NMR-based metabolic profiling and comparison of Japanese persimmon cultivars.

Persimmons are a traditional, autumnal, and healthy fruit commonly consumed in Japan and East Asia based on the saying, "a persimmon a day keeps the doctor away." The differences in metabolites among five major Japanese persimmon cultivars were investigated using a nuclear magnetic resonance (NMR)-based metabolomics approach. By using a broadband water suppression enhanced through T1 effects (WET) method for the sensitive detection of minor metabolites, better discrimination among cultivars and more informative details regarding their metabolic differences have been achieved compared to those achieved in conventional 1H NMR sequences. Among the nonastringent cultivars analyzed, the Taishu cultivar has the highest abundance of amino acids. The Matsumotowase-Fuyu cultivar contains ethyl-ß-glycosides as characteristic components, which may relate to fruit softening. Citric acid concentration is higher in Maekawa Jiro than in other nonastringent cultivars. Among the two astringent cultivars analyzed, ethanol was significantly higher in Hiratanenashi than in Yotsumizo, which indicates different reactivity during deastringency treatments. The present study proposes an efficient and relatively quantitative metabolomics approach based on broadband WET NMR spectra.

Anti-inflammatory activities of astringent persimmons (Diospyros kaki Thunb.) stalks of various cultivar types based on the stages of maturity in the Gyeongnam province.

BACKGROUND: Natural products play a significant role in human health in relation to the prevention and treatment of inflammatory conditions. One of the plants with great medicinal potentials is Diospyros kaki which is mainly cultivated in Asian countries including Korea, Japan, and China. Astringent D. kaki is a wild species with an astringent taste until they are Ripened. kaki calyx is a traditional Korean medicine (TKM) made from the stalks of astringent D. kaki and is used in treating bed-wetting, vomiting, and hiccupping. The present study was designed to investigate the potential anti-inflammatory activities of astringent D. kaki stalks based on cultivar types and stages of maturity. METHODS: The anti-inflammatory effects of the stalk extracts of local astringent D. kaki cultivar species were evaluated on RAW 264.7 cells. Cell viability was measured using a Cell Counting Kit-8 (CCK8) method. The anti-inflammatory effects were determined by measuring the nitric oxide (NO) concentration of the supernatant. Cellular signaling pathways were determined by quantitative polymerase chain reactions of inducible nitric oxide synthase (iNOS). Protein expression of iNOS and phospho-p65 was determined using western blot, and the nuclear localization of p65 was determined using confocal imaging in RAW 264.7 cells. RESULTS: We found that the stage 1 (8-9 month) samples all showed a high percentage of tannic acid content and Gojongsi (Hamyang) stalks had the highest content. The stage 1 samples also showed the highest inhibition of NO production. Decreases in the expression of iNOS and phosphorylated p65, and in the nuclear localization of p65, were dose-dependent. All the extracts were nontoxic under 100 µg/ml concentration. CONCLUSION: This study provides insight into the changes in tannic acid content in astringent D. kaki and their anti-inflammatory effects, in relation to their stage of maturity. These results are expected to be useful in the verification of the efficacy of oriental medicine and the timing of proper harvest for medical use.

DkNAC7, a novel high-CO2/hypoxia-induced NAC transcription factor, regulates persimmon fruit de-astringency.

Artificial high-CO2 atmosphere (AHCA, 95% CO2 and 1% O2) has been widely applied as a postharvest de-astringency treatment for persimmon fruit. AHCA increases expression of transcription factors, including ethylene response factors (DkERF), that target de-astringency genes. Here, the promoter of DkERF9, a previously characterized AHCA-inducible and de-astringency regulator, was utilized to screen a cDNA library by yeast one hybrid assay. A novel NAC transcription factor, named DkNAC7, was identified. Dual-luciferase assay indicated that DkNAC7 could not only trans-activate the promoter of DkERF9, but also activated the previously identified deastringency-related gene DkPDC2. Real-time PCR analysis showed that DkNAC7 was up-regulated by AHCA treatment, in concert with the removal of astringency from persimmon fruit and subcellular localization showed DkNAC7 was located in the nucleus. Thus, these results indicate that DkNAC7 is a putative transcriptional activator involved in regulating persimmon fruit deastringency by trans-activition on both DkERF9 and DkPDC2, which encodes pyruvate decarboxylase.

