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Identification and quantification of phenolic compounds in rapeseed originated lecithin and antioxidant activity evaluation
LWT - Food Science and Technology 73 (2016) 397e405
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Identification and quantification of phenolic compounds in rapeseed originated lecithin and antioxidant activity evaluation Jingbo Li, Zheng Guo* Department of Engineering, Faculty of Science, Aarhus University, Gustav Wieds Vej 10, 8000, Aarhus C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2016 Received in revised form 2 June 2016 Accepted 15 June 2016 Available online 18 June 2016
Phenolic compounds may exist in the gum generated from rapeseed oil refining as rapeseed is rich in phenolics. Therefore, this work identified the main phenolic compounds, followed by tracking their flow according to typical lecithin production procedures. Antioxidant activities of the extracts were also investigated to get a clue of the potential oxidative stability of the final products. Column chromatography, UV spectra, 1H NMR, and HPLC were used to separate and identify the phenolic compounds. Sinapic acid was found to be the dominant phenolic while relatively low contents of sinapine and canolol were also identified. Nonpolar solvent aid extraction was better for extracting more sinapine and sinapic acid while direct ethanol extraction gave more canolol. Almost half of the phenolics in dried gum were removed by acetone during deoiling process, leaving only a small amount in deoiled lecithin. Extracts from deoiled lecithin showed the weakest antioxidant activity while the ethanol solubles showed the strongest. This work demonstrated the occurrences of bioactive phenolic compounds in rapeseed originated gum, which may be an advantage of rapeseed lecithin compared with other types of lecithin. This work could also be a reference for rapeseed lecithin production with different contents of bioactive compounds in industry. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Rapeseed gum Phenolics Phospholipids Antioxidant activity Identification Chemical compounds studied in this article: Sinapic acid (PubChem CID: 637775) Sinapine (PubChem CID: 5280385) Canolol (PubChem CID: 35960)
1. Introduction Soapstock/acid oil, deodorized distillates, and spent bleaching clay are main side-streams from vegetable oil refining (Echim, , De Greyt, & Stevens, 2009). Valorization of these sideVerhe stream products has been investigated either for high value products such as lecithin (Ceci, Constenla, & Crapiste, 2008), vitamin E (Isso & Ryan, 2012), and antioxidants (Chen, Thiyam-Hollander, Barthet, & Aachary, 2014) or for biofuel production (Echim et al., 2009). Phospholipids are either present in a hydratable or a nonhydratable form. The non-hydratable form occurs when the phospholipids are combined with calcium, magnesium or iron cations. Acid treatment must be performed in order to convert them into hydrated gums. Then water is introduced to precipitate the gums (Dumont & Narine, 2007). The best known and the most widely used method to remove phospholipids, free fatty acid, and excess of phosphoric acid is the caustic soda process. The soapstock
* Corresponding author. E-mail addresses: [email protected] (J. Li), [email protected], [email protected] (Z. Guo). http://dx.doi.org/10.1016/j.lwt.2016.06.039 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
accompanied with gum is then continuously separated from crude oil by centrifugation (Dumont & Narine, 2007). Wet gum from degumming process mainly contains phospholipids and soapstock. Phenolics, which are considered as major plant secondary metabolites, are much richer in rapeseed than in the other oilseeds (Yang et al., 2014). Sinapic acid and its derivatives especially sinapine are reported as the main phenolic compounds in rapeseed (Chen et al., 2014; Yang et al., 2014). Consumption of a variety of phenolic compounds present in natural foods may lower the risk of serious health disorders because of their antioxidant activity. Additionally, natural antioxidants are attracting more attention due to safety concerns of synthetic antioxidants (Shahidi & Ambigaipalan, 2015). Some of the phenolics are accompanying with oil when the oil is pressed from rapeseeds (Naczk, Amarowicz, Sullivan, & Shahidi, 1998). Previously, many studies focused on phenolic compounds produced from rapeseed meal (Amarowicz, Naczk, & Shahidi, 2000; Khattab, Eskin, & Thiyam-Hollander, 2013; Szydłowska-Czerniak & Tułodziecka, 2014; Vermorel, Hocouemiller, & Evrard, 1987; Vuorela, Meyer, & Heinonen, 2004) since a large proportion of phenolic compounds are still in rapeseed meal after oil press. Amongst, Chen et al. (2014) investigated sinapic acid derivatives and tocopherols from canola oil refining
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byproducts; where the content and DPPH radicals scavenging equivalency of sinapic acid derivatives in bleaching clay, wash water, soapstock, and deodorization distillate were quantified. However, no relevant data with regards to the phenolics in the gum were reported. Soybean lecithin is now the dominant product on market. However, the concerns on its genetically modified organism have restricted its wide acceptance (Paparini & Romano-Spica, 2004). Rapeseed, on the contrary, does not have this problem, making rapeseed oil as one of the most promising feedstock for lecithin production. As discussed above, a large amount of phenolics existed in rapeseed than the other oilseeds. The phenolics, on one hand, are potent antioxidants (Hassasroudsari, Chang, Pegg, & Tyler, 2009). On the other hand display bitter taste (Clandinin, 1961), leading to an urgent investigation of the phenolics distribution in gums from different processes. This could benefit further commercialization of rapeseed lecithin production. Meanwhile, consumers could make their decision based on the composition of alternative lecithin product. To the best of our knowledge, no systemic investigation about the phenolic compounds in rapeseed gum and the potential antioxidant activity of different products from the gum has been carried out. In the present work, several methods including 1H NMR, UV spectra, and HPLC were employed to identify and elucidate the molecular structures of the phenolics presented in wet gum. Thereafter, direct ethanol extraction (DEE) and nonpolar solvent aid extraction (NPSAE) were compared for determination of phenolic compounds in dried gum. Subsequently, the content of phenolic compounds was tracked following the typical lecithin production procedures. The antioxidant activities of the extracts were also evaluated in order to find out the potential oxidative stability of the resultant products. This work is believed to establish new knowledge basis for advanced utilization of rapeseed originated lecithin. 2. Materials and methods 2.1. Samples and chemicals Rapeseed gum and rapeseed meal were provided by DLG food oil (Alborg, Denmark). The gum is a mixture of phospholipids etc. polar lipids and soapstock and therefore the pH value is around 10.5. HCl (6.0 mol/L) was used to neutralize the gum to pH value of 6.8 and then the neutralized gum was stored at 4 C until use. The neutralized gum was dried under vacuum to obtain dried gum. Hexane was used to dissolve the dried gum and obtain the hexanesolubles. Acetone was then used to deoil the hexane-solubles to obtain deoiled lecithin and recovered oil. The deoiled lecithin was further fractionated by absolute ethanol to obtain both ethanolsolubles. All chemicals used in this study were purchased from Sigma-Aldrich and of HPLC grade.
methanol were used as mobile phase A and B, respectively. Mobile phase B was used to elute the last fraction. Eluates (6 mL) were collected manually and their absorbance was recorded at 300 nm after 10 times dilution. 2.4. Isolation of sinapine and structure determination Sinapine was prepared following a previously reported method (Clandinin, 1961). Briefly, trichloroethylene (TCE) was employed to remove the fat and resinous material from rapeseed meal. The TCE extracted meal was further extracted for 4 h in a Soxhlet apparatus with ethanol. The resultant ethanolic extract was concentrated and 100 mL of 20% KSCN solution was added. The mixture was stored at 4 C for 48 h and the crystals of sinapine SCN were recovered by centrifugation at 3095g. Hot ethanol was used to re-dissolve the recovered sinapine SCN and the solution was stored at 4 C for 48 h again. Crystals were collected by filtration and then dried. The dried sample was dissolved in CD3OD and analyzed with a Bruker Ascend 400 spectrometer for structural confirmation purpose. 2.5. Preparation of canolol and structure determination The synthesis of canolol was performed following the method (Khattab et al., 2013). Sinapic acid solution was put in a 100 mL conical glass flask and 100 mL of 1,8-diazabicy- clo [5.4.0]undec-7ene (DBU), 5 mg hydroquinone, and 2.0 g aluminum oxide were added. The mixture was vortexed and the solvent was completely evaporated under N2. The flask was then treated in a microwave oven for 5 min. Neutralization with 1 mol/L HCl was performed when the flask was cooled down to room temperature. Diethyl ether/ethyl acetate mixture (1:1) was then used to extract the phenolics. Canolol was further isolated from the mixture of phenolic by using TLC plate (TLC silica gel 60, 5 cm 10 cm, Merck, Germany) with diethyl ether: petroleum ether: acetic acid (80:20:1) as the developing solvent. The compounds were visualized by using the DPPH radical solution in pure ethanol based on their DPPH radical scavenging ability. The purified sample was dissolved in CD3OD and analyzed with a Bruker Ascend 400 spectrometer for the purpose of structural confirmation. 2.6. Extraction of sinapic acid derivatives Hexane, heptane, and cyclohexane were selected as typical nonpolar solvents. Briefly, 1 g of sample was dissolved by 5 mL of a nonpolar solvent and mixed by a vortex. Subsequently, 2.5 mL of 70% ethanol (v/v) was employed to extract polar compounds twice. In DEE, 2.5 mL of 70% ethanol was mixed with 1 g of sample and extract for twice. The ethanolic extracts were collected for further analyses.
