Development of an HPLC method to analyze and prepare elsinochrome C and hypocrellin A in the submerged fermentation broth of Shiria sp. SUPER-H168

ABSTRACT

A rapid and sensitive analytical method based on reverse-phase high-performance liquid chromatography was first developed to simultaneously determine elsinochrome C (EC) and hypocrellin A (HA) in the submerged fermentation. The mobile phase consisted of acetonitrile–water 60:40 (v/v) with a flow-rate of 1 mL/min. The calibration curves were as follows: y = 37,625x + 249,775 for EC, y = 30,813x + 556,409 for HA and linear at the investigated concentration. The correlation coefficients (R²) were 0.9989 and 0.9998 respectively for EC and HA. The limits of detection and quantification were 175 and 585 µg/L for EC and 205 and 610 µg/L for HA. The precisions of concentration and retention times were less than 2.5 and 0.3%. The recovery of the method was greater than 95.0%. The methodology was applied to analyze simultaneously EC and HA concentrations in a submerged fermentation, and was adequate for analysis of biosynthesis of perylenequinones. The method was also amplified to separate and purify EC and HA using a semi-preparative C₁₈ column. In addition, elsinochrome C was first identified in the submerged fermentation broth of Shiraia sp. SUPER-H168. Copyright © 2011 John Wiley & Sons, Ltd.

Abbreviations used

EC

elsinochrome C

HA

hypocrellin A

Introduction

Perylenequinones represent a wide range of chemical compounds, including hypocrellins, elsinochromes, phleichrome, cercosporin and calphotins. These compounds are usually isolated from fungi or cultures (Arnone et al., 1985, 1988; Lee et al., 2007; Jimenez et al., 2009) and some of them, such as hypocrellins, elsinochromes and cercosporin, possess promising anticancer and antimicrobial activities (Fang et al., 2006; Fei et al., 2006; Hudson et al., 1997; Ma et al., 2004; Chen and Wan, 1980), protein kinase C inhibition (Yashi et al., 1989; Qu et al., 2003), and human immunodeficiency virus inhibition (Hudson et al., 1994; Hirayama et al., 1997). Furthermore, they exhibit excellent photosensivity; therefore they are expected to be developed into a new generation of photosensitizers instead of porphyrins and phthalocyanines, which are widely used in photodynamic therapy (Allen et al., 2001; Detty et al., 2004). Compared with the widely used hematoporphyrin, hypocrellins have several advantages as photodynamic therapy photosensitizers, including easy preparation and purification, small aggregation tendency, rapid metabolism in vivo and low toxicity to normal cells (Diwu and Lown, 1990, 1994).

Stomata of Shiraia bambusicola are the main source of hypocrellins. Because of the limited natural resource of hypocrellin, we isolated a high-yield hypocrellin-producing strain (Shiraia sp. SUPER-H168) and established solid and submerged fermentation conditions for production of hypocrellins (Liang et al., 2009; Cai et al., 2010, 2011). The study also found that not only does Shiraia sp. SUPER-H168 produce hypocrellins, but a new red compound has also been found in the fermentation broth. The new compound has many similar properties to hypocrellin A and was initially identified as perylenequinone.

So far, determination and analytical methods for perylenequinones have rarely been reported; available analytical methods are usually based on ultraviolet and visible spectrophotometry (Liu et al., 2000; Zhang et al., 1998). There exists a critical disadvantage in that only the total amount of perylenequinones is determined; it is not possible to determine the accurate content of a single compound. Most samples of fungi culture contain several kinds of perylenequinones with similar molecular structures and absorption spectra (Fig. 1). In recent decades, chromatographic techniques, especially high-performance liquid chromatography, have developed into the most important method for the determination and analysis of various samples. However, so far there have been no methods based on high-performance liquid chromatography to determine and analyze perylenequinones similar to hypocrellin A. The objectives of this work were as follows: (1) to establish a new analytical method for perylenequinones in the fermentation broth based on high-performance liquid chromatography instead of ultraviolet and visible spectrophotometry, with shorter analysis times and higher resolutions; (2) to identify the new perylenequinone found in the fermentation broth of Shiraia sp. SUPER-H168; and (3) to separate and purify the new red compound and hypocrellin A (HA) in the fermentation broth using a semi-prepative C₁₈ column.

Figure 1. Wavelengths scanning of elsinochrome C (a) and hypocrellin A (b).

Experimental

Reagents and standards

The elsinochrome C (EC) and HA standards were purchased from Hanbon Science and Technology Co. Ltd. Their purities were both ≥98%. Acetonitrile and methanol (HPLC grade) were also bought from Hanbon Science and Technology Co. Ltd. The other reagents (ethanol, ethyl acetate) were analytical grade and were obtained from Sinopharm Chemical Reagent Co. Ltd.