Effect of particle size on phytochemical composition and antioxidant properties of two persimmon flours from Diospyros kaki Thunb. vars. 'Rojo Brillante' and 'Triumph' co-products.

BACKGROUND: This contribution studies the influence of the particle size of persimmon flours (from two cultivars, 'Rojo Brillante' and 'Triumph') on their primary (sugars and organic acids) and secondary (polyphenols, flavonoids and carotenoids) metabolite content, and also on their antioxidant activity, to assess whether these flours could find applications in the food industry as a potential functional ingredient. RESULTS: The main sugars were fructose and glucose and the principal organic acid was malic acid. The highest content of total phenols, flavonoids and carotenoids was found in flour fractions from cv. 'Rojo Brillante'. The phenol profile determined by HPLC identified six phenolic compounds in both persimmon flours, the most abundant being gallic acid. The greatest ABTS and DPPH radical scavenging capacity and ferric reducing power were found in flour fractions from cv. 'Rojo Brillante'. Although the influence of particle size on all these properties was not always evident, in general, the higher bioactive compound content and antioxidant capacity were in the finest particles. CONCLUSION: Co-products from cvs. 'Rojo Brillante' and 'Triumph' persimmon juice extraction can be processed to obtain flours rich in sugars, organic acids and bioactive compounds, suggesting their possible use as a functional ingredient (with antioxidant properties) in different food products. © 2017 Society of Chemical Industry.

Biochemical Constituents and in Vitro Antioxidant and Anticholinesterase Potential of Seeds from Native Korean Persimmon Genotypes.

In the current study, the functional and biochemical potential of the seeds of four persimmon cultivars (PC1, PC2, PC3 and PC4) and their role against oxidative stress and acetylcholinesterase (AChE) inhibition were evaluated. In terms of biochemical compositions, free amino acids, fatty acids and organic acids analysis was performed. The free amino acids ranged from 2617.31 (PC2) to 3773.01 µg∙g(-1) dry weight (PC4). Oleic acid and linoleic acid were the principal fatty acids, which were significantly higher in PC4 and PC1, respectively. PC4 presented the highest amount of organic acid content (4212 mg∙kg(-1)), whereas PC2 presented the lowest (2498 mg∙kg(-1)). PC2 contained higher total phenolic content and flavonoid content, whereas PC3 had the lowest amount as compared to other cultivars. The in vitro DPPH, ABTS and superoxide anion radicals scavenging activity increased in a dose-dependent manner, whereas PC2 showed significantly higher scavenging activities as compared to PC1, PC2 and PC4 types. In the case of AChE inhibition, PC4 showed a moderate activity (67.34% ± 1.8%). In conclusion, the current findings reveal that the studied persimmon seeds cultivars are a source of bioactive natural antioxidants and AChE inhibitors. Such natural products could be employed in pharmaceutical and food industries, whilst can also be considered for the treatment of neurodegenerative diseases such as Alzheimer's.

Processes Driving the Adaptive Radiation of a Tropical Tree (Diospyros, Ebenaceae) in New Caledonia, a Biodiversity Hotspot.

