Phenolics and Terpenoids Profiling in Diverse Loquat Fruit Varieties and Systematic Assessment of Their Mitigation of Alcohol-Induced Oxidative Stress.

To compare and investigate the phenolic compounds in the peel and flesh of loquat (Eriobotrya japonica) and evaluate their ability to protect against alcohol-induced liver oxidative stress, we employed a combination of ultra-performance liquid chromatography (UPLC) and high-resolution mass spectrometry (HRMS) to qualitatively and quantitatively analyze 22 phenolics and 2 terpenoid compounds in loquat peel and flesh extracts (extraction with 95% ethanol). Among these, six compounds were identified for the first time in loquat, revealing distinct distribution patterns based on variety and tissue. Various chemical models, such as DPPH, FRAP, ORAC, and ABTS, were used to assess free radical scavenging and metal ion reduction capabilities. The results indicate that peel extracts exhibited higher antioxidant capacity compared with flesh extracts. Using a normal mouse liver cell line, AML-12, we explored the protective effects of loquat extracts and individual compounds against ethanol-induced oxidative stress. The findings demonstrate the enhanced cell viability and the induction of antioxidant enzyme activity through the modulation of Nrf2 and Keap1 gene expression. In a C57/BL6 mouse model of alcohol-induced liver damage, loquat extract was found to alleviate liver injury induced by alcohol. The restoration of perturbed serum liver health indicators underscored the efficacy of loquat extract in reclaiming equilibrium. The culmination of these findings significantly bolsters the foundational knowledge necessary to explore the utilization of loquat fruit extract in the creation of health-focused products.

Detection and classification of volatile compounds emitted by three fungi-infected citrus fruit using gas chromatography-mass spectrometry.

Citrus fruit produced some characteristic volatile compounds when infected by fungi compared with the healthy fruit. In the present study, volatile metabolites of postharvest citrus fruit with three different diseases including stem-end rot, blue mold and green mold were detected. Multivariate analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to classify the volatile compounds between the infected and non-infected citrus fruit. The results indicated that volatile compounds of unrotten, unrotten-rotten junction, and rotten tissues were successfully classified. Importantly, eight volatile compounds as biomarkers for stem-end rot and one biomarker for green mold of citrus were screened to discriminate the infected citrus fruit. This study offers the application potential of odor profiling of volatile compounds for detecting the fungi infection in postharvest citrus fruit.

Nobiletin as a chemopreventive natural product against cancer, a comprehensive review.

As a leading cause of death, second only to heart disease, cancer has always been one of the burning topics in medical research. When targeting multiple signal pathways in tumorigenesis chemoprevention, using natural or synthetic anti-cancer drugs is a vital strategy to reduce cancer damage. However, toxic effects, multidrug resistance (MDR) as well as cancer stem cells (CSCs) all prominently limited the clinical application of conventional anticancer drugs. With low side effects, strong biological activity, unique mechanism, and wide range of targets, natural products derived from plants are considered significant sources for new drug development. Nobiletin is one of the most attractive compounds, a unique flavonoid primarily isolated from the peel of citrus fruits. Numerous studies in vitro and in vivo have suggested that nobiletin and its derivatives possess the eminent potential to become effective cancer chemoprevention agents through various cellular and molecular levels. This article aims to comprehensively review the anticancer efficacy and specific mechanisms of nobiletin, enhancing our understanding of its chemoprevention properties and providing the latest research findings. At the end of this review, we also give some discussion and future perspectives regarding the challenges and opportunities in nobiletin efficient exploitation.

Contrasting Roles of Ethylene Response Factors in Pathogen Response and Ripening in Fleshy Fruit.

Fleshy fruits are generally hard and unpalatable when unripe; however, as they mature, their quality is transformed by the complex and dynamic genetic and biochemical process of ripening, which affects all cell compartments. Ripening fruits are enriched with nutrients such as acids, sugars, vitamins, attractive volatiles and pigments and develop a pleasant taste and texture and become attractive to eat. Ripening also increases sensitivity to pathogens, and this presents a crucial problem for fruit postharvest transport and storage: how to enhance pathogen resistance while maintaining ripening quality. Fruit development and ripening involve many changes in gene expression regulated by transcription factors (TFs), some of which respond to hormones such as auxin, abscisic acid (ABA) and ethylene. Ethylene response factor (ERF) TFs regulate both fruit ripening and resistance to pathogen stresses. Different ERFs regulate fruit ripening and/or pathogen responses in both fleshy climacteric and non-climacteric fruits and function cooperatively or independently of other TFs. In this review, we summarize the current status of studies on ERFs that regulate fruit ripening and responses to infection by several fungal pathogens, including a systematic ERF transcriptome analysis of fungal grey mould infection of tomato caused by Botrytis cinerea. This deepening understanding of the function of ERFs in fruit ripening and pathogen responses may identify novel approaches for engineering transcriptional regulation to improve fruit quality and pathogen resistance.

