Fruit extracts of 10 varieties of elderberry (Sambucus nigra L.) interact differently with iron and copper

Přemysl Mladěnka1a*, Michal Říha1a, Jan Martin1b, Barbora Gorová1a, Aleš

Matějíček2 and Jiřina Spilková1b

1Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Czech Republic, aDepartment of Pharmacology and Toxicology, bDepartment of Pharmacognosy,2 Research and Breeding Institute of Pomology Holovousy Ltd., Czech Republic

SUPPLEMENTARY DATA(Supporting Information)

15 pages

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Table S1. Comparison of copper chelation by bathocuproinedisulphonic acid disodium salt (BCS) method and hematoxylin (HEM) method in case of cyanidin.

  pH 4.5 pH 5.5 pH 6.8 pH 7.5Cu+ (BCS) 1.63 ± 0.29 0.48 ± 0.48 5.49 ± 0.87 4.45 ± 0.51Cu2+ (BCS) 0.62 ± 0.21 2.47 ± 0.10 5.49 ± 0.41 1.82 ± 0.85Cu2+ (HEM) - 43.55 ± 2.58 36.38 ± 2.45 22.50 ± 0.15

The data are showing the percentage of Cu chelation in the ratio 1:1 cyanidin to copper.

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Table S2. Complete statistical analysis of iron and copper chelation.

pH 7.5 (Fe2+, 100 ng)

pH 7.5 (Fe2+, 1 mg)

pH 6.8 (Fe2+, 1 mg)

pH 5.5 (Fe2+, 1 mg)

pH 4.5 (Fe2/3+,1 mg)

pH 5.5 (Cu2+)

pH 6.8 (Cu2+)

pH 7.5 (Cu2+)

cyanidin vs. cyanidin-3-glucoside **** **** **** **** ns ns ns ns cyanidin vs. Aurea **** **** **** ns **** **** ns ** cyanidin vs. Dana ns **** ns * *** ns ns ns cyanidin vs. Haschberg **** **** **** **** **** **** **** **** cyanidin vs. Heidegg 13 **** ns ns **** ** * ns ns cyanidin vs. Korsor **** ns ns ns **** ns ns ns cyanidin vs. Pregarten **** **** ** ns **** ** ns ns cyanidin vs. Riese a.V. **** * ns **** ns ns ns ns cyanidin vs. Samdal *** ns ns **** ns ** ns ns cyanidin vs. Samyl **** ns ** **** ns ns ns ns cyanidin vs. Weihenstephan **** **** ns ** ns ns ns ns cyanidin-3-glucoside vs. Aurea ns **** **** **** **** **** ns * cyanidin-3-glucoside vs. Dana **** **** **** *** *** ns ns ns cyanidin-3-glucoside vs. Haschberg **** **** **** **** **** **** **** **** cyanidin-3-glucoside vs. Heidegg 13 ns **** **** ns ** ** ns ns cyanidin-3-glucoside vs. Korsor ns **** **** **** **** ns ns ns cyanidin-3-glucoside vs. Pregarten **** **** **** **** **** * ns ns cyanidin-3-glucoside vs. Riese a.V. * **** **** ns ns ns ns ns cyanidin-3-glucoside vs. Samdal * **** **** ns ns ** ns ns cyanidin-3-glucoside vs. Samyl ns **** **** ns ns ns ns ns cyanidin-3-glucoside vs. Weihenstephan ns ns **** ** ns * ns ns Aurea vs. Dana **** *** **** **** **** **** ns ns Aurea vs. Haschberg **** ns **** **** *** **** **** * Aurea vs. Heidegg 13 ns **** **** **** **** **** ns * Aurea vs. Korsor ns **** **** *** **** **** ns * Aurea vs. Pregarten **** ns **** ns **** *** ns ns Aurea vs. Riese a.V. *** ** **** **** **** **** ns * Aurea vs. Samdal **** *** **** **** **** **** ns * Aurea vs. Samyl ns **** **** **** **** **** ns ** Aurea vs. Weihenstephan ns **** **** **** **** **** ns ns Dana vs. Haschberg **** ns **** **** **** **** **** **** Dana vs. Heidegg 13 **** **** ns ns ns * ns ns Dana vs. Korsor **** **** ns ns ns ns ns ns Dana vs. Pregarten *** ns ns ns ns * ns ns Dana vs. Riese a.V. **** **** ns ns ns ns ns ns Dana vs. Samdal **** **** ns ns * ** ns ns Dana vs. Samyl **** **** **** ** ** ns ns ns Dana vs. Weihenstephan **** **** ** ns ns ns ns ns Haschberg vs. Heidegg 13 **** **** **** **** **** **** **** **** Haschberg vs. Korsor **** **** **** **** **** **** **** **** Haschberg vs. Pregarten **** ns **** **** **** **** **** **** Haschberg vs. Riese a.V. **** **** **** **** **** **** **** **** Haschberg vs. Samdal **** **** **** **** **** **** **** **** Haschberg vs. Samyl **** **** **** **** **** **** **** **** Haschberg vs. Weihenstephan **** **** **** **** **** **** **** **** Heidegg 13 vs. Korsor ns ns ns ** ns ns ns ns Heidegg 13 vs. Pregarten **** **** ns *** ns **** ns ns Heidegg 13 vs. Riese a.V. **** ** ns ns ns ns ns ns Heidegg 13 vs. Samdal **** ns ns ns ns ns ns ns Heidegg 13 vs. Samyl ns ns **** ns * ns ns ns Heidegg 13 vs. Weihenstephan ns **** *** ns ns ns ns ns Korsor vs. Pregarten **** **** ns ns ns **** ns ns Korsor vs. Riese a.V. **** * ns ** ns ns ns ns Korsor vs. Samdal **** ns ns * ** ns ns ns Korsor vs. Samyl ns ns **** **** ** ns ns ns Korsor vs. Weihenstephan ns **** **** ns ns ns ns ns Pregarten vs. Riese a.V. **** **** ns *** ** **** ns ns Pregarten vs. Samdal **** **** * *** *** **** ns ns Pregarten vs. Samyl **** **** **** **** **** **** ns ns Pregarten vs. Weihenstephan **** **** **** * ** **** ns ns Riese a.V. vs. Samdal ns ns ns ns ns ns ns ns Riese a.V. vs. Samyl * **** **** ns ns ns ns ns Riese a.V. vs. Weihenstephan **** **** ** ns ns ns ns ns Samdal vs. Samyl ** *** **** ns ns ns ns ns Samdal vs. Weihenstephan **** **** * ns ns ns ns ns Samyl vs. Weihenstephan ns **** * ** ns ns ns ns