Due to its special geological history, the New Caledonian Archipelago is a mosaic of soil types, and in combination with climatic conditions this results in a heterogeneous environment across relatively small distances. A group of over 20 endemic species of Diospyros (Ebenaceae) has rapidly and recently radiated on the archipelago after a single long-distance dispersal event. Most of the Diospyros species in the radiating group are morphologically and ecologically well differentiated, but they exhibit low levels of DNA variability. To investigate the processes that shaped the diversification of this group we employed restriction site associated DNA sequencing (RADseq). Over 8400 filtered SNPs generally confirm species delimitations and produce a well-supported phylogenetic tree. Our analyses document local introgression, but only a limited potential for gene flow over longer distances. The phylogenetic relationships point to an early regional clustering among populations and species, indicating that allopatric speciation with respect to macrohabitat (i.e., climatic conditions) may have had a role in the initial differentiation within the group. A later, more rapid radiation involved divergence with respect to microhabitat (i.e., soil preference). Several sister species in the group show a parallel divergence in edaphic preference. Searches for genomic regions that are systematically differentiated in this replicated phenotypic divergence pointed to loci potentially involved in ion binding and cellular transport. These loci appear meaningful in the context of adaptations to soil types that differ in heavy-metal and mineral content. Identical nucleotide changes affected only two of these loci, indicating that introgression may have played a limited role in their evolution. Our results suggest that both allopatric diversification and (parapatric) ecological divergence shaped successive rounds of speciation in the Diospyros radiation on New Caledonia.

[Identification of six species of medicinal Diospyros plants based on leaf macro- and micro-morphology].

To establish a method for the identification of five species and one variety of medicinal plants from Diospyros, their leaf veins, epidermis, anatomic and powder characters were observed and compared with macro-morphological and microscopic methods. The results indicated the differences of secondary and tertiary veins among those Diospyros species. The single cell non-glandular hair and glandular hair exist in most species' epidermis while stone cells were only found in the leaf powders of two species. Through the study, the main differences of leaf macro- and micro-morphology of these species were obtained and practical keys were also established, which can provide scientific base not only for identification of these species during their vegetative stages, but also for accuracy authentication of the source of Kaki Folium.

Physico-chemical and structural characterization of mucilage isolated from seeds of Diospyros melonoxylon Roxb

Mucilage was isolated from the seeds of Diospyros melonoxylon Roxb., a plant growing naturally in the forests of India. Various physico-chemical methods like particle analysis, scanning electron microscopy, differential scanning calorimetry, differential thermal analysis, thermogravimetry analysis, molecular weight by gel permeation chromatography, rheometry, elemental analysis, x-ray diffraction spectrometry, zeta potential, fourier transform infrared spectroscopy, 1D(1H and 13C) (NMR) have been employed to characterize this gum in the present study. Particle analyses suggest that mucilage had particle size in nanometer. SEM analysis suggested that the mucilage had irregular particle size. The glass transition temperature of the gum observed was 78 °C and 74 °C by DSC and DTA respectively. The Thermogravimetry analysis suggested that mucilage had good thermal stability with two stage decomposition. The molecular weight of mucilage was determined to be 8760, by gel permeation chromatography, while the viscosity of mucilage was observed to be 219.1 cP. The XRD pattern of the mucilage indicated a complete amorphous nature. Elemental analysis of the gum revealed specific contents of carbon, hydrogen, nitrogen and sulfur. The major functional groups identified from FT-IR spectrum include 3441 cm-1 (-OH), 1632 cm-1 (-COO-), 1414 cm-1 (-COO-) and 1219 cm-1 (-CH3CO). Analysis of mucilage by paper chromatography and 1D NMR indicated the presence of sugars.

Mucilagem foi isolada de sementes de Diospyros melanoxylon Roxb, uma planta que cresce naturalmente nas florestas da Índia. Vários métodos físico-químicos, como análise de partículas, microscopia electrônica, calorimetria diferencial de varredura, análise térmica diferencial, análise termogravimétrica, massa molecular por cromatografia de permeação em gel, viscosidade, análise elementar, espectrometria de difração de raios-x, potencial zeta, espectroscopia no infravermelho com transformada de Fourier, 1D (1H e 13C) (NMR) foram utilizados no presente estudo para caracterizar essa goma. A análise de partículas sugere que a mucilagem tem tamanho de partícula em nm. A análise SEM sugere que a mucilagem tem tamanho de partícula irregular. Observou-se temperatura de transição vítrea da goma de 78 °C e 74 °C por DSC e DTA, respectivamente. A análise termogravimétrica sugeriu que a mucilagem possuía boa estabilidade térmica, com duas fases de decomposição. A massa molecular da mucilagem foi 8760, por meio de cromatografia de permeação em gel, enquanto que a viscosidade foi 219,1 cP. O padrão de DRX da mucilagem indicou natureza completamente amorfa. Os principais grupos funcionais identificados a partir do espectro de FT-IR foram: 3441 cm-1 (-OH), 1632 cm-1 (-COO-), 1414 cm-1 (-COO-) e 1.219 cm-1 (CH3CO-). As análises de mucilagem por cromatografia em papel e 1D RMN indicaram a presença de açúcares.

