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Comparison of fucosterol content in algae using high-performance liquid chromatography

Comparison of fucosterol content in algae using high-performance liquid chromatography Background: Fucosterol is a compound commonly found in algae that has various biological activities. The purpose of this study was to develop a high-performance liquid chromatography (HPLC) validation method for fucosterol and to compare the fucosterol contents of 11 algal species from Ulleungdo, Korea. Method: In this study, we successfully isolated and identified fucosterol from a 70% EtOH extract of Sargassum miyabei, and subsequently conducted specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision analyses for development of an HPLC validation method. Fucosterol contents were compared using the established HPLC validation conditions. Results: We successfully isolated fucosterol from a 70% EtOH extract of S. miyabei and identified it based on spectroscopic analysis. On the basis of HPLC validation using the fucosterol isolated from S. miyabei, we confirmed 2 −1 −1 specificity (8.5 min), linearity (R = 0.9998), LOD (3.20 μgmL ), LOQ (9.77 μgmL ), accuracy (intra-day and inter-day variation, 90–110%), and precision (RSD, 1.07%). Fucosterol contents in the 11 assessed algal species ranged from −1 0.22 to 81.67 mg g , with the highest content being recorded in a 70% EtOH extract of Desmarestia tabacoides −1 −1 (81.67 mg g ), followed by that of Agarum clathratum (78.70 mg g ). Conclusions: The results indicate that 70% EtOH extracts of D. tabacoides and A. clathratum containing fucosterol with various effects can be potential alternative sources of fucosterol. Keywords: Alga, Fucosterol, High-performance liquid chromatography, Validation Background increasing globally (Lee et al. 2011). As aquatic algae Algae are classified into three main classes, namely, can inhabit extreme environments, unlike terrestrial Chlorophyceae, Phaeophyceae, and Rhodophyceae, and organisms, they are known to produce various bio- there are approximately 6000 algal species worldwide, active substances that enable them to adapt to such of which approximately 150 species are used as food environments (Jeon et al. 2012). Owing to the pres- (Devi et al. 2011; Meenakshi et al. 2011). Algae are ence of these bioactive substances, algae have been known to be a rich source of bioactive substances reported to possess anticancer, antioxidant, antibacter- such as carotenoids, dietary fiber, proteins, minerals, ial, antitrypanosomal, antiangiogenic, and anti-HIV vitamins, polyphenols, and low-calorie polyunsaturated activities (Synytsya et al. 2010;Veiga-Santos etal. fatty acids. Research and development of health foods, 2010; Souza et al. 2012;GuerraDoreetal. 2013; cosmetics, and medicines using algae have been Shanmugam et al. 2014;Thuyet al. 2015). Several bioactive substances, including fucoidan (polysac- * Correspondence: daesung@mabik.re.kr charide), fucoxanthin, chlorophyll, xanthophyll (carotenoid), Jeong Min Lee and Jae Hyuk Jeon contributed equally to this work. phlorotannin (tannin), and fucosterol (sterol) have been iso- Department of Genetic Resources Research, National Marine Biodiversity lated from algae (Hosokawa et al. 2004). Fucosterol is Institute of Korea, Seocheon, Republic of Korea © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 2 of 6 commonly found in algae and has been reported to fucosterol contents. In the same year, S. miyabei was lower cholesterol levels and possess various physio- collected in Pohang-si, Korea, for isolation of fucosterol. logical activities, such as antidiabetic, anticancer, and The specimens have been deposited in the Marine Bio- antioxidant activities (Ikeda et al. 1988;Tangetal. diversity Institute of Korea (MABIK). All algal samples 2002; Lee et al. 2004; Ham et al. 2010). were lyophilized, pulverized, and extracted with 70% Currently, fucosterol is mainly isolated from EtOH using a sonicator (WUC-N30H; DAIHAN Scientific Sargassum species. In this study, we isolated fucos- Co., Ltd., Wonju, Korea). terol from Sargassum miyabei and