- Email: [email protected]
Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae)
Phytochemistry xxx (2017) 1e5
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae) Fumi Tatsuzawa a, *, Natsu Tanikawa b, Masayoshi Nakayama b a b
Laboratory of Olericultural and Floricultural Science, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan NARO Institute of Floricultural Science, Tsukuba, Ibaraki 305-0852, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 December 2016 Received in revised form 9 January 2017 Accepted 4 February 2017 Available online xxx
A previously undescribed acylated anthocyanin was extracted from the red-purple flowers of Pueraria lobata with 5% HOAc-H2O, and determined to be petunidin 3-O-(b-glucopyranoside)-5-O-[6-O(malonyl)-b-glucopyranoside], by chemical and spectroscopic methods. In addition, two known acylated anthocyanins, delphinidin 3-O-(b-glucopyranoside)-5-O-[6-O-(malonyl)-b-glucopyranoside] and malvidin 3-O-(b-glucopyranoside)-5-O-[6-O-(malonyl)-b-glucopyranoside] were identified. Delphinidin 3,5di-glucoside, petunidin 3,5-di-glucoside, and malvidin 3,5-di-glucoside, have been known as major components of P. lobata in the former study. However, malonyl esters amounts were detected over 10 times compared with non-malonyl esters amounts. In those anthocyanins the most abundant anthocyanin was petunidin 3-O-(b-glucopyranoside)-5-O-[6-O-(malonyl)-b-glucopyranoside] in total flowers. On the visible absorption spectral curve of fresh red-purple petals, one characteristic absorption maximum was observed at 520 nm, which is similar to those of flowers containing pelargonidin derivatives. In contrast, the absorption spectral curve of old violet petals was observed at 500(sh), 536, 564(sh), and 613(sh) nm, which are similar to those of violet flowers containing delphinidin-type pigments. Pressed juices of both fresh red-purple petals and old violet petals had pH5.2 and 5.5 respectively, and had the same flavonoid constitution. Crude fresh red-purple petal pigments extracted by pH 2.2 and pH 5.2 buffers exhibited the same color and spectral curves as fresh red-purple petals and old violet petals, respectively. Moreover, in a cross-TLC experiment of crude extracted pigments, red-purple color was exhibited by the anthocyanin region and the crossed region of anthocyanins and isoflavone. Thus, it may be assumed that the unusually low pH in the vacuole of fresh petals plays an important role to form red-purple flower color against weak acidic pH in the vacuole of old violet P. lobata petals. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Pueraria lobata Leguminosae Malonylated anthocyanin Red-purple flower color Low pH
1. Introduction Pueraria lobata (Willd.) Ohwi (Leguminosae) is native to Japan and China and has an attractive red-purple flower color. These flowers have been mentioned in poetry, haiku, and other Japanese literature as ornamental plants. P. lobata flowers are also used in Chinese traditional medicine as a remedy for various conditions, including hangover, inappetence, and vomiting, and their alcoholic extract has been shown to contain several isoflavones (Niiho et al., 1990). The methanol extract of these flowers has been shown to contain kakkalide as the main component (Kurihara and Kikuchi, 1975). With regard to the anthocyanin components, the presence
* Corresponding author. E-mail address: [email protected] (F. Tatsuzawa).
of 3,5-diglucosides of delphinidin, petunidin, and malvidin in the methanolic HCl extract of flowers has been observed by paper chromatographic analysis of anthocyanins in the Leguminosae (Ishikura et al., 1978). It is known that the anthocyanins acylated by aliphatic acid are easily hydrolyzed within a short time by methanolic HCl extraction (Harborne and Grayer, 1988). Recently, acylated anthocyanins with aliphatic acid have been reported from several flowers, including carnation, chrysanthemum, tulip, and stock, from which non-acylated anthocyanins were formerly reported (Nakayama et al., 1997, 1999, 2000; Saito et al., 1995). Thus, there is a possibility of the existence of acylated pigments in P. lobata flowers. In the present study, structural elucidation of anthocyanins of P. lobata extracted by a weak acid revealed malonylated anthocyanins as the major component. The color of fresh P. lobata flowers changes from red-purple to violet after a few days in nature. Therefore, we compared anthocyanin components, their
http://dx.doi.org/10.1016/j.phytochem.2017.02.004 0031-9422/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
2
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
co-pigments, and the pH values of flower tissues between fresh red-purple flowers and old violet flowers. Finally, we assumed that unusually low pH in the vacuole is an important factor contributing to the color of P. lobata flowers. 2. Results and discussion Three unknown major peaks 1e3 with three known minor peaks A e C at 530 nm and one known major peak (D) at 350 nm were found in the 5%HOAc (acetic acid-water ¼ 5:95, v/v) (2L) extracts from dried red-purple flowers of Pueraria lobata (100g), by high performance liquid chromatography (HPLC) analysis (method 1) (Fig. 1). The percentage of 1e3 and A e C of Pueraria lobata, calculated as the total content by HPLC vis peak area at 530 nm, was 27.4% (1), 43.4% (2), 12.8% (3), 6.0% (A), 3.8% (B), and 1.7% (C) respectively. Moreover, the percentage of D, calculated as the total content by HPLC peak area at 350 nm, was 41.1%. The six anthocyanin peaks were extracted from the red-purple flowers with 5% HOAc, followed by isolation using Diaion HP-20 (Nippon Rensui Co., Tokyo, Japan) column chromatography (CC), Sephadex™ LH-20 (GE Healthcare UK Ltd., UK) CC and paper chromatography (PC). The chromatographic and spectroscopic properties of 1e3 and A e D are summarized in supplementary information. Acid hydrolysis of 1e3 and A e C yielded delphinidin (1 and A) (Forestal 0.23), petunidin (2 and B) (Forestal 0.39), and malvidin (3 and C) (Forestal 0.56), as their aglycone, respectively (Harborne, 1984), while also affording, glucose (BAW 0.21, EAA 0.33, ETN 0.83, EFW 0.52) was only detected as sugar from 1e3 and A e C (Harborne, 1984). Moreover, malonic acid was detected in the hydrolysates of pigments 1e3 by HPLC (method 2) (Tatsuzawa et al., 2014). These anthocyanidins, glucose, and malonic acid were identified by direct comparison with commercial standards (Wako Pure Chemical Industries Co. Ltd., Tokyo, Japan). Alkaline hydrolysis of 1e3 yielded A e C, respectively. The deacylated anthocyanin structures of 1e3 (¼ A e C) tentatively identified via co-TLC and co-HPLC (method 1) with authentic 3,5diglucosides of delphinidin, petunidin, and malvidin, respectively (Tatsuzawa and Shinoda, 2005). From these results, the structures of A e C were identified to be delphinidin 3, 5-diglucoside,
