HPLC Quantification of Cytotoxic Compounds from Aspergillus niger.
Studies on marine-derived fungi as source of natural products have shown a sharp increase in recent years, due to the discovery of new molecular structures containing a broad of pharmacological property [1-5].
As part of an investigative effort to find promising anticancer agents from marine-derived fungi [6-8], the chemical investigation of a strain of Aspergillus niger (BRF074), recovered from sediments of the Northeast Brazilian coast, yielded the new furan ester derivative (1) and the cyclopeptides malformins A (2) and C (3)  (Figure 1).
Malformins are a group of cyclic pentapeptides with a disulfide bond formed from two cysteine thiols. These compounds show a variety of biological activities [10, 11] such as enhanced fibrinolytic activity  and antimalarial and antitrypanosomal properties . The distinct activity profiles of the malformins A (2) and C (3) against cancer cell lines are also well reported in the literature [14-16]. These compounds have been found to be strongly cytotoxic against the human cancer cell lines NCI-H460 (non-smallcell lung carcinoma, [IC.sub.50] 0.07 [micro]M), MIA Pa Ca-2 (pancreatic inducing root curvatures and malformations in plants [17,18], antibacterial activity cancer, [IC.sub.50] 0.05 [micro]M), MCF-7 (breast cancer, [IC.sub.50] 0.10 [micro]M), and SF-268 (CNS cancer; glioma, [IC.sub.50] 0.07 [micro]M) with slight selectivity towards the pancreatic cancer cell line (MIA Pa Ca-2) compared with the normal human primary fibroblast cells WI-38 ([IC.sub.50] 0.10 [micro]M) . Compound 1, a furan ester derivative, showed cytotoxic activity against HCT-116 tumor cell line with [IC.sub.50] 2.9 [micro]M .
The production of the cytotoxic compounds 1-3 by A. niger (BRF 074), cultured under different growth conditions, was investigated and quantified through high-performance liquid chromatography (HPLC) method using a diode array detector (DAD).
2.1. Samples, Chemicals, and Materials. The furan ester derivative (1) and malformins A (2) and C (3) used for the preparation of standard solutions were isolated from the marine-derived fungus Aspergillus niger (BRF 074) as previously described . HPLC-grade acetonitrile and methanol were purchased from Tedia[R]. All other chemicals were of analytical-grade in the highest purity available. Water was distilled and purified using a Millipore Milli Q Plus system (Bedford, USA). The nutrient media used in this work, malt peptone dextrose, MPD, potato dextrose yeast, PDY, and mannitol peptone yeast, MnPY, were obtained from Himedia[R] Laboratories.
2.2. Fungal Strain. The fungal strain of A. niger (BRF 074) was recovered from sediments collected by autonomous diving in the vicinities of Pecem's offshore port terminal (3[degrees]32'2" S; 38[degrees]47'58" W), state of Ceara, Northeast of Brazil, and its isolation and identification was performed as previously reported [8, 9].
2.3. Cultivation of A. niger (BRF 074) and Extract Preparation. The strain of A. niger (BRF 074) was previously grown in potato dextrose agar (PDA) in reconstructed sea water. Agar pieces with 5 mm diameter of 7-day-old cultures were used as precultures and transferred to a 250 mL Erlenmeyer flask with 100 mL of the nutrient medium: malt peptone dextrose, MPD (malt, 20.0 g[L.sup.-1]; peptone, 2.0 g[L.sup.-1]; dextrose, 20.0 g [L.sup.-1]), potato dextrose yeast, PDY (potato dextrose yeast, 24.0 g [L.sup.-1]; yeast, 3.0 g [L.sup.-1]), or mannitol peptone yeast, MnPY (mannitol, 4.0 g [L.sup.-1]; peptone, 2.0 g [L.sup.-1]; yeast, 2.0 g [L.sup.-1]).
The fungal strain was grown under static conditions at room temperature (28[degrees]C) during 7, 14, 21, and 28 days. The culture broths were separated from mycelium by filtration and submitted to partition with EtOAc (3 x 30 mL) to afford the crude extracts (MPD, PDY, and MnPY) after solvent distillation.