Citrus flavonoids and their antioxidant evaluation.

The antioxidant ability is the link and bridge connecting a variety of biological activities. Citrus flavonoids play an essential role in regulating oxidative stress and are an important source of daily intake of antioxidant supplements. Many studies have shown that citrus flavonoids promote health through antioxidation. In this review, the biosynthesis, composition and distribution of citrus flavonoids were concluded. The detection methods of antioxidant capacity of citrus flavonoids were divided into four categories: chemical, cellular, animal and clinical antioxidant capacity evaluation systems. The modeling methods, applicable scenarios, and their relative merits were compared based on these four systems. The antioxidant functions of citrus flavonoids under different evaluation systems were also discussed, especially the regulation of the Nrf2-antioxidases pathway. Some shortcomings in the current research were pointed out, and some suggestions for progress were put forward.

Selenium-enriched Polysaccharide: an Effective and Safe Selenium Source of C57 Mice to Improve Growth Performance, Regulate Selenium Deposition, and Promote Antioxidant Capacity.

Selenium-enriched polysaccharide (SeEPS) was prepared by reducing Se(IV) to elemental selenium and organic selenium in polysaccharide medium by the obtained Enterobacter cloacae strain Z0206 under aerobic conditions. In the present study, we focused on investigating the role of short-term supplementation of SeEPS at supernutritional doses in the regulation of growth performance, liver damage, antioxidant capacity, and selenium (Se) accumulation in C57 mice. Thirty-two C57 mice were randomly divided into four groups: the control group was gavaged with equal volume of phosphate-buffered saline, while the sodium selenite (Na2SeO3), selenomethionine (SeMet), and SeEPS groups were gavaged with 0.5 mg Se/kg BW of Na2SeO3, SeMet, and selenium-enriched polysaccharide (n = 8), respectively. We examined liver injury indicators, antioxidant capacity in the serum and liver, selenium deposition at different sites, selenoprotein levels, and selenocysteine-synthesizing and degradation-associated gene expression in mouse livers. SeEPS supplementation dramatically increased average daily weight gain but reduced the feed-to-gain ratio (F/G) of mice (P < 0.05). Compared to Na2SeO3 and SeMet supplementation, SeEPS supplementation at supernutritional doses did not cause the liver damage. SeEPS supplementation also markedly enhanced total antioxidant capacity (T-AOC), catalase (CAT), glutathione peroxidase (GSH-PX), and total superoxide dismutase (T-SOD) activities but reduced malondialdehyde (MDA) levels in the liver and serum (P < 0.05), while significantly increasing selenocysteine-synthesizing and degradation-related gene (SEPHS2, SEPSECS, Secisbp, Scly) expression at the mRNA level (P < 0.05), thus upregulating the mRNA levels of selenoproteins (SELENOP, SELENOK) (P < 0.05). We suggest that SeEPS could be a potential replacement for inorganic selenium to improve animals' growth performance, promote antioxidant capacity, and regulate selenium deposition.

Polymethoxyflavones in Citrus Regulate Lipopolysaccharide-Induced Oscillating Decay of Circadian Rhythm Genes by Inhibiting Nlrp3 Expression.

