Physiological and Ultrastructural Alterations Linked to Intrinsic Mastication Inferiority of Segment Membranes in Satsuma Mandarin (Citrus unshiu Marc.) Fruits

Chewing texture is important for fresh citrus fruits, and the mastication trait of a segment directly determines chewing texture. Roughing disorder impairs the quality of Satsuma mandarin fruits, and it is typically correlated with intrinsic mastication inferiority (IMI). This study explored the role of segment membranes (SMs) in IMI. Similar to IMI in roughing-disordered fruits, segment shear force significantly enhanced relative to controls (CK); cell layers and cell wall thickness increased also in inferior masticating SMs. The ‘Miyamoto Wase’ cultivar exhibited larger segment shear force and more SM cell layers than ‘Juxiangzao’. In SMs, vessel cells could be divided into outside layers where segments adjoin and inside layers where juice sacs grow from. The inside vessel cell layers in the inferior masticating SMs were denser. Vessels with a length of 200 to 300 μm and a diameter of 5 to 15 μm predominated in SMs. The average vessel diameter enlarged by 13% to 16.5% in inferior masticating SMs, depending on cultivars. Furthermore, there was a decrease in vessels with a diameter <5 μm and an increase in vessels >10 μm in the inferior masticating SMs. Between phenotypes, protopectin increased significantly throughout development of inferior masticating SMs, while water-soluble pectin increased during the later stages of development. In one inferior masticating SM sample, protopectin and water-soluble pectin levels were higher in the inner-ring area than those in the outer-ring area. Correspondingly, expression of CuPME21 which is involved in pectin hydrolysis was consistently upregulated in the inferior masticating SMs throughout fruit development. The findings in this work provide novel insights into citrus SM structure and its IMI.

Organic Acid-Catalyzed Subcritical Water Hydrolysis of Immature Citrus unshiu Pomace

Immature Citrus unshiu pomace (ICUP) was hydrolyzed under organic acid-catalyzed, subcritical water (SW) conditions to produce flavonoid monoglucosides (hesperetin-7-O-glycoside and prunin) and aglycons (hesperetin and naringenin) with high biological activities. The results of single-factor experiments showed that with 8 h of hydrolysis and an increasing citric acid concentration, the yield of flavonoid monoglucosides (hesperetin-7-O-glycoside and prunin) increased from 0 to 7% citric acid. Afterward, the hesperetin-7-O-glycoside yield remained constant (from 7 to 19% citric acid) while the pruning yield decreased with 19% of citric acid, whereas the aglycon yield increased continuously. In response surface methodology analysis, a citric acid concentration and hydrolysis duration of 13.34% and 7.94 h were predicted to produce the highest monoglucoside yield of 15.41 mg/g, while 18.48% citric acid and a 9.65 h hydrolysis duration produced the highest aglycon yield of 10.00 mg/g. The inhibitory activities of the SW hydrolysates against pancreatic lipase (PL) and xanthine oxidase (XO) were greatly affected by citric acid concentration and hydrolysis duration, respectively. PL and α-glucosidase inhibition rates of 88.2% and 62.7%, respectively, were achieved with 18.48% citric acid and an 8 h hydrolysis duration, compared to 72.8% for XO with 16% citric acid and 12 h of hydrolysis. This study confirms the potential of citric acid-catalyzed SW hydrolysis of ICUP for producing flavonoid monoglucosides and aglycons with enhanced enzyme inhibitory activities.

Morphological Characteristics, Fruit Qualities and Evaluation of Reproductive Functions in Autotetraploid Satsuma Mandarin (Citrus unshiu Marcow)

The morphological characteristics and fruit quality of an autotetraploid plant selected from nucellar seedlings of Satsuma mandarin (Citrus unshiu Marcow.) were investigated. Additionally, in order to evaluate the reproductive potential of male and female gametes of the tetraploid Satsuma mandarin, reciprocal crosses with diploid cultivars were also carried out. The tetraploid had significantly thick and round leaves, as compared to those of the diploid Satsuma mandarin. The sizes of the flowers and pollen grains of the tetraploid were significantly larger than those of the diploid. Pollen fertility of tetraploid was high compared with that of the diploid. The tetraploid produced seedless fruits. The fruit weight of the tetraploid was equal to that of the diploid. Compared to the diploid fruits, the tetraploid fruit had less sugar contents and more organic acid contents. Although the tetraploid fruits showed similar traits to other Citrus tetraploids such as thick and hard peels, the tetraploid had a higher content of carotenoids in the flavedo than the diploid, and the rind color of the tetraploid was much better. In the reciprocal crosses between the tetraploid Satsuma mandarin and diploid cultivars, some seeds were obtained, and triploid progenies were obtained in all cross combinations.

Assessment of Anti-Inflammatory and Antioxidant Effects of Citrus unshiu Peel (CUP) Flavonoids on LPS-Stimulated RAW 264.7 Cells

Citrus unshiu is a popular medicinal herb in several Asian countries, in particular South Korea. C. unshiu peel (CUP) has several biologically active compounds, including flavonoids. Hence, this research aimed to label the flavonoids from CUP by HPLC-MS/MS analysis and examine their anti-inflammatory and antioxidant potential on LPS-stimulated RAW 264.7 macrophages. A total of four flavonoids (Rutin, naringin, hesperidin, and poncirin) were characterized, and their contents were quantified from CUP. It showed that the naringin is rich in CUP. Further, treatment with the flavonoids at concentrations of 2.5 and 5 μg/mL had no effect on the cell viability of RAW 264.7 macrophages. On the other hand, it decreased the production and expression of inflammatory mediators and pro-inflammatory cytokines such as NO, PGE2, TNF-α, IL-1β, iNOS, and COX2 in the LPS-stimulated RAW 264.7 macrophages. In addition, flavonoids treatment inhibited the NF-κB activation by downregulating the p-p65 and p-IκBα proteins expression. Furthermore, reactive oxygen species (ROS) production considerably decreased at the same concentrations while antioxidant enzyme activity increased in the LPS-stimulated RAW 264.7 macrophages. Collectively, our results show that CUP flavonoids have the potential to decrease inflammation and oxidative damage.

Aqueous Extract from Leaves of Citrus unshiu Attenuates Lipopolysaccharide-Induced Inflammatory Responses in a Mouse Model of Systemic Inflammation

Inflammation is related to various life-threatening diseases including cancer, neurodegenerative diseases, and metabolic syndrome. Because macrophages are prominent inflammatory cells, regulation of macrophage activation is a key issue to control the onset of inflammation-associated diseases. In this study, we aimed to evaluate the potential anti-inflammatory activity of Citrus unshiu leaf extract (CLE) and to elucidate the mechanism underlying its anti-inflammatory effect. We found the inhibitory activity of CLE on the secretion of proinflammatory cytokines and a chemokine from mouse macrophage-like RAW 264.7 cells and mouse peritoneal macrophages. The inhibitory activity of CLE was attributed to downregulated JNK, p38 MAPK, and NF-κB signaling pathways, leading to suppressed gene expression of inflammation-associated proteins. Oral administration of CLE significantly decreased the serum level of proinflammatory cytokines IL-6 and TNFα and increased that of anti-inflammatory cytokine IL-10 in lipopolysaccharide-induced systemic inflammation mice. In addition, oral administration of CLE decreased secretion and gene expression of several proinflammatory proteins in the liver and spleen of the model mice. Overall results revealed that C. unshiu leaf is effective to attenuate inflammatory responses in vitro and in vivo.