Fig. 1. HPLC profile of extracts from red-purple flowers of Pueraria lobata. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
petunidin 3,5-diglucoside, and malvidin 3,5-diglucoside, respectively, and 1e3 were presumed to be malonylated delphinidin 3,5diglucoside, malonylated petunidin 3,5-diglucoside, and malonylated malvidin 3,5-diglucoside, respectively. The peak D in Fig. 1 was presumed to be kakkalide (irisolidone 7O-(6-O-xylosyl)-glucoside) which was known as major isoflavone in the flowers of P. lobata (Kurihara and Kikuchi, 1975). The structures of 1e3 and D were further elucidated as follows based on the analyses of their 1H (400 MHz), 13C (100 MHz) and 2D (COSY, NOESY, 1He13C HMQC and 1He13C HMBC) NMR (JNM ALe400, JEOL Ltd., Tokyo, Japan) spectra in CD3ODeCF3COOD (9:1) for 1e3 and DMSO-d6 for D with TMS as an internal standard, as well as their high resolution fast atom bombardment mass spectra (HReFABMS) (LMSe700, JEOL Ltd.). 2.1. Pigment 2 The molecular ion [M]þ of 2 was observed at m/z 727(C31H35O20) indicating that 2 was composed of petunidin with two molecules of glucose and one molecule of malonic acid. The elemental components were confirmed by measuring its HR-FABMS, and the mass data was summarized in section 4.3.1. The structure was elucidated based on the analysis of the NMR spectra (Table 1). The chemical shifts of 5 aromatic protons of petunidin moiety with their coupling constants were assigned as shown in Table 1. The chemical shifts of the sugar moieties were observed in the
Table 1 NMR spectroscopic data of anthocyanin 2 from the flowers of Pueraria lobata in CD3OD-d4/CF3COOD (9:1). 2 1
H (ppm)
Petunidin 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 30 eCH3 Glucose A 1 2 3 4 5 6a 6b Glucose B 1 2 3 4 5 6a 6b Malonic acid eOCH2 COOH COOH
9.11 s 7.02 d(2.0) 7.09 d(2.0)
8.00 d(2.2)
7.83 d(2.2) 4.00 s
13
C (ppm)
164.7 147.0 135.8 156.7 106.2 169.6 97.5 157.3 113.6 119.9 109.8 150.0 146.4 147.7 114.3 57.4
5.36 3.71 3.58 3.42 3.66 3.62 3.99
d(7.8) t(8.5) t(9.0) t(9.3) ddd(2.5, 7.0, 10.0) m dd(2.0, 10.5)
104.0 74.9 77.7 71.5 79.1 62.7
5.19 3.74 3.59 3.48 3.82 4.36 4.57
d(7.8) t(8.2) t(8.9) t(8.8) ddd(2.4, 6.9, 10.0) dd(6.6, 12.0) dd(2.0, 12.0)
102.4 74.5 78.5 71.5 76.0 65.3
3.47s
49.3 168.7 169.6
Coupling constants (J in Hz) in parentheses.
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
region of d 5.36e3.42, where the two anomeric protons exhibited at d 5.36 (d, J ¼ 7.8 Hz, glucose A) and d 5.19 (d, J ¼ 7.8 Hz, glucose B). Based on the observed coupling constants (Table 1), these two glucoses were assumed to be in the b-pyranose form. Based on results of analyses of their COSY spectra, characteristic methylene proton signals [d 4.36 and 4.57] being shifted to lower magnetic field was assigned to H-6a and H-6b protons of glucose B. This result indicated that the OH-6 of glucose B must be acylated with malonic acid. NOESY and HMBC spectra were used to determine the sites of attachment of the sugars and petunidin moieties (Fig. 2). The signal of H-1 (d 5.36) of glucose A was correlated to the signal of C-3 (d 147.0) carbon of petunidin in its HMBC spectrum, and to the signals of the H-4 (d 9.11) proton of petunidin in NOESY spectrum. The signal of H-1 (d 5.19) of glucose B correlated to the signal of the C-5 (d 156.7) carbon of petunidin in the HMBC spectrum, and to the signals of the H-6 (d 7.02) proton of petunidin in NOESY spectrum. These characteristic features revealed that the OH-3 and OH-5 positions are bonded to glucose A and glucose B, respectively (Fig. 2). The signal of H-6a and -6b (d 4.36 and 4.57) of glucose B was correlated to the signal of the COOH (d 168.7) carbon of malonic acid in the HMBC spectrum (Fig. 2). Therefore, the OH-6 of glucose B is bonded with malonic acid. Consequently, the structure of 2 was determined to be petunidin 3-O-(b-glucopyranosyl)-5-O-[6-O-(malonyl)-b-glucopyranoside] (Fig. 2), which is a previously undescribed anthocyanin in plants (Andersen and Jordheim, 2006; Harborne and Baxter, 1999; Veitch and Grayer, 2008, 2011).
3
by paper chromatographic analysis (Ishikura et al., 1978). However, in the present study, we revealed, using HPLC, that 5% HOAc extracts of the red-purple P. lobata flowers contain 3-O-glucoside-5O-[6-O-(malonyl)-glucoside] form of delphinidin, petunidin, and malvidin (1e3) as major anthocyanins and 3,5-diglucosides of delphinidin, petunidin, and malvidin (A e C) as minor anthocyanins (Fig. 1). On the other hand, kakkalide (D), which was extracted with 5%HOAc, did not contain aliphatic acids in its chemical structure similar to the methanol extracts reported previously (Kurihara and Kikuchi, 1975). The florets of P. lobata open from the bottom to the top in an inflorescence (Fig. 3). The color of fresh florets is red-purple. When the florets become older, the petals turn violet (Fig. 3). The fresh florets have red-purple vexilla [Red-Purple 71B by Royal Horticultural Society Colour Chart; chromaticity value, b*(-5.77)/ a*(33.58) ¼ 0.17, L* ¼ 53.45 by a CM-700d Spectro Color Meter (Konica-Minolta, Tokyo, Japan); anthocyanin content by HPLC: 36.2%(1), 45.2%(2), 6.2%(3), 4.9%(A), 1.6%(B), and 0.8%(C) ], and deep red-purple alae [Red-Purple 71A; chromaticity value, b*(2.60)/ a*(3.71) ¼ 0.70, L* ¼ 68.47; anthocyanin content by HPLC: 38.2%(1), 42.3%(2), 5.8%(3), 5.3%(A), 1.4%(B), and 0.5%(C)] and carinas [RedPurple 71A; chromaticity value, b*(-2.82)/a*(13.77) ¼ 0.20, L* ¼ 58.23; anthocyanin content by HPLC: 47.0%(1), 33.3%(2), 4.6%(3), 6.7%(A), 1.5%(B), and 0.3%(C) ] (Fig. 3). The flower's color and anthocyanin components were almost the identical in the vexilla, alae, and carinas. Therefore, for studing flower color reconstruction, we used vexilla as the characteristic structure of
3. Concluding remarks In the present study, we revealed that petunidin 3-O-glucoside5-O-[6-O-(malonyl)-glucoside](2) is a novel anthocyanin. Furthermore, we were the first to identify the two anthocyanins, delphinidin 3-O-glucoside-5-O-[6-O-(malonyl)-glucoside](1) and malvidin 3-O-glucoside-5-O-[6-O-(malonyl)-glucoside](3) in the genus Pueraria (Andersen and Jordheim, 2006; Harborne and Baxter, 1999; Veitch and Grayer, 2008, 2011). In a previous floral anthocyanin study, 3,5-diglucosides of delphinidin, petunidin, and malvidin from 1% methanolic HCl extracts of P. lobata flowers were identified as major components of 14 species of leguminous plants
Fig. 2. Structures of anthocyanins 1e3 (1: R1 ¼ R2 ¼ OH, 2: R1 ¼ OCH3, R2 ¼ OH, 3: R1 ¼ R2 ¼ OCH3) and isoflavone D from the red-purple flowers of Pueraria lobata. Observed main NOEs are indicated by solid arrows. Observed main HMBCs are indicated by dotted arrows.
