Evolution-guided multiomics provide insights into the strengthening of bioactive flavone biosynthesis in medicinal pummelo.

Pummelo (Citrus maxima or Citrus grandis) is a basic species and an important type for breeding in Citrus. Pummelo is used not only for fresh consumption but also for medicinal purposes. However, the molecular basis of medicinal traits is unclear. Here, compared with wild citrus species/Citrus-related genera, the content of 43 bioactive metabolites and their derivatives increased in the pummelo. Furthermore, we assembled the genome sequence of a variety for medicinal purposes with a long history, Citrus maxima 'Huazhouyou-tomentosa' (HZY-T), at the chromosome level with a genome size of 349.07 Mb. Comparative genomics showed that the expanded gene family in the pummelo genome was enriched in flavonoids-, terpenoid-, and phenylpropanoid biosynthesis. Using the metabolome and transcriptome of six developmental stages of HZY-T and Citrus maxima 'Huazhouyou-smooth' (HZY-S) fruit peel, we generated the regulatory networks of bioactive metabolites and their derivatives. We identified a novel MYB transcription factor, CmtMYB108, as an important regulator of flavone pathways. Both mutations and expression of CmtMYB108, which targets the genes PAL (phenylalanine ammonia-lyase) and FNS (flavone synthase), displayed differential expression between Citrus-related genera, wild citrus species and pummelo species. This study provides insights into the evolution-associated changes in bioactive metabolism during the origin process of pummelo.

[Fruit variation and geographical distribution of citron].

The ripe dried fruit of citron(Citrus medica) is one of the important sources of Chinese herb Citri Fructus. At the same time, it is also grown for edible and ornamental uses. There are many species and abundant genetic variation. To clarify the intraspecific variation and resource distribution of citron, this study investigated the variation in 11 citron fruits, basically covering the main species in China, including Xiaoguo citron(C. medica var. ethrog), Goucheng(C. medica var. yunnanensis), Muli citron(C.medica var. muliensis), Dehong citron(C.medica×Citrus spp.), Fuzhou citron(C.medica×C.grandis?), Mawu(C.medica×C.grandis?), Cangyuan citron, Binchuan citron, Sweet citron, Big citron, and Small citron. The natural communities of citron were proved to be mainly distributed in the southwestern and western Yunnan and southeastern Tibet of China, with Yunnan, Sichuan, Guangxi, Chongqing, Hubei, and Zhejiang identified as the main production areas. Citron has also been widely grown in India, the Mediterranean region, and the Caribbean coast countries. The field investigation revealed the large-scale intraspecific variation of citron fruits. Most of the fruits are oval-like or sphere-like in shape. The fruits are green when raw and yellow when ripe, with oil cell dots on the skin, stripe-likes running from top to bottom, and bulge at the top. Usually, in the smaller citron fruits, the pulp and juice vesicles are better developed and the central columella is tighter. By contrast, the juice vesicles and central columella in larger fruits became more vacant, with carpels visible, and the apex segregation and development of the carpels is one of the reasons for variation. These variations should be given top priority in the future variety selection and breeding, and the quality differences of different citron species and their mechanisms should be further studied. In particular, variety selection and classification management according to their medicinal or edible purposes will provide scientific and technological supports for the orderly, safe, and effective production of citron products consumed as food and medicine.

The Anti-Inflammatory Properties of Citrus wilsonii Tanaka Extract in LPS-Induced RAW 264.7 and Primary Mouse Bone Marrow-Derived Dendritic Cells.

