Anthocyanin Pigments in Redbud (Cercis spp) Flowers

Redbud (Cercis spp.) is used as a spring flowering ornamental tree and is found wild in much of North America. Typically flowers are light purple although there are selected cultigens that are white, rose, or red-purple. Flowers from cultigens common to the eastern U.S. and from wild Eastern redbud (C. canadensis) were collected and tested for color and anthocyanin pigment composition. The anthocyanins cyanidin 3-glucoside, petunidin 3-glucoside, peonidin 3-glucoside, and malvidin 3-glucoside were most aboundant in purple, rose, and red-purple redbud flowers and total anthocyanin content was 2263 to 8730 mg.kg DW-1. Small amounts of delphinidin, cyanidin, and petunidin 3, diglucosides were also present. Most of the typical purple-flowered redbuds contained cyanidin 3-glucoside as the dominant pigment, while the red-purple flowered ‘Appalachian Red’ and ‘Crosswicks Red’ contained malvidin 3,5-diglucoside as the dominant anthocyanin. An unknown anthocyanin was present in all redbud flowers, and was higher in the red-purple flowered phenotypes. These results show that the color of redbud flowers is from anthocyanins, predominantly cyanidin 3-glucoside and malvidin 3,5-diglucoside, with malvidin 3,5-diglucoside as the primary pigment in red-purple flowers and cyanidin 3-glucoside dominant in purple flowers.


Introduction
Use of plants as sources of anthocyanins for new uses is an emerging area of research. Certain anthocyanins, such as cyanidins, are thought to be more resistant to oxidation and important in human bioactive defense reactions, while other pigments such as malvidins are valued for imparting stable color to fruits and vegetables, as well as for anti-inflammatory effects [1]. Use of plantbased anthocyanins, regarded as safer alternatives to synthetic colorants, has become increasingly common in the food industry [2]. In addition, anthocyanins extracted from flowers are being explored for use as sensitizer dyes in solar cells [3].
Redbud (Cercis spp), is a small tree of the legume family indigenous to the Americas, Europe, and China. It can be found as a wild spring flowering tree in much of North America [4]. C. canadensis var canadensis, the Eastern redbud, is found from the eastern and Midwestern US, north to Toronto and south to Oklahoma and north Texas [5]. Botanical varieties "texensis" and "mexicana" are found in Oklahoma/northern Texas, and in south Texas into Mexico, respectively. Various C. canadensis phenotypes have been selected from the wild for unusual leaf or flower color or modified tree architecture [6]. 'Appalachian Red' (C. canadensis var canadensis) (AR) has flowers with a strong reddish purple color, in contrast to the more common pink-purple color of Eastern redbud ( Figure 1). 'Oklahoma' (OK), a cultivar of C. canadensis var. texaniana, in contrast, has a dark purple-magenta flower color. Weeping cultigens such as 'Traveller' (C. canadensis var. texaniana) (TRV) and 'Ruby Falls' (RF) have flowers that show typical purple color. An understanding of the pigments in redbud flowers will help establish inheritance of flower color in crosses of redbud cultigens.
Information on the anthocyanin content of redbud (Cercis spp.) flowers is lacking. Anti-malarial properties of the leaf and bark of C. siliquastrum have been reported and may be from flavonoids or galloyl triterpenes [7].
This study was done to characterize the amount and type of anthocyanins in redbud flowers among cultigens prevalent in eastern North America.

Plant material
Redbud flowers were collected from 12 cultigens in North Carolina in 2015 and 2016. Flowers from Appalachian Red (AR), Crosswicks Red (CR), Alba (AB), Ace of Hearts (AOH), Flame (FL), Big John (BJ), Kays Early Hope (KEH) and Oklahoma (OK) were collected from the JC Raulston Arboretum in Raleigh NC. Flowers from Forest Pansy (FP), Traveller (TRV), Ruby Falls (RF) and a seedling Eastern redbud (ERB) were collected from 3-5 year old trees in Kannapolis NC. Flowers were carefully selected to make sure they were at optimum bloom stage, with full extension of calyx tube but before petals started to flex. Samples of 50 to 100 g were collected each year, with the exception of FL and AOH, where a sample of 30 g was collected only in 2016. All flowers were held in plastic zipper bags on ice until return to the laboratory. Pedicels and sepals were removed and flowers were weighed.