Fig. 3. Visible spectra of flower tissue pigments of Pueraria lobata. Fresh vexilla [520 nm]. Crude extracted pigments in the pH 2.2 buffer solution (phosphate-citrate buffer (MacIlvaine)) [520 nm]. Crude extracted pigments in the pH 5.2 buffer solution [500sh, 536sh, 564, 613sh nm]. Crude extracted pigments in the pH 5.6 buffer solution [500sh, 536sh, 564, 613sh nm]. Pressed fresh vexilla [500sh, 536, 564sh, 613sh nm]. Vexilla of old flowers [500sh, 536, 564sh, 613sh nm].
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
4
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
P. lobata. The spectral absorption curves of red-purple vexilla of P. lobata exhibited an absorption maximum at 520 nm, which is similar to the value of flowers containing pelargonidin derivatives (Saito et al., 2015). This was the same absorption maximum exhibited by the crude extract solution of red-purple vexilla in a buffer solution of pH 2.2 [the lowest pH for McIlvaine buffer (phosphate-citrate buffer)] (Elving et al., 1956) containing flavylium cation form of anthocyanins (Fig. 3). However, the pH value of fresh vexilla homogenate was pH 5.2 ± 0.0 (n ¼ 5). Fresh red-purple vexilla turned violet after mashing [Violet N87A by RHS CC. and hue: b*(-13.97)/a*(12.65) ¼ 1.07, L* ¼ 63.21]. The spectral absorption curves of mashed vexilla and those of old-flowers [Violet 83D by RHS CC. and hue: b*(-14.12)/a*(12.67) ¼ 1.12, L* ¼ 63.12; pH 5.5 ± 0.0 (n ¼ 5); anthocyanin content by HPLC: 25.5%(1), 45.8%(2), 11.4%(3), 5.5%(A), 3.7%(B), and 1.0%(C)] exhibited one major absorption peak (536 nm: flavylium cation) and two shoulders (546 nm: neutral quinonoidal form, and 613 nm: ionized quinonoidal form, Fig. 3). Absorption spectral curves with three absorption maxima are mainly observed in the absorption spectral curves of violet to blue flowers and in those of their anthocyanin solutions under weak acidic conditions, caused by co-pigmentation or self-association of delphinidin-type pigments (Saito et al., 2015). These results suggested that the pH of cell vacuole of fresh P. lobata flowers is more acidic than that of freshly mashed petals and old violet flowers. An additional possibility is that the flower color and spectral curve were changed by cytoplasmic substances, which provided a co-pigment effect, when petals were mashed. The cross-TLC experiment is an effective technique to easily and comprehensively confirm co-pigmentation between anthocyanins and co-pigments (Shimizu-Yumoto et al., 2012). Slantingly cross loading the pigment mixture or crude extracted pigments on TLC enables compounds to symmetrically develop at various angular lines from the upper origin to individual Rf values and cross each other in an orderly manner, while the mixing is simultaneously performed with separation (Shimizu-Yumoto et al., 2012). The occurrence of co-pigmentation can be detected as a change in color on the developed line of anthocyanin. Crude extracted pigments of P. lobata were analyzed by cross-TLC, and a pale blue spot different from flower color on the anthocyanin line (Rf value: 0.64) was detected under visible ray. This pale blue spot observed under ultraviolet rays crossed with hydroxycinnamic acids, such as caffeic acid derivatives (Rf value: 0.73). It is known that hydroxycinnamic acids, such as caffeic acid derivatives, exist, however, they are absent in the vacuole of superficial cells of petals containing anthocyanins. Therefore, the bluing effect by hydroxycinnamic acids, such as caffeic acid derivatives, occurred because the crude extract was mixed with hydroxycinnamic acids present outside the vacuole of P. lobata flowers. On the other hand, during the cross-TLC experiment of crude extract of P. lobata under visible ray, the crossing points of anthocyanin and kakkalide (Rf value: 0.82), which are the major components of crude extract (Fig. 1), exhibited red-purple colors. These colors were similar to those exhibited by TLC on anthocyanins of fresh petals. Moreover, the spectral absorption curve of mixture of the purified anthocyanins (1e3) and kakkalide (D) exhibited only one major absorption maximum at 520 nm, which is the same absorption maximum of crude extracted pigments in the pH 2.2 buffer and fresh vexilla. Therefore, evidence for kakkalide acting as a co-pigment was not obtained. Thus, based on the red-purple flower color of P. lobata, it can be concluded that the anthocyanins present in the vacuole do not interact with other compounds. From the above mentioned results, we concluded that the violet flower colors of fresh mashed petals and old violet flowers were caused by mixture of hydroxycinnamic acids, such as caffeic acid derivatives of the cytoplasm, and/or by the weak acidic conditions
of the vacuole. Therefore, it may be assumed that the unusually low pH condition in the vacuole is an important factor contributing to the red-purple color of P. lobata flower. 4. Experimental 4.1. General procedures TLC was carried out on plastic coated cellulose sheets (Merck) using eight mobile phases: BAW (n-BuOH-HOAc-H2O, 4:1:2, v/v/v), BuHCl (n-BuOH-2N HCl, 1:1, v/v, upper layer), AHW (HOAc-HClH2O, 15:3:82, v/v/v), 1% HCl for anthocyanins and isoflavone with UV light, Forestal (HOAc-HCl-H2O, 30:3:10, v/v/v) for anthocyanidin and BAW, EAA (EtOAc-HOAc-H2O, 3:1:1, v/v/v), ETN (EtOH-NH4OHH2O, 16:1:3, v/v/v) and EFW (EtOAc-HCOOH-H2O, 5:2:1, v/v/v) for sugars with aniline hydrogen phthalate spray reagent (Harborne, 1984). Analytical HPLC was performed on a LC 10A system (Shimadzu), using a Waters C18 (4.6 250 mm) column at 40 C with a flow rate of 1 mL/min and monitoring at 530 nm for anthocyanins and 350 nm for isoflavone and other flavonoids. The eluant was applied as a linear gradient elution for 40 min from 20 to 85% solvent B (1.5% H3PO4, 20% HOAc, 25% MeCN in H2O) in solvent A (1.5% H3PO4 in H2O) with 5 min of re-equilibration at 20% solvent B (method 1). The other eluant for malonic acid was applied as an isocratic elution of solvent A for 10 min and monitoring at 210 nm (Tatsuzawa et al., 2014; method 2). UV-Vis spectra were recorded on UV-Vis Multi Purpose Spectrophotometer (MPS-2450, Shimadzu, Kyoto, Japan) in 0.1% HClMeOH (from 200 to 700 nm) for anthocyanins and in MeOH, MeOH þ NaOMe, MeOH þ AlCl3, MeOH þ AlCl3þHCl, MeOH þ NaOAc, MeOH þ NaOAc þ H3BO3 (from 200 to 500 nm) for isoflavone. Spectral absorption of flowers directly measured on intact petals sandwiched in a pierced (5 mm) black holder using a recording spectrophotometer operated as a double-beam instrument (Type MPS-2450). High resolution FAB mass (FABMS) spectra were determined on a JEOL JMSe700 Mass spectrometer (JEOL, Ltd.) operating in the positive ion mode using 1:1 mixture of dithiothreitol and 3nitrobenzyl alcohol as a matrix. 