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Composition of phenolics and anthocyanins in a sweet potato cell suspension culture
Biochemical Engineering Journal 14 (2003) 155–161
Composition of phenolics and anthocyanins in a sweet potato cell suspension culture Izabela Konczak-Islam a,∗ , Shigenori Okuno b , Makoto Yoshimoto b , Osamu Yamakawa b a
b
Food Science Australia, CRC for Bioproducts, Riverside Life Sciences Centre, 16 Julius Avenue, Riverside Corporate Park, North Ryde, NSW 2113, Australia Department of Upland Farming Research, National Agricultural Research Centre for Kyushu Okinawa Region, Miyakonojo, Miyazaki 885-0091, Japan Received 8 June 2002; accepted after revision 30 August 2002
Abstract Accumulation of selected phenolic acids and anthocyanins and changes in their compositions were monitored in a sweet potato cell line (PL) suspension culture during one growth period of 24 days in the dark in modified Murashige and Skoog (MS) medium for high-anthocyanin production. The total amount of phenolic compounds increased 3-fold over 4 days after transfer into a modified medium and remained at the same level over the whole growth period. Chlorogenic acid and caffeic acid were among the phenolic compounds identified. Dynamic changes in the composition of anthocyanin pigments were detected. The relative concentrations of non-acylated anthocyanins YGM-0a and -0b, which significantly dominated pigment extract in the tissue produced in non-modified MS medium, dropped drastically after transfer into a high-anthocyanin production medium from 40.5 to 5.7% and from 19.8 to 3.9%, respectively. These decreases were concomitant with increases in the relative concentrations of acylated pigments. Within 9 days of growth in the high-anthocyanin production medium the level of pigment accumulation increased 3-fold. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Anthocyanin; Caffeic acid; Chlorogenic acid; Plant cell culture; Phenolics; Sweet potato
1. Introduction Plant cell culture is considered to be a potential means of producing valuable plant products in a factory setting. Among these products food additives such as anthocyanins, shikonin compounds, safflower yellow, saffron and madder colorants are of high interest [1]. Anthocyanins, a large group of water-soluble pigments responsible for red to purple and red to blue colours of fruits and vegetables, are commonly used in acidic solutions as a red pigment in soft drinks, jams, confectionery and bakery products. They also possess physiological activities such as antioxidative, antimutagenic and antihypertensive potential and reduction of liver injury, that improve the beneficial health properties of food [2]. Plant cell cultures have been successfully applied to produce anthocyanin pigments in vitro. Anthocyanin accumulation in cell lines from various species was frequently reported as a model system for secondary product biosynthesis, because their colour allows accumulation to be ∗ Corresponding author. Tel.: +61-2-9490-8563; fax: +61-2-9490-8524. E-mail address: [email protected] (I. Konczak-Islam).
easily visualised [3]. Induction of anthocyanin accumulating cell lines [3–6], kinetics of tissue growth and pigment accumulation [7], optimal conditions for anthocyanin biosynthesis [8–15], addition of precursors [16], elicitors [17], conditioned medium [18] and the effect of viscous additive-supplemented medium [19] have been studied. Large-scale production of anthocyanin has been proposed [20] and image analysis techniques to estimate anthocyanin content within a cell have been developed [21]. Chemical structure of anthocyanin pigments accumulated in plant cell cultures [22–24] and the composition of pigment extract compared with that of the field-grown plant [25,26] have been reported. However, research on regulation of the composition of anthocyanin pigments accumulated in vitro is limited. Previously, we have identified that composition of culture medium or environmental factors influence not only the level of anthocyanin accumulation in the sweet potato (Ipomoea batatas L.) PL suspension culture, but also induce changes in the composition of crude pigment extract [27]. Decreasing the level of NH4 + ions in Murashige and Skoog (MS) basal medium promoted accumulation of acylated compounds. An increase of culture temperature brought higher concentration of non-acylated compounds. Changes
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in composition of anthocyanin extract may reflect on its colour characteristics, stability and nutraceutical properties. Therefore, the ability to regulate the quality of anthocyanin pigments accumulated in vitro can be of high value to the food colorant industry. The biosynthetic pathway of polyphenolics is closely related to that of anthocyanins [28] and it may be presumed that in parallel with accumulation of anthocyanin in the PL suspension culture phenolic compounds are also present. These compounds may influence pigment stability as well as nutraceutical properties of tissue extract. Recent literature data revealed that numerous polyphenolics possess strong health promoting properties such as antioxidative, antimutagenic, antihypertensive, antihemorrhagic and antivirus activities and protection from UV radiation [2,29]. Occurrence of phenolic substances in plant cell cultures such as tannins [30] or procyanidines [31] have been reported. Phenols accumulated in cell culture of Vitis vinifera were identified as potential cancer-chemopreventive components [32]. Therefore, the presence of polyphenolics in PL suspension culture could increase the value of potential food additives produced by this cell line. In this paper we report on identification in crude pigment extract of the PL suspension culture of two phenolic acids, caffeic acid (CA) and chlorogenic acid (CH), and their accumulation within one growth period in a high-anthocyanin producing medium. Changes in the composition of anthocyanin pigments under the same culture conditions are also reported. 