2.2. Preparation of extracts for column chromatography
2.7. HPLC analysis of sinapic acid derivatives
Neutralized gum (20 g) was extracted with ethyl acetate in a ratio of 1:10 (w/v) for 20 min. The extracts were dried in a rotary evaporator at 50 C and then re-dissolved in 5 mL of 70% ethanol. Hexane was used in a ratio of 1:1 to extract hydrophobic compounds thereafter. After separation, the lower phase was collected and condensed for column separation.
Sinapic acid derivatives were quantified using a reversed-phase HPLC-PDA, with a 25 cm 4.6 mm, 5 mm, SUPELCOSIL LC-18 column (Sigma-Aldrich Co., St Louis, MO). Gradient elution with two mobile phases, namely A (0.08 mol/L KH2PO4:CH3CN:85% H3PO4 (85:15:0.25)), and B (60% MeOH: 85%H3PO4 (100:0.05)), was adopted for the separation with the following strategy: t0-t18 100% A, t23-t320%A, and t37-t42100%A (t0 … t42 means elution at 0 … 42 min). Standards of sinapine, sinapic acid, and canolol were used to authenticate the retention time and UV adsorption spectra. The contents of all sinapic acid derivatives were expressed as sinapic acid equivalents (SAE), wherein sinapine and sinapic acid were detected at 330 nm; while canolol was detected at 270 nm.
2.3. Column chromatography Silica gel (high-purity grade, pore size 60 Å, 220e440 mesh particle size, 35e75 mm particle size) was used as stationary phase and diethyl ether:petroleum ether:acetic acid (80:20:1) and
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2.8.
31
P NMR analysis of phospholipids
Phospholipids were quantified by 31P NMR. Triisobutyl phosphate (TiBP, Merk) was used as internal standard. The solvent was composed of CDCl3, MeOH, and 1 mol/L aqueous CDTA (1,2-Diaminocyclohexanetetraacetic acid) with the ratio of 5:4:1. The pH of CDTA was adjusted by using CsOH to 10.5. Appropriate amount of the extract was dissolved by 1 mL of the above mentioned solvent and 25 mL of 80 mg/mL TiBP in the same solvent was added. The mixture was vortexed and centrifuged at 9700g for 3 min. Liquid phase was placed in a 5 mm NMR tube. The parameters for the NMR were: number of scan 16, delay time 18 s, disk temperature 300 K. 2.9. Determination of total phenolic content Total phenolic content was estimated by following the modified Folin-Ciocalteu method (FC method) as described elsewhere (Li et al., 2013) and was expressed as SAE. 2.10. Estimation of antioxidant activity DPPH radical scavenging activity was measured exactly by following the method as described elsewhere (Li et al., 2012) and the results were expressed as ASE. Hydroxyl radical scavenging activity was estimated by following the method as described elsewhere (Li et al., 2013) and the results were expressed as vitamin C equivalents (VCE). Ferrous chelating activity was measured according to a previous report (Falkeborg et al., 2014) and the results were expressed as EDTA equivalents (EDTAE). Reducing powder was determined following the method of Li et al. (2014) and the results were expressed as SAE.
399
Table 1 1 H NMR results of the synthesized canolol and the isolated sinapine. Position
Chemical shift (ppm)
2 3 4 5 6
6.719 6.627 5.617 5.083 3.858
(2H, (1H, (1H, (1H, (6H,
s, AreH) dd, eCH]) d, ]CH2) d, ]CH2) s, eOCH3)
2 3 4 5 6 7 8
6.946 7.690 6.465 4.675 3.795 3.275 3.885
(2H, (1H, (1H, (2H, (2H, (9H, (6H,
s, AreH) d, eCH]) d, ]CH2) d, eCH2) d, eCH2) s, eCH3) s, eOCH3)
and is shown in Fig. 1. Four peaks were clearly observed and the eluates were classified into 6 fractions in order to identify the phenolic compounds subsequently. The first 5 fractions were eluted by the solvent mixture of diethyl ether:petroleum ether:acetic acid (80:20:1) while the last fraction was eluted by pure methanol. It could be expected that the polarity of the eluted phenolic compounds increases with the fraction number. Sinapine and its derivatives are reported to be the dominating phenolic compounds in rapeseeds (Chen et al., 2014; Yang et al., 2014). The sinapic acid and vinylsyringol (canolol) are the two main existing forms of sinapine derivatives. The polarity of sinapine is the highest, followed by sinapic acid and then canolol, which is probably correlated with the different fractions. UV spectra, 1H NMR, and HPLC were employed to identify them in the following sections.
2.11. Statistical analyses 3.3. Identification of phenolic compounds All experiments were conducted in duplicate and the data are presented as the mean values ± standard deviation (SD). Statistical analyses were carried out using the IBM SPSS statistics 19 software using one-way ANOVA and Duncan’s multiple range tests, and the results were considered statistically significant at a 95% confidence interval (p < 0.05). 3. Results and discussion 3.1. Confirmation of self-prepared standards The standard compounds of sinapine and canolol are not commercially available. Thus both compounds were prepared from rapeseed meal and sinapic acid, respectively. The molecular structures of both isolated sinapine and synthesized canolol were confirmed by 1H NMR and UV spectroscopy analyses. The adsorption maxima of canolol and sinapine were reported at 270 nm and 330 nm, respectively (Khattab, Eskin, Aliani, & Thiyam, 2010; Spielmeyer, Wagner, & Jahreis, 2009), which agreed well with the present study (Fig. 2). Moreover, 1H NMR results supported the success of isolation and synthesis of both standards as shown in Table 1, Figs. S1 and S2.
It has been reported that phenolic acids in rapeseed are present in free, esterified and insoluble-bound forms and are the derivatives of benzoic and cinnamic acids. Additionally, sinapic acid is the predominant free phenolic acid found in rapeseed meanwhile sinapine is the predominant esterified phenolic acid (Naczk et al., 1998). Vinylsyringol (canolol) was discovered by Koski, € l€ Pekkarinen, Hopia, W€ aha a, and Heinonen (2003) and was suspected to be a derivative of sinapic acid during the press process or roasting of the seeds. In some previous reports (Chen et al., 2014; Szydłowska-Czerniak, Trokowski, Karlovits, & Szłyk, 2010), only
3.2. Column chromatography Column chromatography has been used widely for separation of a complex phenolic mixture (Amarowicz, Wanasundara, Karama c, & Shahidi, 1996; Wanasundara, Amarowicz, & Shahidi, 1994). The sample prepared by extracting from wet gum was separated using silica gel column chromatography. The UV absorption intensity of isolated fractions after 10 times dilution at 300 nm was recorded
Fig. 1. Separation of the extract of wet gum from rapeseed oil refinery on a silica gel column.
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Fig. 2. UV spectra of individual fractions of the extract from wet gum of rapeseed oil refinery and standard compounds. A: F1; B: F2; C: F3; D: F4; E: F5; F: F6; G: Sinapic acid; H: Sinapine; I: Canolol.