Analyses of amplified fragment length polymorphisms (AFLP) indicate rapid radiation of Diospyros species (Ebenaceae) endemic to New Caledonia.

BACKGROUND: Radiation in some plant groups has occurred on islands and due to the characteristic rapid pace of phenotypic evolution, standard molecular markers often provide insufficient variation for phylogenetic reconstruction. To resolve relationships within a clade of 21 closely related New Caledonian Diospyros species and evaluate species boundaries we analysed genome-wide DNA variation via amplified fragment length polymorphisms (AFLP). RESULTS: A neighbour-joining (NJ) dendrogram based on Dice distances shows all species except D. minimifolia, D. parviflora and D. vieillardii to form unique clusters of genetically similar accessions. However, there was little variation between these species clusters, resulting in unresolved species relationships and a star-like general NJ topology. Correspondingly, analyses of molecular variance showed more variation within species than between them. A Bayesian analysis with BEAST produced a similar result. Another Bayesian method, this time a clustering method, Structure, demonstrated the presence of two groups, highly congruent with those observed in a principal coordinate analysis (PCO). Molecular divergence between the two groups is low and does not correspond to any hypothesised taxonomic, ecological or geographical patterns. CONCLUSIONS: We hypothesise that such a pattern could have been produced by rapid and complex evolution involving a widespread progenitor for which an initial split into two groups was followed by subsequent fragmentation into many diverging populations, which was followed by range expansion of then divergent entities. Overall, this process resulted in an opportunistic pattern of phenotypic diversification. The time since divergence was probably insufficient for some species to become genetically well-differentiated, resulting in progenitor/derivative relationships being exhibited in a few cases. In other cases, our analyses may have revealed evidence for the existence of cryptic species, for which more study of morphology and ecology are now required.

Molecular phylogenetics of New Caledonian Diospyros (Ebenaceae) using plastid and nuclear markers.

To clarify phylogenetic relationships among New Caledonian species of Diospyros, sequences of four plastid markers (atpB, rbcL, trnK-matK and trnS-trnG) and two low-copy nuclear markers (ncpGS and PHYA) were analysed. New Caledonian Diospyros species fall into three clades, two of which have only a few members (1 or 5 species); the third has 21 closely related species for which relationships among species have been mostly unresolved in a previous study. Although species of the third group (NC clade III) are morphologically distinct and largely occupy different habitats, they exhibit little molecular variability. Diospyros vieillardii is sister to the rest of the NC clade III, followed by D. umbrosa and D. flavocarpa, which are sister to the rest of this clade. Species from coastal habitats of western Grande Terre (D. cherrieri and D. veillonii) and some found on coralline substrates (D. calciphila and D. inexplorata) form two well-supported subgroups. The species of NC clade III have significantly larger genomes than found in diploid species of Diospyros from other parts of the world, but they all appear to be diploids. By applying a molecular clock, we infer that the ancestor of the NC clade III arrived in New Caledonia around 9 million years ago. The oldest species are around 7 million years old and the youngest ones probably much less than 1 million years.

Carotenoids in fruits of different persimmon cultivars.

Carotenoids in the peel and the flesh of persimmon fruit were identified, and the contents of carotenoids in the fleshes of 46 different persimmon cultivars were analyzed. The results indicated that 31 specific carotenoids were detected in both cultivars of persimmons, among which nine specific carotenoids were characterized. ß-cryptoxanthin was the most abundant carotenoid among all individual components in both the peel and the flesh, accounting for about 20-30% of the total carotenoids in both cultivars. The contents of total carotenoids in the fleshes of different persimmon cultivars were between 194.61 µg/100g FW and 1,566.30 µg/100g FW. Zeaxanthin was also the most abundant in all persimmon fleshes besides ß-cryptoxanthin, and the total amount of these two components accounted for 37.84-85.11% of the total carotenoids. The RE values in the fleshes of different cultivars also differed greatly. Besides, the stage of maturation was also important factor which could influence the carotenoid content and RE value in the fleshes.