'Zhique' (Citrus wilsonii Tanaka) is a traditional Chinese medicine. Its fruits have been used to treat inflammation-related symptoms, such as cough and sputum, though the underlying mechanism remains poorly understood. The aim of this study was to investigate the anti-inflammatory properties of 'Zhique' pulp extract (ZQE) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and primary mouse bone marrow-derived dendritic cells (BMDCs). The flavonoid profiles of the ZQE were determined by high performance liquid chromatography. The anti-inflammatory activity was evaluated in LPS-induced inflammatory RAW 264.7 macrophages and BMDCs through enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and Western blot assays. Naringin was a predominant flavonoid occurring in ZQE, followed by eriocitrin, hesperidin, neohesperidin, rhoifolin, naringenin, and poncirin. ZQE exhibited a very low cytotoxicity in LPS-stimulated RAW 264.7 macrophages. Meanwhile, ZQE significantly inhibited the production of prostaglandins E2 and secretion of cyclooxygenase-2 protein in LPS-stimulated RAW 264.7 macrophages, and markedly suppressed the mRNA expression of inflammatory mediators, such as cyclooxygenase-2, tumor necrosis factor alpha, interleukin-1 beta (IL-1ß), and IL-6 in LPS-induced RAW 264.7 macrophages and/or primary BMDCs. The ZQE inhibited the inflammatory responses in RAW 264.7 macrophages and BMDCs triggered by LPS. The results suggested that 'Zhique' has a high potential as a novel therapeutic agent to treat chronic inflammatory diseases.

Exploration of D-limonene as a sex pheromone for males of Bactrocera minax (Diptera: Tephritidae).

BACKGROUND: Bactrocera minax is a devastating pest of citrus fruits. However, there have been no effective control measures before. Few reports on the sex pheromones of B. minax are available. RESULTS: In this study, nine of the volatile compounds in adult females were identified using headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS). Among them, d-limonene, caprolactam, 2-Nitro-1H-imidazole, and creatinine could evoke antennal responses in males. Field bioassays showed that only d-limonene could lure male flies, with a relative lure rate of 78.18% in all tested samples, which was significantly higher than that of paraffin oil control, while all volatile compounds did not have any lure effective to female flies. Moreover, d-limonene was diluted with paraffin oil into differential concentrations, the lure effect on males was better at 100, 500, and 800 µL d-limonene mL-1 than pure d-limonene (1000 µL mL-1 ). The relative male lure rate of d-limonene at 100 µL mL-1 was 85.88%, which was significantly higher than that of food-baits (14.12%) on day 3. However, d-limonene was unattractive to female and male Bactrocera dorsalis and Zeugodacus tau. Further kinetic analysis showed that female adults released d-limonene around 15-day post eclosion. Electroantennography 1 results showed that 500 µL mL-1 d-limonene evoked the strongest responses to antennae of 10- to 25-day-old male flies. CONCLUSION: Our results indicated that d-limonene could be a sex pheromone from female flies of B. minax, and it could be used as a male-specific sex attractant for B. minax. © 2023 Society of Chemical Industry.

Changes in health-promoting metabolites associated with high-altitude adaptation in honey.

The levels of metabolites in honey are influenced by floral origin, production region, and bee species. However, how environmental factors affect honey quality remains unclear. Based on untargeted metabolomics and using UPLC Q-Orbitrap MS, we analyzed 3596 metabolites in 51 honey samples from Yunnan and Shennongjia. Comparative analysis revealed that geniposidic acid, kynurenic acid and caffieine accumulated at significantly different levels between Shennongjia and Yunnan honey. Based on cluster structure analysis, 36 Yunnan honey samples were divided into two distinct groups by altitude. Notably, quercetin, hyperoside, taxifolin, rutin, tryptophan, astragalin and phenylalanine were higher levels in high-altitude honey (>1700 m), whereas abscisic acid was higher levels in low-altitude honey (≤1700 m). Among these, significantly elevated levels of hyperoside, taxfolin, astragalin, and tryptophan were observed in honey collected from high-altitude areas in Shennongjia. Our findings highlight the effect of altitude on honey health-promoting components, providing valuable insights into honey quality.

Integration of metabolomics and subcellular organelle expression microarray to increase understanding the organic acid changes in post-harvest citrus fruit.