developed a high- performance liquid chromatography (HPLC) valid- Instrument and reagents ation method for this compound. Furthermore, we Medium-pressure liquid chromatography (MPLC) and compared the content of fucosterol in 11 algal spe- HPLC were performed using the Buchi Sepacore Flash cies from Ulleungdo, Korea, using the established system (Flawil, Switzerland) consisting of a UV photom- HPLC validation conditions. eter C-640 and Agilent 1260 Infinity system (Tokyo, Japan) equipped with a diode array detector (DAD). Mass spec- Methods trometry (MS) was conducted using a JEOL JMS-600-W Samples of algae Spectrometer (Tokyo, Japan), and nuclear magnetic In 2015, eleven species of algae (Agarum clathratum, resonance (NMR) spectra were recorded using a Bruker Caulerpa okamurae, Codium fragile, Desmarestia taba- AVANCE 500 NMR spectrometer (Rheinstetten, coides, Dictyopteris divaricata, Ecklonia cava, Eisenia Germany) in CDCl with tetramethylsilane used as the in- bicyclis, Myagropsis myagroides, Sargassum horneri, S. ternal standard. Chemical shifts are reported in parts per serratifolium,and Sporochnus radiciformis) were col- million (δ) and the coupling constant (J) is expressed in lected in Ulleungdo Island, Korea, for comparison of Hertz (Hz). All reagents used were of analytical grade. Fig. 1 Fucosterol isolated from Sargassum miyabei Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 3 of 6 Isolation of fucosterol Validation A dried sample of S. miyabei (1.2 kg) was powdered and The HPLC method was validated for specificity, extracted with 70% EtOH (12 L × 3) using a sonicator linearity, limit of detection (LOD), limit of quantifica- and then evaporated under vacuum. The 70% EtOH ex- tion (LOQ), accuracy, and precision according to the tract (91.7 g) was suspended in water and partitioned International Conference on Harmonization (ICH; 1997, with n-hexane, chloroform, ethyl acetate, and n-butanol. 2005) guidelines. A portion of the n-hexane fraction (15.6 g) was subjected The linearity of the method was established using to chromatographic analysis using an MPLC system and triplicate injections at six concentrations in the −1 eluted in a gradient solvent system (100% n-hexane and range 3.91–125.00 μgmL . A calibration curve for up to 100% ethyl acetate) to yield eight subfractions fucosterol was constructed using peak area (Y), −1 (H1–H8). Subfraction H5 (8.1 g, n-hexane:ethyl acet- concentration (X, μg10 μL ), and mean (n =3)± ate = 60:40) was re-chromatographed in an MPLC standard deviation values. Correlation coefficient system and eluted in a gradient solvent system (70% values (R ) were determined from the calibration n-hexane and up to 100% ethyl acetate) to yield five curve. The LOD and LOQ of fucosterol were calcu- subfractions (H5-1 to H5-5). H5-3 was recrystallized lated using the following formulae: LOD = 3.3 × σ/ with MeOH to obtain fucosterol (Fig. 1). S and LOQ = 10 × σ/S;where, σ =the deviation Fucosterol was obtained as a white powder; EI-MS m/ and S = the slope of the calibration curve. The z: 412 [M] (13.0), 314 (100), 299 (19.2), 271 (11.3), 229 accuracy of the method was determined based on (15.5); H-NMR (500 MHz, CDCl δ ) 5.35 (1H, d, J = intra- and inter-day variations. Intra-day variation 3, H 5.5 Hz, H-6), 5.18 (1H, q, J = 6.5, 13.5 Hz, H-28), 3.53 was determined by analyzing triplicate samples at (1H, m, H-3), 1.58 (3H, d, J = 7.5 Hz, H-29), 1.02 (3H, s, three different concentrations (15.63, 31.25, −1 H-19), 1.00 (3H, d, J = 7.0 Hz, H-21), 0.98 (3H, d, J = 62.50 μgmL ) in a single day. Inter-day variation 6.5 Hz, H-27), 0.97 (3H, d, J = 7.0 Hz, H-26), 0.69 (3H, s, wasdeterminedbyanalyzing asinglesampleat H-18); C-NMR (125 MHz, CDCl , δ ) 147.2 (C-24), three different concentrations (15.63–62.50 μg 3 C −1 141.0 (C-5), 121.9 (C-6), 115.8 (C-28), 72.0 (C-3), 56.9 mL ) for 3 days. Variations are expressed as per- (C-14), 56.0 (C-17), 50.3 (C-9), 42.5 (C-13), 42.4 (C-4), centage recoveries. Precision was assessed based on 39.9 (C-12), 37.5 (C-1), 36.7 (C-10), 36.6 (C-20), 35.4 repeatability. Repeatability data were obtained from (C-22), 35.0 (C-25), 32.1 (C-7,8), 