Effects of rootstocks and developmental time on the dynamic changes of main functional substances in 'Orah' (Citrus reticulata Blanco) by HPLC coupled with UV detection

Abstract

Introduction: Citrus fruit is rich in important functional constituents such as flavonoids, phenolic acids terpenes and other functional substances that play an important role for treating clinical diseases or controlling major agricultural diseases and pests. Plant secondary metabolites have become one of the most important resources of novel lead compounds, especially young citrus fruits contain multiple functional substances. 'Orah', a type of citrus reticulata, is known for its fine appearance, productivity, delicious sweetness, late-maturing characteristics, and is widely cultivated in China. Fruit thinning and rootstock selection are commonly used agronomic measures in its production to ensure its quality and tree vigor. However, few studies have demonstrated the effects of these agronomic measures on the functional substances of 'Orah'.

Methods: In this study, we used HPLC coupled with UV to detect the dynamic changes of fruit quality, 13 main flavonoids, 7 phenolic acids, 2 terpenes, synephrine and antioxidant capacity in both peel and pulp of citrus fruits grafted on four rootstocks (Red orange Citrus reticulata Blanco cv. red tangerine, Ziyang xiangcheng Citrus junos Sieb. ex Tanaka, Trifoliate orange Poncirus trifoliata L. Raf, and Carrizo citrange Citrus sinensis Osb.×P.trifoliate Raf) at six different developmental stages (from 90 DAF to 240 DAF).

Results: The results indicated that rootstock can significantly affect the contents of functional constituents and antioxidant capacity in 'Orah'. Additionally, it was found that pruning at either 90 DAF (days after flowering) or 150 DAF produced the most favorable outcomes for extracting functional substances. We also identified rootstock 'Trifoliate orange' has the highest total soluble solids (TSS) and 'Ziyang xiangcheng' to be the optimal in terms of comprehensive sensory of fruit quality, while 'Red orange' and 'Ziyang xiangcheng' are optimal in terms of functional substance quality, and 'Red orange' excels in antioxidant capacity.

Discussion: Overall, the findings demonstrate the important role of rootstocks and developmental stage in shaping fruit sensory quality and functional substance synthesis, providing valuable insights for guiding rootstock selection, determining thinning time, and utilizing pruned fruits in a more informed manner.

Keywords: antioxidant activity; bioactive compounds; fruit quality; mandarin; phenolic compounds.

Figure 1

Effect of different rootstocks on fruit and quality of ‘Orah’. (A) persentage of peel weight. (B) persentage of pulp weight. (C) TSS. (D) Acid. (E) RTT. (F) VC. Z: P. trifoliata (L.) Raf, ZC: C. sinensis Osb.× P. trifoliate Raf. H: C. reticulata Blanco cv. Red tangerine, X: C. junos Sieb. ex Tanaka, 90, 120, 150, 180, 210, 240 DAF: Fruit at 90d, 120d, 150d, 180d, 210d, 240d after flowering. The samples in panels (C–F) were from the fruit pulp from 150 to 240 DAF. Different lowercase letters in the same rootstock at different times represented significant difference (P<0.05), while capital letters represent significant differences in the different rootstocks at the same time period (P<0.05).

Figure 2

Changes in the content of functional substances in ‘Orah’ under grafting on different rootstocks. HP: Peel of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, HR: Pulp of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, XP: Peel of ‘Orah’ grafted on C. junos Sieb. ex Tanaka, XR: Pulp of ‘Orah’ grafted on C. junos Sieb. ex Tanaka, ZP: Peel of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZR: Pulp of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZCP: Peel of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf, ZCR: Pulp of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf. 90, 120, 150, 180, 210, 240 DAF: Fruit at 90d, 120d, 150d, 180d, 210d, 240d after flowering. All samples were fresh samples. Different lowercase letters in the same rootstock at different times represented significant difference (P<0.05), while capital letters represent significant differences in the different rootstocks at the same time period (P<0.05). (A) Total phenol. (B) Tatol flavonoids. (C) Tatole amino acid contents.