Sample preparation

Shiraia sp. SUPER-H168 was used to produce EC and HA. The culture medium was described in our previous work (Cai et al., 2011) The fermentation broth was extracted with ethanol at 30 °C for 2.4 h. The ratio of fermentation and ethanol was 1:7. To ensure the greatest extraction was achieved, the sample was extracted twice under the same conditions and the two parts of the extraction were merged. The combined extract was concentrated in a vacuum to an oily mass. The oily mass was dissolved with ethyl acetate saturated with water, and an equal volume of water saturated with ethyl acetate was subsequently added. Some water-soluble impurities were removed. The organic solvent layer was concentrated in vacuum to an oily mass, then dissolved with methanol and filtered in 0.45 µm membrane.

Optimization of chromatographic conditions

A new-style analytical high-performance liquid chromatography system (Hanbon Science and Technology Co. Ltd) equipped with an NP7000 series pump (maximum flow-rate 10 mL/min), NU3000 series multiple-wavelength UV detector and injector valve VI-11 (FLOM Co. Ltd) with a 20 μL sample loop was used. Chromatographic separation of EC and HA was performed on a Phecda RP-C₁₈ column (5 µm, 250 × 4.6 mm, Hanbon Science and Technology Co. Ltd).

The chromatographic conditions were optimized using the single factor method, where one variable is changed at a time. An isocratic-mode elution was used in the optimization assay. Two factors were considered in the experiments: category of organic modifier and composition of the mobile phase. The results of optimization were evaluated by resolution, indicating the degree of separation between adjacent peaks and the total analysis time. Detection wavelength was selected as 265 nm. Ethanol, methanol and acetonitrile were used as organic modifiers in the mobile phase. The flow-rate and ratio of organic modifier and water were fixed followed by the definition of the most suitable modifier. In the assay, the percentage of acetonitrile was changed from 50 to 70% and the flow-rate was varied between 0.80 and 1.0 mL/min.

Quantification and validation of the method

Stock solution of EC was prepared by dissolving 20.5 mg in 20.0 mL methanol; 7.5 mg HA was dissolved in 10.0 mL methanol. Two stock solutions were diluted with methanol to prepared different concentration standard solutions (EC: 14.0–220.0 mg/L; HA: 37.7–754.4 mg/L).

Quantification of EC and HA were performed using an external standard. The peak area was used to construct a calibration curve against the corresponding concentration. The calibration curves of EC and HA were established respectively at 269 and 262 nm. Each standard solution was injected into HPLC column in triplicate. The validation of the method was evaluated by linearity, limit of detection, limit of quantification and precision calculated as relative standard deviation (RSD).

Amplification of analytical method and preparation of peak 2 and HA

The chromatographic conditions were amplified to prepare EC and HA. A new-style semi-preparative high-performance liquid chromatography system (Hanbon Science and Technology Co. Ltd) equipped with an NP7000 series pump (maximum flow-rate 100 mL/min) and an NU3000 series multiple-wavelength UV detector was used. Semi-preparation of EC and HA was run on a Phecda RP-C₁₈ column (10 µm, 250 × 30 mm, Hanbon Science and Technology Co. Ltd). The extract was concentrated under vacuum to one-quarter of original volume followed by treatment sample as described above. The injection volume was the only considered factor affecting the semi-preparation process and varied between 5 and 20 mL. To ensure the purity of EC and HA, their purification was repeated three times. The collected HA and EC were concentrated under vacuum to aqueous solution, followed by extraction with ethyl acetate. The organic solvent layer was concentrated in vacuum to an oily mass.

Identification of EC

The purified EC was identified by color reaction, electrospray ionization mass spectrometry and nuclear magnetic resonance. The electrospray ionization mass spectrometry data for EC were recorded using a Maldi Synapt QT of MS (Waters, USA). NMR experiments were performed on an Avance III 400 MHz Digital NMR Spectrometer (Bruker, Germany) operated at 400 MHz for ¹H and ¹³ C using CDCl₃ as a solvent.

Application of the analytical method

The established method was applied to analysis of the fermentation broth. A 10 g aliquot fermentation was treated as described above. The treated samples were first analyzed at 269 and 262 nm. The concentration of EC was calculated in accordance with the calibration curve for EC when the wavelength was 269 nm. The concentration of HA was calculated in accordance with calibration curve for HA when the wavelength was in 262 nm. Then the treated samples were analyzed at 266 nm. All experiments were run independently in triplicate.

Results and discussion

Optimization of chromatographic conditions

Resolution (Rs) was calculated based on Rs = 2(t₂ – t₁)/(w_b1 + w_b2), where t₂ and t₁ are retention times, and w_b1 and w_b2 are the widths at the base of the corresponding peaks. The chromatographic resolution is not less than 1.0 to ensure baseline separation in the analysis.