Citric acid plays an important role in fresh fruit flavor and its adaptability to post-harvest storage conditions. In order to explore organic acid regulatory mechanisms in post-harvest citrus fruit, systematic biological analyses were conducted on stored Hirado Buntan Pummelo (HBP; Citrus grandis) fruits. High-performance capillary electrophoresis, subcellular organelle expression microarray, real-time quantitative reverse transcription polymerase chain reaction, gas chromatography mass spectrometry (GC-MS), and conventional physiological and biochemical analyses were undertaken. The results showed that the concentration of organic acids in HBP underwent a regular fluctuation. GC-MS-based metabolic profiling indicated that succinic acid, γ-aminobutyric acid (GABA), and glutamine contents increased, but 2-oxoglutaric acid content declined, which further confirmed that the GABA shunt may have some regulatory roles in organic acid catabolism processes. In addition, the concentration of organic acids was significantly correlated with senescence-related physiological processes, such as hydrogen peroxide content as well as superoxide dismutase and peroxidase activities, which showed that organic acids could be regarded as important parameters for measuring citrus fruit post-harvest senescence processes.

Panax ginseng abuse exhibits a pro-inflammatory effect by activating the NF-κB pathway.

P. ginseng (Panax ginseng C. A. Meyer) is a well-known traditional medicine that has been used for thousands of years to treat diseases. However, "ginseng abuse syndrome" (GAS) often occurs due to an inappropriate use such as high-dose or long-term usage of ginseng; information about what causes GAS and how GAS occurs is still lacking. In this study, the critical components that potentially caused GAS were screened through a step-by-step separation strategy, the pro-inflammatory effects of different extracts on messenger RNA (mRNA) or protein expression levels were evaluated in RAW 264.7 macrophages through quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot, respectively. It was found that high-molecular water-soluble substances (HWSS) significantly increased the expression of cytokines (cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and interleukin 6 (IL-6)) and cyclooxygenase 2 (COX-2) protein; gel filtration chromatography fraction 1 (GFC-F1) further purified from HWSS showed prominent pro-inflammatory effects by increasing the transcription of cytokines (COX-2, iNOS, tumor necrosis factor alpha (TNF-α), and interleukin 1ß (IL-1ß)) as well as the expression of COX-2 and iNOS protein. Moreover, GFC-F1 activated nuclear factor-kappa B (NF-кB) (p65 and inhibitor of nuclear factor-kappa B alpha (IκB-α)) and the p38/MAPK (mitogen-activated protein kinase) signaling pathways. On the other hand, the inhibitor of the NF-κB pathway (pyrrolidine dithiocarbamate (PDTC)) reduced GFC-F1-induced nitric oxide (NO) production, while the inhibitors of the MAPK pathways did not. Taken together, GFC-F1 is the potential composition that caused GAS through the production of inflammatory cytokines by activating the NF-кB pathway.

The content and diversity of carotenoids associated with high-altitude adaptation in Tibetan peach fruit.

Carotenoids are important secondary metabolites that may participate in response to extreme environments. Fruit color changes were observed in peaches growing at altitude on the Tibetan Plateau. Here, we qualitatively and quantitatively analyzed 43 kinds of carotenoids in 96 Tibetan peach and 12 cultivated peach fruit samples. Comparative analysis revealed that 25 kinds of carotenoids accumulated at significantly different levels between Tibetan peaches and cultivated peaches. Based on a population structure analysis, the carotenoid levels of Tibetan peaches were divided into two groups, which are mainly affected by the environmental factors light and temperature. The correlation analysis implied that the levels of 9 carotenoids were significantly correlated with altitude. qRT-PCR results showed that PSY, CCD4 and BCH were significantly differently expressed between the low and high altitude Tibetan peaches. In summary, this study showed that the abundant variation in carotenoids was highly associated with high-altitude adaptations in Tibetan peach fruit.

Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citrus fruits.