2.4. Quantitative High-Performance Liquid Chromatography Analysis (HPLC Analysis). Samples were filtered through a 0.45 [micro]m Millex-HV PVDF membrane (Millipore, New Bedford, MA, USA). HPLC analyses were performed on a Shimadzu chromatographer equipped with a ternary pump (Shimadzu LC-20AT) and DAD detector (Diode Assay Detector) (Shimadzu SPD-M20A, Kyoto, Japan) and carried out on an analytical column (Phenomenex[R] ODS 100 A 250 mm x 4.60 mm, 5 [micro]m) preceded by an C18 guard column (2.0 cm x 4.0 mm; 5 [micro]m), also from Phenomenex. LC solutions software (version 1.25) was used for data processing. Chromatographic conditions were gradient using acetonitrile and water as mobile phase, initially consisting of acetonitrile/water (2: 8 v/v) and increasing up to acetonitrile/water (8: 2 v/v) in 30 minutes at a flow rate of 1 mL x [min.sup.-1]. The mobile phase was prepared daily and degassed by sonication before use. The column temperature was 25[degrees]C and the injection volume was 20 [micro]L. The UV spectra were monitored over a range of 450 to 200 nm, while the chromatograms were recorded at 273 nm to detect the furan ester derivative (1), and to detect malformins A (2) and C (3) the PAD detector was set at [lambda] = 203 nm.
Methanol was used to prepare all standard solutions and extracts. The standard solutions were used in six different concentrations, as follows: 12.5, 25.0, 100.0, 150.0, 250.0, and 500.0 [micro]g x [mL.sup.-1], and the concentration of the solution extracts was 2,000 [micro]g x [mL.sup.-1].
2.5. Analytical Method Validation. The validation of the analytical method was performed in the HPLC-DAD system and the validated parameters evaluated were specificity, linearity, accuracy, precision (repeatability and intermediate precision), limit of quantification (LOQ), and limit of detection (LOD), which are in accordance with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines .
The linearity was determined by the correlation coefficients of the analytical curves, which were built by analyzing the working solutions at six different concentrations. The method linearity for the three compounds was tested through linear least-square regression analysis based on calibration curves constructed by using 12.5,25.0,100.0,150.0,250.0, and 500.0 [micro]g x [mL.sup.-1] solutions (Graphpad Prism[R] 5.03 software).
The limits of detection (LOD) and quantification (LOQ) were calculated based on the standard deviation and slope of the regression curves.
The precision was evaluated in two steps: repeatability and intermediate precision. The repeatability was determined by injecting in triplicate, on the same day, standard solutions at three different concentrations (25.0, 100.0, and 500.0 [micro]g [mL.sup.-1]). Intermediate precision was estimated by analyzing, in triplicate, the same solutions employed in the repeatability test on three consecutive days. The results obtained were expressed in terms of relative standard deviation (RSD).
The accuracy was determined from recovery tests with solutions of the standard compounds having low, medium, and high concentration levels, and the recoveries of the tree standard compounds were calculated from the corresponding calibration curve according to the following equation:
Recovery (%) = (amount found/amount added) x 100. (1)
The test concentrations used were 140.0, 186.0, and 233.0 [micro]g [mL.sup.-1] for the furan ester derivative (1); 70.0,92.0, and 115.0 [micro]g [mL.sup.-1] for malformin A (2); and 101.0, 134.0, and 166.0 [micro]g [mL.sup.-1] for malformin C (3). All the samples were analyzed in quintuplicate.
Accuracy was expressed as the percentage of deviation between the measured value and the reference value.
The selectivity of the method was evaluated by analyzing sterile medium (blank) and fungal culture broth under the conditions previously established.
2.6. Quantification and Validation Procedures. To ensure the linearity, calibration curves of the three compounds were obtained. Standard solutions of the furan ester derivative (1) and malformins A (2) and C (3) were prepared at a concentration range of 12.5 to 500.0 [micro]g [mL.sup.-1]. All the curves showed a linear response with [r.sup.2] > 0.998 in the selected range for each sample (Table 1).
The furan ester derivative (1) showed LOD of 46.0 [micro]g [mL.sup.-1] and LOQ of 67.9 [micro]g [mL.sup.-1]. Malformin A (2) showed LOD of 22.7 [micro]g [mL.sup.-1] and LOQ of 33.6 [micro]g [mL.sup.-1], while malformin C (3) showed LOD of 34.7 [micro]g [mL.sup.-1] and LOQ of 51.3 [micro]g [mL.sup.-1].
The precision was estimated by measuring repeatability (intraday assay) and intermediate precision (interday assay), both in triplicate. All the values of relative standard deviation (RSD) in the repeatability and intermediate precision estimates were below 5% (Table 2), which did not exceed the limits recommended in the literature [19, 20].
The accuracy of the method was evaluated by recovery experiments with the standard solutions at three different concentration levels. The recovery values ranged from 95.8 to 106.4% (Table 3) and were also in line with ICH parameters.
2.7. Method Application: Growth Behavior of Aspergillus niger (BRF 074). The method developed showed being efficient for the quantitative analyses of three bioactive compounds produced by A. niger (BRF 074) in three fermentative mediums: malt peptone dextrose (MPD), potato dextrose yeast (PDY), and mannitol peptone yeast (MnPY).
