Comparison of Bioactive Compounds and Antioxidant Activities in Differentially Pigmented Cerasus humilis Fruits

Abstract

Chinese dwarf cherry (Cerasus humilis) is a wild fruit tree and medicinal plant endemic to China. Its fruits are rich in various bioactive compounds, such as flavonoids and carotenoids, which contribute greatly to their high antioxidant capacity. In this study, the contents of bioactive substances (chlorophyll, carotenoids, ascorbic acid, anthocyanin, total flavonoids, and total phenols), antioxidant capacities, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS⁺) scavenging ability, and ferric-reducing antioxidant power (FRAP)) in differentially pigmented C. humilis fruits of four varieties were determined and compared. The results revealed that anthocyanin, total flavonoids and total phenols were the three main components responsible for the antioxidant activity of C. humilis fruits. 'Jinou No.1' fruits with dark red peel and red flesh had the highest contents of anthocyanin, total flavonoids, and total phenols, as well as the highest antioxidant capacities; 'Nongda No.5' fruits with yellow-green peel and yellow flesh had the highest contents of carotenoids and chlorophyll, while 'Nongda No.6' fruit had the highest ascorbic acid content. To further reveal the molecular mechanism underlying differences in the accumulation of carotenoids and flavonoids among differentially pigmented C. humilis fruits, the expression patterns of structural genes involved in the biosynthesis of the two compounds were investigated. Correlation analysis results revealed that the content of carotenoids in C. humilis fruits was very significantly positively correlated with the expression of the ChCHYB, ChZEP, ChVDE, ChNSY, ChCCD1, ChCCD4, ChNCED1, and ChNCED5 genes (p < 0.01) and significantly negatively correlated with the expression of ChZDS (p < 0.05). The anthocyanin content was very significantly positively correlated with ChCHS, ChFLS, and ChUFGT expression (p < 0.01). The total flavonoid content was very significantly positively correlated with the expression of ChCHS, ChUFGT, and ChC4H (p < 0.01) and significantly positively correlated with ChFLS expression (p < 0.05). This study can provide a basis for understanding the differences in the accumulation of bioactive substances, and is helpful for clarifying the mechanisms underlying the accumulation of various carotenoids and flavonoids among differentially pigmented C. humilis fruits.

Keywords: Cerasus humilis; antioxidant capacity; bioactive substances; carotenoids; flavonoids.

Figure 1

Mature fruits of the four C. humilis varieties used in this study.

Figure 2

PCA results for the fruit parameters of the four different C. humilis varieties. Chl: Chlorophyll; Chl a: chlorophyll a; Chl b: chlorophyll b; Car: carotenoids; ABA: abscisic acid; Ant: anthocyanin; TFC: total flavonoids; TPC: total phenols; AA: ascorbic acid; FRAP: ferric reducing antioxidant power; ABTS⁺: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging ability; DPPH: 2,2-diphenyl-1-picrylhydrazyl free radical scavenging ability.

Figure 3

Carotenoid metabolism comparison in fruits of the four different C. humilis varieties: (A) carotenoid synthesis and metabolic pathways; (B) comparison of carotenoid contents in four different C. humilis varieties; (C) comparison of ABA content in four different C. humilis varieties; (D) expression heatmap for carotenoid biosynthesis-related structural genes. MEP: 2-C-methyl-D-erythritol-4-phosphate; IPP: isopentenyl diphosphate; DMAPP: dimethylallyl diphosphate; GGPP: geranylgeranyl diphosphate; PSY: phytoene synthase; PDS: phytoene desaturase; Z-ISO: ζ-carotene isomerase; ZDS: ζ-carotene desaturase; CRTISO: carotene isomerase; LCYB: Lycopene β-cyclase; LCYE: lycopene ε-cyclase; CHYB: β-carotene hydroxylase; CHYE: ε-carotene hydroxylase; ZEP: zeaxanthin epoxidase; VDE: violaxanthin de-epoxidase; NSY: neoxanthin synthase; CCD: carotenoid cleavage dioxygenase; NCED: 9-cis-epoxycarotenoid dioxygenase. The different letters above the columns in (B,C) indicate significant differences at the p < 0.05 level.

Figure 4

Correlation (A) and PCA (B) analysis results for the of carotenoid and ABA contents and the expression levels of carotenoid metabolism-related genes. Car: carotenoids; ABA: abscisic acid. * and ** indicate significant correlation (p < 0.05) and very significant correlation (p < 0.01), respectively.

Figure 5

Fruit flavonoid metabolism comparison of the four different C. humilis varieties. (A) Anthocyanin extract solution of C. humilis fruits. The color of anthocyanin extract of ‘Jinou No.1’ was the reddest, the anthocyanin extract of ‘Nongda No.5’ was yellow-green, the anthocyanin extract of ‘Nongda No.6’ and ‘Nongda No.7’ was orange, and the color of ‘Nongda No.6’ was slightly darker than that of ‘Nongda No.7’. (B) Anthocyanin contents in fruits of four C. humilis varieties: (C) total flavonoid content in fruits of four C. humilis varieties; (D) flavonoid biosynthesis pathway; (E) expression heatmap for flavonoid biosynthesis-related structural genes. PAL: phenylalanine ammonia lyase; C4H: cinnamate 4-hydroxylase; CHS: chalcone synthase; CHI: chalcone isomerase; FLS: flavonol synthase; F3H: flavanone 3-hydroxylase; DFR: dihydroflavonol 4-reductase; ANS: anthocyanin synthase; UFGT: UDP-glucose: flavonoid 3-O-glucosyltransferase. The different letters above the columns in (B,C) indicate significant differences at the p < 0.05 level.

Figure 6

Correlation analysis (A) and PCA (B) results for the contents of anthocyanin and total flavonoids and expression levels of flavonoid metabolism-related genes. Ant: anthocyanin; TFC: total flavonoids. * and ** indicate significant correlation (p < 0.05) and very significant correlation (p < 0.01), respectively.