Baseline separation was not achieved when methanol was used as the organic modifier and ethanol led to high pressure in the HPLC system. Considering the degree of separation between adjacent peaks and total analysis time, the most appropriate chromatographic conditions were as follows: suitable Rs and total analysis time were achieved with acetonitrile as the modifier. The ratio of acetonitrile and water was 6:4. The flow-rate was 1.0 mL/min. The column temperature was ambient. As shown in Fig. 2(B), every component in the fermentation broth was separated well under the optimized conditions.

Figure 2. Chromatograms of separations of elsinochrome C and hypocrellin A for (a) standards and (b) sample extracted from fermentation broth.

Linearity

An equation of linear regression was constructed between peak area (y) and concentration (x) to check linearity of response of detector. The equations were as follows: y = 37,625x + 249,775 for EC, y = 30,813x + 556,409 for HA. The squared correlation coefficients (R²) were used to evaluate the linearity of the calibration curves. The squared correlation coefficients of EC and HA were 0.9989 and 0.9998, respectively, which suggested that the linearity of the developed method was excellent over the whole investigated concentration range.

Limit of detection and quantification

The sensitivity of the developed method was evaluated by limit of detection (LOD) and limit of quantification (LOQ). The LOD is the lowest concentration that is detected by the detector. The LOQ is the minimum concentration that is quantifiable for samples. The LOD was calculated as 3 × (SD/slope); the LOD was calculated as 10 × (SD/slope), where SD is the standard deviation of five response values assigned to zero concentration, and slope is the slope of the calibration curve. In the study, the LOD of EC and HA was 175 and 205 µg/L, and the LOQ of EC and HA was 585 and 610 µg/L. The results indicated that the developed method had enough sensitivity for determination of EC and HA in the fermentation broth.

Precision

The precision was validated using the relative standard deviation of samples in the investigated concentrations. Intraday precision was defined as precision within investigated concentrations analyzed in one day, and interday precision as the precision between individual runs on five different days. For EC and HA, the intraday RSD of concentrations was lower than 1.7%, and the interday RSD of concentrations lower than 2.5%. The intraday RSD of retention time was <0.1%, and the interday RSD of the retention time <0.3%. The results are shown in Table 1.

Table 1. Analytical results of intra- and inter-day test

Analytes	Concentration (mg/L)	Intra-day				Inter-day
		Concentration (mg/L)		Retention times (min)		Concentration (mg/mL)		Retention times (min)
		Mean	RSD	Mean	RSD	Mean	RSD	Mean	RSD
EC	219.00	218.33	1.6%	11.25	0.09%	218.47	2.0%	11.25	0.14%
	109.00	108.6	1.1%	11.25	0.04%	109.57	1.7%	11.24	0.06%
HA	150.88	150.69	1.7%	35.31	0.07%	151.21	2.5%	35.23	0.30%
	80.44	81.31	1.1%	35.32	0.03%	81.79	1.8%	35.36	0.20%

Accuracy

The accuracy of the method was verified by the percentage recovery of EC and HA. The recovery test was carried out by adding two identified concentrations of standard solution to a known quantity of EC and HA sample, and the mixed sample was treated according to the method described above and analyzed by HPLC. This experiment was performed by three independent runs at each concentration and each sample was injected five times. The RSD of recovery test was <3.5%. The results are shown in Table 2.

Table 2. Analytical results of recovery test of EC and HA by using established method

Analytes	Original (mg)	Spiked (mg)	Found (mg)	Recovery (%)	RSD (%)
elsinochrome C (EC) and hypocrellin A (HA).
HA	0.22	0.30	0.50	96.2	2.5
		0.50	0.70	97.2	2.2
EC	0.38	0.10	0.46	95.8	3.5
		0.20	0.56	98.3	3.0

Amplification of analytical method and preparation of the peak 2 and HA

The study indicated that the analytical chromatographic conditions were appropriate for semi-preparation of perylenequinones. In the semi-preparation process, five perylenequinones were isolated, and further purification of peak 2 and HA was performed. Under these conditions, the injection volume was the main affluence factor. As shown in Fig. 3, a small injection volume was beneficial to the separation of the components; however, the peak 2 and HA productivity was low. It increased with increased injection volume, but the degree of separation between components decreased. In view of the productivity of the two main components and the degree of separation, the most appropriate injection volume was 15 mL. The purities of peak 2 and HA were respectively about 98 and 95%.

Figure 3. Chromatograms of semi-prepare process: (a) injection volume 5 mL; (b) injection volume 15 mL; (c) injection volume 20 mL.

Identification of EC

In our published work, one of the main components has been identified as HA (Liang et al., 2009; Cai et al., 2011). In this study, we found that another main component (peak 2) also showed the characteristic color reaction of perylenequinones. In addition, the retention time of peak 2 was in accordance with EC in the chromatograph (Fig. 2). Therefore peak 2 was further identified to confirm whether the peak 2 was EC. Peak 2 turned green under alkaline conditions and dark purple when FeCl₃ was added, which were characteristic color reactions of perylenequinones.