31.9 (C-2), 28.4 (C- six injections of samples at a concentration of −1 16), 25.9 (C-23), 24.5 (C-15), 22.4 (C-26), 22.3 (C-27), 125.00 μgmL on the same day. Repeatability is 21.3 (C-11), 19.6 (C-19), 19.0 (C-21), 13.4 (C-29), expressed as the percentage relative standard 12.1 (C-18). deviation (%RSD). Results and discussion Sample preparation and HPLC condition Isolation of fucosterol A stock solution of fucosterol (1 mg) was dissolved The fucosterol extracted from S. miyabei was ob- in 1 mL of acetonitrile and was diluted to give the tained as a white powder with a molecular formula of desired concentrations (3.91, 7.82, 15.63, 31.25, C H O based on EI-MS analysis. In the H-NMR 29 48 −1 62.50, and 125.00 μgmL ). To analyze fucosterol spectrum of fucosterol, we detected two olefinic content, samples of the 11 aforementioned algal proton signals [δ 5.35 (1H, d, J =5.5Hz,H-6), 5.18 species were extracted with 70% EtOH for 1 h by (1H, q, J = 6.5, 13.5 Hz, H-28)], one oxygenated sonication (three times) and evaporated under vac- methine proton signal [δ 3.53 (1H, m, H-3)], and six uum. The residues were dissolved in 1 mL of 50% methyl groups [δ 1.58 (3H, d, J = 7.5 Hz, H-29), MeOH andfilteredusing a0.45-μm syringe filter. 1.02 (3H, s, H-19), 1.00 (3H, d, J = 7.0 Hz, H-21), The resulting solution was subjected to HPLC ana- 0.98 (3H, d, J =6.5Hz,H-27),0.97(3H,d, J =7.0 lysis, using a Kinetex C18 (4.6 mm × 100 mm, Hz, H-26), 0.69 (3H, s, H-18)]. In the C-NMR 2.6 μm) column, with a mobile phase comprising a spectrum of fucosterol, we observed 29 carbon solution of MeOH (solvent A) and 0.1% acetic acid signals, including two olefin quaternary carbons [δ (solvent B). The gradient solvent system was as fol- 147.2 (C-24), 141.0 (C-5)], two olefin methine carbons lows: from 50 to 0% B for 5 min, and then retained [δ 121.9 (C-6), 115.8 (C-28)], one oxygenated at 0% B for 10 min. The injection volume was 10 μL methine carbon [δ 72.0 (C-3)], and six methyl car- −1 and the flow rate was 1 mL min .The column bons [δ 22.4 (C-26), 22.3 (C-27), 19.6 (C-19), 19.0 temperature was maintained at 25 °C and detection (C-21), 13.4 (C-29), 12.1 (C-18)]. Fucosterol was wavelength was 210 nm. All injections were performed elucidated by comparison of the EI-MS and NMR 1 13 1 1 in triplicate. ( H-NMR, C-NMR, DEPT 45, 90, 135, H- HCOSY, Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 4 of 6 Fig. 2 High-performance liquid chromatography chromatograms of the fucosterol extracted from Agarum clathratum (a) and Desmarestia tabacoides (b) HSQC, HMBC) spectral data with those previously fucosterol was greater than 0.9995, thereby verifying published (Bang et al. 2011). the linearity of fucosterol. The LOD and LOQ of −1 fucosterol were 3.20 and 9.77 μgmL , respectively Validation (Table 1). The intra- and inter-day accuracy ranges In the HPLC data, fucosterol showed a single peak were 95.76–103.21% and 96.49–101.46%, respectively. unaffected by solvent and other components at 8.5 The intra- and inter-day variations were within the min (Fig. 2a). Good resolution of fucosterol and spe- range of 90–110%, which confirmed the accuracy of cificity of the method were confirmed. In the calibra- the method (Table 2). The precision of the HPLC tion curve at six different concentrations (3.91– −1 125.00 μgmL ) of fucosterol for determining linear- 2 2 Table 2 Accuracy of the high-performance liquid ity, y was 5.70x−23.90 and R was 0.9998. The R of chromatography validation method for fucosterol from S. miyabei Table 1 High-performance liquid chromatography calibration a b Concentration Intra-day variation Inter-day variation data for fucosterol from S. miyabei −1 (μgmL ) Recovery (%) SD (%) Recovery (%) SD (%) Linear range Regression Correlation LOD LOQ −1 a 2 −1 b −1 c (μgmL ) equation coefficient (R ) (μgmL ) (μgmL ) 62.50 101.05 0.71 101.46 0.59 3.91–125.00 y = 5.7x + 23.9 0.9998 3.20 9.77 31.25 103.22 0.13 99.55 0.28 Triplicate injections at six concentrations 15.63 95.76 2.25 96.49 0.97 a −1 Regression equation is y = Ax + B [y, the peak area; x, concentration (μg10 μL )] b a Limit of detection Triplicate injections at three different concentrations in a single day c b Limit of quantification One injection at three different concentration for 3 days Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 5 of 6 Table 3 Precision of the high-performance liquid Terasaki et al. 2009;Zhenetal. 