HPLC-DAD was used for phenolic acids quantification directly without identification, which is in question because the method is based on separation, and different compounds might show the same retention time if the mobile phases are not sufficient to separate them. To circumvent with this problem, in the present study, the phenolic compounds in the gum were identified by 1H NMR, UV spectra, and HPLC retention time at the first place and then quantification was carried out based on the established HPLC method.
polarities. A peak at 330 nm could be observed in fraction 6 although it was not very apparent. Sinapic acid has an absorption maximum at 324 nm (Fig. 2), which is in line with another report (Sun et al., 2007). Canolol adsorption maximum was 270 nm as shown in Fig. 2; while it was 275 nm in a previous report (Koski et al., 2003). The spectrum of sinapine showed a peak at 330 nm (Fig. 2). Therefore, canolol might exist in fraction 1 and 2 and sinapic acid may present in fraction 3 and 4. Fraction 6 might contain sinapine.
3.3.1. UV spectra of different fractions UV spectrophotometry is a commonly used method for identification and characterization of phenolics (Chen et al., 2014; Gil, s-Barbera n, Hess-Pierce, Holcroft, & Kader, 2000) because Toma different phenolic compounds display various UV maximum absorbance wavelength. For example, gallic acid shows an absorption maximum at 272 nm while gentisic acid shows it at 326 nm although they share similar chemical structure (Sun, Liang, Bin, Li, & Duan, 2007). Thus the UV spectra of the different fractions were recorded and are shown in Fig. 2. Fraction 1 showed an adsorption maxima peak at 277 nm and the same peak was also observed in fraction 2. In addition to the peak at 277 nm, another peak at 320 nm could be observed in fraction 2. Compared with fraction 2, fraction 3 and 4 showed only one peak at 320 nm. A peak at 280 nm was displayed in fraction 5 and 6. However, the compound in fraction 5 and 6 should be different from that in fraction 1 because these fractions were separated according to their different
3.3.2. 1H NMR results NMR including 1H NMR and 13C NMR is a commonly used method for elucidating the structure of chemicals. NMR is also the most convincing technique for identifying unknown compounds. All the 6 fractions were subjected to 1H NMR but only fractions 3 and 4 showed relatively higher signal. The spectra of 1H NMR of fractions 3 and 4 are shown in Fig. 3. The peaks from 0.8 ppm to 1.7 ppm are generally assigned to the resonances of alkane groups s, Vargas, Vielfaure, & Schuchardt, 1995) which is (Gelbard, Bre abundant in fatty acids. The signal at 2.3 ppm is assigned to a-CH2 in all lipids and the signal at 5.3 ppm is assigned to the protons of eCH]CHe(Gelbard et al., 1995), suggesting that the extract contained fatty acids and part of the fatty acids were unsaturated. The chemical shifts of protons in phenolic compounds are downfield and do not overlap with signals of fatty acids protons. The chemical shifts of protons on the two methoxy group and the aromatic ring were 3.96 and 6.92 ppm, respectively. The other two protons
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401
Fig. 3. Spectra of 1H NMR of fractions 3 and 4 of the extract from wet gum of rapeseed oil refinery.
resonated at 7.61 and 6.38 ppm, respectively. Combined with integral results, it was obvious that the resonances were from sinapic acid, confirming the results from UV spectra further.
3.3.3. Reversed phase HPLC results As a qualitative and quantitative method, HPLC is widely used because the retention time of specific compound is constant and the peak height or area is proportional to its content under the same conditions. Therefore, the six fractions were analyzed individually by HPLC on a C-18 column and the corresponding chromatographs are shown in Fig. 4. Isolated sinapine, synthesized canolol, and commercial sinapic acid were used as standards. Canolol showed absorption maxima at 270 nm while no absorption at 330 nm (Fig. 2). Combined with the retention time of the canolol standard, canolol could be differentiated from the cluster of peaks within 26e29 min. All fractions contained sinapic acid but with different contents. Only fraction 6 contained sinapine while only fraction 1 contained canolol. The contents of sinapic acid in fractions 3 and 4 were much higher than the other fractions, which could also be concluded from 1H NMR results. The polarity of sinapine is higher than that of sinapic acid and therefore it is reasonable that sinapine existed in the last fraction eluted by pure methanol (Fig. 1). Apparently, sinapic acid was the most abundant phenolic compounds in the extract from wet gum although there were some other unknown phenolics. Meanwhile, it should be noted that some compounds other than phenolic also absorb UV light, resulting in peaks in the chromatography. Sinapine derivatives retain in different fractions during traditional oil refining processes including degumming, neutralization, washing, bleaching, and deodorization. Sinapine, sinapic acid, and canolol were mainly concentrated in bleaching clay, soapstock, and
wash water, respectively as reported (Chen et al., 2014). In the present work, the gum was generated from a combined process of neutralization and degumming. Therefore, soapstock was one of the main components of the waste gum. In line with the work (Chen et al., 2014), sinapic acid was found to be the most abundant phenolic compound in the gum. Different from the results of soapstock (Chen et al., 2014), sinapine and canolol were also detected in the gum, suggesting sinapine and canolol flowed into gum fraction during degumming process.
3.4. Effect of different extraction approaches on the content of phenolic compounds In order to compare the effects of extraction approaches on sinapine derivatives content, several nonpolar solvents were employed to pre-dissolve the substrate and extracted by aqueous ethanol subsequently (NPSAE). Meanwhile, direct aqueous ethanol extraction (DEE) was performed. As shown in Fig. 5, NPSAE is more efficient than DEE for sinapine and sinapic acid extraction from dried gum; whereas DEE extracted significantly higher amount of canolol than NPSAE. It is probably because the sinapine and sinapic acid are more polar than canolol, making the nonpolar solvents force the polar sinapine and sinapic acid to the aqueous ethanol phase. Canolol displayed relatively higher solubility in aqueous ethanol than sinapine and sinapic acid. Total phenolic content obtained by DEE as measured by Folin-Ciocalteu assay (TPC-FC) was significantly higher than that in hexane and cyclohexane aid extracts. Among the selected nonpolar solvents, hexane and cyclohexane seemed to perform better than heptane with regards to sinapic acid extraction, whereas the extraction with the aid of heptane showed the highest TPC-FC. Different extraction
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Fig. 4. HPLC chromatography of different fractions of the extract from wet gum of rapeseed oil refinery on a C-18 column. 330 nm; F1, 330 nm; F1, 270 nm; F6, 330 nm.
F2, 330 nm;
F3, 330 nm;
F4, 330 nm;
F5,
therefore it may be another component in the extract that can be detected by the TPC-FC method. Another possible reason is that the wash water and deodistillates did not contain sinapine and sinapic acid. In conclusion, more polar compounds could be obtained when presolvent solubilization by nonpolar solvents was performed while direct ethanol extraction gave higher total phenolic content. 3.5. Tracking of phenolic compounds in different processed gums
Fig. 5. Effect of different extraction approaches on the contents of phenolic compounds in the dried gum. Hexane (blank, black); Heptane (sparse, red); Cyclohexane (medium, blue); Direct ethanol extraction (dense, green). Different letters within the same group indicate significant difference (P < 0.05, n ¼ 2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
approaches were investigated for extracting phenolic compounds from wash water and deodistillates generated from canola oil refining procedures (Chen et al., 2014). Hexane and cyclohexane assisted extraction resulted in the same total phenolic content as direct methanol extraction from wash water and deodistillates. The difference between the current study and the previous study (Chen et al., 2014) might be due to the different substrate. Phosphatidylcholine is the major phospholipid in the gum and it is easily to be extracted by ethanol (Cabezas, Diehl, & Tom as, 2009) and
Crude lecithin could be produced from the gum generated from degumming process of plant oil by drying. Hexane could be used to improve the purity of crude lecithin, following which deoiling could be performed to produce deoiled lecithin. Ethanolic fractionation is also a commonly used method to obtain phosphatidylcholine (PC) and phosphatidylinositol (PI) enriched products (Fig. 6). Rapeseed gum contained phenolic compounds as identified above and therefore it is meaningful to track how it flows during processes. Table 2 shows the content of phenolic compounds in each substrate. The content of sinapine derivatives in the hexane solubles was decreased compared with those in the dried gum (see Fig. 5), indicating hexane could only dissolve part of the phenolics. Deoiling process by using acetone changed the distribution of sinapine derivatives significantly. Most of them were dissolved by acetone and thus appeared in the extracts of recovered oils (Table 2). It suggests that the recovered oil can be a rich source for extracting abundant sinapine derivatives. Correspondingly, the content of sinapine derivatives in deoiled lecithin was very low. Ethanol solubles (PC fraction) did not contain sinapine and sinapic acid but canolol. However, the total phenolic content of ethanol solubles measured by the Folin-Ciocalteu method was the highest, which might be attributed to the interference of phospholipids in the extract. When different processed samples were compared,
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403
Fig. 6. Flowchart of the gum processing.