The aim of this study is to compare the regulatory abilities of citrus flavonoids on the oscillating expression of circadian genes. Seven varieties of citrus fruits and twenty-five citrus flavonoids were selected and evaluated. Per2 luciferase bioluminescence report system and serum shock were used to induce circadian gene expression in mouse microglia BV-2 cells. In vivo experiments were carried out using C57BL6/J mice to evaluate the regulation of flavonoids on the oscillatory expression of liver biorhythm genes. Lipopolysaccharide was used to interfere the gene oscillating expression. QRT-PCR was performed to detect the expression of circadian rhythm-related genes, including Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbß, Rorα, Dbp, and Npas2. The results show that the polymethoxyflavones (PMFs) exerted stronger circadian gene regulatory capability, while the flavonoids containing glycosides showed no biological activity. Also, all tested flavonoids decreased LPS-induced nitric oxide release, but only polymethoxyflavones inhibited circadian rhythm disorder. PMFs inhibited Nlrp3 inflammasome-related genes and proteins, including Nlrp3, IL-1ß, ASC, and Caspase1, while other flavonoids only affected IL-1ß and Caspase1 expression. This mechanism was preliminarily verified using the Nlrp3 inhibitor INF39.

Tangeretin maintains antioxidant activity by reducing CUL3 mediated NRF2 ubiquitination.

To explore the antioxidant capacity of citrus flavonoids under different evaluation systems, chemical and biological methods were engaged to determine the antioxidant abilities of flavanones and polymethoxyflavones. Results showed that flavanones exhibited good antioxidant activity, while polymethoxyflavones had a weak ability to scavenge free radicals. Both flavanones and polymethoxyflavones exerted the ability to inhibit H2O2-induced oxidative stress, but the effective concentration of polymethoxyflavones was lower. Further exploration showed that neohesperidin and tangeretin selectively regulated antioxidant enzyme activity, both in vitro and in vivo. Tangeretin also maintained the expression of antioxidant enzymes in L02 cells and in ICR mice liver. The mechanism exploration showed that both neohesperidin and tangeretin promoted the expression of NRF2 and inhibit the expression of KEAP1, but tangeretin could inhibit the ubiquitination of NRF2 by inhibiting CUL3. The mechanism was verified by CUL3 gene silencing. This study demonstrates a novel antioxidant mechanism of natural products.

Inorganic material based macrophage regulation for cancer therapy: basic concepts and recent advances.

Macrophages with the M1 phenotype are a type of immune cell with exciting prospects for cancer therapy; however, when these macrophages infiltrate into tumours, many of them are induced by the tumour microenvironment to transform into the M2 type, which can enable tumour defence against external therapeutic strategies, assisting in tumour development. Macrophages have strong plasticity and functional heterogeneity, and their phenotypic transformation is complex and still poorly understood in relation to cancer therapy. Recent material advances in inorganic nanomaterials, especially inorganic elements in vivo, have accelerated the development of macrophage regulation-based cancer treatments. This review summarizes the basics of recent research on macrophage phenotype transformation and discusses the current challenges in macrophage type regulation. Then, the current achievements involving inorganic material-based macrophage regulation and the related anticancer effects of induced macrophages and their extracellular secretions are reviewed systematically. Importantly, inorganic nanomaterial-based macrophage phenotype regulation is flexible and can be adapted for different types of cancer therapies, presenting a possible novel approach for the generation of immune materials for cancer therapy.

Limonin induces apoptosis of HL-60 cells by inhibiting NQO1 activity.

Limonin is an important bioactive substance in citrus fruits, especially in seeds, which has great potential in cancer prevention and treatment. In order to explore the anticancer activity based on interaction between limonin and NQO1, Human promyelocytic leukemia cells (HL-60) were studied in vitro. We found that limonin could inhibit proliferation and promote apoptosis of HL-60 cells, and the effect was positively correlated with its dosage. Western blot results showed that limonin could activate the endogenous apoptosis pathway mediated by mitochondria via up-regulating pro-apoptotic proteins (Bax, cytochrome c, Caspase3, and Caspase9) and down-regulating anti-apoptotic proteins (Bcl-2), thus inhibiting the proliferation of HL-60 cells and promoting apoptosis, which further proved the anticancer activity of limonin from the molecular mechanism. At the same time, limonin down-regulated the expression of NQO1, indicating that limonin may indirectly act on the apoptosis pathway by regulating the expression activity of antioxidant enzymes in vivo, thus exerting its inhibitory effect on tumor cells, which provides an idea for the molecular mechanism that natural products can indirectly exert their anticancer effect by regulating the activity of antioxidant enzymes.

Polymethoxyflavones from citrus inhibited gastric cancer cell proliferation through inducing apoptosis by upregulating RARß, both in vitro and in vivo.