2015; Majik et al. 2015;Per- chromatography validation method for fucosterol from S. miyabei umal et al. 2018). Although Sargassum species are a good Concentration Number of Area Mean RSD (%) source of fucosterol, our present study results confirmed −1 (μgmL ) measurements that 70% EtOH extracts of D. tabacoides and A. clathratum 125.00 1 682.8 685.4 1.05 could replace Sargassum species as a potential source of 2 674.5 fucosterol. Therefore, we anticipate that D. tabacoides and A. clathratum, which have high fucosterol contents with 3 681.7 the various aforementioned properties, will have a high 4 688.0 industrial value. 5 692.0 6 693.6 Conclusions We successfully isolated fucosterol from S. miyabei and de- veloped an HPLC validation method for this compound for method, calculated as %RSD, was evaluated for re- comparison of the fucosterol contents of 11 selected algal −1 peatability (125.00 μgmL ). The RSD was less than species. The recorded fucosterol contents in 70% EtOH 2.0% (1.07%), which confirmed the precision of the extracts of D. tabacoides and A. clathratum were 81.67 and −−1 method (Table 3). 78.70 mg g , respectively. These results indicate that 70% EtOH extracts of D. tabacoides and A. clathratum contain- Determination of fucosterol contents ing high contents of fucosterol with various beneficial The fucosterol contents in the 11 assessed algal properties can be potential alternative sources of fucosterol. −1 species ranged from 0.22 to 81.67 mg g (Table 4). Acknowledgements Specifically, the content of fucosterol in A. clathra- This work was supported by the National Marine Biodiversity Institute of tum, D. tabacoides, E. cava,and S. radiciformis was Korea (2020M00500). approximately four times higher than that in Sargas- Authors’ contributions sum species. The highest content of fucosterol was JML isolated the compounds, elucidated the structures, and wrote the detected in the 70% EtOH extract of D. tabacoides manuscript. JHJ performed validations of the HPLC method and investigated −1 (81.67 mg g ,Fig. 2b), followed by that of A. clathratum the content of fucosterol in the algae. MJY and GC collected algal samples −1 and assisted in the preparation of the manuscript. YGP and MSL contributed (78.70 mg g ). to plant material preparation. DSL designed and managed the research Fucosterol has been reported to have various effect such program. All authors read and approved the final manuscript. as antioxidant, antidepressant, anticonvulsant, antipredator, Funding and antiplasmodial activities, and it has been isolated from This work was supported by the National Marine Biodiversity Institute of the Sargassum species S. micracanthum, S. tenerrimum, S. Korea Research Program 2019M00700. fusiforme, S. horneri,and S. linearifolium (Ham et al. 2010; Availability of data and materials All data generated or analyzed during this study are included in this published article. Table 4 Content of fucosterol in 70% EtOH extracts of 11 algal species Ethics approval and consent to participate Sample Content of Not applicable fucosterol −1 (mg g ) Consent for publication a Not applicable Agarum clathratum 78.70 ± 0.01 Caulerpa okamurae 0.22 ± 0.02 Competing interests The authors declare that they have no competing interests. Codium fragile N.D. Desmarestia tabacoides 81.67 ± 0.07 Received: 18 October 2019 Accepted: 26 February 2020 Dictyopteris divaricata Trace Ecklonia cava 48.03 ± 0.14 References Bang MH, Kim HH, Lee DY, Han MW, Baek YS, Chung DK, Baek NI. Anti- Eisenia bicyclis 13.10 ± 0.07 osteoporotic activities of fucosterol from sea mustard (Undaria pinnatifida). Myagropsis myagroides 3.13 ± 0.01 Food Sci Biotechnol. 2011;20:343–7. Devi GK, Manivannan K, Thirumaran G, Rajathi FA, Anantharaman P. In vitro Sargassum horneri 2.63 ± 0.02 antioxidant activities of selected seaweeds from Southeast coast of India. 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Comparison of fucosterol content in algae using high-performance liquid chromatography