As shown in Fig. 1, the UV–vis absorption curve shows that one of the two compounds had six absorption peaks at 575, 544, 459, 338, 269 and 215 nm. The ESI-MS data (Fig. 4) of EC provided the molecular weight (m/z [M + H] 549, m/z [M − H] 547.05). Its ¹HNMR spectra were as follows (CHCl₃; δ): 1.10–1.11 (6H, d, H-18 and H-16), 3.50 (2H, s, 15-OH and 17-OH), 3.69 (2H, m, H-15 and H-17), 4.06 (6H, s,6-OCH₃ and 7-OCH₃), 4.14–4.15 (2H, d, H-13 and H-14), 4.22 (6H, s, 2- and 11-OCH₃), 6.61(2H, s, H-5 and H-8), and 16.14 (2H, s, 3-and 10-OH). Its ¹³CNMR spectra were as follows: 21.97 (q, C-16 and C-18), 42.24 (d, C-13 and C-14), 56.42 (q, 6-OCH₃ and 7-OCH₃ ), 61.14 (q, 2-OCH₃ and 11-OCH₃), 70.40 (d, C-15 and C-17), 102.24 (d, C-5 and C-8), 107.39 (s, C-3a and C-9a), 118.26 (s, C-6a and C-7a), 122.66 (s, C-3b and C-9b or C-1a and C-12a), 122.82 (s, C-3b and C-9b or C-1a and C-12a), 134.41 (s, C-1 and C-12), 150.53 (s, C-2 and C-11), 167.25 (C-6 and C-7), 172.25 (s, C-3 and C-10), and 179.35 (s, C-4 and C-9). Compared with EC standard and references (Liu et al., 2001; Lousberg et al., 1969; Kurobane et al., 1981), this compound was identified as EC based on the data of MS, ¹HNMR and ¹³CNMR. The molecular structure of EC is shown in Fig. 5.

Figure 4. Spectrum of ESI-MS of elsinochrome C:(a): time-of-flight (TOF) MS ES⁻, negative-ion spectra with one lost proton, m/z [M − H] 547.05; (b) TOF MS ES⁺, positive-ion spectra with one adduct proton, m/z [M + H] 549.

Figure 5. Molecular structure of elsinochrome C.

The elsinochromes are produced by many species of ascomycetous fungi of the genus Elsinoë and its imperfect stage Sphaceloma (Kurobane et al., 1981; Weiss et al., 1957, 1965). In a recent report, elsinochrome C was also found in the solid culture of Hypomyces (Fr·) Tul·sp· (Liu et al., 2001). Elsinochrome C was first found and identified in the liquid culture of Shiraia sp. SUPER-H168.

Analysis of the fermentation broth

The developed HPLC method was applied for determination of EC and HA. The other components in the fermentation broth had no effect on separation of EC and HA. The concentrations of EC and HA were calculated based on calibration curves. At 269 nm, the average concentration of EC was 430 ± 10 mg/L based on the calibration curve of EC. In 262 nm, the average concentration of HA was 220 ± 10 mg/L based on the calibration curve of HA. At 266 nm, the average concentrations of EC and HA were respectively 420 ± 15 and 214 ± 12 mg/L. The results suggest that the method can be applied to simultaneous determination of EC and HA in the fermentation broth based on HPLC-UV.

In the previous study, ultraviolet and visible spectrophotometry was the main methodology (Liu et al., 2000; Zhang et al., 1998); however, only the amount of perylenequinones was determined by the method and the composition of perylenequinones was unknown. The described method was applied to not only determine the amount but also to analyze the composition of perylenequinones in the fermentation broth. More significantly, the effect of nutrient substance on biosynthesis of perylenequinones was also initially analyzed in terms of composition of the fermentation broth.

Conclusions

In this work, elsinochrome C was first identified in the submerged fermentation of Shiraia sp. SUPER-H168 based on mass spectrometry, ¹HNMR and ¹³CNMR data. Moreover, a new analytical method of perylenequinones based on RP-HPLC/UV, which was more reliable and accurate than the method based on ultraviolet and visible spectrophotometry, was developed and applied to simultaneous analysis of EC and HA in the fermentation. In addition, EC and HA were separated and purified by semi-preparative high-performance liquid chromatography.

Acknowledgment

This work was financially supported by the National High Technology and Special Funds of Science and Technology, Innovation of Science and Technology Department of Jiangsu Province (grant no. BY2010117), the National Natural Science Foundation of China (grant no. 21045007), the Fundamental Research Funds for the Central Universities (JUSRP21120), and the Development Program of China (863 Program, grant no. 2010AA101501).