* p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Data are related to the Figure 2.

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Table S3. Reduction of iron at pH 5.5

reduction slope at pH 5.5 fold less reduction than at pH 4.5*Aurea 0,1164 ± 0,02291 40Dana 0,1872 ± 0,02102 40Haschberg 0,06027 ± 0,04230 23Heidegg 0,2724 ± 0,02429 27Kosor 0,2506 ± 0,02570 24Pregarten 0,1881 ± 0,01243 31Riese 0,3115 ± 0,01011 28Samdal 0,1461 ± 0,01983 36Samyl 0,1110 ± 0,01918 89Weihenstefan 0,09901 ± 0,01258 67

*Ratio between the reduction slope at pH 4.5 and pH 5.5

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Table S4. Origin of the varieties tested in the study

Cultivar Country of origin Latin name

‘Aurea‘ unknown Sambucus nigra ‘Aurea‘

‘Dana‘ Slovakia Sambucus nigra ‘Dana‘

‘Haschberg‘ Austria Sambucus nigra ‘Haschberg‘

‘Heidegg 13‘ Austria Sambucus nigra ‘Heidegg 13‘

‘Korsør‘ Denmark Sambucus nigra ‘Korsør‘

‘Pregarten‘ Austria Sambucus nigra ‘Pregarten‘

‘Riese aus Voβloch‘ Germany Sambucus nigra ‘Riese aus Voβloch‘

‘Samdal‘ Denmark Sambucus nigra ‘Samdal‘

‘Samyl‘ Denmark Sambucus nigra ‘Samyl‘

‘Weihenstephan‘ Germany Sambucus nigra ‘Weihenstephan‘

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Table S5. Anthocyanin concentration (expressed as cyanidin-3-glucoside) in extracts, extracts after drying and in dry fruits.

Cultivar Cextract (mg/ml) Cdry extract (mg/g) Cdry fruits (mg/100 g)

‘Aurea‘ 0.52 ± 0.00 120.10 ± 0.06 650.97 ± 0.31

‘Dana‘ 0.87 ± 0.09 407.93 ± 41.95 1539.27 ± 158.29

‘Haschberg‘ 0.67 ± 0.03 183.04 ± 12.13 1634.14 ± 91.96

‘Heidegg 13‘ 1.39 ± 0.03 551.33 ± 12.13 3621.67 ± 79.69

‘Korsør‘ 1.09 ± 0.10 267.90 ± 23.40 1978.94 ± 172.82

‘Pregarten‘ 0.69 ± 0.03 247.25 ± 12.36 1508.52 ± 75.40

‘Riese aus Voβloch‘ 1.61 ± 0.03 416.74 ± 8.01 3225.54 ± 60.01

‘Samdal‘ 2.02 ± 0.05 661.53 ± 17.37 5153.06 ± 135.34

‘Samyl‘ 2.12 ± 0.12 546.01 ± 31.86 4398.61 ± 256.68

‘Weihenstephan‘ 1.35 ± 0.04 402.35 ± 13.31 2449.12 ± 81.01

Data are averages ± s.d.

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Table S6. Anthocyanin concentration in extracts measured by HPLC in mg/mL.

Cultivar cyanin cyanidin-3-glucoside delphinidin malvidin cyanidin peonidin pelargonidin

‘Aurea‘ 0,084 ± 0,008 0,254 ± 0,011 <0,001 0,022 ± 0,004 0,006 ± 0,003 <0,001 <0,001

‘Dana‘ 0,255 ± 0,005 0,592 ± 0,040 0,006 ± 0,002 0,047 ± 0,004 0,025 ± 0,004 0,007 ± 0,002 0,003 ± 0,002

‘Haschberg‘ 0,401 ± 0,005 0,700 ± 0,020 0,009 ± 0,003 0,040 ± 0,005 0,012 ± 0,003 0,001 ± 0,002 <0,001

‘Heidegg 13‘ 0,185 ± 0,007 0,582 ± 0,008 0,003 ± 0,001 0,012 ± 0,002 0,005 ± 0,001 <0,001 0,003 ± 0,001

‘Korsør‘ 0,254 ± 0,014 0,458 ± 0,008 0,005 ± 0,001 0,034 ± 0,003 0,004 ± 0,001 0,005 ± 0,001 0,003 ± 0,001

‘Pregarten‘ 0,305 ± 0,009 0,379 ± 0,011 0,028 ± 0,004 0,019 ± 0,002 0,005 ± 0,001 0,003 ± 0,000 0,001 ± 0,000

‘Riese aus Voβloch‘ 0,160 ± 0,006 0,640 ± 0,020 0,037 ± 0,002 0,022 ± 0,002 0,002 ± 0,001 0,013 ± 0,002 0,007 ± 0,003

‘Samyl‘ 0,201 ± 0,008 0,779 ± 0,012 0,011 ± 0,002 0,050 ± 0,005 0,080 ± 0,004 0,015 ± 0,002 0,007 ± 0,002

‘Samdal‘ 0,113 ± 0,004 0,473 ± 0,017 0,001 ± 0,001 0,009 ± 0,001 0,047 ± 0,004 0,003 ± 0,001 0,002 ± 0,000

‘Weihenstephan‘ 0,377 ± 0,014 0,553 ± 0,028 0,003 ± 0,000 0,055 ± 0,004 0,004 ± 0,000 0,016 ± 0,002 <0,001

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Table S7. Correlation between total cyanidin (aglycone + glycosides) content of extracts measured by HPLC and iron/copper chelation or reduction.

pH 4.5 pH 5.5 pH 6.8 pH 7.5Fe chelation Fe3+: 0.73 (0.02) 0.89 (0.0006) 0.77 (0.009) 100 ng: 0.82 

(0.003),1 μg: 0.57 (0.08)

Fe reduction -0.79  (0.007) - - -Cu chelation (hematoxylin)

- 0.86 (0.002) 0.89 (0.0007) 0.83 (0.003)

Cu reduction 0.00 (0.99) 0.11 (0.75) -0.12 (0.73) -0.06 (0.86)Data in table are Pearson correlation coefficients with the p value in parentheses.

Table S8. Correlation between total anthocyanin content of extracts measured by HPLC and iron/copper chelation or reduction.

pH 4.5 pH 5.5 pH 6.8 pH 7.5Fe chelation Fe3+: 0.72 (0.02) 0.88 (0.0008) 0.76 (0.01) 100 ng: 0.83 

(0.003),1 μg: 0.57 (0.08)

Fe reduction -0.78  (0.007) - - -Cu chelation (hematoxylin)

- 0.85 (0.001) 0.88 (0.0009) 0.82 (0.003)

Cu reduction -0.01 (0.98) 0.11 (0.76) -0.12 (0.73) -0.06 (0.88)Data in table are Pearson correlation coefficients with the p value in parentheses.

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Fig. S1. Interaction of cyanidin-3-glucoside with iron and copper.   A: iron chelation,  B: copper chelation (hematoxylin method),  C: iron reduction and D: copper reduction.

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Fig. S2. Relationship between amounts of chelated metal and dose of cyanidin.  A: ferrous chelation at   pH  6.8   and  B:  cupric   chelation   at   pH  6.8.   There   is   linear   relationship  between   iron/copper chelation and the dose of cyanidin (red lines, left y axis). However, only in case of cupric ions, the ratio of the amount of chelated metal to the dose of cyanidin was approximately constant (blue line, right y axis) suggesting linear relationship.

Fig S3. Very low ferrous chelation at pH 4.5.

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Fig. S4. Cupric reduction at different pH conditions. Cyanidin, its 3-glucoside and some extracts with different reducing potential were selected. Lines are shown with SD. Data after reaching maximum chelation are not shown.

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Fig. S5. Cupric ions reduction by cyaniding, its 3-glucoside and extracts at different pH: 7.5 (A), 6.8 (B), 5.5 (C) and 4.5 (D). Data show the slope ± 95% confidence intervals of reduction line depicting the relationship between the amount of reduced iron and added μg of the extract.

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Fig. S6. Relationship between copper chelation and reduction.  At all tested pH, the relationships were insignificant. Exclusion of ‘Haschberg‘ and ‘Aurea‘ with high standard deviation (see result part for reason) did not lead to a significant relationship.

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Fig. S7. Relationship between cyanidin-3-glucuronide content in extracts and metal chelation/reduction. A: iron   chelation, B: Cu2+-chelation (hematoxylin), C: iron reduction at pH 4.5 and D: copper reduction. Correlation coefficients are  shown in Table 1 in the main article.

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Fig. S8. Chromatogram of standards

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