Table 2 Content of phenolic compounds in different processed gums. Samples
Sinapine (mg/g)a
Sinapic acid (mg/g)a
Canolol (mg/g)a
Hexane solubles Deoiled lecithin Recovered oil Ethanol solubles
46.34 ± 3.34a 4.89 ± 1.37b 41.41 ± 2.58a BDL
212.98 ± 22.82a 18.38 ± 4.49b 255.58 ± 16.38a BDL
27.93 13.31 44.78 22.84
a
± ± ± ±
4.76a 1.33b 15.60c 1.66d
TPC-FC (mgSAE/g)a 4.21 3.68 4.50 7.05
± ± ± ±
0.02a 0.30b 0.03c 0.35d
Direct ethanol extraction was employed for analyses of phenolic compounds. Different letters within the same column indicate significant difference (P < 0.05, n ¼ 2).
Table 3 Antioxidant activity of the resultant extracts from different processed gums. Substrate
DPPH (mgSAE/g)
Dried gum Hexane solubles Deoiled lecithin Recovered oil Ethanol solubles
3.64 3.68 2.40 4.22 6.20
± ± ± ± ±
0.11a 0.13a 0.07b 0.07c 0.60d
HRSA (mgVCE/g)a 14.35 13.01 11.15 11.80 12.40
± ± ± ± ±
0.02a 0.19b 0.27c 0.12c 0.47b
FCA (mgEDTAE/g)a 0.78 0.64 0.96 0.74 4.89
± ± ± ± ±
0.02a 0.01b 0.03c 0.06a 0.07d
RP (mgSAE/g)a 6.06 5.38 3.51 5.14 7.08
± ± ± ± ±
0.03a 1.64a 0.49b 0.02c 0.44d
a HRSA means hydroxyl radical scavenging activity, FCA means ferrous chelating activity, and RP means reducing power. Different letters within the same column indicate significant difference (P < 0.05, n ¼ 2). Extracts were obtained by direct ethanol extraction.
most of sinapine derivatives removed by acetone and presented in the recovered oil (Tables 2). 3.6. Antioxidant activity of the resultant extracts from different processed gums Antioxidant activity of the extracts from rapeseed related materials has been studied extensively (Amarowicz et al., 1996, 2000; Hassasroudsari et al., 2009; Koski et al., 2003; Szydłowska-Czerniak et al., 2010; Sørensen et al., 2013). Antioxidant activity of the extracts from different processed gums should also be evaluated because it is related to the oxidative stability of the final products and the results are shown in Table 3. Compared with dried gum, extract from hexane solubles showed the same antioxidant activity, suggesting hexane refining could not decrease the oxidative
stability of the product. However, the results of DPPH, HRSA, and RP of deoiled lecithin were the lowest amongst the studied substrates, indicating deoiling process may reduce the oxidative stability of the lecithin. The results of DPPH, FCA, and RP of extract from ethanol solubles were the highest, meaning ethanol fractionation concentrated the active compound in ethanol solubles. However, no sinapine and sinapic acid but canolol were detected in the extract from ethanol solubles (Table 2). Additionally, it should be noted that the content of PC, LPC, and GPC in the extract from ethanol solubles were the highest (Table 4). Therefore, such fantastic antioxidant activity should be mainly attributed to the synergistic action between phospholipids and canolol. Except for phenolic compounds, phospholipids were also found to be kind of antioxidant because supplementation of phospholipids enhanced the oxidation stability of virgin olive oil (Koprivnjak et al., 2008).
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dx.doi.org/10.1016/j.lwt.2016.06.039.
Table 4 Phospholipid content in extracts from different processed gums. Substrate
PC (mg/g)
Dried gum Hexane solubles Deoiled lecithin Recovered oil Ethanol solubles
10.77 10.40 17.79 12.86 48.48
± ± ± ± ±
2.18a 0.61a 0.37b 0.26a 2.96c
LPC (mg/g)
GPC (mg/g)
6.14 ± 1.37a 6.18 ± 0.17a 15.46 ± 0.80b 2.35 ± 0.34c 69.92 ± 3.20d
0.95 1.78 1.59 ND 6.37
± 0.03a ± 0.11b ± 0.21b ± 0.11c
PA (mg/g) 1.92 ± 1.01a 2.20 ± 0.16a 1.94 ± 0.15a ND ND
PC: phosphatidylcholine, LPC: lyso-phosphatidylcholine, GPC: glycerophosphocholine, PA: phosphatidic acid. Different letters within the same column indicate significant difference (P < 0.05, n ¼ 2).
Soybean PC was found to display strong DPPH scavenging activity (data not shown). Sugino et al. (1997) investigated the antioxidative activity of egg yolk phospholipids and found that higher concentration of phospholipids displayed stronger antioxidant activity in docosahexaenoic acid enriched oil. Egg yolk PC and its hydrogenated derivatives were also examined and indicated that antioxidant activity was decreased with an increase in the degree of saturation of fatty acid chains within the phospholipids. Oleic and linoleic acid are main fatty acid chains in phospholipids from rapeseed (data not shown). It was found that oxidative stabilities of models enriched with quercetin-phosphatidylcholine complex formulations were better than in the models containing lecithin or quercetin alone, most likely as a consequence of synergism between polar lipids and quercetin as well as tocopherols (Ramadan, 2012). Therefore, the rapeseed based lecithin products are unique because they contain not only phospholipid bearing unsaturated fatty acids but also phenolics compounds with potential synergistic effect for strong antioxidant activity. 4. Conclusion In conclusion, this work identified the major phenolic compounds in rapeseed waste gum and it was found that sinapic acid was the most abundant phenolic compound followed by sinapine and canolol. The flow of the identified phenolic compounds was tracked following the typical processes of lecithin related products. Part of the phenolics was lost during hexane refining and a large part of sinapic acid derivatives were removed by acetone extraction during deoiling process. Deoiling is not recommended if a product enriched with bioactive phenolic compounds is required. The extracts obtained in the present work displayed good antioxidant activity which most likely resulted from a synergic effect of phenolic and phospholipid components. The strong antioxidant activity of the extract reflected the oxidative stability of substrate to some extent. The co-presence of phenolics and phospholipids in rapeseed gum originated products endowed them with excellent ion-chelating activity and antioxidant property, which may distinguish rapeseed lecithin from other types of lecithin. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements Financial support from the Graduate School of Science and Technology (GSST), Aarhus University and DLG (Dansk Landbrugs Grovvareselskab) Food Oil, Denmark is gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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Public health issues related with the consumption of food obtained from genetically modified organisms. Biotechnology Annual Review, 10, 85e122. Ramadan, M. F. (2012). Antioxidant characteristics of phenolipids (quercetinenriched lecithin) in lipid matrices. Industrial Crops and Products, 36(1), 363e369. Shahidi, F., & Ambigaipalan, P. (2015). Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health effects e A review. Journal of Functional Foods, 18, 820e897. Sørensen, A.-D. M., Friel, J., Winkler-Moser, J. K., Jacobsen, C., Huidrom, D., Reddy, N., et al. (2013). Impact of endogenous canola phenolics on the oxidative stability of oil-in-water emulsions. European Journal of Lipid Science and Technology, 115(5), 501e512.