1H (400 MHz) and 13C (100 MHz) NMR spectra were measured on a JEOL ALe400 NMR spectrometer (JEOL, Ltd.) using CD3ODeCF3COOD (9:1) and DMSO-d6 as a solvent. Chemical shifts are reported on the d-scale from tetramethylsilane as the internal standard, and coupling constants (J) are in Hz. 4.2. Plant materials The red-purple flowers of Pueraria lobata (Willd.) Ohwi (Legminosae) [Red-Purple 71B by Royal Horticultural Society Colour Chart and chromaticity value, b*(-5.77)/a*(33.58) ¼ 0.17, L* ¼ 53.45 by a CM-700d Spectro Color Meter (Konica-Minolta, Tokyo, Japan)] were collected in autumun (from 2015 to 2016) in The Botanical Garden, Faculty of Agriculture, Iwate University. The petals were trimmed from flowers by hand and dried by air for 1 day at 45 C. Then they were kept at 20 C until used. 4.3. Isolation and purification of 1e3 and AeD Dried flowers of P. lobata (100 g) were immersed in 5% HOAc (acetic acid-water ¼ 5:95, v/v) (5 L), respectively, at room temperature for 24 h and extracted. 1e3 and A e D were isolated and purified by Diaion HP-20 (Nippon Rensui Co. Ltd., Tokyo, Japan) column (90 150 mm) chromatography (CC), Sephadex™ LH-20 (GE Healthcare UK Ltd., UK) CC and paper chromatography (PC). The purified 1e3 and A e D were obtained from the flowers as
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
follows; 1 (ca. 10 mg), 2 (ca. 27 mg), 3 (ca. 8 mg), A (ca. 2 mg), B (ca. 3 mg), C (ca. 1 mg), and D (ca. 130 mg). 4.3.1. Petunidin 3-O- (b-glucopyranosyl)-5-O-[6-O-(malonyl)-bglucopyranoside] (2) Red powder: UV-VIS (in 0.1% HCl-MeOH): lmax 536, 273 nm, E440/E536 (%) ¼ 14, AlCl3 shift þ; TLC: (Rf-values) BAW 0.50, BuHCl 0.11, 1% HCl 0.10, AHW 0.39; HPLC: Rt (min) 21.1. HReFABMS; [M]þ calc. for C31H35O20: 727.1722. found: 727.1734. 4.4. Measurement of pH of petal homogenates Fresh red-purple vexilla and old violet vexilla excluding nectar guide potions (yellow-coloured potions) were used for a pH measurement. Vexilla from 7 to 10 florets per inflorescence were mashed, and the pH of the homogenate was measured using a compact pH meter (B-212; Horiba, Ltd., Kyoto, Japan). Five inflorescences were measured and the average ± SE was obtained. 4.5. Cross-TLC Petals (48 mg dry weight) were extracted with 2 mL of 10% acetic acid. A cellulose TLC plate (200 mm 200 mm, Merck, Germany) was cut into a 100 mm 100 mm square. The extract was slantingly cross loaded on the TLC plate. About 25 mL of the extract was loaded using a 10 mL micro syringe, keeping 15 mm margins in four corners. The TLC plate was developed with solvent of EtOH:10% acetic acid ¼ 3:7. Observation under ultraviolet lights was performed in a dark chamber with an ultraviolet light irradiation system (CSN-15 AC/AC, Cosmo Bio, Japan). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.02.004. References Elving, P.J., Markowitz, J.M., Rosenthal, I., 1956. Preparation of buffer systems of constant ionic strength. Anal. Chem. 28 (7), 1179e1180.
5
Harborne, J.B., 1984. Phytochemical Methods, second ed. Chapman and Hall, London. Harborne, J.B., Baxter, H., 1999. The Handbook of Natural Flavonoids, vol. 2. John Wiley & Sons, Chichester. Harborne, J.B., Grayer, R.J., 1988. The flavonoids. In: Harborne, J.B. (Ed.), Advance in Research Since 1980. Chapman and Hall, London, pp. 1e20. Ishikura, N., Ito, S., Shibata, M., 1978. Paper Chromatographic Survey of Anthocyanins in Leguminosae. In: III. Identification and Distribution Pattern of Anthocyanins in Twenty-two Legumes. Bot. Mag, Tokyo 91, pp. 25e30. Andersen, Ø.M., Jordheim, M., 2006. The Anthocyanins. In: Andersen, Ø.M., Markham, K.R. (Eds.), Flavonoids: Chemistry, Biochemistry and Applications. CRC Press, Boca Raton, pp. 471e551. Kurihara, T., Kikuchi, M., 1975. Studies on the constituents of flowers. V. On the components of flower of Pueraria thunbergiana Benth. (2). Isolation of a new isoflavone glycoside. Yakugaku Zasshi 95 (11), 1283e1285 (Japanese). Nakayama, M., Koshioka, M., Shibata, M., Hiradate, S., Sugie, H., Ymaguchi, M., 1997. Identification of cyanidin 3-O-(3”,6”-O-dimalonyl-b-glucopyranoside) as a flower pigment of chrysanthemum (Dendranthema grandiflorum). Biosci. Biotech. Biochem. 61 (9), 1607e1608. Nakayama, M., Ymaguchi, M., Urashima, O., Kan, Y., Fukui, Y., Yamaguchi, Y., Koshioka, M., 1999. Anthocyanins in the dark purple anthers of Tulipa gesneriana: identification of two novel delphinidin 3-O-(6-O-(acetyl-a-rhamnopyranosyl)-b-glucopyranosides). Biosci. Biotech. Biochem. 63 (8), 1509e1511. Nakayama, M., Koshioka, M., Yoshida, H., Kan, Y., Fukui, Y., Koike, A., Ymaguchi, M., 2000. Cyclic malyl anthocyanins in Dianthus caryophyllus. Phytochemistry 55, 937e939. Niiho, Y., Yamazaki, T., Nakajima, Y., Itoh, H., Takeshita, T., Kinjo, J., Nohara, T., 1990. Pharmacological study on Puerariae flos. II. The effects of Puerariae flos on alcohol-induced unusual metabolism and experimental liver injury in mice. Yakugaku zasshi 110 (8), 604e611 (Japanese). Saito, N., Tatsuzawa, F., Nishiyama, A., Yokoi, M., Shigihara, A., Honda, T., 1995. Acylated cyanidin 3-sambubioside-5-glucosides in Matthiola incana. Phytochemistry 38, 1027e1032. Saito, N., Tatsuzawa, F., Honda, T., 2015. Light absorption spectral patterns of intact garden flowers in relation to the flower colors and anthocyanin pigments. Heterocycles 90 (1), 41e84. Shimizu-Yumoto, H., Hayashi, N., Ichimura, K., Nakayama, M., 2012. Slantingly cross loading sample system enables simultaneous performance of separation and mixture to detect molecular interactions on thin-layer chromatography. J. Chromatogr. A 1245, 183e189. Tatsuzawa, F., Shinoda, K., 2005. Comparison between identification of anthocyanin by HPLC analysis with a photodiode array detector and that using TLC combined with UV-Vis spectral analysis. Hort. Res. 4 (2), 225e228 (Japanese). Tatsuzawa, F., Saito, N., Yukawa, T., Honda, T., Shinoda, K., Kato, K., Miyoshi, K., 2014. Acylated cyaniding 3,7-diglucosides in the red-purple flowers of Sophronitis wittigiana (Orchidaceae). J. Jpn. Soc. Hort. Sci. 83 (1), 64e71. Veitch, N.C., Grayer, R.J., 2008. Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 25, 555e611. Veitch, N.C., Grayer, R.J., 2011. Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 28, 1626e1695.