Figure 3

Changes of limonin, nomilin and synephrine content in ‘Orah’ under grafting of different rootstocks-scion combinations. HP: Peel of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, HR: Pulp of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, XP: Peel of ‘Orah’ grafted on C. junos Sieb. ex Tanaka, XR: Pulp of ‘Orah’ grafted on C. junos Sieb. ex Tanaka, ZP: Peel of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZR: Pulp of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZCP: Peel of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf, ZCR: Pulp of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf. 90, 120, 150, 180, 210, 240 DAF: Fruit at 90d, 120d, 150d, 180d, 210d, 240d after flowering. FW: Fresh weight. Different lowercase letters in the same rootstock at different times represented significant difference (P<0.05), while capital letters represent significant differences in the different rootstocks at the same time period (P<0.05). (A) Limonin. (B) Nomilin. (C) Synephrine.

Figure 4

Changes in the content of four phenolic acid in ‘Orah’ under grafting on different rootstocks. HP: Peel of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, HR: Pulp of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, XP: Peel of ‘Orah’ grafted on C. junos Sieb. ex Tanaka,XR: Pulp of ‘Orah’ grafted on C. junos Sieb. ex Tanaka, ZP: Peel of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZR: Pulp of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZCP: Peel of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf, ZCR: Pulp of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf. 90, 120, 150, 180, 210, 240 DAF: Fruit at 90d, 120d, 150d, 180d, 210d, 240d after flowering. FW, Fresh weight. Different lowercase letters in the same rootstock at different times represented significant difference (P<0.05), while capital letters represent significant differences in the different rootstocks at the same time period (P<0.05). (A) Protocatechin. (B) Caffeic acid. (C) Ferulic acid. (D) Sinapic acid.

Figure 5

Changes in the content of six main flavonoids in ‘Orah’ under grafting on different rootstocks. HP: Peel of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, HR: Pulp of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, XP: Peel of ‘Orah’ grafted on C. junos Sieb. ex Tanaka,XR: Pulp of ‘Orah’ grafted on C. junos Sieb. ex Tanaka, ZP: Peel of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZR: Pulp of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZCP: Peel of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf, ZCR: Pulp of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf. 90, 120, 150, 180, 210, 240 DAF: Fruit at 90d, 120d, 150d, 180d, 210d, 240d after flowering. FW, Fresh weight. Different lowercase letters in the same rootstock at different times represented significant difference (P<0.05), while capital letters represent significant differences in the different rootstocks at the same time period (P<0.05). (A) sinensetin. (B) Nobiletin. (C) Narirutin. (D) Hesperidin. (E) Neohesperidin. (F) Poncirin.

Figure 6

Changes in antioxidant capacity of ‘Orah’ under under grafting on different rootstocks. HP: Peel of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, HR: Pulp of ‘Orah’ grafted on C. reticulata Blanco cv. Red tangerine, XP: Peel of ‘Orah’ grafted on C. junos Sieb. ex Tanaka,XR: Pulp of ‘Orah’ grafted on C. junos Sieb. ex Tanaka, ZP: Peel of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZR: Pulp of ‘Orah’ grafted on P. trifoliata (L.) Raf, ZCP: Peel of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf, ZCR: Pulp of ‘Orah’ grafted on C. sinensis Osb.× P. trifoliate Raf. 90, 120, 150, 180, 210, 240 DAF: Fruit at 90d, 120d, 150d, 180d, 210d, 240d after flowering; FW, Fresh weight. Different lowercase letters in the same rootstock at different times represented significant difference (P<0.05), while capital letters represent significant differences in the different rootstocks at the same time period (P<0.05). (A) FRAP free radical scavenging capacity. (B) DPPH free radical scavenging activity. (C) ABTS free radical scavenging activity.

Figure 7

Principal component analysis (PCA) of the 16 parameters analyzed for ‘Orah’. (A), fruit at 90d after flowering. (B), fruit at120 d after flowering. (C), fruit at 150d after flowering. (D), fruit at 180d after flowering. (E) fruit at 210d after flowering. (F), fruit at 240d after flowering. Antioxidant activity, limonin, flavone compounds and phenolic compounds are coloured as categories as specified in the figure legend.