J. Li, Z. Guo / LWT - Food Science and Technology 73 (2016) 397e405 Spielmeyer, A., Wagner, A., & Jahreis, G. (2009). Influence of thermal treatment of rapeseed on the canolol content. Food Chemistry, 112(4), 944e948. Sugino, H., Ishikawa, M., Nitoda, T., Koketsu, M., Juneja, L. R., Kim, M., et al. (1997). Antioxidative activity of egg yolk phospholipids. Journal of Agricultural and Food Chemistry, 45(3), 551e554. Sun, J., Liang, F., Bin, Y., Li, P., & Duan, C. (2007). Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet and mass spectrometry/ mass spectrometry libraries. Molecules, 12(3), 679e693. Szydłowska-Czerniak, A., Trokowski, K., Karlovits, G., & Szłyk, E. (2010). Determination of antioxidant capacity, phenolic acids, and fatty acid composition of rapeseed varieties. Journal of Agricultural and Food Chemistry, 58(13), 7502e7509. Szydłowska-Czerniak, A., & Tułodziecka, A. (2014). Antioxidant capacity of rapeseed extracts obtained by conventional and ultrasound-assisted extraction. Journal of
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Identification and quantification of phenolic compounds in rapeseed originated lecithin and antioxidant activity evaluation Jingbo Li, Zheng Guo* Department of Engineering, Faculty of Science, Aarhus University, Gustav Wieds Vej 10, 8000, Aarhus C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 April 2016 Received in revised form 2 June 2016 Accepted 15 June 2016 Available online 18 June 2016
Phenolic compounds may exist in the gum generated from rapeseed oil refining as rapeseed is rich in phenolics. Therefore, this work identified the main phenolic compounds, followed by tracking their flow according to typical lecithin production procedures. Antioxidant activities of the extracts were also investigated to get a clue of the potential oxidative stability of the final products. Column chromatography, UV spectra, 1H NMR, and HPLC were used to separate and identify the phenolic compounds. Sinapic acid was found to be the dominant phenolic while relatively low contents of sinapine and canolol were also identified. Nonpolar solvent aid extraction was better for extracting more sinapine and sinapic acid while direct ethanol extraction gave more canolol. Almost half of the phenolics in dried gum were removed by acetone during deoiling process, leaving only a small amount in deoiled lecithin. Extracts from deoiled lecithin showed the weakest antioxidant activity while the ethanol solubles showed the strongest. This work demonstrated the occurrences of bioactive phenolic compounds in rapeseed originated gum, which may be an advantage of rapeseed lecithin compared with other types of lecithin. This work could also be a reference for rapeseed lecithin production with different contents of bioactive compounds in industry. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Rapeseed gum Phenolics Phospholipids Antioxidant activity Identification Chemical compounds studied in this article: Sinapic acid (PubChem CID: 637775) Sinapine (PubChem CID: 5280385) Canolol (PubChem CID: 35960)
1. Introduction Soapstock/acid oil, deodorized distillates, and spent bleaching clay are main side-streams from vegetable oil refining (Echim, , De Greyt, & Stevens, 2009). Valorization of these sideVerhe stream products has been investigated either for high value products such as lecithin (Ceci, Constenla, & Crapiste, 2008), vitamin E (Isso & Ryan, 2012), and antioxidants (Chen, Thiyam-Hollander, Barthet, & Aachary, 2014) or for biofuel production (Echim et al., 2009). Phospholipids are either present in a hydratable or a nonhydratable form. The non-hydratable form occurs when the phospholipids are combined with calcium, magnesium or iron cations. Acid treatment must be performed in order to convert them into hydrated gums. Then water is introduced to precipitate the gums (Dumont & Narine, 2007). The best known and the most widely used method to remove phospholipids, free fatty acid, and excess of phosphoric acid is the caustic soda process. The soapstock
* Corresponding author. E-mail addresses: [email protected] (J. Li), [email protected], [email protected] (Z. Guo). http://dx.doi.org/10.1016/j.lwt.2016.06.039 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
accompanied with gum is then continuously separated from crude oil by centrifugation (Dumont & Narine, 2007). Wet gum from degumming process mainly contains phospholipids and soapstock. Phenolics, which are considered as major plant secondary metabolites, are much richer in rapeseed than in the other oilseeds (Yang et al., 2014). Sinapic acid and its derivatives especially sinapine are reported as the main phenolic compounds in rapeseed (Chen et al., 2014; Yang et al., 2014). Consumption of a variety of phenolic compounds present in natural foods may lower the risk of serious health disorders because of their antioxidant activity. Additionally, natural antioxidants are attracting more attention due to safety concerns of synthetic antioxidants (Shahidi & Ambigaipalan, 2015). Some of the phenolics are accompanying with oil when the oil is pressed from rapeseeds (Naczk, Amarowicz, Sullivan, & Shahidi, 1998). Previously, many studies focused on phenolic compounds produced from rapeseed meal (Amarowicz, Naczk, & Shahidi, 2000; Khattab, Eskin, & Thiyam-Hollander, 2013; Szydłowska-Czerniak & Tułodziecka, 2014; Vermorel, Hocouemiller, & Evrard, 1987; Vuorela, Meyer, & Heinonen, 2004) since a large proportion of phenolic compounds are still in rapeseed meal after oil press. Amongst, Chen et al. (2014) investigated sinapic acid derivatives and tocopherols from canola oil refining
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byproducts; where the content and DPPH radicals scavenging equivalency of sinapic acid derivatives in bleaching clay, wash water, soapstock, and deodorization distillate were quantified. However, no relevant data with regards to the phenolics in the gum were reported. Soybean lecithin is now the dominant product on market. However, the concerns on its genetically modified organism have restricted its wide acceptance (Paparini & Romano-Spica, 2004). Rapeseed, on the contrary, does not have this problem, making rapeseed oil as one of the most promising feedstock for lecithin production. As discussed above, a large amount of phenolics existed in rapeseed than the other oilseeds. The phenolics, on one hand, are potent antioxidants (Hassasroudsari, Chang, Pegg, & Tyler, 2009). On the other hand display bitter taste (Clandinin, 1961), leading to an urgent investigation of the phenolics distribution in gums from different processes. This could benefit further commercialization of rapeseed lecithin production. Meanwhile, consumers could make their decision based on the composition of alternative lecithin product. To the best of our knowledge, no systemic investigation about the phenolic compounds in rapeseed gum and the potential antioxidant activity of different products from the gum has been carried out. In the present work, several methods including 1H NMR, UV spectra, and HPLC were employed to identify and elucidate the molecular structures of the phenolics presented in wet gum. Thereafter, direct ethanol extraction (DEE) and nonpolar solvent aid extraction (NPSAE) were compared for determination of phenolic compounds in dried gum. Subsequently, the content of phenolic compounds was tracked following the typical lecithin production procedures. The antioxidant activities of the extracts were also evaluated in order to find out the potential oxidative stability of the resultant products. This work is believed to establish new knowledge basis for advanced utilization of rapeseed originated lecithin. 2. Materials and methods 2.1. Samples and chemicals Rapeseed gum and rapeseed meal were provided by DLG food oil (Alborg, Denmark). The gum is a mixture of phospholipids etc. polar lipids and soapstock and therefore the pH value is around 10.5. HCl (6.0 mol/L) was used to neutralize the gum to pH value of 6.8 and then the neutralized gum was stored at 4 C until use. The neutralized gum was dried under vacuum to obtain dried gum. Hexane was used to dissolve the dried gum and obtain the hexanesolubles. Acetone was then used to deoil the hexane-solubles to obtain deoiled lecithin and recovered oil. The deoiled lecithin was further fractionated by absolute ethanol to obtain both ethanolsolubles. All chemicals used in this study were purchased from Sigma-Aldrich and of HPLC grade.
