Printer Friendly

HPLC Quantification of Cytotoxic Compounds from Aspergillus niger.

1. Introduction

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) [9] (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 [12] and antimalarial and antitrypanosomal properties [13]. 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) [16]. Compound 1, a furan ester derivative, showed cytotoxic activity against HCT-116 tumor cell line with [IC.sub.50] 2.9 [micro]M [9].

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. Experimental

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 [9]. 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 [19].

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.

https://doi.org/ 10.1155/2017/6969358

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

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.

References

[1] B. S. Davidson, "New dimensions in natural products research: cultured marine microorganisms," Current Opinion in Biotechnology, vol. 6, no. 3, pp. 284-291, 1995.

[2] C. Bailly, "Ready for a comeback of natural products in oncology," Biochemical Pharmacology, vol. 77, no. 9, pp. 1447-1457, 2009.

[3] J. W. Blunt, B. R. Copp, R. A. Keyzers, M. H. G. Munro, and M. R. Prinsep, "Marine natural products," Natural Product Reports, vol. 31, no. 2, pp. 160-258, 2014.

[4] C.-C. Chang, W.-C. Chen, T.-F. Ho, H.-S. Wu, and Y.-H. Wei, "Development of natural anti-tumor drugs by microorganisms," Journal of Bioscience and Bioengineering, vol. 111, no. 5, pp. 501-511, 2011.

[5] W. H. Gerwick and B. S. Moore, "Lessons from the past and charting the future of marine natural products drug discovery and chemical biology," Chemistry and Biology, vol. 19, no. 1, pp. 85-98, 2012.

[6] T. G. C. Montenegro, F. A. R. Rodrigues, P. C. Jimenez et al., "Cytotoxic activity of fungal strains isolated from the ascidian eudistoma vannamei," Chemistry and Biodiversity, vol. 9, no. 10, pp. 2203-2209, 2012.

[7] N. N. Saraiva, B. S. F. Rodrigues, P. C. Jimenez et al., "Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast," Natural Product Research, vol. 29, no. 16, pp. 1545-1550, 2015.

[8] B. S. F. Rodrigues, B. D. B. Sahm, P. C. Jimenez et al., "Bioprospection of cytotoxic compounds in fungal strains recovered from sediments of the brazilian coast," Chemistry and Biodiversity, vol. 12, no. 3, pp. 432-442, 2015.

[9] P. K. Uchoa, A. T. Pimenta, R. Braz-Filho et al., "New cytotoxic furan from the marine sediment-derived fungi Aspergillus niger," Natural Product Research, pp. 1-5, 2017.

[10] S. Suda and R. W. Curtis, "Antibiotic properties of malformin," Applied Microbiology, vol. 14, no. 3, pp. 475-476, 1966.

[11] B. Kobbe, M. Cushman, G. N. Wogan, and A. L. Demain, "Production and antibacterial activity of malforming C, a toxic metabolite of Aspergillus niger" Applied and Environmental Microbiology, vol. 33, no. 4, pp. 996-997, 1977.

[12] Y. Koizumi and K. Hasumi, "Enhancement of fibrinolytic activity of U937 cells by malformin A1," Journal of Antibiotics, vol. 55, no. 1, pp. 78-82, 2002.

[13] Y. Kojima, T. Sunazuka, K. Nagai et al., "Solid-phase synthesis and biological activity of malformin C and its derivatives," Journal of Antibiotics, vol. 62, no. 12, pp. 681-686, 2009.

[14] K. Hagimori, T. Fukuda, Y. Hasegawa, S. Omura, and H. Tomoda, "Fungal malformins inhibit bleomycin-induced G2 checkpoint in Jurkat cells," Biological and Pharmaceutical Bulletin, vol. 30, no. 8, pp. 1379-1383, 2007.

[15] M. Varoglu and P. Crews, "Biosynthetically diverse compounds from a saltwater culture of sponge- derived Aspergillus niger," Journal of Natural Products, vol. 63, no. 1, pp. 41-43, 2000.

[16] J. Zhan, G. M. K. B. Gunaherath, E. M. K. Wijeratne, and A. A. L. Gunatilaka, "Asperpyrone D and other metabolites of the plant-associated fungal strain Aspergillus tubingensis," Phytochemistry, vol. 68, no. 3, pp. 368-372, 2007.

[17] R. W. Curtis, "Curvatures and malformations in bean plants caused by culture filtrate of Aspergillus niger," Plant Physiology, vol. 33, no. 1, pp. 17-22, 1958.

[18] R. W. Curtis, "Root curvatures induced by culture filtrates of Aspergillus niger" Science, vol. 128, no. 3325, pp. 661-662, 1958.

[19] ICH, "Validation of analytical procedures: text and methodology--Q2(R1)," in Proceedings of the International Conference on Harmonization (ICH '05), London, UK, 2005.

[20] Q. B. Cass and A. L. G. Degani, Desenvolvimento de Metodos por HPLC: Fundamentos, Estrategias e Validacao, UFSCar, Sao Carlos, Brazil, 2001.

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; mary@dqoi.ufc.br

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).
COPYRIGHT 2017 Hindawi Limited
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2017 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
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
Words:3615
Previous Article:Ranitidine Loaded Biopolymer Floats: Designing, Characterization, and Evaluation.
Next Article:Application of Central Composite Design in the Adsorption of Ca(II) on Metakaolin Zeolite. (Research Article).
Topics:

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters