Quantitation of Anthocyanins in an Exotic Myrmecophyte via UV/Vis SpectroscopyV. George, J. Camillo, F. James, M. Dotseth, and V. Carmona-Galindo

Biology Department | Loyola Marymount University | Los Angeles, CA 90045

LMU|LAFrank R. Seaver Collegeof Science and Engineering

Figure 6. Method 4

Figure 1. Castor bean plants prefer disturbed sites. Figure 2. Biotic & Chemical defense strategies.

Figure 3. Action Spectrum of Anthocyanins

Introduction

Methods

Optimal Plant Defense Theory

Literature Cited

• The Optimal Defense Theory (ODT) suggest that plants exert energy to protect themselves through either chemical (poison, repellents, etc.) or physical means (thorns, hard exterior, etc.)•Plants may also use animals such as ants as a protection by

providing the ants with nutrients and/or protection• The theory assumes that the energy put forth into protection

cannot be used simultaneously for another function• Essentially the plant must choose whether to divert its nutrient

resources into growth or defense•Defenses are beneficial in comparison to an undefended organism

and enhance fitness when predators and parasites are present.•Defenses can be costly and reduce fitness in comparison to an

undefended plant when no predators and parasites are present• The allocation to defenses within a plant will depend on whether,

and how often, the plant part is attacked and how valuable the plant part is to fitness.

•Anthocyanins are vacuolar pigments that help plants in terms of physiology and defense.• Photoinhibition, which causes lignin degradation and high-light

stress, is prevented by anthocyanin production.•Anthocyanins absorb UV radiation.• Exotic myrmecophyte (ant-loving plant), Ricinus communis L.,

commonly called the castor bean plant, produces anthocyanins to survive high levels of UV bombardment.•Anthocyanins in castor bean stems and leaves give the plant a

characteristic red coloration.• Castor bean plants also produce specialized extra-floral nectar

glands along their leaves and stems, which attract ants.•Ants provide protection for myrmecophytes such as the castor

bean plant.• Castor bean plants follow the principle of allocation, which means

that resources must be allotted between chemical defense (anthocyanins) and biotic defense (ant-attraction).• The intent of this study is to evaluate investment tradeoffs in

chemical and biotic plant-defense strategies in Castor bean growing in non-native habitats in southern California.• Four methodologies are being compared to determine which is

most effective in extraction of anthocyanins and measuring anthocyanin concentration in castor bean leaves and stems via UV/VIS Spectroscopy.

"Anthocyanin Content in Bilberry by pH-Differential Spectrophotometry INA Method 116.000." NSF International. NSF International, n.d. Web. 2 Mar. 2010.

Abdel-Aal, E.-S. M., and P. Hucl. "A Rapid Method for Quantifying Total Anthocyanins in Blue Aleurone and Purple Pericarp Wheats." Cereal Chemistry 76.3 (1999): 350-54. Print.

Barto, E. K., and Don Cipollini. "Testing the Optimal Defense Theory and the Growth-differentiation Balance Hypothesis in Arabidopsis Thaliana." Oecologia 146.2 (2005): 169-78. Link.springer.com. Springer, Part of Springer Science+Business Media, 01 Dec. 2005. Web. 23 Mar. 2014.

Fuleki, T. and Francis, F. J. (1968), Quantitative Methods for Anthocyanins. Journal of Food Science, 33: 72–77.

"Optimal Defense." Life.illinois.edu. National Science Foundation, 12 May 2001. Web. 23 Mar. 2014.Syed Jaafar, S. N., Baron, J., Siebenhandl-Ehn, S., Rosenau, T., Böhmdorfer, S., Grausgruber, H. (2013),

Increased anthocyanin content in purple pericarp × blue aleurone wheat crosses. Plant Breeding, 132: 546–552.

Table 4. Extraction Method by Fuleki et al. 1968

•Prior to extraction: Castor bean plants (3 small bags of stems and leaves) were

collected near University Hall on the LMU campus. Stems and leaves were enveloped in aluminum foil and freeze-

dried for 2.5 days Stems and leaves were ground into powder

•The four methods being compared all include•Extraction solutions consisting of various ratios of ethanol and hydrochloric acid•Adjustment to pH 1.0•Measure absorbance via UV/VIS Spectrometer

•Anthocyanin λmax in the range of 510-535 nm

•Select methods also include•Adjustment to pH 4.5 or pH 5•Shaking•Centrifugation

Extraction Solution Preparation pH 1.0 buffer Dissolve 1.49g KCl in 100mL DI water

Transfer 1.7 mL concentrated HCl to 100 mL DI water

Mix 25 mL KCl solution with 67 mL HCl solution Adjust to pH 1.0 ± 0.1

pH 4.5 buffer Dissolve 1.64 g sodium acetate in 100 mL DI water Adjust to pH 4.5 ± 0.1 with HCl

Extraction Solution

Preparation

o Mix 85 mL 95% ethanol and 15 mL 1.5N HCl

o Blend 100 g frozen sample and 100 mL extraction solution in a Waring blendor.

o Adjust to pH 1.0

o Wash blendor jar with 50 mL extraction solution and transfer mixture to 400 mL beaker.

o Cover beaker with parafilm and store overnight at 4°C.

o Filter mixture on Whatman No. 1 paper through No. 2 Buchner funnel.

o Wash beaker and filter repeatedly with extraction solution until 450 mL extract is obtained.

o Transfer extracts to 500 mL volumetric flask and make up to volume.

o Filter 25 mL extract through a fine porosity sintered glass filter and make up to volume

o store extract in darkness for 2 hr.o Measure absorbance at 535 nm.o T (total) O.D. = O.D. x DV x VFWhere O.D. = absorbance reading of diluted

sampleDV = Diluted VolumeVF = Volume Factor which corrects for the

differences in size between original volume (OV = 100 mL) and sample volume (SV): OV/SV = 100/SV

Extraction Procedure Weigh 75 mg powdered extract. Transfer to 100 mL volumetric flask. Add 80 mL distilled H2O. Sonicate 15 min.

Cool to room temperature. Dilute to volume with H2O, then mix.

Transfer 1 mL solution to 25 mL vol. flask. Dilute to 25 mL with pH 1.0 buffer, mix. Repeat with 1 mL solution for pH 4.5 buffer, mix. Measure absorbance of both solutions using

spectrophotometer at 510 nm and 700 nm.

Calculate absorbance difference between both solutions:Absorbance = (A510nm pH 1 – A700nm pH 1) – (A510nm pH 4.5 – A700nm pH

4.5)

Calculate %w/w total anthocyanins in sample:%w/w = A/ƐL x MW x DF x V/Wt x 100%Where A = AbsorbanceƐ = Cyd-3-glu absorbance (26,900)MW = anthocyanin molecular weight (449.2)DF = dilution factorV = final volume (mL)Wt = sample weight (mg)L = cell pathlength (1 cm)

ExtractionPrepare solutions composed of 85mL ethanol and 15mL of 0.1, 1.0 and 1.5N HCl to vary levels of pH.

Set solution to pH=1 using 4N HCl

Shake solution for 15min. Readjust pH to 1 (if necessary). Shake for additional 15min.

Centrifuge tube at 27,200 x g for 15min.

Pour supernatant into 50-mL volumetric flask. Fill remaining 50-mL with ethanol.

Measure absorbance at 535nm with reagent blank.

Continue to record absorbance for a series of varying solutions.

Calculate concentration of anthocyanins from absorbance by the following equation:C=A/E x (vol/1000) x MW x (1/sample wt.) x 10^6C= concentration, A=absorbance, vol=total vol. anthocyanin extract (50mL)

ExtractionPrepare solvent (methanol/1M HCl, 85:15, pH=0.95 ± 0.05).

Add 8mL of solvent to 1g of sample in 15mL centrifuge tube.

Pre-mix sample on vortex mixer.Put on a shaker for 30min (150 rpm).Centrifuge samples for 5min. at 4000rpm. Decant to separate extract from solids. Repeat for each sample 3 times.

Collect supernatants in 25mL volumetric flask. Adjust volume to 25mL using extraction solvent.

Use spectrophotometer to measure anthocyanin content. Take measurements at 525nm.

Table 1. Extraction Method by Abdel-Aal et al. 1999 Table 2. Extraction Method by Syed Jaafar et al. 2013

Growth-Differentiation Balance Hypothesis • The Growth-Differentiation Balance Hypothesis (GDBH) states

that slow growing plant parts will have more resources available for defense and thus will have higher defense levels than faster growing tissues.

Table 3. Extraction INA Method 116.000 (2010)