2. Materials and methods 2.1. Plant material and culture conditions Callus culture has been established from the sweet potato storage root, cv. Ayamurasaki, as described previously [26]. Suspended cell cultures were initiated by transferring about 1 g (fresh weight) of callus to 25 ml of liquid medium in 100 ml Erlenmayer flasks. MS basal medium supplemented with 1.0 mg/l 2,4-D was used as a maintenance medium. The cultures were incubated on a rotary shaker (130 rpm) at 25 ◦ C in the dark. The medium was changed weekly. Seven-day-old subcultures were transferred into a liquid high-anthocyanin producing medium which was a modified MS medium with 9.4 mM KNO3 and 5% sucrose and without NH4 NO3 nor growth regulators [27]. Cell aggregates (100 mg) were placed in 50 ml Erlenmayer flasks containing 10 ml medium (pH 5.8 before autoclaving). The samples (six replications) were harvested in 2–3–days intervals for a period of 24 days (growth period). 2.2. Determination of growth Growth was measured by removing the aggregates from the medium, rinsing them with distilled water, separating
them from the liquid by vacuum filtration and weighing. The growth index was defined as W/W0 , where W0 and W denote fresh weight of the aggregates before and after the cultivation, respectively. 2.3. Extraction of phenolics and anthocyanins Cell aggregates separated from the culture medium by vacuum filtration were ground and steeped in 15% acetic acid for 16 h. The volume of acetic acid solution was adjusted to 20 times equivalent of the sample weight. The samples were centrifuged at 12,000 × g for 10 min. The supernatants were used for identification of anthocyanin and phenolic compounds and quality analysis. 2.4. Identification of phenolic acids and HPLC analysis The supernatants filtered through a 0.2 m filter membrane (DISMIC-13cp, Advantec, Japan) were injected (10 l) into HPLC system consisting of two LC-10AT pumps, SIL-10AXL auto-injector, CTO-10AC column oven and SPD-M10AVP photodiode array UV-VIS detector (Shimadzu, Kyoto, Japan). The system was controlled by a CLASS-LC10 workstation (Shimadzu, Kyoto, Japan). The analysis was conducted using YMC-Pack ODS-AM AM-302 column (150 mm × 4.6 mm, 5 m particles; YMC, Kyoto, Japan) at 40 ◦ C. The mobile phase, supplied at a flow rate of 1 ml/min, consisted of 0.2% (v/v) formic acid in water (solvent A) and methanol (solvent B). The elution profile was a linear gradient elution with 2% solvent B from 0 to 15 min, 2–45% from 15 to 50 min, and 45% from 50 to 65 min in solvent A [33]. The standards used in this analysis: CH and CA were purchased from Wako Pure Chemical Industries (Osaka, Japan). The chromatograms were recorded and the absolute concentrations of phenolic acids were calculated from the peak areas. The total peak area index was defined as A/A0 , where A0 and A denote total peak area at day 0 and sampling days, respectively. 2.5. Identification of anthocyanins and HPLC analysis The supernatant diluted 4-fold with a McIlvain’s buffer solution and pH adjusted to 3 was used for the measurement of the optical densities at 530 nm with spectrophotometer CS-9300PC (Shimadzu, Kyoto, Japan). A colour value (CV) of the pigment extract, which is a commercial indicator of total anthocyanins, was calculated by the following formula: CV = 0.1 × OD530 × D1 × D2 /g-FW, where OD530 is a spectrophotometric reading at 530 nm, D1 and D2 are the levels of dilution, and FW the tissue fresh weight [26]. For HPLC analysis the supernatants were filtered through a 0.2 m filter membrane (DISMIC-13cp, Advantec, Tokyo, Japan). The HPLC system consisted of two LC-10AD pumps, SPD-M10A diode array detector, CTO-10AS column oven, DGV-12A degasser, SIL-10AD auto-injector
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and SCL-10A system controller (Shimadzu, Kyoto, Japan) equipped with Luna (3 m C18(2), 4.6 mm × 100 mm, Phenomenex, USA) column at 35 ◦ C. The following solvents in water with a flow rate of 1 ml/min were used: A—1.5% phosphoric acid and B—1.5% phosphoric acid, 20% acetic acid, 25% acetonitrile. The elution profile was a linear gradient elution with 25–85% solvent B in solvent A for 100 min. The chromatograms were monitored at 530 nm and recorded and the relative concentrations of pigments were calculated from the peak areas. Identification of anthocyanins was carried out comparing the peaks with YGM-0a (cyanidin-3-sophoroside-5-glucoside) and YGM-0f (cyanidin-3-(E)-p-coumaroyl-sophoroside-5-glucoside) standards isolated from the PL cell line [23] and standard peaks of purple-fleshed sweet potato YGM anthocyanins: YGM-1a [cyanidin-3-(6,6 -caffeoyl-p-hydroxybenzoylsophoroside)-5-glucoside], YGM-1b [cyanidin-3-(6, 6 -dicaffeoylsophoroside)-5-glucoside], YGM-3 [cyanidin3-(6,6 -caffeoylferuloylsophoroside)-5-glucoside], YGM4b [peonidin-3-(6,6 -dicaffeoylsophoroside)-5-glucoside], YGM-5a [peonidin-3-(6,6 -caffeoyl-p-hydroxybenzoylsophoroside)-5-glucoside], and YGM-6 [peonidin-3-(6,6 caffeoylferuloylsophoroside)-5-glucoside] [34].