The orange subfamily (Aurantioideae) contains several Citrus species cultivated worldwide, such as sweet orange and lemon. The origin of Citrus species has long been debated and less is known about the Aurantioideae. Here, we compiled the genome sequences of 314 accessions, de novo assembled the genomes of 12 species and constructed a graph-based pangenome for Aurantioideae. Our analysis indicates that the ancient Indian Plate is the ancestral area for Citrus-related genera and that South Central China is the primary center of origin of the Citrus genus. We found substantial variations in the sequence and expression of the PH4 gene in Citrus relative to Citrus-related genera. Gene editing and biochemical experiments demonstrate a central role for PH4 in the accumulation of citric acid in citrus fruits. This study provides insights into the origin and evolution of the orange subfamily and a regulatory mechanism underpinning the evolution of fruit taste.

Short-term pretreatment of naringin isolated from Citrus wilsonii Tanaka attenuates rat myocardial ischemia/reperfusion injury.

Pretreatment or treatment with anti-apoptotic, anti-inflammatory, or anti-oxidative approaches could be critical for attenuated the severity of myocardial ischemia/reperfusion (I/R) injury. Naringin, a natural flavonoid, plays important roles in inflammation-related diseases. Immature dry fruits of Citrus wilsonii Tanaka (Xiang Yuan) are rich in naringin that can be used as traditional Chinese medicine to treat inflammation-related symptoms. However, its roles in cardioprotective role remain unclear. This study aimed to isolate naringin from Citrus wilsonii Tanaka fruit and tested their cardioprotective effect. The dry fruits of Citrus wilsonii Tanaka were extracted with boiling water and then supernatants were freeze-dried to yield aqueous extract (ZQAE). The extract was chemoprofiled using UPLC-MS/MS to stand for major constituents, and then subjected to different chromatographic separation steps, and naringin was isolated in a high yield. The cardioprotective effects of the aqueous extract of ZQAE and naringin were investigated in a myocardial I/R rat model and to elucidate the mechanism underlying its cardioprotective effect. Our results indicated that 5-day ZQAE and naringin pretreatments both promoted histopathological changes and reduced myocardial enzymes (cTnl, CK-MB, CK and LDH) induced by I/R. Moreover, the 50 mg/kg and 100 mg/kg ZQAE dose pretreatments presented a significantly decreased infarct size as well as myocardial enzyme levels but also inhibited myocardial apoptosis (cleaved-caspase3 protein expression), the inflammatory response (IL-23, IL-6, and TNF-α) and oxidative stress (MDA and SOD). The cardioprotective effect of 5 mg/kg dose of naringin pretreatment is comparable with that of 5 mg/kg drug ditiazem pretreatment. Additionally, naringin pretreatment exhibited striking decreases in the apoptosis index and downregulation of the protein expression levels of cleaved-Caspase3, Bcl2 and Bax. Meanwhile, naringin downregulated HMGB1 expression and upregulated SIRT1 expression in the myocardium. These findings suggest that short-term pretreatments with ZQAE and naringin both protect against myocardial I/R injury by suppressing myocardial apoptosis, the inflammatory response, and oxidative stress. The cardioprotective effect of naringin involves SIRT1 activation and may interact with HMGB1 and inhibit the release of HMGB1.

Citrus fruits are rich in flavonoids for immunoregulation and potential targeting ACE2.

The most recent outbreak of 2019 novel coronavirus, named as COVID-19, caused pneumonia epidemic in Wuhan with 2121 deaths cases as of February 20th 2020. Identification of effective antiviral agents to combat the novel coronavirus is urgently needed. Citrus fruit peel or wild citrus are rich in flavonoids, and clinically documented for roles in relief of cough and promotion of digestive health. Therefore, citrus fruits are assumed to possess antivirus activities or enhance the host immunity. A previous study found that hesperetin could act as a high potent inhibitor of SARS-CoV 3CLpro. We determined six flavonoid compounds' content in three citrus species by using LC-MS technique. The content of naringin and naringenin was at higher levels in pummelo. Hesperetin and hesperidin were highly accumulated in mandarin and sweet orange. The subsequent in vitro and in vivo experiments indicated that naringin could inhibit the expression of the proinflammatory cytokines (COX-2, iNOS, IL-1ß and IL-6) induced by LPS in Raw macrophage cell line, and may restrain cytokine through inhibiting HMGB1 expression in a mouse model. The results revealed that naringin may have a potential application for preventing cytokine storm. We simulated molecular docking to predict the binding affinity of those flavonoids to bind Angiotensin-converting enzyme 2 (ACE 2), which is a receptor of the coronavirus. Consideration of the potential anti-coronavirus and anti-inflammatory activity of flavonoids, the citrus fruit or its derived phytochemicals are promising in the use of prevention and treatment of SARS-CoV-2 infection.