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Abstract

Background: Fucosterol is a compound commonly found in algae that has various biological activities. The purpose of this study was to develop a high-performance liquid chromatography (HPLC) validation method for fucosterol and to compare the fucosterol contents of 11 algal species from Ulleungdo, Korea. Method: In this study, we successfully isolated and identified fucosterol from a 70% EtOH extract of Sargassum miyabei, and subsequently conducted specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision analyses for development of an HPLC validation method. Fucosterol contents were compared using the established HPLC validation conditions. Results: We successfully isolated fucosterol from a 70% EtOH extract of S. miyabei and identified it based on spectroscopic analysis. On the basis of HPLC validation using the fucosterol isolated from S. miyabei, we confirmed 2 −1 −1 specificity (8.5 min), linearity (R = 0.9998), LOD (3.20 μgmL ), LOQ (9.77 μgmL ), accuracy (intra-day and inter-day variation, 90–110%), and precision (RSD, 1.07%). Fucosterol contents in the 11 assessed algal species ranged from −1 0.22 to 81.67 mg g , with the highest content being recorded in a 70% EtOH extract of Desmarestia tabacoides −1 −1 (81.67 mg g ), followed by that of Agarum clathratum (78.70 mg g ). Conclusions: The results indicate that 70% EtOH extracts of D. tabacoides and A. clathratum containing fucosterol with various effects can be potential alternative sources of fucosterol. Keywords: Alga, Fucosterol, High-performance liquid chromatography, Validation Background increasing globally (Lee et al. 2011). As aquatic algae Algae are classified into three main classes, namely, can inhabit extreme environments, unlike terrestrial Chlorophyceae, Phaeophyceae, and Rhodophyceae, and organisms, they are known to produce various bio- there are approximately 6000 algal species worldwide, active substances that enable them to adapt to such of which approximately 150 species are used as food environments (Jeon et al. 2012). Owing to the pres- (Devi et al. 2011; Meenakshi et al. 2011). Algae are ence of these bioactive substances, algae have been known to be a rich source of bioactive substances reported to possess anticancer, antioxidant, antibacter- such as carotenoids, dietary fiber, proteins, minerals, ial, antitrypanosomal, antiangiogenic, and anti-HIV vitamins, polyphenols, and low-calorie polyunsaturated activities (Synytsya et al. 2010;Veiga-Santos etal. fatty acids. Research and development of health foods, 2010; Souza et al. 2012;GuerraDoreetal. 2013; cosmetics, and medicines using algae have been Shanmugam et al. 2014;Thuyet al. 2015). Several bioactive substances, including fucoidan (polysac- * Correspondence: daesung@mabik.re.kr charide), fucoxanthin, chlorophyll, xanthophyll (carotenoid), Jeong Min Lee and Jae Hyuk Jeon contributed equally to this work. phlorotannin (tannin), and fucosterol (sterol) have been iso- Department of Genetic Resources Research, National Marine Biodiversity lated from algae (Hosokawa et al. 2004). Fucosterol is Institute of Korea, Seocheon, Republic of Korea © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 2 of 6 commonly found in algae and has been reported to fucosterol contents. In the same year, S. miyabei was lower cholesterol levels and possess various physio- collected in Pohang-si, Korea, for isolation of fucosterol. logical activities, such as antidiabetic, anticancer, and The specimens have been deposited in the Marine Bio- antioxidant activities (Ikeda et al. 1988;Tangetal. diversity Institute of Korea (MABIK). All algal samples 2002; Lee et al. 2004; Ham et al. 2010). were lyophilized, pulverized, and extracted with 70% Currently, fucosterol is mainly isolated from EtOH using a sonicator (WUC-N30H; DAIHAN Scientific Sargassum species. In this study, we isolated fucos- Co., Ltd., Wonju, Korea). terol from Sargassum miyabei and developed a high- performance liquid chromatography (HPLC) valid- Instrument and reagents ation method for this compound. Furthermore, we Medium-pressure liquid chromatography (MPLC) and compared the content of fucosterol in 11 algal spe- HPLC were performed using the Buchi Sepacore Flash cies from Ulleungdo, Korea, using the established system (Flawil, Switzerland) consisting of a UV photom- HPLC validation conditions. eter C-640 and Agilent 1260 Infinity system (Tokyo, Japan) equipped with a diode array detector (DAD). Mass spec- Methods trometry (MS) was conducted using a JEOL JMS-600-W Samples of algae Spectrometer (Tokyo, Japan), and nuclear magnetic In 2015, eleven species of algae (Agarum clathratum, resonance (NMR) spectra were recorded using a Bruker Caulerpa okamurae, Codium fragile, Desmarestia taba- AVANCE 500 NMR spectrometer (Rheinstetten, coides, Dictyopteris divaricata, Ecklonia cava, Eisenia Germany) in CDCl with tetramethylsilane used as the in- bicyclis, Myagropsis myagroides, Sargassum horneri, S. ternal standard. Chemical shifts are reported in parts per serratifolium,and Sporochnus radiciformis) were col- million (δ) and the coupling constant (J) is expressed in lected in Ulleungdo Island, Korea, for comparison of Hertz (Hz). All reagents used were of analytical grade. Fig. 1 Fucosterol isolated from Sargassum miyabei Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 3 of 6 Isolation of fucosterol Validation A dried sample of S. miyabei (1.2 kg) was powdered and The HPLC method was validated for specificity, extracted with 70% EtOH (12 L × 3) using a sonicator linearity, limit of detection (LOD), limit of quantifica- and then evaporated under vacuum. The 70% EtOH ex- tion (LOQ), accuracy, and precision according to the tract (91.7 g) was suspended in water and partitioned International Conference on Harmonization (ICH; 1997, with n-hexane, chloroform, ethyl acetate, and n-butanol. 2005) guidelines. A portion of the n-hexane fraction (15.6 g) was subjected The linearity of the method was established using to chromatographic analysis using an MPLC system and triplicate injections at six concentrations in the −1 eluted in a gradient solvent system (100% n-hexane and range 3.91–125.00 μgmL . A calibration curve for up to 100% ethyl acetate) to yield eight subfractions fucosterol was constructed using peak area (Y), −1 (H1–H8). Subfraction H5 (8.1 g, n-hexane:ethyl acet- concentration (X, μg10 μL ), and mean (n =3)± ate = 60:40) was re-chromatographed in an MPLC standard deviation values. Correlation coefficient system and eluted in a gradient solvent system (70% values (R ) were determined from the calibration n-hexane and up to 100% ethyl acetate) to yield five curve. The LOD and LOQ of fucosterol were calcu- subfractions (H5-1 to H5-5). H5-3 was recrystallized lated using the following formulae: LOD = 3.3 × σ/ with MeOH to obtain fucosterol (Fig. 1). S and LOQ = 10 × σ/S;where, σ =the deviation Fucosterol was obtained as a white powder; EI-MS m/ and S = the slope of the calibration curve. The z: 412 [M] (13.0), 314 (100), 299 (19.2), 271 (11.3), 229 accuracy of the method was determined based on (15.5); H-NMR (500 MHz, CDCl δ ) 5.35 (1H, d, J = intra- and inter-day variations. Intra-day variation 3, H 5.5 Hz, H-6), 5.18 (1H, q, J = 6.5, 13.5 Hz, H-28), 3.53 was determined by analyzing triplicate samples at (1H, m, H-3), 1.58 (3H, d, J = 7.5 Hz, H-29), 1.02 (3H, s, three different concentrations (15.63, 31.25, −1 H-19), 1.00 (3H, d, J = 7.0 Hz, H-21), 0.98 (3H, d, J = 62.50 μgmL ) in a single day. Inter-day variation 6.5 Hz, H-27), 0.97 (3H, d, J = 7.0 Hz, H-26), 0.69 (3H, s, wasdeterminedbyanalyzing asinglesampleat H-18); C-NMR (125 MHz, CDCl , δ ) 147.2 (C-24), three different concentrations (15.63–62.50 μg 3 C −1 141.0 (C-5), 121.9 (C-6), 115.8 (C-28), 72.0 (C-3), 56.9 mL ) for 3 days. Variations are expressed as per- (C-14), 56.0 (C-17), 50.3 (C-9), 42.5 (C-13), 42.4 (C-4), centage recoveries. Precision was assessed based on 39.9 (C-12), 37.5 (C-1), 36.7 (C-10), 36.6 (C-20), 35.4 repeatability. Repeatability data were obtained from (C-22), 35.0 (C-25), 32.1 (C-7,8), 31.9 (C-2), 28.4 (C- six injections of