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Fig. 1. HPLC chromatograms of phenolic components detected in crude extract of the PL suspension culture over one growth period of 24 days in a high-anthocyanin production medium (CA—caffeic acid, CH—chlorogenic acid).
3. Results and discussion 3.1. Accumulation of phenolic acids The HPLC patterns of phenolic components monitored in the PL suspension culture after tissue transfer from MS basal medium into a high-anthocyanin production medium over one growth period of 24 days indicated dynamic changes in composition over time (Fig. 1). These changes occurred in parallel with a high increase of total level of phenolic components as indicated by the total peak area index (Fig. 2A). The total amount increased 3.2-fold by day 4 and was maintained at the same level over the whole growth period. The biosynthesis of phenolics occurred in parallel with increased accumulation of total anthocyanins (Fig. 2B) and linear tissue growth (Fig. 2C). Among phenolic components, two phenolic acids have been identified: CA and CH, as shown in Fig. 1. These acids are represented by peaks which appeared on ODS-column chromatography under the specified elution conditions with retention time of 31.8 and 33.2 min, respectively. In the tissue extract of suspension culture obtained from MS basal medium only trace amounts of CA and CH were detected (Fig. 3A and B). Transfer into a high-anthocyanin production medium induced significant increase of CA level from 4.0 to 9.4 mg/100 g-FW in 2 days. This level was maintained during an active tissue growth. At the stationary phase of growth period (days 18–24) a further 3-fold increase of CA level occurred. A drastic 10-fold increase of CH level (from 0.86 to 8.9 mg/100 g-FW) was observed within only 2 days after
transfer into a high-anthocyanin production medium. The accumulation decreased to about 6.0 mg/100 g-FW between days 4 and 15 of growth period and a further decrease occurred during the stationary phase of tissue growth. The major peak in tissue extract during an active growth of suspension culture was indicated on the HPLC chromatogram as X (Fig. 1). This peak eluted on the ODScolumn chromatography under specified conditions with retention time of 47.2 min. The relative concentration of this component, as calculated according to the peak area, increased about 10-fold during the first 4 days following the decrease of CA and CH accumulation and was maintained at a similar level up to day 15 (data not presented). Accumulation of this component decreased at the stationary phase of tissue growth when a sharp increase of the CA content was observed. It can be speculated that the accumulation of CA and the unknown component are closely related and CA might be a precursor for its biosynthesis. This component is under identification. Various groups of phenolic compounds such as hydroxycinnamoyl esters (e.g. CH) and flavonoids were identified to form non-covalent complexes with other electron-deficient aromatic systems such as anthocyanins (intermolecular copigmentation). A result of this interaction is manifested as the intensification and change in colour of the anthocyanin and its stabilisation [28]. Therefore, the presence of CA and CH in the PL suspension culture extract may have a significant impact on the characteristics of anthocyanin extract produced by this cell line such as colour characteristics and pigment stability—both of a high importance to the food
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Fig. 3. Accumulation of (A): caffeic acid (CA) and (B): chlorogenic acid (CH) in the PL suspension culture over one growth period in a high-anthocyanin production medium. Bars in the figure represent standard deviations of six replications.