These analyses were carried out after inoculation of the mycelia obtained from seed medium into MPD, PDY, and MnPY. In order to evaluate the kinetic production of the compounds, different periods of incubation were analyzed (7,14,21, and 28 days), and their concentrations in each period were obtained in triplicate (n = 3). The contents of the three cytotoxic compounds in the crude extracts, MPD and PDY, are shown in Table 4.
3. Results and Discussion
The focus of the study was to quantify the amount of the cytotoxic metabolites, furan ester derivative (1), and malformins A (2) and C (3), produced by the marine-derived strain of A. niger (BRF 074) grown in varied nutrient medium and time of cultivation.
The validation method was carried out simultaneously for the bioactive compounds, and the retention time was found to be 13.7 min for 1 and 21.4 min and 22.1 min for 2 and 3, respectively. Although the analysis showed a relative long run time (30 min), the secondary metabolites could be analyzed with good separation and baseline resolution between all peaks (Figure 2).
The chromatographic analysis of the organic extracts (MPD, PDY, and MnPY) revealed a similar HPLC profile for MPD and PDY and suggested that the bioactive compounds were produced by A. niger in both culture media. No significative amounts of the analyzed compounds 1-3 were found in the extract from the MnPY medium.
In our analysis, the highest content (274.0 [micro]g [mL.sup.-1]) of 1 was found when using MPD as culture medium, during a growth period of 28 days. In this same condition, the contents of 2 and 3 were 86.3 [micro]g[mL.sup.-1] and 125.9 [micro]g[mL.sup.-1], respectively. On the other hand, the best condition for the production of 2 and 3 was achieved when using PDY as culture medium, within 21 and 28 days of growth. Within 21 days of growth, the concentrations of 2 and 3 were 179.0 and 233.8 [micro]g [mL.sup.-1], respectively, while the concentration of 1 was 44.4 [micro]g [mL.sup.-1]. With 28 days of fermentation, also in the PDY medium, the concentrations of 2 and 3 did not change considerably when compared with 21 days of growth, and their respective contents were 163.1 and 235.4 [micro]g [mL.sup.-1]. However, the concentration of 1 increased almost twice (74.3 [micro]g [mL.sup.-1]) when comparing the periods of 28 and 21 days of fermentation.
The developed chromatographic method herein described proved to be efficient in the quantitative analyses of the cytotoxic compounds 1-3 in the extracts from A. niger cultured under different conditions, as confirmed by its validated analytical parameters by HPLC-PDA system. Considering these aspects, the analytical quantitative method can be a useful and important tool for monitoring the production of the analyzed cytotoxic compounds by A. niger.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
The authors acknowledge the Fundacao Cearense de Apoio ao Desenvolvimento Cientifico e Tecnologico (FUNCAP), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), and Coordenacao de Aperfeicoamento de Ensino Superior (CAPES) for the fellowships and financial support.
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Paula Karina S. Uchoa, Leandro Bezerra de Lima, Antonia T. A. Pimenta, Maria da Conceicao F. de Oliveira, Jair Mafezoli, and Mary Anne S. Lima
Departamento de Quimica Organica e Inorganica, Centro de Ciencias, Universidade Federal do Ceara, Fortaleza, CE, Brazil
Correspondence should be addressed to Mary Anne S. Lima; firstname.lastname@example.org
Received 5 December 2016; Revised 14 March 2017; Accepted 19 March 2017; Published 30 April 2017
Academic Editor: Marco Radi
Caption: FIGURE 1: Cytotoxic compounds 1-3 produced by Aspergillus niger (BRF074).
Caption: FIGURE 2: HPLC chromatograms of the (a) standard compounds 1-3, (b) extract obtained from A. niger (BRF 074) cultured during 28 days in MPD medium, (c) extract obtained from A. niger (BRF 074) cultured during 28 days in PDY medium, and (d) extract obtained from A. niger (BRF 074) cultured during 28 days in MnPY medium. Chromatography conditions: reversed-phase column ODS (250 x 4.6 mm, 5 [micro]m); gradient elution going from [H.sub.2]O/MeCN 80: 20 to [H.sub.2]O/MeCN 20: 80 in 30 minutes; flow rate of 1.0 mL [min.sup.-1]; sample concentration of 2.0 [micro]g [mL.sup.-1].
