Figure 3. Anthocyanin assay. (A) Standard curve of increasing amounts of anthocyanin in solution measured in the presence of 1 M HCl at 525 nm. Mean ± SD of 5 assays. Eight µg of anthocyanin gave an absorbance reading of approximately 1.00, and therefore this amount was used in the hydroxyapatite binding assay (Fig. 3B). (B) Anthocyanin (AN) and parotid saliva (S) sequentially or simultaneously bound to hydroxyapatite. Mean ± SD of 6 assays. Saliva first, then anthocyanin sequentially (S/AN); anthocyanin first, then saliva sequentially (AN/S); saliva and anthocyanin simultaneously (S+AN). Background absorbance due to saliva has been subtracted from the results. ^aS/AN and S+AN were statistically significantly greater than anthocyanin alone, p < 0.05 by paired t test. (C,D) Anthocyanin and salivary fractions sequentially or simultaneously bound to hydroxyapatite. Total amount of histatin (H) fraction in solution added to hydroxyapatite was 0.42 µg or 1.7 µg. Proline-rich protein (P) fraction in solution added to hydroxyapatite was 25 µg or 100 µg. Total amount of anthocyanin added to hydroxyapatite was 8 µg. Histatin first, then anthocyanin sequentially (H/AN); histatin and anthocyanin simultaneously (H+AN); anthocyanin only (AN). Proline-rich proteins first, then anthocyanin sequentially (P/AN); proline-rich proteins and anthocyanin simultaneously (P+AN); anthocyanin only (AN). Mean ± SD of 6 assays. Background absorbance due to histatins and proline-rich proteins has been subtracted from the results. Binding of anthocyanin was statistically different by ANOVA (p < 0.05) in the presence of histatins or proline-rich proteins. ^bAt 1.7 µg protein, H/AN and H+AN were statistically significantly lower than anthocyanin alone (p < 0.05, paired t test). ^aP/AN and P+AN were statistically significantly greater than anthocyanin alone (p < 0.05, paired t test).