Fig. 2. Accumulation of phenolics (A) and anthocyanins (B) and tissue growth (C) in the PL suspension culture over one growth period in a high-anthocyanin production medium. Bars in the figure represent standard deviations of six replications.
colorant industry. Additionally, both these components were found to be strong antioxidants and CH was also identified to be an effective inhibitor of potentially mutagenic and carcinogenic reactions in vivo [35], which may enhance nutraceutical properties of the food additives produced by the PL suspension culture. 3.2. Accumulation of anthocyanins Pigment extract of the PL suspension culture obtained in MS basal medium represented a mixture of anthocyanins in which two pigments dominated: YGM-0a and YGM-0b (Fig. 4, day 0). The YGM-0a appeared on the reverse phase column chromatography with the shortest retention time of 10.8 min and comprised 40.5% of the total anthocyanins as calculated by the peak area (Fig. 5). This pigment was previously identified as a cyanidin-3-sophoroside-5-glucoside,
a non-acylated anthocyanin [23]. The second major peak, YGM-0b, which appeared with the retention time of 18.3 min, comprised 19.8% of the total anthocyanins. At present this peak is under identification and is estimated to be peonidin-3-sophoroside-5-glucoside, another non-acylated anthocyanin (Terahara, personal communication). Transfer of suspension culture into a high-anthocyanin production medium resulted in drastic changes in pigment composition over time (Figs. 4 and 5). Within 4 days the relative concentration of YGM-0a dropped to 20.8% and that of YGM-0b dropped to 12.4%. A further gradual decrease in the relative concentrations of YGM-0a and YGM-0b continued until the end of the growth period and reached 5.7 and 3.9%, respectively. The decreases in the level of non-acylated components were concomitant with significant increases in the relative concentrations of YGM-0f and YGM-0g which appeared with longer retention times. The peaks YGM-0f and YGM-0g were identified to be cell line specific pigments, which are not biosynthesised in the sweet potato storage root tissue [26]. Terahara et al. [23] have identified the peak YGM-0f as cyanidin-3-O-((E)-p-coumaroyl-sophoroside)-5-glucoside, the first anthocyanin acylated with p-coumaric acid found among sweet potato pigments. Within the first 4 days of the growth period the relative concentrations of YGM-0f and YGM-0g reached their maximums of 19.2 and 9.9%, respectively. These levels were maintained during the next 5 days, while the tissue continued a linear growth and the total accumulation of anthocyanins within a cell continued to increase (Fig. 1C and B). At the stationary phase of the PL suspension culture growth the relative concentrations of YGM-0f and YGM-0g slightly decreased to 15.5 and 6.9%,
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Fig. 4. HPLC chromatograms of anthocyanins detected in crude extract of the PL suspension culture over one growth period of 24 days in a high-anthocyanin production medium. The characters in the chromatogram are the YGM numbers of sweet potato anthocyanins [26].
respectively. A steady increase in the relative concentrations of pigments YGM-7a and YGM-7e was observed during the whole growth period. While only trace amounts of these anthocyanins were detected in the tissue maintained on MS medium, in the high-anthocyanin production medium their relative concentrations increased over time to 7.5 and 3.7%,
respectively. The peaks YGM-7a and YGM-7e are not yet identified. They appear on the ODS-column HPLC with the longest retention time of 69.5 and 80.2 min, respectively. We estimate these pigments to have more complicated structures than pigments appearing with shorter retention times such as YGM-0f .
Fig. 5. Relative concentrations of selected YGM anthocyanins in the PL suspension culture over one growth period in a high-anthocyanin production medium. Bars in the figure represent standard deviations of six replications.
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Anthocyanin composition of the PL suspension culture maintained in the MS basal medium appeared to be relatively constant during the whole growth period [26]. Similarly, no changes in the intracellular concentrations of the major anthocyanins during one growth cycle were reported for Ajuga reptans cultures grown on MS basal medium [36]. From the data presented above it can be concluded that the high-anthocyanin producing medium condition promotes accumulation of anthocyanin pigments with complex molecular structures, such as acylated YGM-0f . Knowledge of these changes in the composition of anthocyanins may allow regulation of the quality of anthocyanin pigment as a food colorant produced in this system. Previously, we have identified that the presence of NH4 + ion in the culture medium inhibits acylation of cyanidin-3-sophoroside-5-glucoside in the PL suspension culture [27]. It can be suspected that under the condition of modified NH4 + -free culture medium the activity of acyltransferases in our system increases. The mechanism of these changes is yet to be identified. Acylation of anthocyanins with aromatic/aliphatic acids is important for the stabilisation of anthocyanin pigments [37,38]. Acylation can also modify food colour by favouring intramolecular and intermolecular co-pigmentation [38]. Therefore the ability to promote pigment acylation in tissue culture can be of interest to the food colorant industry.
Acknowledgements Funding of this project through JSPS post-doctoral fellowship to Izabela Konczak-Islam by the Japan International Science and Technology Exchange Centre and by the National Agricultural Research Centre for Kyushu Okinawa Region, Japan, is thankfully acknowledged.