HGFDB: a collective database of helmeted guinea fowl genomics.

As a vigorous and hardy and an almost disease-free game bird, the domestic helmeted guinea fowl (Numida meleagris, hereafter HGF) has attracted considerable attention in a large number of genetic study projects. However, none of the current/recent avian databases are related to this agriculturally and commercially important poultry species. To address this data gap, we developed Helmeted Guinea Fowl Database (HGFDB), which manages and shares HGF genomic and genetic data. By processing the data of genome assembly, sequencing reads and genetic variations, we organized them into eight modules, which correspond to 'Home', 'Genome', 'Re-sequence', 'Gene', 'Variation', 'Download', 'Tools' and 'Help', HGFDB provides the most comprehensive view of the HGF genome to date and will be relevant for future studies on HGF structural and functional genomics and genetic improvement. Database URL: http://hgfdb.ynau.edu.cn/.

Effect of temperature treatment on fruit quality and immunoregulation of Satsuma (Citrus unshiu Marc.) during storage.

Satsuma (Citrus unshiu Marc.) is rich in high levels of nutrients and popular for its unique flavor, but the consumption of satsuma is limited by some adverse reactions in human body. Previous studies have mainly focused on the effects of storage temperature on the postharvest quality of satsumas, and little attention has paid to the effect of postharvest satsumas on human body immunoregulation. The purpose of this study was to explore the differences in fruit quality, and the effect of satsuma fruits stored at different temperatures on human health. Satsumas stored at low temperature (5.8°C, LT) and room temperature (23 ± 2°C, RT) for 60 days were sampled every 10 days to measure the fruit quality. Sixty volunteers were recruited for the oral stimulation experiment of satsumas, and then the effect of satsumas on human health was examined through the immunoregulation of RAW 264.7 macrophages. The results showed that compared with RT treatment, LT treatment could delay the degradation of satsuma fruit quality. Both the results of the volunteer experiment and cell experiment indicated that postharvest temperature treatments could reduce the adverse effects of satsuma fruits on human body. These findings indicated that 10-day storage at room temperature plus subsequent storage at low temperature was the optimal treatment to maintain fruit quality and functional components of postharvest satsumas. This study provides useful information on satsuma consumption and research work from the perspective of immunoregulation evaluation.

Porous and nonporous silk fibroin (SF) membranes wrapping for Achilles tendon (AT) repair: Which one is a better choice?

Two types of silk fibroin (SF) membranes were developed for tendon repair: porous and nonporous SF membranes. The objective of this study was to compare the efficiency of these two films according to the ability of tendon regeneration using a rat Achilles tendon (AT) rupture suture wrapping model. The in vitro tests were conducted, and theSF membranes were proved to be with ultimate-biodegradability, good-biocompatibility and without toxicity. In vivo, 12 Sprague Dawley rats were used to create a rat AT rupture suture model wrapped by SF membranes. They were randomly divided into six groups. The results revealed that the nonporous SF membrane wrapping group was shown to reduce the inflammatory effect and induce the proliferation of fibroblast-like cells at one week and four weeks post-operatively. After four weeks, the nonporous SF membrane wrapping group exhibited more organized collagen structures and had increased expression of tendon repair proteins. Hence, our nonporous SF membrane improved the efficacy of tendon regeneration by decreasing inflammatory cells, growing fibroblast-like cells, and promoting extracellular matrix production. Nonporous SF membrane can, therefore, be regarded as a better functional membrane for tendon repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 733-740, 2019.