methanol were used as mobile phase A and B, respectively. Mobile phase B was used to elute the last fraction. Eluates (6 mL) were collected manually and their absorbance was recorded at 300 nm after 10 times dilution. 2.4. Isolation of sinapine and structure determination Sinapine was prepared following a previously reported method (Clandinin, 1961). Briefly, trichloroethylene (TCE) was employed to remove the fat and resinous material from rapeseed meal. The TCE extracted meal was further extracted for 4 h in a Soxhlet apparatus with ethanol. The resultant ethanolic extract was concentrated and 100 mL of 20% KSCN solution was added. The mixture was stored at 4 C for 48 h and the crystals of sinapine SCN were recovered by centrifugation at 3095g. Hot ethanol was used to re-dissolve the recovered sinapine SCN and the solution was stored at 4 C for 48 h again. Crystals were collected by filtration and then dried. The dried sample was dissolved in CD3OD and analyzed with a Bruker Ascend 400 spectrometer for structural confirmation purpose. 2.5. Preparation of canolol and structure determination The synthesis of canolol was performed following the method (Khattab et al., 2013). Sinapic acid solution was put in a 100 mL conical glass flask and 100 mL of 1,8-diazabicy- clo [5.4.0]undec-7ene (DBU), 5 mg hydroquinone, and 2.0 g aluminum oxide were added. The mixture was vortexed and the solvent was completely evaporated under N2. The flask was then treated in a microwave oven for 5 min. Neutralization with 1 mol/L HCl was performed when the flask was cooled down to room temperature. Diethyl ether/ethyl acetate mixture (1:1) was then used to extract the phenolics. Canolol was further isolated from the mixture of phenolic by using TLC plate (TLC silica gel 60, 5 cm 10 cm, Merck, Germany) with diethyl ether: petroleum ether: acetic acid (80:20:1) as the developing solvent. The compounds were visualized by using the DPPH radical solution in pure ethanol based on their DPPH radical scavenging ability. The purified sample was dissolved in CD3OD and analyzed with a Bruker Ascend 400 spectrometer for the purpose of structural confirmation. 2.6. Extraction of sinapic acid derivatives Hexane, heptane, and cyclohexane were selected as typical nonpolar solvents. Briefly, 1 g of sample was dissolved by 5 mL of a nonpolar solvent and mixed by a vortex. Subsequently, 2.5 mL of 70% ethanol (v/v) was employed to extract polar compounds twice. In DEE, 2.5 mL of 70% ethanol was mixed with 1 g of sample and extract for twice. The ethanolic extracts were collected for further analyses.
2.2. Preparation of extracts for column chromatography
2.7. HPLC analysis of sinapic acid derivatives
Neutralized gum (20 g) was extracted with ethyl acetate in a ratio of 1:10 (w/v) for 20 min. The extracts were dried in a rotary evaporator at 50 C and then re-dissolved in 5 mL of 70% ethanol. Hexane was used in a ratio of 1:1 to extract hydrophobic compounds thereafter. After separation, the lower phase was collected and condensed for column separation.
Sinapic acid derivatives were quantified using a reversed-phase HPLC-PDA, with a 25 cm 4.6 mm, 5 mm, SUPELCOSIL LC-18 column (Sigma-Aldrich Co., St Louis, MO). Gradient elution with two mobile phases, namely A (0.08 mol/L KH2PO4:CH3CN:85% H3PO4 (85:15:0.25)), and B (60% MeOH: 85%H3PO4 (100:0.05)), was adopted for the separation with the following strategy: t0-t18 100% A, t23-t320%A, and t37-t42100%A (t0 … t42 means elution at 0 … 42 min). Standards of sinapine, sinapic acid, and canolol were used to authenticate the retention time and UV adsorption spectra. The contents of all sinapic acid derivatives were expressed as sinapic acid equivalents (SAE), wherein sinapine and sinapic acid were detected at 330 nm; while canolol was detected at 270 nm.
2.3. Column chromatography Silica gel (high-purity grade, pore size 60 Å, 220e440 mesh particle size, 35e75 mm particle size) was used as stationary phase and diethyl ether:petroleum ether:acetic acid (80:20:1) and
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2.8.
31
P NMR analysis of phospholipids
Phospholipids were quantified by 31P NMR. Triisobutyl phosphate (TiBP, Merk) was used as internal standard. The solvent was composed of CDCl3, MeOH, and 1 mol/L aqueous CDTA (1,2-Diaminocyclohexanetetraacetic acid) with the ratio of 5:4:1. The pH of CDTA was adjusted by using CsOH to 10.5. Appropriate amount of the extract was dissolved by 1 mL of the above mentioned solvent and 25 mL of 80 mg/mL TiBP in the same solvent was added. The mixture was vortexed and centrifuged at 9700g for 3 min. Liquid phase was placed in a 5 mm NMR tube. The parameters for the NMR were: number of scan 16, delay time 18 s, disk temperature 300 K. 2.9. Determination of total phenolic content Total phenolic content was estimated by following the modified Folin-Ciocalteu method (FC method) as described elsewhere (Li et al., 2013) and was expressed as SAE. 2.10. Estimation of antioxidant activity DPPH radical scavenging activity was measured exactly by following the method as described elsewhere (Li et al., 2012) and the results were expressed as ASE. Hydroxyl radical scavenging activity was estimated by following the method as described elsewhere (Li et al., 2013) and the results were expressed as vitamin C equivalents (VCE). Ferrous chelating activity was measured according to a previous report (Falkeborg et al., 2014) and the results were expressed as EDTA equivalents (EDTAE). Reducing powder was determined following the method of Li et al. (2014) and the results were expressed as SAE.
399
Table 1 1 H NMR results of the synthesized canolol and the isolated sinapine. Position
Chemical shift (ppm)
2 3 4 5 6
6.719 6.627 5.617 5.083 3.858
(2H, (1H, (1H, (1H, (6H,
s, AreH) dd, eCH]) d, ]CH2) d, ]CH2) s, eOCH3)
2 3 4 5 6 7 8
6.946 7.690 6.465 4.675 3.795 3.275 3.885
(2H, (1H, (1H, (2H, (2H, (9H, (6H,
s, AreH) d, eCH]) d, ]CH2) d, eCH2) d, eCH2) s, eCH3) s, eOCH3)
and is shown in Fig. 1. Four peaks were clearly observed and the eluates were classified into 6 fractions in order to identify the phenolic compounds subsequently. The first 5 fractions were eluted by the solvent mixture of diethyl ether:petroleum ether:acetic acid (80:20:1) while the last fraction was eluted by pure methanol. It could be expected that the polarity of the eluted phenolic compounds increases with the fraction number. Sinapine and its derivatives are reported to be the dominating phenolic compounds in rapeseeds (Chen et al., 2014; Yang et al., 2014). The sinapic acid and vinylsyringol (canolol) are the two main existing forms of sinapine derivatives. The polarity of sinapine is the highest, followed by sinapic acid and then canolol, which is probably correlated with the different fractions. UV spectra, 1H NMR, and HPLC were employed to identify them in the following sections.
2.11. Statistical analyses 3.3. Identification of phenolic compounds All experiments were conducted in duplicate and the data are presented as the mean values ± standard deviation (SD). Statistical analyses were carried out using the IBM SPSS statistics 19 software using one-way ANOVA and Duncan’s multiple range tests, and the results were considered statistically significant at a 95% confidence interval (p < 0.05). 3. Results and discussion 3.1. Confirmation of self-prepared standards The standard compounds of sinapine and canolol are not commercially available. Thus both compounds were prepared from rapeseed meal and sinapic acid, respectively. The molecular structures of both isolated sinapine and synthesized canolol were confirmed by 1H NMR and UV spectroscopy analyses. The adsorption maxima of canolol and sinapine were reported at 270 nm and 330 nm, respectively (Khattab, Eskin, Aliani, & Thiyam, 2010; Spielmeyer, Wagner, & Jahreis, 2009), which agreed well with the present study (Fig. 2). Moreover, 1H NMR results supported the success of isolation and synthesis of both standards as shown in Table 1, Figs. S1 and S2.
It has been reported that phenolic acids in rapeseed are present in free, esterified and insoluble-bound forms and are the derivatives of benzoic and cinnamic acids. Additionally, sinapic acid is the predominant free phenolic acid found in rapeseed meanwhile sinapine is the predominant esterified phenolic acid (Naczk et al., 1998). Vinylsyringol (canolol) was discovered by Koski, € l€ Pekkarinen, Hopia, W€ aha a, and Heinonen (2003) and was suspected to be a derivative of sinapic acid during the press process or roasting of the seeds. In some previous reports (Chen et al., 2014; Szydłowska-Czerniak, Trokowski, Karlovits, & Szłyk, 2010), only
3.2. Column chromatography Column chromatography has been used widely for separation of a complex phenolic mixture (Amarowicz, Wanasundara, Karama c, & Shahidi, 1996; Wanasundara, Amarowicz, & Shahidi, 1994). The sample prepared by extracting from wet gum was separated using silica gel column chromatography. The UV absorption intensity of isolated fractions after 10 times dilution at 300 nm was recorded
Fig. 1. Separation of the extract of wet gum from rapeseed oil refinery on a silica gel column.