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Composition of phenolics and anthocyanins in a sweet potato cell suspension culture Izabela Konczak-Islam a,∗ , Shigenori Okuno b , Makoto Yoshimoto b , Osamu Yamakawa b a
b
Food Science Australia, CRC for Bioproducts, Riverside Life Sciences Centre, 16 Julius Avenue, Riverside Corporate Park, North Ryde, NSW 2113, Australia Department of Upland Farming Research, National Agricultural Research Centre for Kyushu Okinawa Region, Miyakonojo, Miyazaki 885-0091, Japan Received 8 June 2002; accepted after revision 30 August 2002
Abstract Accumulation of selected phenolic acids and anthocyanins and changes in their compositions were monitored in a sweet potato cell line (PL) suspension culture during one growth period of 24 days in the dark in modified Murashige and Skoog (MS) medium for high-anthocyanin production. The total amount of phenolic compounds increased 3-fold over 4 days after transfer into a modified medium and remained at the same level over the whole growth period. Chlorogenic acid and caffeic acid were among the phenolic compounds identified. Dynamic changes in the composition of anthocyanin pigments were detected. The relative concentrations of non-acylated anthocyanins YGM-0a and -0b, which significantly dominated pigment extract in the tissue produced in non-modified MS medium, dropped drastically after transfer into a high-anthocyanin production medium from 40.5 to 5.7% and from 19.8 to 3.9%, respectively. These decreases were concomitant with increases in the relative concentrations of acylated pigments. Within 9 days of growth in the high-anthocyanin production medium the level of pigment accumulation increased 3-fold. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Anthocyanin; Caffeic acid; Chlorogenic acid; Plant cell culture; Phenolics; Sweet potato
1. Introduction Plant cell culture is considered to be a potential means of producing valuable plant products in a factory setting. Among these products food additives such as anthocyanins, shikonin compounds, safflower yellow, saffron and madder colorants are of high interest [1]. Anthocyanins, a large group of water-soluble pigments responsible for red to purple and red to blue colours of fruits and vegetables, are commonly used in acidic solutions as a red pigment in soft drinks, jams, confectionery and bakery products. They also possess physiological activities such as antioxidative, antimutagenic and antihypertensive potential and reduction of liver injury, that improve the beneficial health properties of food [2]. Plant cell cultures have been successfully applied to produce anthocyanin pigments in vitro. Anthocyanin accumulation in cell lines from various species was frequently reported as a model system for secondary product biosynthesis, because their colour allows accumulation to be ∗ Corresponding author. Tel.: +61-2-9490-8563; fax: +61-2-9490-8524. E-mail address: [email protected] (I. Konczak-Islam).
easily visualised [3]. Induction of anthocyanin accumulating cell lines [3–6], kinetics of tissue growth and pigment accumulation [7], optimal conditions for anthocyanin biosynthesis [8–15], addition of precursors [16], elicitors [17], conditioned medium [18] and the effect of viscous additive-supplemented medium [19] have been studied. Large-scale production of anthocyanin has been proposed [20] and image analysis techniques to estimate anthocyanin content within a cell have been developed [21]. Chemical structure of anthocyanin pigments accumulated in plant cell cultures [22–24] and the composition of pigment extract compared with that of the field-grown plant [25,26] have been reported. However, research on regulation of the composition of anthocyanin pigments accumulated in vitro is limited. Previously, we have identified that composition of culture medium or environmental factors influence not only the level of anthocyanin accumulation in the sweet potato (Ipomoea batatas L.) PL suspension culture, but also induce changes in the composition of crude pigment extract [27]. Decreasing the level of NH4 + ions in Murashige and Skoog (MS) basal medium promoted accumulation of acylated compounds. An increase of culture temperature brought higher concentration of non-acylated compounds. Changes
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in composition of anthocyanin extract may reflect on its colour characteristics, stability and nutraceutical properties. Therefore, the ability to regulate the quality of anthocyanin pigments accumulated in vitro can be of high value to the food colorant industry. The biosynthetic pathway of polyphenolics is closely related to that of anthocyanins [28] and it may be presumed that in parallel with accumulation of anthocyanin in the PL suspension culture phenolic compounds are also present. These compounds may influence pigment stability as well as nutraceutical properties of tissue extract. Recent literature data revealed that numerous polyphenolics possess strong health promoting properties such as antioxidative, antimutagenic, antihypertensive, antihemorrhagic and antivirus activities and protection from UV radiation [2,29]. Occurrence of phenolic substances in plant cell cultures such as tannins [30] or procyanidines [31] have been reported. Phenols accumulated in cell culture of Vitis vinifera were identified as potential cancer-chemopreventive components [32]. Therefore, the presence of polyphenolics in PL suspension culture could increase the value of potential food additives produced by this cell line. In this paper we report on identification in crude pigment extract of the PL suspension culture of two phenolic acids, caffeic acid (CA) and chlorogenic acid (CH), and their accumulation within one growth period in a high-anthocyanin producing medium. Changes in the composition of anthocyanin pigments under the same culture conditions are also reported. 