samples at a concentration of −1 16), 25.9 (C-23), 24.5 (C-15), 22.4 (C-26), 22.3 (C-27), 125.00 μgmL on the same day. Repeatability is 21.3 (C-11), 19.6 (C-19), 19.0 (C-21), 13.4 (C-29), expressed as the percentage relative standard 12.1 (C-18). deviation (%RSD). Results and discussion Sample preparation and HPLC condition Isolation of fucosterol A stock solution of fucosterol (1 mg) was dissolved The fucosterol extracted from S. miyabei was ob- in 1 mL of acetonitrile and was diluted to give the tained as a white powder with a molecular formula of desired concentrations (3.91, 7.82, 15.63, 31.25, C H O based on EI-MS analysis. In the H-NMR 29 48 −1 62.50, and 125.00 μgmL ). To analyze fucosterol spectrum of fucosterol, we detected two olefinic content, samples of the 11 aforementioned algal proton signals [δ 5.35 (1H, d, J =5.5Hz,H-6), 5.18 species were extracted with 70% EtOH for 1 h by (1H, q, J = 6.5, 13.5 Hz, H-28)], one oxygenated sonication (three times) and evaporated under vac- methine proton signal [δ 3.53 (1H, m, H-3)], and six uum. The residues were dissolved in 1 mL of 50% methyl groups [δ 1.58 (3H, d, J = 7.5 Hz, H-29), MeOH andfilteredusing a0.45-μm syringe filter. 1.02 (3H, s, H-19), 1.00 (3H, d, J = 7.0 Hz, H-21), The resulting solution was subjected to HPLC ana- 0.98 (3H, d, J =6.5Hz,H-27),0.97(3H,d, J =7.0 lysis, using a Kinetex C18 (4.6 mm × 100 mm, Hz, H-26), 0.69 (3H, s, H-18)]. In the C-NMR 2.6 μm) column, with a mobile phase comprising a spectrum of fucosterol, we observed 29 carbon solution of MeOH (solvent A) and 0.1% acetic acid signals, including two olefin quaternary carbons [δ (solvent B). The gradient solvent system was as fol- 147.2 (C-24), 141.0 (C-5)], two olefin methine carbons lows: from 50 to 0% B for 5 min, and then retained [δ 121.9 (C-6), 115.8 (C-28)], one oxygenated at 0% B for 10 min. The injection volume was 10 μL methine carbon [δ 72.0 (C-3)], and six methyl car- −1 and the flow rate was 1 mL min .The column bons [δ 22.4 (C-26), 22.3 (C-27), 19.6 (C-19), 19.0 temperature was maintained at 25 °C and detection (C-21), 13.4 (C-29), 12.1 (C-18)]. Fucosterol was wavelength was 210 nm. All injections were performed elucidated by comparison of the EI-MS and NMR 1 13 1 1 in triplicate. ( H-NMR, C-NMR, DEPT 45, 90, 135, H- HCOSY, Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 4 of 6 Fig. 2 High-performance liquid chromatography chromatograms of the fucosterol extracted from Agarum clathratum (a) and Desmarestia tabacoides (b) HSQC, HMBC) spectral data with those previously fucosterol was greater than 0.9995, thereby verifying published (Bang et al. 2011). the linearity of fucosterol. The LOD and LOQ of −1 fucosterol were 3.20 and 9.77 μgmL , respectively Validation (Table 1). The intra- and inter-day accuracy ranges In the HPLC data, fucosterol showed a single peak were 95.76–103.21% and 96.49–101.46%, respectively. unaffected by solvent and other components at 8.5 The intra- and inter-day variations were within the min (Fig. 2a). Good resolution of fucosterol and spe- range of 90–110%, which confirmed the accuracy of cificity of the method were confirmed. In the calibra- the method (Table 2). The precision of the HPLC tion curve at six different concentrations (3.91– −1 125.00 μgmL ) of fucosterol for determining linear- 2 2 Table 2 Accuracy of the high-performance liquid ity, y was 5.70x−23.90 and R was 0.9998. The R of chromatography validation method for fucosterol from S. miyabei Table 1 High-performance liquid chromatography calibration a b Concentration Intra-day variation Inter-day variation data for fucosterol from S. miyabei −1 (μgmL ) Recovery (%) SD (%) Recovery (%) SD (%) Linear range Regression Correlation LOD LOQ −1 a 2 −1 b −1 c (μgmL ) equation coefficient (R ) (μgmL ) (μgmL ) 62.50 101.05 0.71 101.46 0.59 3.91–125.00 y = 5.7x + 23.9 0.9998 3.20 9.77 31.25 103.22 0.13 99.55 0.28 Triplicate injections at six concentrations 15.63 95.76 2.25 96.49 0.97 a −1 Regression equation is y = Ax + B [y, the peak area; x, concentration (μg10 μL )] b a Limit of detection Triplicate injections at three different concentrations in a single day c b Limit of quantification One injection at three different concentration for 3 days Lee et al. Fisheries and Aquatic Sciences (2020) 23:9 Page 5 of 6 Table 3 Precision of the high-performance liquid Terasaki et al. 2009;Zhenetal. 