 2009, 116, 198−201. (26) Schimidt, K.; Riese, U.; Li, Z. Z.; Hamburger, M. Noval tetramic acids and pyridone alkaloids, militarinones B, C and D, from the insect pathogenic fungus Paecilomyces militaris. J. Nat. Prod. 2003, 66, 378− 383. (27) Cheng, Y. X.; Li, Z. Z.; Hamburger, M. Farinosones A−C, neurotrophic alkaloidal metabolites from the entomopathogenous deuteromycete Paecilomyces farinosus. J. Nat. Prod. 2004, 67, 1854− 1858. (28) Lu, R. L.; Bao, G. H.; Hu, F. L.; Huang, B.; Li, C. R.; Li, Z. Z. Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana. Food Chem. 2014, 145, 1066−1071. (29) Lu, R. L.; Luo, F. F.; Hu, F. L.; Huang, B.; Li, C. R.; Bao, G. H. Identification and production of a novel natural pigment, cordycepoid A, from Cordyceps bifusispora. Appl. Microbiol. Biotechnol. 2013, 97, 6241−6249. (30) Luo, F.; Lu, R.; Zhou, H.; Hu, F.; Bao, G.; Huang, B.; Li, Z. Metabolic effect of an exogenous gene on transgenic Beauveria bassiana using liquid chromatography−mass spectrometry-based metabolomics. J. Agric. Food Chem. 2013, 61, 7008−7017. (31) Valadez-Lira, J. A.; Alcocer-Gonzalez, J. M.; Damas, G.; NunezMejia, G.; Oppert, B.; Rodriguez-Padilla, C.; Tamez-Guerra, P. Comparative evaluation of phenoloxidase activity in different larval stages of four lepidopteran pests after exposure to Bacillus thuringiensis. J. Insect Sci. 2012, 12, 801−811. (32) Costa, L. G.; Giordano, G.; Guizzetti, M.; Vitalone, A. Neurotoxicity of pesticides: A brief review. Front. Biosci. 2008, 13, 1240−1249.
11923
dx.doi.org/10.1021/jf504128c | J. Agric. Food Chem. 2014, 62, 11917−11923