In order to discover the active anti-tumor ingredients during the flavonoids separation process of Ougan (Citrus reticulata cv. Suavissima), gastric cancer cell lines including AGS, BGC-823, and SGC-7901 were employed to evaluate the proliferation inhibition abilities of Ougan extracts, flavanone components, polymethoxyflavone components, neohesperidin, nobiletin, tangeretin, and 5-demethylnobiletin. Quantitative real-time PCR was used to detect the expression of three retinoic acid receptor genes, including RARA, RARB, and RARG. Western blot and immunohistochemistry were used to detect protein expressions. The results showed that the polymethoxyflavone components and the PMFs monomers inhibited the proliferation of three gastric cancer cell lines and induced apoptosis. The mechanism exploration found that PMFs up-regulated the expression of the RARB gene selectively and activated the Caspase3, 9, and PARP1 proteins. In addition to 5-demethylnobiletin, other PMFs also upregulated the expression of cleaved Caspase8. The mechanism was preliminarily verified by a RARß inhibitor AGN 193109. Moreover, a nude mice tumor xenograft model confirmed the tangeretin could exhibit in vivo anti-tumor effect through inducing apoptosis and upregulating RARß protein. All result suggested that tangeretin may be a potentially novel, safe and effective drugs with less toxicity and lesser side effects for gastric cancer therapeutics.

Analysis of Phenolic Components and Related Biological Activities of 35 Apple (Malus pumila Mill.) Cultivars.

Apple (Malus pumila Mill.) is a popular fruit with high economic values and various biological activities that are beneficial to human health. In this study, 35 apple cultivars were collected and were evaluated for their basic quality indexes, phenolic compositions, antioxidant activity, anti-tumour, and anti-diabetic activities. The compositions of phenolics were detected by using high-performance liquid chromatography (HPLC) and high-resolution mass spectroscopy (HRMS) assays. The antioxidant activities of peel and pulp extracts from 35 apple cultivars were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and ferric reducing antioxidant power (FRAP) assay. Results showed that the contents of phenolic acids and proanthocyanidins showed significant correlations with the antioxidant activities. Phenolic-rich extracts significantly inhibited HepG2 cell proliferation, with the inhibition activity varied significantly between cultivars. 'Gold Delicious' pulp extract, 'Xiboliyabaidian' peel and pulp extracts showed protective effects on H2O2-induced injury of human umbilical vein endothelial cells (HUVEC). 'Red Fuji' peel extract, 'Xiboliyabaidian' peel and pulp extracts, as well as 'Gold Delicious' peel extract, significantly increased glucose consumption of HepG2 cells, in a dose-dependent manner. This research may provide theoretical guidance for further nutritional investigation of the apple resources.

Beneficial Regulatory Effects of Polymethoxyflavone-Rich Fraction from Ougan (Citrus reticulata cv. Suavissima) Fruit on Gut Microbiota and Identification of Its Intestinal Metabolites in Mice.

Polymethoxyflavones (PMFs) are special flavonoids in citrus fruits that have been suggested to be beneficial to human health. However, whether PMFs in citrus fruit alter human gut microbiota is not well understood. The aim of the present study was to investigate the effects of PMF-rich fraction from Ougan (Citrus reticulata cv. Suavissima) on gut microbiota and evaluate the intestinal metabolic profile of PMFs in Institute of Cancer Research mice. The main components of the PMF-rich fraction were nobiletin, tangeretin, and 5-demethylnobiletin. The composition of the gut microbiota was analyzed using 16S ribosomal DNA sequencing. The results showed that after oral administration, the composition of mice gut microbiota was significantly altered. The relative abundance of two probiotics, Lactobacillus and Bifidobacterium, were found to increase significantly. A total of 21 metabolites of PMFs were detected in mice intestinal content by high performance liquid chromatography electrospray ionization tandem mass spectrometry, and they were generated through demethylation, demethoxylation, hydroxylation, and glucuronidation. Our results provided evidence that PMFs have potential beneficial regulatory effects on gut microbiota that in turn metabolize PMFs, which warrants further investigation in human clinical trials.

Characterization of a Flavonoid 3'/5'/7-O-Methyltransferase from Citrus reticulata and Evaluation of the In Vitro Cytotoxicity of Its Methylated Products.