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae) Fumi Tatsuzawa a, *, Natsu Tanikawa b, Masayoshi Nakayama b a b
Laboratory of Olericultural and Floricultural Science, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan NARO Institute of Floricultural Science, Tsukuba, Ibaraki 305-0852, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 December 2016 Received in revised form 9 January 2017 Accepted 4 February 2017 Available online xxx
A previously undescribed acylated anthocyanin was extracted from the red-purple flowers of Pueraria lobata with 5% HOAc-H2O, and determined to be petunidin 3-O-(b-glucopyranoside)-5-O-[6-O(malonyl)-b-glucopyranoside], by chemical and spectroscopic methods. In addition, two known acylated anthocyanins, delphinidin 3-O-(b-glucopyranoside)-5-O-[6-O-(malonyl)-b-glucopyranoside] and malvidin 3-O-(b-glucopyranoside)-5-O-[6-O-(malonyl)-b-glucopyranoside] were identified. Delphinidin 3,5di-glucoside, petunidin 3,5-di-glucoside, and malvidin 3,5-di-glucoside, have been known as major components of P. lobata in the former study. However, malonyl esters amounts were detected over 10 times compared with non-malonyl esters amounts. In those anthocyanins the most abundant anthocyanin was petunidin 3-O-(b-glucopyranoside)-5-O-[6-O-(malonyl)-b-glucopyranoside] in total flowers. On the visible absorption spectral curve of fresh red-purple petals, one characteristic absorption maximum was observed at 520 nm, which is similar to those of flowers containing pelargonidin derivatives. In contrast, the absorption spectral curve of old violet petals was observed at 500(sh), 536, 564(sh), and 613(sh) nm, which are similar to those of violet flowers containing delphinidin-type pigments. Pressed juices of both fresh red-purple petals and old violet petals had pH5.2 and 5.5 respectively, and had the same flavonoid constitution. Crude fresh red-purple petal pigments extracted by pH 2.2 and pH 5.2 buffers exhibited the same color and spectral curves as fresh red-purple petals and old violet petals, respectively. Moreover, in a cross-TLC experiment of crude extracted pigments, red-purple color was exhibited by the anthocyanin region and the crossed region of anthocyanins and isoflavone. Thus, it may be assumed that the unusually low pH in the vacuole of fresh petals plays an important role to form red-purple flower color against weak acidic pH in the vacuole of old violet P. lobata petals. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Pueraria lobata Leguminosae Malonylated anthocyanin Red-purple flower color Low pH
1. Introduction Pueraria lobata (Willd.) Ohwi (Leguminosae) is native to Japan and China and has an attractive red-purple flower color. These flowers have been mentioned in poetry, haiku, and other Japanese literature as ornamental plants. P. lobata flowers are also used in Chinese traditional medicine as a remedy for various conditions, including hangover, inappetence, and vomiting, and their alcoholic extract has been shown to contain several isoflavones (Niiho et al., 1990). The methanol extract of these flowers has been shown to contain kakkalide as the main component (Kurihara and Kikuchi, 1975). With regard to the anthocyanin components, the presence
* Corresponding author. E-mail address: [email protected] (F. Tatsuzawa).
of 3,5-diglucosides of delphinidin, petunidin, and malvidin in the methanolic HCl extract of flowers has been observed by paper chromatographic analysis of anthocyanins in the Leguminosae (Ishikura et al., 1978). It is known that the anthocyanins acylated by aliphatic acid are easily hydrolyzed within a short time by methanolic HCl extraction (Harborne and Grayer, 1988). Recently, acylated anthocyanins with aliphatic acid have been reported from several flowers, including carnation, chrysanthemum, tulip, and stock, from which non-acylated anthocyanins were formerly reported (Nakayama et al., 1997, 1999, 2000; Saito et al., 1995). Thus, there is a possibility of the existence of acylated pigments in P. lobata flowers. In the present study, structural elucidation of anthocyanins of P. lobata extracted by a weak acid revealed malonylated anthocyanins as the major component. The color of fresh P. lobata flowers changes from red-purple to violet after a few days in nature. Therefore, we compared anthocyanin components, their
http://dx.doi.org/10.1016/j.phytochem.2017.02.004 0031-9422/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
2
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
co-pigments, and the pH values of flower tissues between fresh red-purple flowers and old violet flowers. Finally, we assumed that unusually low pH in the vacuole is an important factor contributing to the color of P. lobata flowers. 2. Results and discussion Three unknown major peaks 1e3 with three known minor peaks A e C at 530 nm and one known major peak (D) at 350 nm were found in the 5%HOAc (acetic acid-water ¼ 5:95, v/v) (2L) extracts from dried red-purple flowers of Pueraria lobata (100g), by high performance liquid chromatography (HPLC) analysis (method 1) (Fig. 1). The percentage of 1e3 and A e C of Pueraria lobata, calculated as the total content by HPLC vis peak area at 530 nm, was 27.4% (1), 43.4% (2), 12.8% (3), 6.0% (A), 3.8% (B), and 1.7% (C) respectively. Moreover, the percentage of D, calculated as the total content by HPLC peak area at 350 nm, was 41.1%. The six anthocyanin peaks were extracted from the red-purple flowers with 5% HOAc, followed by isolation using Diaion HP-20 (Nippon Rensui Co., Tokyo, Japan) column chromatography (CC), Sephadex™ LH-20 (GE Healthcare UK Ltd., UK) CC and paper chromatography (PC). The chromatographic and spectroscopic properties of 1e3 and A e D are summarized in supplementary information. Acid hydrolysis of 1e3 and A e C yielded delphinidin (1 and A) (Forestal 0.23), petunidin (2 and B) (Forestal 0.39), and malvidin (3 and C) (Forestal 0.56), as their aglycone, respectively (Harborne, 1984), while also affording, glucose (BAW 0.21, EAA 0.33, ETN 0.83, EFW 0.52) was only detected as sugar from 1e3 and A e C (Harborne, 1984). Moreover, malonic acid was detected in the hydrolysates of pigments 1e3 by HPLC (method 2) (Tatsuzawa et al., 2014). These anthocyanidins, glucose, and malonic acid were identified by direct comparison with commercial standards (Wako Pure Chemical Industries Co. Ltd., Tokyo, Japan). Alkaline hydrolysis of 1e3 yielded A e C, respectively. The deacylated anthocyanin structures of 1e3 (¼ A e C) tentatively identified via co-TLC and co-HPLC (method 1) with authentic 3,5diglucosides of delphinidin, petunidin, and malvidin, respectively (Tatsuzawa and Shinoda, 2005). From these results, the structures of A e C were identified to be delphinidin 3, 5-diglucoside,
Fig. 1. HPLC profile of extracts from red-purple flowers of Pueraria lobata. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
petunidin 3,5-diglucoside, and malvidin 3,5-diglucoside, respectively, and 1e3 were presumed to be malonylated delphinidin 3,5diglucoside, malonylated petunidin 3,5-diglucoside, and malonylated malvidin 3,5-diglucoside, respectively. The peak D in Fig. 1 was presumed to be kakkalide (irisolidone 7O-(6-O-xylosyl)-glucoside) which was known as major isoflavone in the flowers of P. lobata (Kurihara and Kikuchi, 1975). The structures of 1e3 and D were further elucidated as follows based on the analyses of their 1H (400 MHz), 13C (100 MHz) and 2D (COSY, NOESY, 1He13C HMQC and 1He13C HMBC) NMR (JNM ALe400, JEOL Ltd., Tokyo, Japan) spectra in CD3ODeCF3COOD (9:1) for 1e3 and DMSO-d6 for D with TMS as an internal standard, as well as their high resolution fast atom bombardment mass spectra (HReFABMS) (LMSe700, JEOL Ltd.). 2.1. Pigment 2 The molecular ion [M]þ of 2 was observed at m/z 727(C31H35O20) indicating that 2 was composed of petunidin with two molecules of glucose and one molecule of malonic acid. The elemental components were confirmed by measuring its HR-FABMS, and the mass data was summarized in section 4.3.1. The structure was elucidated based on the analysis of the NMR spectra (Table 1). The chemical shifts of 5 aromatic protons of petunidin moiety with their coupling constants were assigned as shown in Table 1. The chemical shifts of the sugar moieties were observed in the
Table 1 NMR spectroscopic data of anthocyanin 2 from the flowers of Pueraria lobata in CD3OD-d4/CF3COOD (9:1). 2 1
H (ppm)
Petunidin 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 30 eCH3 Glucose A 1 2 3 4 5 6a 6b Glucose B 1 2 3 4 5 6a 6b Malonic acid eOCH2 COOH COOH
9.11 s 7.02 d(2.0) 7.09 d(2.0)
8.00 d(2.2)
7.83 d(2.2) 4.00 s
13
C (ppm)
164.7 147.0 135.8 156.7 106.2 169.6 97.5 157.3 113.6 119.9 109.8 150.0 146.4 147.7 114.3 57.4
5.36 3.71 3.58 3.42 3.66 3.62 3.99
d(7.8) t(8.5) t(9.0) t(9.3) ddd(2.5, 7.0, 10.0) m dd(2.0, 10.5)
104.0 74.9 77.7 71.5 79.1 62.7
5.19 3.74 3.59 3.48 3.82 4.36 4.57
d(7.8) t(8.2) t(8.9) t(8.8) ddd(2.4, 6.9, 10.0) dd(6.6, 12.0) dd(2.0, 12.0)
102.4 74.5 78.5 71.5 76.0 65.3
3.47s
49.3 168.7 169.6
Coupling constants (J in Hz) in parentheses.