TABLE 1: Parameters of the analytical curves utilized for quantification of the bioactive compounds 1-3 produced by A. niger (BRF 074). Compound Linearity range Regression equation (a) ([micro]g [mL.sup.-1]) Furan ester (1) 12.5-500.0 7 = 11717.0x - 136817.5 Malformin A (2) 12.5-500.0 7 = 26362.2x - 246504.3 Malformin C (3) 12.5-500.0 7 = 26160.0x - 246504.3 Compound Correlation factor ([r.sup.2]) Furan ester (1) 0.9984 Malformin A (2) 0.9996 Malformin C (3) 0.9991 (a) Six data points (n = 3). TABLE 2: Repeatability and intermediate precision data of the bioactive compounds 1-3 produced by A. niger (BRF 074). Compound Repeatability (n = 3) Concentration Measured values Mean RSD ([micro]g ([micro]g ([micro]g (%) [mL.sup.-1]) [mL.sup.-1]) [mL.sup.-1]) Furan ester 50.0 54.2892 (1) 54.3437 54.3067 0.06 54.2872 250.0 233.8591 234.3952 234.1195 0.11 234.1043 500.0 509.2980 509.2856 509.2795 0.01 509.2549 Malformin A 50.0 51.7388 (2) 51.7246 51.6893 0.14 51.6044 250.0 246.2154 246.0825 246.1638 0.03 246.1936 500.0 503.6637 502.9370 503.2790 0.07 503.2363 Malformin C 50.0 52.8589 (3) 52.7992 52.8315 0.06 52.8364 250.0 241.1501 241.3867 241.3161 0.06 241.4114 500.0 505.8446 506.9641 505.9971 0.18 505.1826 Compound Intermediate precision (n = 3) Concentration Measured values Mean RSD ([micro]g ([micro]g ([micro]g (%) [mL.sup.-1]) [mL.sup.-1]) [mL.sup.-1]) Furan ester 50.1338 (1) 50.0 50.2537 50.1595 0.17 50.0912 245.7478 250.0 245.7127 245.7319 0.01 245.7350 500.9200 500.0 500.8662 501.0567 0.06 501.3840 Malformin A 49.3617 (2) 50.0 49.4094 49.3967 0.06 49.4188 239.8196 250.0 240.0045 239.9550 0.05 240.0410 505.5801 500.0 505.6485 505.6792 0.02 505.8089 Malformin C 51.1270 (3) 50.0 51.0972 51.0618 0.17 50.9613 231.0606 250.0 231.2140 231.1973 0.06 231.3173 510.3529 500.0 510.2716 510.2467 0.02 510.1154 TABLE 3: Accuracy data of the bioactive compounds 1-3 produced by A. niger (BRF 074). Accuracy (n = 5) Compound Concentration Measured values Mean Recovery ([micro]g ([micro]g ([micro]g (%) [mL.sup.-1]) [mL.sup.-1]) [mL.sup.-1]) Furan 140.0 134.1553 134.0627 95.8 ester (1) 133.9125 134.1571 134.2570 133.8316 186.0 178.5342 178.5453 95.9 178.6454 178.5064 178.4201 178.6202 232.0 224.0426 223.9634 96.4 223.6944 223.9213 224.0262 224.1322 Malformin 70.0 72.7681 72.9632 104.2 A (2) 73.0678 73.0619 72.9432 72.9750 92.0 98.0389 97.9246 106.4 97.8777 97.9863 97.9622 97.7580 115.0 117.8192 117.6743 102.3 117.5671 117.6607 117.7728 117.5518 Malformin 101.0 102.5096 102.5837 101.5 C (3) 102.4978 102.7050 102.5853 102.6210 134.0 130.4046 130.5013 97.4 130.5980 130.5686 130.6417 130.2936 166.0 160.7364 160.6178 96.8 160.7337 160.5222 160.4871 160.6094 TABLE 4: Contents of the bioactive compounds 1-3 in MPD and PDY extracts produced by A. niger (BRF 074). Concentration of the extracts = 2,000 [micro]g [mL.sup.-1]. Growth Furan ester (1) Malformin A (2) Malformin C (3) time (days) (Rt = 13.7 min) (Rt = 21.4 min) (Rt = 22.1 min) MPD extracts 07 14.7 37.5 60.1 14 17.0 26.5 34.9 21 92.3 46.2 67.5 28 274.0 86.3 125.9 PDY extracts 07 13.8 37.4 50.1 14 24.8 112.7 166.7 21 44.4 179.0 233.8 28 74.3 163.1 235.4 Concentration expressed as [micro]g [mL.sup.-1] (n = 3).
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|Title Annotation:||Research Article; high-performance liquid chromatography|
|Author:||Uchoa, Paula Karina S.; de Lima, Leandro Bezerra; Pimenta, Antonia T.A.; de Oliveira, Maria da Conce|
|Publication:||Journal of Chemistry|
|Article Type:||Technical report|
|Date:||Jan 1, 2017|
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