Flower color parameters and pH
Flower color was determined by filling a 20 ml glass cuvette (93-G-10, Starna, Atascadero CA USA) with flowers. The cuvette was fitted with black cloth containing a hole of 8 cm diameter cut in the center. A Konica Minolta CR400 (Konica Minolta, Ramsey, NJ) was calibrated with a white tile, placed so the aperture was centered over the hole and color measured using Hunter coordinates L * a * b * , illuminant C, and diffuse illumination/0º viewing angle and 2° observer angle. Hue and chroma were calculated using the formula of McGuire [11]. Each cultigen was measured in duplicate except for FL.
Flower and petal pH were determined by microelectrode in triplicate to determine if pH was related to phenotypic color. Petals were removed from subsamples of redbuds when sufficient sample was available to compare to flowers (calyx, petals, stamens, styles), to determine if petal pH differed from that of the whole flower. Petals and flowers were frozen at -20 ºC. Subsamples of frozen flowers or petals were placed in microcentrifuge tubes and crushed with micropestles to release juice. The pH of the juice was determined by placing the tip of a steel pH probe (Hach Co., Loveland CO USA) into the tube.

HPLC analysis
Redbud flowers (5-10 g per sample) were freeze-dried using a VirTis LyoTroll (SP Scientific, Warminster, PA, USA) then were ground with stainless steel balls with  15  a homogenizer (Geno-Grinder, SPEX, Metuchen, NJ USA). Powders were extracted following the method of Bradish, et al. [12] with slight modification. Briefly, 0.04 g of redbud powder was extracted with 1.5 mL of acidified methanol (formic acid:methanol:deionized water, 3:60:37 v/v/v). Samples were vortexed and centrifuged at 10,600 g for 20 min at 4 °C. All samples were re-extracted and supernatants combined to achieve 98% of total anthocyanin present in samples. Supernatant aliquots of 1 ml were filtered through 0.2 µm PTFE membranes (Fisher Scientific, Pittsburg, PA) into 2 ml amber vials (Agilent), flushed with nitrogen gas, capped, and loaded onto an autosampler coupled to a Hitachi LaChrom HPLC (Hitachi Ltd., Tokoyo), equipped with a UV-VS diode array detector (DAD), controlled temperature auto sampler (4 °C), and column compartment (30 °C). D-2000 software (Hitachi Ltd., Tokoyo) was used as the system run controller and for data processing. Three individual samples were extracted for each cultigen.
Data were subjected to one way ANOVA to determine significance of variables relative to genotype. Where significant, genotype means were separated using HSD or Student's t test, P < 0.05. Correlation of anthocyanin and color parameters was done using Pearson's correlation, P < 0.05.