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
region of d 5.36e3.42, where the two anomeric protons exhibited at d 5.36 (d, J ¼ 7.8 Hz, glucose A) and d 5.19 (d, J ¼ 7.8 Hz, glucose B). Based on the observed coupling constants (Table 1), these two glucoses were assumed to be in the b-pyranose form. Based on results of analyses of their COSY spectra, characteristic methylene proton signals [d 4.36 and 4.57] being shifted to lower magnetic field was assigned to H-6a and H-6b protons of glucose B. This result indicated that the OH-6 of glucose B must be acylated with malonic acid. NOESY and HMBC spectra were used to determine the sites of attachment of the sugars and petunidin moieties (Fig. 2). The signal of H-1 (d 5.36) of glucose A was correlated to the signal of C-3 (d 147.0) carbon of petunidin in its HMBC spectrum, and to the signals of the H-4 (d 9.11) proton of petunidin in NOESY spectrum. The signal of H-1 (d 5.19) of glucose B correlated to the signal of the C-5 (d 156.7) carbon of petunidin in the HMBC spectrum, and to the signals of the H-6 (d 7.02) proton of petunidin in NOESY spectrum. These characteristic features revealed that the OH-3 and OH-5 positions are bonded to glucose A and glucose B, respectively (Fig. 2). The signal of H-6a and -6b (d 4.36 and 4.57) of glucose B was correlated to the signal of the COOH (d 168.7) carbon of malonic acid in the HMBC spectrum (Fig. 2). Therefore, the OH-6 of glucose B is bonded with malonic acid. Consequently, the structure of 2 was determined to be petunidin 3-O-(b-glucopyranosyl)-5-O-[6-O-(malonyl)-b-glucopyranoside] (Fig. 2), which is a previously undescribed anthocyanin in plants (Andersen and Jordheim, 2006; Harborne and Baxter, 1999; Veitch and Grayer, 2008, 2011).
3
by paper chromatographic analysis (Ishikura et al., 1978). However, in the present study, we revealed, using HPLC, that 5% HOAc extracts of the red-purple P. lobata flowers contain 3-O-glucoside-5O-[6-O-(malonyl)-glucoside] form of delphinidin, petunidin, and malvidin (1e3) as major anthocyanins and 3,5-diglucosides of delphinidin, petunidin, and malvidin (A e C) as minor anthocyanins (Fig. 1). On the other hand, kakkalide (D), which was extracted with 5%HOAc, did not contain aliphatic acids in its chemical structure similar to the methanol extracts reported previously (Kurihara and Kikuchi, 1975). The florets of P. lobata open from the bottom to the top in an inflorescence (Fig. 3). The color of fresh florets is red-purple. When the florets become older, the petals turn violet (Fig. 3). The fresh florets have red-purple vexilla [Red-Purple 71B by Royal Horticultural Society Colour Chart; chromaticity value, b*(-5.77)/ a*(33.58) ¼ 0.17, L* ¼ 53.45 by a CM-700d Spectro Color Meter (Konica-Minolta, Tokyo, Japan); anthocyanin content by HPLC: 36.2%(1), 45.2%(2), 6.2%(3), 4.9%(A), 1.6%(B), and 0.8%(C) ], and deep red-purple alae [Red-Purple 71A; chromaticity value, b*(2.60)/ a*(3.71) ¼ 0.70, L* ¼ 68.47; anthocyanin content by HPLC: 38.2%(1), 42.3%(2), 5.8%(3), 5.3%(A), 1.4%(B), and 0.5%(C)] and carinas [RedPurple 71A; chromaticity value, b*(-2.82)/a*(13.77) ¼ 0.20, L* ¼ 58.23; anthocyanin content by HPLC: 47.0%(1), 33.3%(2), 4.6%(3), 6.7%(A), 1.5%(B), and 0.3%(C) ] (Fig. 3). The flower's color and anthocyanin components were almost the identical in the vexilla, alae, and carinas. Therefore, for studing flower color reconstruction, we used vexilla as the characteristic structure of
3. Concluding remarks In the present study, we revealed that petunidin 3-O-glucoside5-O-[6-O-(malonyl)-glucoside](2) is a novel anthocyanin. Furthermore, we were the first to identify the two anthocyanins, delphinidin 3-O-glucoside-5-O-[6-O-(malonyl)-glucoside](1) and malvidin 3-O-glucoside-5-O-[6-O-(malonyl)-glucoside](3) in the genus Pueraria (Andersen and Jordheim, 2006; Harborne and Baxter, 1999; Veitch and Grayer, 2008, 2011). In a previous floral anthocyanin study, 3,5-diglucosides of delphinidin, petunidin, and malvidin from 1% methanolic HCl extracts of P. lobata flowers were identified as major components of 14 species of leguminous plants
Fig. 2. Structures of anthocyanins 1e3 (1: R1 ¼ R2 ¼ OH, 2: R1 ¼ OCH3, R2 ¼ OH, 3: R1 ¼ R2 ¼ OCH3) and isoflavone D from the red-purple flowers of Pueraria lobata. Observed main NOEs are indicated by solid arrows. Observed main HMBCs are indicated by dotted arrows.
