Journal of Chemistry
Volume 2017 (2017), Article ID 6969358, 7 pages
https://doi.org/10.1155/2017/6969358

Research Article

HPLC Quantification of Cytotoxic Compounds from Aspergillus niger

Paula Karina S. Uchoa, Leandro Bezerra de Lima, Antonia T. A. Pimenta, Maria da Conceição F. de Oliveira, Jair Mafezoli, and Mary Anne S. Lima

Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, 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

Copyright © 2017 Paula Karina S. Uchoa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

A high-performance liquid chromatography method was developed and validated for the quantification of the cytotoxic compounds produced by a marine strain of Aspergillus niger. The fungus was grown in malt peptone dextrose (MPD), potato dextrose yeast (PDY), and mannitol peptone yeast (MnPY) media during 7, 14, 21, and 28 days, and the natural products were identified by standard compounds. The validation parameters obtained were selectivity, linearity (coefficient of correlation > 0.99), precision (relative standard deviation below 5%), and accuracy (recovery > 96).

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).

Figure 1: Cytotoxic compounds 1–3 produced by Aspergillus niger (BRF074).

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-small-cell lung carcinoma, IC₅₀ 0.07 μM), MIA Pa Ca-2 (pancreatic inducing root curvatures and malformations in plants [17, 18], antibacterial activity cancer, IC₅₀ 0.05 M), MCF-7 (breast cancer, IC₅₀ 0.10 μM), and SF-268 (CNS cancer; glioma, IC₅₀ 0.07 μM) with slight selectivity towards the pancreatic cancer cell line (MIA Pa Ca-2) compared with the normal human primary fibroblast cells WI-38 (IC₅₀ 0.10 μM) [16]. Compound 1, a furan ester derivative, showed cytotoxic activity against HCT-116 tumor cell line with IC₅₀ 2.9 μM [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®. 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® 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 Pecém’s offshore port terminal (3°32′2′′ S; 38°47′58′′ W), state of Ceará, 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⁻¹; peptone, 2.0 g L⁻¹; dextrose, 20.0 g L⁻¹), potato dextrose yeast, PDY (potato dextrose yeast, 24.0 g L⁻¹; yeast, 3.0 g L⁻¹), or mannitol peptone yeast, MnPY (mannitol, 4.0 g L⁻¹; peptone, 2.0 g L⁻¹; yeast, 2.0 g L⁻¹).

The fungal strain was grown under static conditions at room temperature (28°C) during 7, 14, 21, and 28 days. The culture broths were separated from mycelium by filtration and submitted to partition with EtOAc (3 × 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 μm 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® ODS 100 A 250 mm × 4.60 mm, 5 μm) preceded by an C18 guard column (2.0 cm × 4.0 mm; 5 μm), 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·min⁻¹. The mobile phase was prepared daily and degassed by sonication before use. The column temperature was 25°C and the injection volume was 20 μL. 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 λ = 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 μg·mL⁻¹, and the concentration of the solution extracts was 2,000 μg·mL⁻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 μg·mL⁻¹ solutions (Graphpad Prism® 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 μg mL⁻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:

The test concentrations used were 140.0, 186.0, and 233.0 μg mL⁻¹ for the furan ester derivative (1); 70.0, 92.0, and 115.0 μg mL⁻¹ for malformin A (2); and 101.0, 134.0, and 166.0 μg mL⁻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 μg mL⁻¹. All the curves showed a linear response with in the selected range for each sample (Table 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
(µg mL⁻¹)
Regression equation
Correlation factor ()

Furan ester (1)
12.5–500.0

0.9984

Malformin A (2)
12.5–500.0

0.9996

Malformin C (3)
12.5–500.0

0.9991

Six data points ().

The furan ester derivative (1) showed LOD of 46.0 μg mL⁻¹ and LOQ of 67.9 μg mL⁻¹. Malformin A (2) showed LOD of 22.7 μg mL⁻¹ and LOQ of 33.6 μg mL⁻¹, while malformin C (3) showed LOD of 34.7 μg mL⁻¹ and LOQ of 51.3 μg mL⁻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].