2015; Majik et al. 2015;Per- chromatography validation method for fucosterol from S. miyabei umal et al. 2018). Although Sargassum species are a good Concentration Number of Area Mean RSD (%) source of fucosterol, our present study results confirmed −1 (μgmL ) measurements that 70% EtOH extracts of D. tabacoides and A. clathratum 125.00 1 682.8 685.4 1.05 could replace Sargassum species as a potential source of 2 674.5 fucosterol. Therefore, we anticipate that D. tabacoides and A. clathratum, which have high fucosterol contents with 3 681.7 the various aforementioned properties, will have a high 4 688.0 industrial value. 5 692.0 6 693.6 Conclusions We successfully isolated fucosterol from S. miyabei and de- veloped an HPLC validation method for this compound for method, calculated as %RSD, was evaluated for re- comparison of the fucosterol contents of 11 selected algal −1 peatability (125.00 μgmL ). The RSD was less than species. The recorded fucosterol contents in 70% EtOH 2.0% (1.07%), which confirmed the precision of the extracts of D. tabacoides and A. clathratum were 81.67 and −−1 method (Table 3). 78.70 mg g , respectively. These results indicate that 70% EtOH extracts of D. tabacoides and A. clathratum contain- Determination of fucosterol contents ing high contents of fucosterol with various beneficial The fucosterol contents in the 11 assessed algal properties can be potential alternative sources of fucosterol. −1 species ranged from 0.22 to 81.67 mg g (Table 4). Acknowledgements Specifically, the content of fucosterol in A. clathra- This work was supported by the National Marine Biodiversity Institute of tum, D. tabacoides, E. cava,and S. radiciformis was Korea (2020M00500). approximately four times higher than that in Sargas- Authors’ contributions sum species. The highest content of fucosterol was JML isolated the compounds, elucidated the structures, and wrote the detected in the 70% EtOH extract of D. tabacoides manuscript. JHJ performed validations of the HPLC method and investigated −1 (81.67 mg g ,Fig. 2b), followed by that of A. clathratum the content of fucosterol in the algae. MJY and GC collected algal samples −1 and assisted in the preparation of the manuscript. YGP and MSL contributed (78.70 mg g ). to plant material preparation. DSL designed and managed the research Fucosterol has been reported to have various effect such program. All authors read and approved the final manuscript. as antioxidant, antidepressant, anticonvulsant, antipredator, Funding and antiplasmodial activities, and it has been isolated from This work was supported by the National Marine Biodiversity Institute of the Sargassum species S. micracanthum, S. tenerrimum, S. Korea Research Program 2019M00700. fusiforme, S. horneri,and S. linearifolium (Ham et al. 2010; Availability of data and materials All data generated or analyzed during this study are included in this published article. Table 4 Content of fucosterol in 70% EtOH extracts of 11 algal species Ethics approval and consent to participate Sample Content of Not applicable fucosterol −1 (mg g ) Consent for publication a Not applicable Agarum clathratum 78.70 ± 0.01 Caulerpa okamurae 0.22 ± 0.02 Competing interests The authors declare that they have no competing interests. Codium fragile N.D. Desmarestia tabacoides 81.67 ± 0.07 Received: 18 October 2019 Accepted: 26 February 2020 Dictyopteris divaricata Trace Ecklonia cava 48.03 ± 0.14 References Bang MH, Kim HH, Lee DY, Han MW, Baek YS, Chung DK, Baek NI. Anti- Eisenia bicyclis 13.10 ± 0.07 osteoporotic activities of fucosterol from sea mustard (Undaria pinnatifida). Myagropsis myagroides 3.13 ± 0.01 Food Sci Biotechnol. 2011;20:343–7. Devi GK, Manivannan K, Thirumaran G, Rajathi FA, Anantharaman P. In vitro Sargassum horneri 2.63 ± 0.02 antioxidant activities of selected seaweeds from Southeast coast of India. 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