Fig. 3. Visible spectra of flower tissue pigments of Pueraria lobata. Fresh vexilla [520 nm]. Crude extracted pigments in the pH 2.2 buffer solution (phosphate-citrate buffer (MacIlvaine)) [520 nm]. Crude extracted pigments in the pH 5.2 buffer solution [500sh, 536sh, 564, 613sh nm]. Crude extracted pigments in the pH 5.6 buffer solution [500sh, 536sh, 564, 613sh nm]. Pressed fresh vexilla [500sh, 536, 564sh, 613sh nm]. Vexilla of old flowers [500sh, 536, 564sh, 613sh nm].
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
4
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
P. lobata. The spectral absorption curves of red-purple vexilla of P. lobata exhibited an absorption maximum at 520 nm, which is similar to the value of flowers containing pelargonidin derivatives (Saito et al., 2015). This was the same absorption maximum exhibited by the crude extract solution of red-purple vexilla in a buffer solution of pH 2.2 [the lowest pH for McIlvaine buffer (phosphate-citrate buffer)] (Elving et al., 1956) containing flavylium cation form of anthocyanins (Fig. 3). However, the pH value of fresh vexilla homogenate was pH 5.2 ± 0.0 (n ¼ 5). Fresh red-purple vexilla turned violet after mashing [Violet N87A by RHS CC. and hue: b*(-13.97)/a*(12.65) ¼ 1.07, L* ¼ 63.21]. The spectral absorption curves of mashed vexilla and those of old-flowers [Violet 83D by RHS CC. and hue: b*(-14.12)/a*(12.67) ¼ 1.12, L* ¼ 63.12; pH 5.5 ± 0.0 (n ¼ 5); anthocyanin content by HPLC: 25.5%(1), 45.8%(2), 11.4%(3), 5.5%(A), 3.7%(B), and 1.0%(C)] exhibited one major absorption peak (536 nm: flavylium cation) and two shoulders (546 nm: neutral quinonoidal form, and 613 nm: ionized quinonoidal form, Fig. 3). Absorption spectral curves with three absorption maxima are mainly observed in the absorption spectral curves of violet to blue flowers and in those of their anthocyanin solutions under weak acidic conditions, caused by co-pigmentation or self-association of delphinidin-type pigments (Saito et al., 2015). These results suggested that the pH of cell vacuole of fresh P. lobata flowers is more acidic than that of freshly mashed petals and old violet flowers. An additional possibility is that the flower color and spectral curve were changed by cytoplasmic substances, which provided a co-pigment effect, when petals were mashed. The cross-TLC experiment is an effective technique to easily and comprehensively confirm co-pigmentation between anthocyanins and co-pigments (Shimizu-Yumoto et al., 2012). Slantingly cross loading the pigment mixture or crude extracted pigments on TLC enables compounds to symmetrically develop at various angular lines from the upper origin to individual Rf values and cross each other in an orderly manner, while the mixing is simultaneously performed with separation (Shimizu-Yumoto et al., 2012). The occurrence of co-pigmentation can be detected as a change in color on the developed line of anthocyanin. Crude extracted pigments of P. lobata were analyzed by cross-TLC, and a pale blue spot different from flower color on the anthocyanin line (Rf value: 0.64) was detected under visible ray. This pale blue spot observed under ultraviolet rays crossed with hydroxycinnamic acids, such as caffeic acid derivatives (Rf value: 0.73). It is known that hydroxycinnamic acids, such as caffeic acid derivatives, exist, however, they are absent in the vacuole of superficial cells of petals containing anthocyanins. Therefore, the bluing effect by hydroxycinnamic acids, such as caffeic acid derivatives, occurred because the crude extract was mixed with hydroxycinnamic acids present outside the vacuole of P. lobata flowers. On the other hand, during the cross-TLC experiment of crude extract of P. lobata under visible ray, the crossing points of anthocyanin and kakkalide (Rf value: 0.82), which are the major components of crude extract (Fig. 1), exhibited red-purple colors. These colors were similar to those exhibited by TLC on anthocyanins of fresh petals. Moreover, the spectral absorption curve of mixture of the purified anthocyanins (1e3) and kakkalide (D) exhibited only one major absorption maximum at 520 nm, which is the same absorption maximum of crude extracted pigments in the pH 2.2 buffer and fresh vexilla. Therefore, evidence for kakkalide acting as a co-pigment was not obtained. Thus, based on the red-purple flower color of P. lobata, it can be concluded that the anthocyanins present in the vacuole do not interact with other compounds. From the above mentioned results, we concluded that the violet flower colors of fresh mashed petals and old violet flowers were caused by mixture of hydroxycinnamic acids, such as caffeic acid derivatives of the cytoplasm, and/or by the weak acidic conditions
of the vacuole. Therefore, it may be assumed that the unusually low pH condition in the vacuole is an important factor contributing to the red-purple color of P. lobata flower. 4. Experimental 4.1. General procedures TLC was carried out on plastic coated cellulose sheets (Merck) using eight mobile phases: BAW (n-BuOH-HOAc-H2O, 4:1:2, v/v/v), BuHCl (n-BuOH-2N HCl, 1:1, v/v, upper layer), AHW (HOAc-HClH2O, 15:3:82, v/v/v), 1% HCl for anthocyanins and isoflavone with UV light, Forestal (HOAc-HCl-H2O, 30:3:10, v/v/v) for anthocyanidin and BAW, EAA (EtOAc-HOAc-H2O, 3:1:1, v/v/v), ETN (EtOH-NH4OHH2O, 16:1:3, v/v/v) and EFW (EtOAc-HCOOH-H2O, 5:2:1, v/v/v) for sugars with aniline hydrogen phthalate spray reagent (Harborne, 1984). Analytical HPLC was performed on a LC 10A system (Shimadzu), using a Waters C18 (4.6 250 mm) column at 40 C with a flow rate of 1 mL/min and monitoring at 530 nm for anthocyanins and 350 nm for isoflavone and other flavonoids. The eluant was applied as a linear gradient elution for 40 min from 20 to 85% solvent B (1.5% H3PO4, 20% HOAc, 25% MeCN in H2O) in solvent A (1.5% H3PO4 in H2O) with 5 min of re-equilibration at 20% solvent B (method 1). The other eluant for malonic acid was applied as an isocratic elution of solvent A for 10 min and monitoring at 210 nm (Tatsuzawa et al., 2014; method 2). UV-Vis spectra were recorded on UV-Vis Multi Purpose Spectrophotometer (MPS-2450, Shimadzu, Kyoto, Japan) in 0.1% HClMeOH (from 200 to 700 nm) for anthocyanins and in MeOH, MeOH þ NaOMe, MeOH þ AlCl3, MeOH þ AlCl3þHCl, MeOH þ NaOAc, MeOH þ NaOAc þ H3BO3 (from 200 to 500 nm) for isoflavone. Spectral absorption of flowers directly measured on intact petals sandwiched in a pierced (5 mm) black holder using a recording spectrophotometer operated as a double-beam instrument (Type MPS-2450). High resolution FAB mass (FABMS) spectra were determined on a JEOL JMSe700 Mass spectrometer (JEOL, Ltd.) operating in the positive ion mode using 1:1 mixture of dithiothreitol and 3nitrobenzyl alcohol as a matrix. 