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Fig. 2. UV spectra of individual fractions of the extract from wet gum of rapeseed oil refinery and standard compounds. A: F1; B: F2; C: F3; D: F4; E: F5; F: F6; G: Sinapic acid; H: Sinapine; I: Canolol.
HPLC-DAD was used for phenolic acids quantification directly without identification, which is in question because the method is based on separation, and different compounds might show the same retention time if the mobile phases are not sufficient to separate them. To circumvent with this problem, in the present study, the phenolic compounds in the gum were identified by 1H NMR, UV spectra, and HPLC retention time at the first place and then quantification was carried out based on the established HPLC method.
polarities. A peak at 330 nm could be observed in fraction 6 although it was not very apparent. Sinapic acid has an absorption maximum at 324 nm (Fig. 2), which is in line with another report (Sun et al., 2007). Canolol adsorption maximum was 270 nm as shown in Fig. 2; while it was 275 nm in a previous report (Koski et al., 2003). The spectrum of sinapine showed a peak at 330 nm (Fig. 2). Therefore, canolol might exist in fraction 1 and 2 and sinapic acid may present in fraction 3 and 4. Fraction 6 might contain sinapine.
3.3.1. UV spectra of different fractions UV spectrophotometry is a commonly used method for identification and characterization of phenolics (Chen et al., 2014; Gil, s-Barbera n, Hess-Pierce, Holcroft, & Kader, 2000) because Toma different phenolic compounds display various UV maximum absorbance wavelength. For example, gallic acid shows an absorption maximum at 272 nm while gentisic acid shows it at 326 nm although they share similar chemical structure (Sun, Liang, Bin, Li, & Duan, 2007). Thus the UV spectra of the different fractions were recorded and are shown in Fig. 2. Fraction 1 showed an adsorption maxima peak at 277 nm and the same peak was also observed in fraction 2. In addition to the peak at 277 nm, another peak at 320 nm could be observed in fraction 2. Compared with fraction 2, fraction 3 and 4 showed only one peak at 320 nm. A peak at 280 nm was displayed in fraction 5 and 6. However, the compound in fraction 5 and 6 should be different from that in fraction 1 because these fractions were separated according to their different
3.3.2. 1H NMR results NMR including 1H NMR and 13C NMR is a commonly used method for elucidating the structure of chemicals. NMR is also the most convincing technique for identifying unknown compounds. All the 6 fractions were subjected to 1H NMR but only fractions 3 and 4 showed relatively higher signal. The spectra of 1H NMR of fractions 3 and 4 are shown in Fig. 3. The peaks from 0.8 ppm to 1.7 ppm are generally assigned to the resonances of alkane groups s, Vargas, Vielfaure, & Schuchardt, 1995) which is (Gelbard, Bre abundant in fatty acids. The signal at 2.3 ppm is assigned to a-CH2 in all lipids and the signal at 5.3 ppm is assigned to the protons of eCH]CHe(Gelbard et al., 1995), suggesting that the extract contained fatty acids and part of the fatty acids were unsaturated. The chemical shifts of protons in phenolic compounds are downfield and do not overlap with signals of fatty acids protons. The chemical shifts of protons on the two methoxy group and the aromatic ring were 3.96 and 6.92 ppm, respectively. The other two protons
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401
Fig. 3. Spectra of 1H NMR of fractions 3 and 4 of the extract from wet gum of rapeseed oil refinery.
resonated at 7.61 and 6.38 ppm, respectively. Combined with integral results, it was obvious that the resonances were from sinapic acid, confirming the results from UV spectra further.
3.3.3. Reversed phase HPLC results As a qualitative and quantitative method, HPLC is widely used because the retention time of specific compound is constant and the peak height or area is proportional to its content under the same conditions. Therefore, the six fractions were analyzed individually by HPLC on a C-18 column and the corresponding chromatographs are shown in Fig. 4. Isolated sinapine, synthesized canolol, and commercial sinapic acid were used as standards. Canolol showed absorption maxima at 270 nm while no absorption at 330 nm (Fig. 2). Combined with the retention time of the canolol standard, canolol could be differentiated from the cluster of peaks within 26e29 min. All fractions contained sinapic acid but with different contents. Only fraction 6 contained sinapine while only fraction 1 contained canolol. The contents of sinapic acid in fractions 3 and 4 were much higher than the other fractions, which could also be concluded from 1H NMR results. The polarity of sinapine is higher than that of sinapic acid and therefore it is reasonable that sinapine existed in the last fraction eluted by pure methanol (Fig. 1). Apparently, sinapic acid was the most abundant phenolic compounds in the extract from wet gum although there were some other unknown phenolics. Meanwhile, it should be noted that some compounds other than phenolic also absorb UV light, resulting in peaks in the chromatography. Sinapine derivatives retain in different fractions during traditional oil refining processes including degumming, neutralization, washing, bleaching, and deodorization. Sinapine, sinapic acid, and canolol were mainly concentrated in bleaching clay, soapstock, and
wash water, respectively as reported (Chen et al., 2014). In the present work, the gum was generated from a combined process of neutralization and degumming. Therefore, soapstock was one of the main components of the waste gum. In line with the work (Chen et al., 2014), sinapic acid was found to be the most abundant phenolic compound in the gum. Different from the results of soapstock (Chen et al., 2014), sinapine and canolol were also detected in the gum, suggesting sinapine and canolol flowed into gum fraction during degumming process.
3.4. Effect of different extraction approaches on the content of phenolic compounds In order to compare the effects of extraction approaches on sinapine derivatives content, several nonpolar solvents were employed to pre-dissolve the substrate and extracted by aqueous ethanol subsequently (NPSAE). Meanwhile, direct aqueous ethanol extraction (DEE) was performed. As shown in Fig. 5, NPSAE is more efficient than DEE for sinapine and sinapic acid extraction from dried gum; whereas DEE extracted significantly higher amount of canolol than NPSAE. It is probably because the sinapine and sinapic acid are more polar than canolol, making the nonpolar solvents force the polar sinapine and sinapic acid to the aqueous ethanol phase. Canolol displayed relatively higher solubility in aqueous ethanol than sinapine and sinapic acid. Total phenolic content obtained by DEE as measured by Folin-Ciocalteu assay (TPC-FC) was significantly higher than that in hexane and cyclohexane aid extracts. Among the selected nonpolar solvents, hexane and cyclohexane seemed to perform better than heptane with regards to sinapic acid extraction, whereas the extraction with the aid of heptane showed the highest TPC-FC. Different extraction
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Fig. 4. HPLC chromatography of different fractions of the extract from wet gum of rapeseed oil refinery on a C-18 column. 330 nm; F1, 330 nm; F1, 270 nm; F6, 330 nm.