2. Materials and methods 2.1. Plant material and culture conditions Callus culture has been established from the sweet potato storage root, cv. Ayamurasaki, as described previously [26]. Suspended cell cultures were initiated by transferring about 1 g (fresh weight) of callus to 25 ml of liquid medium in 100 ml Erlenmayer flasks. MS basal medium supplemented with 1.0 mg/l 2,4-D was used as a maintenance medium. The cultures were incubated on a rotary shaker (130 rpm) at 25 ◦ C in the dark. The medium was changed weekly. Seven-day-old subcultures were transferred into a liquid high-anthocyanin producing medium which was a modified MS medium with 9.4 mM KNO3 and 5% sucrose and without NH4 NO3 nor growth regulators [27]. Cell aggregates (100 mg) were placed in 50 ml Erlenmayer flasks containing 10 ml medium (pH 5.8 before autoclaving). The samples (six replications) were harvested in 2–3–days intervals for a period of 24 days (growth period). 2.2. Determination of growth Growth was measured by removing the aggregates from the medium, rinsing them with distilled water, separating
them from the liquid by vacuum filtration and weighing. The growth index was defined as W/W0 , where W0 and W denote fresh weight of the aggregates before and after the cultivation, respectively. 2.3. Extraction of phenolics and anthocyanins Cell aggregates separated from the culture medium by vacuum filtration were ground and steeped in 15% acetic acid for 16 h. The volume of acetic acid solution was adjusted to 20 times equivalent of the sample weight. The samples were centrifuged at 12,000 × g for 10 min. The supernatants were used for identification of anthocyanin and phenolic compounds and quality analysis. 2.4. Identification of phenolic acids and HPLC analysis The supernatants filtered through a 0.2 m filter membrane (DISMIC-13cp, Advantec, Japan) were injected (10 l) into HPLC system consisting of two LC-10AT pumps, SIL-10AXL auto-injector, CTO-10AC column oven and SPD-M10AVP photodiode array UV-VIS detector (Shimadzu, Kyoto, Japan). The system was controlled by a CLASS-LC10 workstation (Shimadzu, Kyoto, Japan). The analysis was conducted using YMC-Pack ODS-AM AM-302 column (150 mm × 4.6 mm, 5 m particles; YMC, Kyoto, Japan) at 40 ◦ C. The mobile phase, supplied at a flow rate of 1 ml/min, consisted of 0.2% (v/v) formic acid in water (solvent A) and methanol (solvent B). The elution profile was a linear gradient elution with 2% solvent B from 0 to 15 min, 2–45% from 15 to 50 min, and 45% from 50 to 65 min in solvent A [33]. The standards used in this analysis: CH and CA were purchased from Wako Pure Chemical Industries (Osaka, Japan). The chromatograms were recorded and the absolute concentrations of phenolic acids were calculated from the peak areas. The total peak area index was defined as A/A0 , where A0 and A denote total peak area at day 0 and sampling days, respectively. 2.5. Identification of anthocyanins and HPLC analysis The supernatant diluted 4-fold with a McIlvain’s buffer solution and pH adjusted to 3 was used for the measurement of the optical densities at 530 nm with spectrophotometer CS-9300PC (Shimadzu, Kyoto, Japan). A colour value (CV) of the pigment extract, which is a commercial indicator of total anthocyanins, was calculated by the following formula: CV = 0.1 × OD530 × D1 × D2 /g-FW, where OD530 is a spectrophotometric reading at 530 nm, D1 and D2 are the levels of dilution, and FW the tissue fresh weight [26]. For HPLC analysis the supernatants were filtered through a 0.2 m filter membrane (DISMIC-13cp, Advantec, Tokyo, Japan). The HPLC system consisted of two LC-10AD pumps, SPD-M10A diode array detector, CTO-10AS column oven, DGV-12A degasser, SIL-10AD auto-injector
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and SCL-10A system controller (Shimadzu, Kyoto, Japan) equipped with Luna (3 m C18(2), 4.6 mm × 100 mm, Phenomenex, USA) column at 35 ◦ C. The following solvents in water with a flow rate of 1 ml/min were used: A—1.5% phosphoric acid and B—1.5% phosphoric acid, 20% acetic acid, 25% acetonitrile. The elution profile was a linear gradient elution with 25–85% solvent B in solvent A for 100 min. The chromatograms were monitored at 530 nm and recorded and the relative concentrations of pigments were calculated from the peak areas. Identification of anthocyanins was carried out comparing the peaks with YGM-0a (cyanidin-3-sophoroside-5-glucoside) and YGM-0f (cyanidin-3-(E)-p-coumaroyl-sophoroside-5-glucoside) standards isolated from the PL cell line [23] and standard peaks of purple-fleshed sweet potato YGM anthocyanins: YGM-1a [cyanidin-3-(6,6 -caffeoyl-p-hydroxybenzoylsophoroside)-5-glucoside], YGM-1b [cyanidin-3-(6, 6 -dicaffeoylsophoroside)-5-glucoside], YGM-3 [cyanidin3-(6,6 -caffeoylferuloylsophoroside)-5-glucoside], YGM4b [peonidin-3-(6,6 -dicaffeoylsophoroside)-5-glucoside], YGM-5a [peonidin-3-(6,6 -caffeoyl-p-hydroxybenzoylsophoroside)-5-glucoside], and YGM-6 [peonidin-3-(6,6 caffeoylferuloylsophoroside)-5-glucoside] [34].