O-methylation of flavonoids is an important modification reaction that occurs in plants. O-methylation contributes to the structural diversity of flavonoids, which have several biological and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase and could effectively catalyze the methylation of 3'-, 5'-, and 7-OH of flavonoids with vicinal hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901 and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated C2-C3 bond was increased after being methylated at position 3'. These combined results provide biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products methylated by its catalytic reaction.

Antioxidant Capacity, Anticancer Ability and Flavonoids Composition of 35 Citrus (Citrus reticulata Blanco) Varieties.

Citrus (Citrus reticulate Blanco) is one of the most commonly consumed and widely distributed fruit in the world, which is possessing extensive bioactivities. Present study aimed to fully understand the flavonoids compositions, antioxidant capacities and in vitro anticancer abilities of different citrus resources. Citrus fruits of 35 varieties belonging to 5 types (pummelos, oranges, tangerines, mandarins and hybrids) were collected. Combining li quid chromatography combined with electrospray ionization mass spectrometry (LC-ESI-MS/MS) and ultra-performance liquid chromatography combined with diode array detector (UPLC-DAD), a total of 39 flavonoid compounds were identified, including 4 flavones, 9 flavanones and 26 polymethoxylated flavonoids (PMFs). Each citrus fruit was examined and compared by 4 parts, flavedo, albedo, segment membrane and juice sacs. The juice sacs had the lowest total phenolics, following by the segment membrane. Four antioxidant traits including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and cupric reducing antioxidant capacity (CUPRAC) were applied for the antioxidant capacities evaluation. Three gastric cancer cell lines, SGC-7901, BGC-823 and AGS were applied for the cytotoxicity evaluation. According to the results of correlation analysis, phenolics compounds might be the main contributor to the antioxidant activity of citrus extracts, while PMFs existing only in the flavedo might be closely related to the gastric cancer cell line cytotoxicity of citrus extracts. The results of present study might provide a theoretical guidance for the utilization of citrus resources.

The Growth of SGC-7901 Tumor Xenografts Was Suppressed by Chinese Bayberry Anthocyanin Extract through Upregulating KLF6 Gene Expression.

To investigate the antitumor effect of anthocyanins extracted from Chinese bayberry fruit (Myrica rubra Sieb. et Zucc.), a nude mouse tumor xenograft model was established. Treatments with C3G (cyanidin-3-glucoside, an anthocyanin) significantly suppressed the growth of SGC-7901 tumor xenografts in a dose-dependent manner. Immunohistochemical staining showed a significant increase in p21 expression, indicating that the cell cycle of tumor xenografts was inhibited. qPCR screening showed that C3G treatment up-regulated the expression of the KLF6 gene, which is an important tumor suppressor gene inactivated in many human cancers. Western blot showed that C3G treatments markedly increased KLF6 and p21 protein levels, inhibited CDK4 and Cyclin D1 expression, but did not notably change the expression of p53. These results indicated that KLF6 up-regulates p21 in a p53-independent manner and significantly reduces tumor proliferation. This study provides important information for the possible mechanism of C3G-induced antitumor activity against gastric adenocarcinoma in vivo.

Bioassay-based isolation and identification of phenolics from sweet cherry that promote active glucose consumption by HepG2 cells.

A variety of phenolics had been found to be functional in promoting cellular glucose consumption that is important for blood glucose regulation. Sweet cherry (Prunus avium) is rich in such kinds of phenolics, including hydrocinnamic acids, anthocyanins, flavonols, and flavan-3-ols. Furthermore, a sweet cherry phenolics-rich extract (PRE) was found to be effective in promoting HepG2 glucose consumption. Seventeen components were preliminarily identified by HPLC-ESI-MS, including 9 hydrocinnamic acids, 4 anthocyanins, 3 flavonols, and 1 flavan-3-ol. To investigate the cellular glucose consumption-promotion activity of different phneolics subclasses, the phenolics were further fractionated into an anthocyanin-rich fraction (ARF), hydrocinnamic acid-rich fraction (HRF), and flavonol-rich fraction (FRF) through liquid-liquid extraction and mix-mode cation-exchange solid-phase extraction. The 3 fractions promoted HepG2 glucose consumption to different levels, with the promotion effects of HRF and FRF stronger than that of the ARF. The results provide guidance on the use of sweet cherry as a functional fruit.