Flower weight, color and pH
Fresh flower weight was highest for FL (0.288 g), followed by KEH, and lowest for AOH (0.012 g) ( Table 1). The remaining cultigens were of similar flower weights. Percent dry weight of flowers averaged 15% for all cultigens (data not shown).
Colorimeter data of redbud flowers showed differences primarily in L * values, with AB highest (most white) with L * of 65 and OK lowest with L * of 38. The values of a, b, chroma and hue did not clearly delineate differences in visual color. CR and AR flowers were highest in hue, about 355° (more red purple) while other values (other than those of 'AB') indicated values that were more purple (about 343°). OK, a visually darker purplemagenta flower Figure 2, could not be distinguished from other cultigens using colorimeter values of a, b, or hue. Chroma was lower for the purple flowered redbuds Petal pH has been implicated in various flowers as important to maintain pigment color in red or blue forms [17]. The pH of all redbud flowers and petals measured was acidic, under a pH of 5.0 table 2 and was slightly higher than that of petals. Among the redbud flowers, the pH was highest for AOH, AB, and AR and lowest for FL. Petal pH ranged from 4.13 in TRV to 4.48 in AB.
The relative amounts of pigments varied widely among redbud cultigens (Table 3 and Figure 2). In AB, only trace amounts of total anthocyanin (CG, UK) were found (Table 3). Total anthocyanin content was highest for RF, followed by OK and AR. TRV, ERB, FP, and AOH were similar in total anthocyanin content. BJ, KEH and FL were lowest in total anthocyanin content. The cultigens KEH and BJ had proportionally more CG and MDG and less MG than the other purple-red flowered    (Table 3).
AR and CR had distinctly reddish-purple flowers, and high amounts of MDG and UK compared to other cultigens ( Figure 2 and Table 3). The visual phenotype resembles that of cyclamen flowers with MDG as the dominant pigment [19]. The unknown pigment may further enhance the reddish color of AR and CR. In contrast, the higher amounts of CG combined with MG in may explain the purple-red cultigen color of the other redbuds. Although DDG can confer a blue or purple color to plant tissues [18], the low amount of DDG among redbud cultigens table 3 may have had little effect on color phenotype.
Cultigens exhibiting red-purple blooms were dominant in MDG (60% of total pigment) and in UK (30% of total pigment) ( Figure 2). Redbud flowers of the typical purple-red color were higher in CG (54-71%) and MG (6 to 20% of total anthocyanin). RF, although similar in color phenotype to other purple-red flowers, had an anthocyanin profile that was between the redpurple CR and AR and the purple-red cultigens ( Figure  2 and Table 3). MG and CG as percent total anthocyanin in RF were about half that of OK, but much higher than that of AR ( Figure 2). In contrast, the percent of MDG in RF was three times higher than OK yet only half that of AR. The more purple color of OK compared to RF may be the result of a high total anthocyanin content with proportionally less MDG and more CG than RF.
Use of reflectance colorimetry sometimes correlates well to total anthocyanin or the major anthocyanins present. Correlations of color values with anthocyanins showed that as total anthocyanin increased, L (darkness) decreased and UK increased (Table 4). Hue was not significantly correlated to any of the anthocyanins. Chroma, or distance from gray tone, was positively but weakly correlated to MDG and UK. Colorimeter a * values were positively correlated with UK and MDG while b * values were negatively correlated with CG and MG and positively correlated to UK. The color parameter a/b, sometimes used for distinguishing color changes with ripeness (red to green/yellow to blue), had the highest correlations with UK and MDG table 4, as the cultigens highest in UK and MDG also were most red and least blue.
The more acidic pH of redbud would indicate that CG and MG would be expressed as a red color [18]. Copigmentation of anthocyanins with flavonols commonly occurs in flowers, and was found in Rhododendron, a flower with cyanidin and malvidin mono and diglucosides [20]. In the present study, only Cercis, anthocya-   Table 3: Anthocyanins identified in redbud flowers, mg·kg DW -1 . * Means separated within column by HSD. Different letters indicate significant differences between means, P < 0.05. nins were analyzed and only with HPLC; use of liquid chromatography-mass spectrometry will be needed to identify flavanols contributing to redbud pigmentation.

Conclusions
The pigment profile of anthocyanins in redbud is presented for several cultigens commonly found in North America. Relative total anthocyanin ranged from almost none in AB to 9.3 g·kg DW -1 among pigmented flowers. Three cultigens with the purple flower phenotype were found to have an anthocyanin profile containing predominantly CG and MG while the reddish-purple phenotype of AR and the pink-red phenotype of CR was found to contain predominantly MDG and an unknown pigment. To our knowledge, this is the first detailed information on anthocyanin pigments in Cercis cultigens found in North America.