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Fig. 1. HPLC chromatograms of phenolic components detected in crude extract of the PL suspension culture over one growth period of 24 days in a high-anthocyanin production medium (CA—caffeic acid, CH—chlorogenic acid).
3. Results and discussion 3.1. Accumulation of phenolic acids The HPLC patterns of phenolic components monitored in the PL suspension culture after tissue transfer from MS basal medium into a high-anthocyanin production medium over one growth period of 24 days indicated dynamic changes in composition over time (Fig. 1). These changes occurred in parallel with a high increase of total level of phenolic components as indicated by the total peak area index (Fig. 2A). The total amount increased 3.2-fold by day 4 and was maintained at the same level over the whole growth period. The biosynthesis of phenolics occurred in parallel with increased accumulation of total anthocyanins (Fig. 2B) and linear tissue growth (Fig. 2C). Among phenolic components, two phenolic acids have been identified: CA and CH, as shown in Fig. 1. These acids are represented by peaks which appeared on ODS-column chromatography under the specified elution conditions with retention time of 31.8 and 33.2 min, respectively. In the tissue extract of suspension culture obtained from MS basal medium only trace amounts of CA and CH were detected (Fig. 3A and B). Transfer into a high-anthocyanin production medium induced significant increase of CA level from 4.0 to 9.4 mg/100 g-FW in 2 days. This level was maintained during an active tissue growth. At the stationary phase of growth period (days 18–24) a further 3-fold increase of CA level occurred. A drastic 10-fold increase of CH level (from 0.86 to 8.9 mg/100 g-FW) was observed within only 2 days after
transfer into a high-anthocyanin production medium. The accumulation decreased to about 6.0 mg/100 g-FW between days 4 and 15 of growth period and a further decrease occurred during the stationary phase of tissue growth. The major peak in tissue extract during an active growth of suspension culture was indicated on the HPLC chromatogram as X (Fig. 1). This peak eluted on the ODScolumn chromatography under specified conditions with retention time of 47.2 min. The relative concentration of this component, as calculated according to the peak area, increased about 10-fold during the first 4 days following the decrease of CA and CH accumulation and was maintained at a similar level up to day 15 (data not presented). Accumulation of this component decreased at the stationary phase of tissue growth when a sharp increase of the CA content was observed. It can be speculated that the accumulation of CA and the unknown component are closely related and CA might be a precursor for its biosynthesis. This component is under identification. Various groups of phenolic compounds such as hydroxycinnamoyl esters (e.g. CH) and flavonoids were identified to form non-covalent complexes with other electron-deficient aromatic systems such as anthocyanins (intermolecular copigmentation). A result of this interaction is manifested as the intensification and change in colour of the anthocyanin and its stabilisation [28]. Therefore, the presence of CA and CH in the PL suspension culture extract may have a significant impact on the characteristics of anthocyanin extract produced by this cell line such as colour characteristics and pigment stability—both of a high importance to the food
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Fig. 3. Accumulation of (A): caffeic acid (CA) and (B): chlorogenic acid (CH) in the PL suspension culture over one growth period in a high-anthocyanin production medium. Bars in the figure represent standard deviations of six replications.