Effect of lemon water soluble extract on hyperuricemia in a mouse model.

Lemon is a healthy fruit with high medicinal value. This study found that lemon water soluble extract (LET) can reduce uric acid levels in mice with potassium oxonate induced hyperuricemia. Histopathological analysis suggested that LET caused little damage to the kidneys of mice. It affected mABCG2 and mGLUT9 mRNA expression only in hyperuricemic mice, but not in healthy mice. Our further results show that potassium citrate, rather than citric acid, is the main ingredient in LET with a hypouricemic effect. This study also indicates that lemon does have unique medicinal value for the treatment of hyperuricemia, and that potassium citrate has the potential to be developed as a drug for hyperuricemia. Lowering uric acid through LET and potassium citrate may directly promote the degradation of excessive uric acid in patients with hyperuricemia.

Immunoblotting Quantification Approach for Identifying Potential Hypoallergenic Citrus Cultivars.

The inherent allergens of citrus fruits, such as Cit s 1, Cit s 2, Cit s 3 can cause allergic reactions. A better understanding of the genetic factors (cultivar to cultivar) affecting the allergenic potential of citrus fruits would be beneficial for further identification of hypoallergenic genotypes. In the present study, an immunoblotting quantification approach was adopted to assess the potential allergenicity of 21 citrus cultivars, including nine subgroups (tangerine, satsuma, orange, pummelo, grapefruit, lemon, kumquat, tangor, and tangelo). To prepare highly sensitive and specific rabbit polyclonal antibodies, antigenicity of purified rCit s 1.01, rCit s 2.01, and rCit s 3.01 peptides were enhanced with high epitope density in a single protein molecule. The data integration of three citrus allergen quantifications demonstrated that the four pummelo cultivars (Kao Phuang Pummelo, Wanbai Pummelo, Shatian Pummelo, and Guanxi Pummelo) were potential hypoallergenic, compared with other 8 subgroups. Moreover, the immunological analyses with sera of allergic subjects revealed that Shatian Pummelo and Guanxi Pummelo showed the lowest immunoreactivity in 8 representative citrus cultivars. These potential hypoallergenic genotypes are of great significance to not only allergic consumers but also citrus breeders in the genetic improvement of hypoallergenic citrus as breeding resources.

A Facile Pathway to Modify Cellulose Composite Film by Reducing Wettability and Improving Barrier towards Moisture.

The hydrophilic property of cellulose is a key limiting factor for its wide application. Here, a novel solution impregnation pathway was developed to increase the hydrophobic properties of cellulose. When compared with the regenerated cellulose (RC), the composite films showed a decrease in water uptake ability towards water vapor, and an increase of the water contact angle from 29° to 65° with increasing resin content in the composites, with only a slight change in the transmittance. Furthermore, the Young's modulus value increased from 3.2 GPa (RC film) to 5.1 GPa (RCBEA50 film). The results indicated that the composites had combined the advantages of cellulose and biphenyl A epoxy acrylate prepolymer (BEA) resin. The presented method has great potential for the preparation of biocomposites with improved properties. The overall results suggest that composite films can be used as high-performance packaging materials.

Erratum: Pro-inflammatory effects of a litchi protein extract in murine RAW264.7 macrophages.

[This corrects the article DOI: 10.1038/hortres.2016.17.].

Data on water consumption in streptozotocin-induced diabetic mice by a novel peach gum-derived polysaccharide.

The data presented in this article are related to the article entitled "The impact of a novel peach gum-derived polysaccharide on postprandial blood glucose control in streptozotocin-induced diabetic mice" (Wang et al., 2017) [1]. Polydipsia was one of the most important symptoms of diabetic mellitus (DM) mice, which showed more water consumption than normal ones. The water consumption of DM mice in different groups administrated with metformin hydrochloride or a novel polysaccharide (coded as PGPSD) were exhibited in this article (Fig. 1). The field data set is made publicly available to enable critical or extended analyzes.