1H (400 MHz) and 13C (100 MHz) NMR spectra were measured on a JEOL ALe400 NMR spectrometer (JEOL, Ltd.) using CD3ODeCF3COOD (9:1) and DMSO-d6 as a solvent. Chemical shifts are reported on the d-scale from tetramethylsilane as the internal standard, and coupling constants (J) are in Hz. 4.2. Plant materials The red-purple flowers of Pueraria lobata (Willd.) Ohwi (Legminosae) [Red-Purple 71B by Royal Horticultural Society Colour Chart and chromaticity value, b*(-5.77)/a*(33.58) ¼ 0.17, L* ¼ 53.45 by a CM-700d Spectro Color Meter (Konica-Minolta, Tokyo, Japan)] were collected in autumun (from 2015 to 2016) in The Botanical Garden, Faculty of Agriculture, Iwate University. The petals were trimmed from flowers by hand and dried by air for 1 day at 45 C. Then they were kept at 20 C until used. 4.3. Isolation and purification of 1e3 and AeD Dried flowers of P. lobata (100 g) were immersed in 5% HOAc (acetic acid-water ¼ 5:95, v/v) (5 L), respectively, at room temperature for 24 h and extracted. 1e3 and A e D were isolated and purified by Diaion HP-20 (Nippon Rensui Co. Ltd., Tokyo, Japan) column (90 150 mm) chromatography (CC), Sephadex™ LH-20 (GE Healthcare UK Ltd., UK) CC and paper chromatography (PC). The purified 1e3 and A e D were obtained from the flowers as
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004
F. Tatsuzawa et al. / Phytochemistry xxx (2017) 1e5
follows; 1 (ca. 10 mg), 2 (ca. 27 mg), 3 (ca. 8 mg), A (ca. 2 mg), B (ca. 3 mg), C (ca. 1 mg), and D (ca. 130 mg). 4.3.1. Petunidin 3-O- (b-glucopyranosyl)-5-O-[6-O-(malonyl)-bglucopyranoside] (2) Red powder: UV-VIS (in 0.1% HCl-MeOH): lmax 536, 273 nm, E440/E536 (%) ¼ 14, AlCl3 shift þ; TLC: (Rf-values) BAW 0.50, BuHCl 0.11, 1% HCl 0.10, AHW 0.39; HPLC: Rt (min) 21.1. HReFABMS; [M]þ calc. for C31H35O20: 727.1722. found: 727.1734. 4.4. Measurement of pH of petal homogenates Fresh red-purple vexilla and old violet vexilla excluding nectar guide potions (yellow-coloured potions) were used for a pH measurement. Vexilla from 7 to 10 florets per inflorescence were mashed, and the pH of the homogenate was measured using a compact pH meter (B-212; Horiba, Ltd., Kyoto, Japan). Five inflorescences were measured and the average ± SE was obtained. 4.5. Cross-TLC Petals (48 mg dry weight) were extracted with 2 mL of 10% acetic acid. A cellulose TLC plate (200 mm 200 mm, Merck, Germany) was cut into a 100 mm 100 mm square. The extract was slantingly cross loaded on the TLC plate. About 25 mL of the extract was loaded using a 10 mL micro syringe, keeping 15 mm margins in four corners. The TLC plate was developed with solvent of EtOH:10% acetic acid ¼ 3:7. Observation under ultraviolet lights was performed in a dark chamber with an ultraviolet light irradiation system (CSN-15 AC/AC, Cosmo Bio, Japan). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.02.004. References Elving, P.J., Markowitz, J.M., Rosenthal, I., 1956. Preparation of buffer systems of constant ionic strength. Anal. Chem. 28 (7), 1179e1180.
5
Harborne, J.B., 1984. Phytochemical Methods, second ed. Chapman and Hall, London. Harborne, J.B., Baxter, H., 1999. The Handbook of Natural Flavonoids, vol. 2. John Wiley & Sons, Chichester. Harborne, J.B., Grayer, R.J., 1988. The flavonoids. In: Harborne, J.B. (Ed.), Advance in Research Since 1980. Chapman and Hall, London, pp. 1e20. Ishikura, N., Ito, S., Shibata, M., 1978. Paper Chromatographic Survey of Anthocyanins in Leguminosae. In: III. Identification and Distribution Pattern of Anthocyanins in Twenty-two Legumes. Bot. Mag, Tokyo 91, pp. 25e30. Andersen, Ø.M., Jordheim, M., 2006. The Anthocyanins. In: Andersen, Ø.M., Markham, K.R. (Eds.), Flavonoids: Chemistry, Biochemistry and Applications. CRC Press, Boca Raton, pp. 471e551. Kurihara, T., Kikuchi, M., 1975. Studies on the constituents of flowers. V. On the components of flower of Pueraria thunbergiana Benth. (2). Isolation of a new isoflavone glycoside. Yakugaku Zasshi 95 (11), 1283e1285 (Japanese). Nakayama, M., Koshioka, M., Shibata, M., Hiradate, S., Sugie, H., Ymaguchi, M., 1997. Identification of cyanidin 3-O-(3”,6”-O-dimalonyl-b-glucopyranoside) as a flower pigment of chrysanthemum (Dendranthema grandiflorum). Biosci. Biotech. Biochem. 61 (9), 1607e1608. Nakayama, M., Ymaguchi, M., Urashima, O., Kan, Y., Fukui, Y., Yamaguchi, Y., Koshioka, M., 1999. Anthocyanins in the dark purple anthers of Tulipa gesneriana: identification of two novel delphinidin 3-O-(6-O-(acetyl-a-rhamnopyranosyl)-b-glucopyranosides). Biosci. Biotech. Biochem. 63 (8), 1509e1511. Nakayama, M., Koshioka, M., Yoshida, H., Kan, Y., Fukui, Y., Koike, A., Ymaguchi, M., 2000. Cyclic malyl anthocyanins in Dianthus caryophyllus. Phytochemistry 55, 937e939. Niiho, Y., Yamazaki, T., Nakajima, Y., Itoh, H., Takeshita, T., Kinjo, J., Nohara, T., 1990. Pharmacological study on Puerariae flos. II. The effects of Puerariae flos on alcohol-induced unusual metabolism and experimental liver injury in mice. Yakugaku zasshi 110 (8), 604e611 (Japanese). Saito, N., Tatsuzawa, F., Nishiyama, A., Yokoi, M., Shigihara, A., Honda, T., 1995. Acylated cyanidin 3-sambubioside-5-glucosides in Matthiola incana. Phytochemistry 38, 1027e1032. Saito, N., Tatsuzawa, F., Honda, T., 2015. Light absorption spectral patterns of intact garden flowers in relation to the flower colors and anthocyanin pigments. Heterocycles 90 (1), 41e84. Shimizu-Yumoto, H., Hayashi, N., Ichimura, K., Nakayama, M., 2012. Slantingly cross loading sample system enables simultaneous performance of separation and mixture to detect molecular interactions on thin-layer chromatography. J. Chromatogr. A 1245, 183e189. Tatsuzawa, F., Shinoda, K., 2005. Comparison between identification of anthocyanin by HPLC analysis with a photodiode array detector and that using TLC combined with UV-Vis spectral analysis. Hort. Res. 4 (2), 225e228 (Japanese). Tatsuzawa, F., Saito, N., Yukawa, T., Honda, T., Shinoda, K., Kato, K., Miyoshi, K., 2014. Acylated cyaniding 3,7-diglucosides in the red-purple flowers of Sophronitis wittigiana (Orchidaceae). J. Jpn. Soc. Hort. Sci. 83 (1), 64e71. Veitch, N.C., Grayer, R.J., 2008. Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 25, 555e611. Veitch, N.C., Grayer, R.J., 2011. Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep. 28, 1626e1695.
Please cite this article in press as: Tatsuzawa, F., et al., Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae), Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.004