Fig. 2. Accumulation of phenolics (A) and anthocyanins (B) and tissue growth (C) in the PL suspension culture over one growth period in a high-anthocyanin production medium. Bars in the figure represent standard deviations of six replications.
colorant industry. Additionally, both these components were found to be strong antioxidants and CH was also identified to be an effective inhibitor of potentially mutagenic and carcinogenic reactions in vivo [35], which may enhance nutraceutical properties of the food additives produced by the PL suspension culture. 3.2. Accumulation of anthocyanins Pigment extract of the PL suspension culture obtained in MS basal medium represented a mixture of anthocyanins in which two pigments dominated: YGM-0a and YGM-0b (Fig. 4, day 0). The YGM-0a appeared on the reverse phase column chromatography with the shortest retention time of 10.8 min and comprised 40.5% of the total anthocyanins as calculated by the peak area (Fig. 5). This pigment was previously identified as a cyanidin-3-sophoroside-5-glucoside,
a non-acylated anthocyanin [23]. The second major peak, YGM-0b, which appeared with the retention time of 18.3 min, comprised 19.8% of the total anthocyanins. At present this peak is under identification and is estimated to be peonidin-3-sophoroside-5-glucoside, another non-acylated anthocyanin (Terahara, personal communication). Transfer of suspension culture into a high-anthocyanin production medium resulted in drastic changes in pigment composition over time (Figs. 4 and 5). Within 4 days the relative concentration of YGM-0a dropped to 20.8% and that of YGM-0b dropped to 12.4%. A further gradual decrease in the relative concentrations of YGM-0a and YGM-0b continued until the end of the growth period and reached 5.7 and 3.9%, respectively. The decreases in the level of non-acylated components were concomitant with significant increases in the relative concentrations of YGM-0f and YGM-0g which appeared with longer retention times. The peaks YGM-0f and YGM-0g were identified to be cell line specific pigments, which are not biosynthesised in the sweet potato storage root tissue [26]. Terahara et al. [23] have identified the peak YGM-0f as cyanidin-3-O-((E)-p-coumaroyl-sophoroside)-5-glucoside, the first anthocyanin acylated with p-coumaric acid found among sweet potato pigments. Within the first 4 days of the growth period the relative concentrations of YGM-0f and YGM-0g reached their maximums of 19.2 and 9.9%, respectively. These levels were maintained during the next 5 days, while the tissue continued a linear growth and the total accumulation of anthocyanins within a cell continued to increase (Fig. 1C and B). At the stationary phase of the PL suspension culture growth the relative concentrations of YGM-0f and YGM-0g slightly decreased to 15.5 and 6.9%,
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Fig. 4. HPLC chromatograms of anthocyanins detected in crude extract of the PL suspension culture over one growth period of 24 days in a high-anthocyanin production medium. The characters in the chromatogram are the YGM numbers of sweet potato anthocyanins [26].
respectively. A steady increase in the relative concentrations of pigments YGM-7a and YGM-7e was observed during the whole growth period. While only trace amounts of these anthocyanins were detected in the tissue maintained on MS medium, in the high-anthocyanin production medium their relative concentrations increased over time to 7.5 and 3.7%,
respectively. The peaks YGM-7a and YGM-7e are not yet identified. They appear on the ODS-column HPLC with the longest retention time of 69.5 and 80.2 min, respectively. We estimate these pigments to have more complicated structures than pigments appearing with shorter retention times such as YGM-0f .
Fig. 5. Relative concentrations of selected YGM anthocyanins in the PL suspension culture over one growth period in a high-anthocyanin production medium. Bars in the figure represent standard deviations of six replications.
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Anthocyanin composition of the PL suspension culture maintained in the MS basal medium appeared to be relatively constant during the whole growth period [26]. Similarly, no changes in the intracellular concentrations of the major anthocyanins during one growth cycle were reported for Ajuga reptans cultures grown on MS basal medium [36]. From the data presented above it can be concluded that the high-anthocyanin producing medium condition promotes accumulation of anthocyanin pigments with complex molecular structures, such as acylated YGM-0f . Knowledge of these changes in the composition of anthocyanins may allow regulation of the quality of anthocyanin pigment as a food colorant produced in this system. Previously, we have identified that the presence of NH4 + ion in the culture medium inhibits acylation of cyanidin-3-sophoroside-5-glucoside in the PL suspension culture [27]. It can be suspected that under the condition of modified NH4 + -free culture medium the activity of acyltransferases in our system increases. The mechanism of these changes is yet to be identified. Acylation of anthocyanins with aromatic/aliphatic acids is important for the stabilisation of anthocyanin pigments [37,38]. Acylation can also modify food colour by favouring intramolecular and intermolecular co-pigmentation [38]. Therefore the ability to promote pigment acylation in tissue culture can be of interest to the food colorant industry.
Acknowledgements Funding of this project through JSPS post-doctoral fellowship to Izabela Konczak-Islam by the Japan International Science and Technology Exchange Centre and by the National Agricultural Research Centre for Kyushu Okinawa Region, Japan, is thankfully acknowledged.
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