F2, 330 nm;
F3, 330 nm;
F4, 330 nm;
F5,
therefore it may be another component in the extract that can be detected by the TPC-FC method. Another possible reason is that the wash water and deodistillates did not contain sinapine and sinapic acid. In conclusion, more polar compounds could be obtained when presolvent solubilization by nonpolar solvents was performed while direct ethanol extraction gave higher total phenolic content. 3.5. Tracking of phenolic compounds in different processed gums
Fig. 5. Effect of different extraction approaches on the contents of phenolic compounds in the dried gum. Hexane (blank, black); Heptane (sparse, red); Cyclohexane (medium, blue); Direct ethanol extraction (dense, green). Different letters within the same group indicate significant difference (P < 0.05, n ¼ 2). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
approaches were investigated for extracting phenolic compounds from wash water and deodistillates generated from canola oil refining procedures (Chen et al., 2014). Hexane and cyclohexane assisted extraction resulted in the same total phenolic content as direct methanol extraction from wash water and deodistillates. The difference between the current study and the previous study (Chen et al., 2014) might be due to the different substrate. Phosphatidylcholine is the major phospholipid in the gum and it is easily to be extracted by ethanol (Cabezas, Diehl, & Tom as, 2009) and
Crude lecithin could be produced from the gum generated from degumming process of plant oil by drying. Hexane could be used to improve the purity of crude lecithin, following which deoiling could be performed to produce deoiled lecithin. Ethanolic fractionation is also a commonly used method to obtain phosphatidylcholine (PC) and phosphatidylinositol (PI) enriched products (Fig. 6). Rapeseed gum contained phenolic compounds as identified above and therefore it is meaningful to track how it flows during processes. Table 2 shows the content of phenolic compounds in each substrate. The content of sinapine derivatives in the hexane solubles was decreased compared with those in the dried gum (see Fig. 5), indicating hexane could only dissolve part of the phenolics. Deoiling process by using acetone changed the distribution of sinapine derivatives significantly. Most of them were dissolved by acetone and thus appeared in the extracts of recovered oils (Table 2). It suggests that the recovered oil can be a rich source for extracting abundant sinapine derivatives. Correspondingly, the content of sinapine derivatives in deoiled lecithin was very low. Ethanol solubles (PC fraction) did not contain sinapine and sinapic acid but canolol. However, the total phenolic content of ethanol solubles measured by the Folin-Ciocalteu method was the highest, which might be attributed to the interference of phospholipids in the extract. When different processed samples were compared,
J. Li, Z. Guo / LWT - Food Science and Technology 73 (2016) 397e405
403
Fig. 6. Flowchart of the gum processing.
Table 2 Content of phenolic compounds in different processed gums. Samples
Sinapine (mg/g)a
Sinapic acid (mg/g)a
Canolol (mg/g)a
Hexane solubles Deoiled lecithin Recovered oil Ethanol solubles
46.34 ± 3.34a 4.89 ± 1.37b 41.41 ± 2.58a BDL
212.98 ± 22.82a 18.38 ± 4.49b 255.58 ± 16.38a BDL
27.93 13.31 44.78 22.84
a
± ± ± ±
4.76a 1.33b 15.60c 1.66d
TPC-FC (mgSAE/g)a 4.21 3.68 4.50 7.05
± ± ± ±
0.02a 0.30b 0.03c 0.35d
Direct ethanol extraction was employed for analyses of phenolic compounds. Different letters within the same column indicate significant difference (P < 0.05, n ¼ 2).
Table 3 Antioxidant activity of the resultant extracts from different processed gums. Substrate
DPPH (mgSAE/g)
Dried gum Hexane solubles Deoiled lecithin Recovered oil Ethanol solubles
3.64 3.68 2.40 4.22 6.20
± ± ± ± ±
0.11a 0.13a 0.07b 0.07c 0.60d
HRSA (mgVCE/g)a 14.35 13.01 11.15 11.80 12.40
± ± ± ± ±
0.02a 0.19b 0.27c 0.12c 0.47b
FCA (mgEDTAE/g)a 0.78 0.64 0.96 0.74 4.89
± ± ± ± ±
0.02a 0.01b 0.03c 0.06a 0.07d
RP (mgSAE/g)a 6.06 5.38 3.51 5.14 7.08
± ± ± ± ±
0.03a 1.64a 0.49b 0.02c 0.44d
a HRSA means hydroxyl radical scavenging activity, FCA means ferrous chelating activity, and RP means reducing power. Different letters within the same column indicate significant difference (P < 0.05, n ¼ 2). Extracts were obtained by direct ethanol extraction.
most of sinapine derivatives removed by acetone and presented in the recovered oil (Tables 2). 3.6. Antioxidant activity of the resultant extracts from different processed gums Antioxidant activity of the extracts from rapeseed related materials has been studied extensively (Amarowicz et al., 1996, 2000; Hassasroudsari et al., 2009; Koski et al., 2003; Szydłowska-Czerniak et al., 2010; Sørensen et al., 2013). Antioxidant activity of the extracts from different processed gums should also be evaluated because it is related to the oxidative stability of the final products and the results are shown in Table 3. Compared with dried gum, extract from hexane solubles showed the same antioxidant activity, suggesting hexane refining could not decrease the oxidative
stability of the product. However, the results of DPPH, HRSA, and RP of deoiled lecithin were the lowest amongst the studied substrates, indicating deoiling process may reduce the oxidative stability of the lecithin. The results of DPPH, FCA, and RP of extract from ethanol solubles were the highest, meaning ethanol fractionation concentrated the active compound in ethanol solubles. However, no sinapine and sinapic acid but canolol were detected in the extract from ethanol solubles (Table 2). Additionally, it should be noted that the content of PC, LPC, and GPC in the extract from ethanol solubles were the highest (Table 4). Therefore, such fantastic antioxidant activity should be mainly attributed to the synergistic action between phospholipids and canolol. Except for phenolic compounds, phospholipids were also found to be kind of antioxidant because supplementation of phospholipids enhanced the oxidation stability of virgin olive oil (Koprivnjak et al., 2008).
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dx.doi.org/10.1016/j.lwt.2016.06.039.
Table 4 Phospholipid content in extracts from different processed gums. Substrate
PC (mg/g)
Dried gum Hexane solubles Deoiled lecithin Recovered oil Ethanol solubles
10.77 10.40 17.79 12.86 48.48
± ± ± ± ±
2.18a 0.61a 0.37b 0.26a 2.96c
LPC (mg/g)
GPC (mg/g)
6.14 ± 1.37a 6.18 ± 0.17a 15.46 ± 0.80b 2.35 ± 0.34c 69.92 ± 3.20d
0.95 1.78 1.59 ND 6.37
± 0.03a ± 0.11b ± 0.21b ± 0.11c
PA (mg/g) 1.92 ± 1.01a 2.20 ± 0.16a 1.94 ± 0.15a ND ND
PC: phosphatidylcholine, LPC: lyso-phosphatidylcholine, GPC: glycerophosphocholine, PA: phosphatidic acid. Different letters within the same column indicate significant difference (P < 0.05, n ¼ 2).
Soybean PC was found to display strong DPPH scavenging activity (data not shown). Sugino et al. (1997) investigated the antioxidative activity of egg yolk phospholipids and found that higher concentration of phospholipids displayed stronger antioxidant activity in docosahexaenoic acid enriched oil. Egg yolk PC and its hydrogenated derivatives were also examined and indicated that antioxidant activity was decreased with an increase in the degree of saturation of fatty acid chains within the phospholipids. Oleic and linoleic acid are main fatty acid chains in phospholipids from rapeseed (data not shown). It was found that oxidative stabilities of models enriched with quercetin-phosphatidylcholine complex formulations were better than in the models containing lecithin or quercetin alone, most likely as a consequence of synergism between polar lipids and quercetin as well as tocopherols (Ramadan, 2012). Therefore, the rapeseed based lecithin products are unique because they contain not only phospholipid bearing unsaturated fatty acids but also phenolics compounds with potential synergistic effect for strong antioxidant activity. 4. Conclusion In conclusion, this work identified the major phenolic compounds in rapeseed waste gum and it was found that sinapic acid was the most abundant phenolic compound followed by sinapine and canolol. The flow of the identified phenolic compounds was tracked following the typical processes of lecithin related products. Part of the phenolics was lost during hexane refining and a large part of sinapic acid derivatives were removed by acetone extraction during deoiling process. Deoiling is not recommended if a product enriched with bioactive phenolic compounds is required. The extracts obtained in the present work displayed good antioxidant activity which most likely resulted from a synergic effect of phenolic and phospholipid components. The strong antioxidant activity of the extract reflected the oxidative stability of substrate to some extent. The co-presence of phenolics and phospholipids in rapeseed gum originated products endowed them with excellent ion-chelating activity and antioxidant property, which may distinguish rapeseed lecithin from other types of lecithin. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements Financial support from the Graduate School of Science and Technology (GSST), Aarhus University and DLG (Dansk Landbrugs Grovvareselskab) Food Oil, Denmark is gratefully acknowledged. Appendix A. Supplementary data Supplementary data related to this article can be found at http://
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