Table 2: Repeatability and intermediate precision data of the bioactive compounds 1–3 produced by A. niger (BRF 074).

Compound
Repeatability ()
Intermediate precision ()

Concentration
(µg mL⁻¹)
Measured values
(µg mL⁻¹)
Mean
(µg mL⁻¹)
RSD
(%)
Concentration
(µg mL⁻¹)
Measured values
(µg mL⁻¹)
Mean
(µg mL⁻¹)
RSD
(%)

Furan ester (1)
50.0
54.2892
54.3437
54.2872
54.3067
0.06
50.0
50.1338
50.2537
50.0912
50.1595
0.17

250.0
233.8591
234.3952
234.1043
234.1195
0.11
250.0
245.7478
245.7127
245.7350
245.7319
0.01

500.0
509.2980
509.2856
509.2549
509.2795
0.01
500.0
500.9200
500.8662
501.3840
501.0567
0.06

Malformin A (2)
50.0
51.7388
51.7246
51.6044
51.6893
0.14
50.0
49.3617
49.4094
49.4188
49.3967
0.06

250.0
246.2154
246.0825
246.1936
246.1638
0.03
250.0
239.8196
240.0045
240.0410
239.9550
0.05

500.0
503.6637
502.9370
503.2363
503.2790
0.07
500.0
505.5801
505.6485
505.8089
505.6792
0.02

Malformin C (3)
50.0
52.8589
52.7992
52.8364
52.8315
0.06
50.0
51.1270
51.0972
50.9613
51.0618
0.17

250.0
241.1501
241.3867
241.4114
241.3161
0.06
250.0
231.0606
231.2140
231.3173
231.1973
0.06

500.0
505.8446
506.9641
505.1826
505.9971
0.18
500.0
510.3529
510.2716
510.1154
510.2467
0.02

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.

Table 3: Accuracy data of the bioactive compounds 1–3 produced by A. niger (BRF 074).

Compound
Accuracy ()

Concentration
(µg mL⁻¹)
Measured values
(µg mL⁻¹)
Mean
(µg mL⁻¹)
Recovery
(%)

Furan ester (1)
140.0
134.1553
133.9125
134.1571
134.2570
133.8316
134.0627
95.8

186.0
178.5342
178.6454
178.5064
178.4201
178.6202
178.5453
95.9

232.0
224.0426
223.6944
223.9213
224.0262
224.1322
223.9634
96.4

Malformin A (2)
70.0
72.7681
73.0678
73.0619
72.9432
72.9750
72.9632
104.2

92.0
98.0389
97.8777
97.9863
97.9622
97.7580
97.9246
106.4

115.0
117.8192
117.5671
117.6607
117.7728
117.5518
117.6743
102.3

Malformin C (3)
101.0
102.5096
102.4978
102.7050
102.5853
102.6210
102.5837
101.5

134.0
130.4046
130.5980
130.5686
130.6417
130.2936
130.5013
97.4

166.0
160.7364
160.7337
160.5222
160.4871
160.6094
160.6178
96.8

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 (). The contents of the three cytotoxic compounds in the crude extracts, MPD and PDY, are shown in Table 4.

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  µg mL⁻¹.

Growth time (days)
Furan ester (1) 
(Rt = 13.7 min)
Malformin A (2) 
(Rt = 21.4 min)
Malformin C (3) 
(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 µg mL⁻¹ ().

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).

(a)

(b)

(c)

(d)

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 × 4.6 mm, 5 μm); gradient elution going from H₂O/MeCN 80 : 20 to H₂O/MeCN 20 : 80 in 30 minutes; flow rate of 1.0 mL min⁻¹; sample concentration of 2.0 μg mL⁻¹.

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 μg mL⁻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 μg mL⁻¹ and 125.9 μg mL⁻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 μg mL⁻¹, respectively, while the concentration of 1 was 44.4 μg mL⁻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 μg mL⁻¹. However, the concentration of 1 increased almost twice (74.3 μg mL⁻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.

Acknowledgments

The authors acknowledge the Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Coordenação de Aperfeiçoamento de Ensino Superior (CAPES) for the fellowships and financial support.

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