Influence of two cultivars of persimmon on atherosclerosis indices in rats fed cholesterol-containing diets: Investigation in vitro and in vivo.

OBJECTIVE: To assess the influence of two persimmon cultivars on some atherosclerosis indices in rats fed cholesterol (Chol)-containing diets. METHODS: Persimmon cultivars "Fuyu" and "Jiro" as supplementation to rats' diets were investigated in vitro to compare the contents of their bioactive compounds (polyphenols, flavonoids, flavanols, tannins, carotenoids, and ascorbic acid) and antioxidant potentials. In the in vivo investigation, 36 male Wistar rats were randomly divided into six diet groups, each with six rats: control, control/Fuyu, control/Jiro, Chol, Chol/Fuyu, and Chol/Jiro. During a period of 47 d (42 d of feeding and 5-d adaptation before the experiment) of the trial, rats in the control group were fed a basal diet and two additional control groups (control/Fuyu and control/Jiro) a basal diet plus 5% of lyophilized Fuyu and Jiro, respectively. The Chol, Chol/Fuyu, and Chol/Jiro rat groups were fed a basal diet supplemented with 1% Chol (Chol group) and 1% Chol plus 5% lyophilized Fuyu (Chol/Fuyu group) and plus 5% lyophilized Jiro (Chol/Jiro group), respectively. After completion of the experiment, the rats were anesthetized using Narcotan (halothane) and sacrificed and the atherosclerotic lesions in the aorta were assessed. The obtained results of the investigation of all six groups were compared. Testing of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triacylglycerols, total cholesterol in the liver, electrophoretic patterns of liver tissue, and three-dimensional fluorescence of serum protein fractions was performed. RESULTS: The polyphenols and tannins were significantly higher in the Fuyu cultivar (P<0.05). The antioxidant potential of persimmon Fuyu was higher than in the Jiro cultivar, but the difference was significant only according to the 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay (P<0.05). Supplementation of diets with 5% of the lyophilized Fuyu and Jiro hindered the increase in plasma lipids versus the Chol group (total cholesterol 19.4% and 9.5%, low-density lipoprotein cholesterol 25.6% and 13.1%, respectively, P<0.05) and hindered the decrease in plasma antioxidant activity versus the Chol group by 40.0% and 16.8% and by 39.6% and 11.3% for the ABTS and 1,1-diphenyl-2-picrylhydrazyl assays, respectively. The atherosclerotic lesions in the aortas of the Chol/Fuyu and Chol/Jiro groups were significantly less than in the Chol group (P<0.05). Electrophoresis of the proteins from rats' liver tissue showed changes in 14-kDa bands after persimmon supplementation. A shift in maximum wavelengths in three-dimensional fluorescence of serum protein fractions after persimmon supplementation was found in comparison with the control group and an increase in fluorescence intensity compared with the Chol groups. CONCLUSION: The contents of polyphenols and tannins were significantly higher in the Fuyu cultivar (P<0.05). The antioxidant potentials of Fuyu were higher than those of the Jiro cultivar, but the difference was significant only according to the ABTS assay (P<0.05). Supplementation of 5% lyophilized Fuyu and Jiro to diets of rats fed Chol-containing diets 1) hindered the increase in plasma lipids levels and the decrease in plasma antioxidant activity and 2) significantly decreased the atherosclerotic lesions in the aorta (P<0.05). Electrophoretic patterns of liver tissue and fluorescence spectra can be used as additional biomarkers for determination of atherosclerosis indices.

Diospyrone, crassiflorone and plumbagin: three antimycobacterial and antigonorrhoeal naphthoquinones from two Diospyros spp.

The aim of this study was to evaluate the antimycobacterial and antigonorrhoeal activities of three naphthoquinones (diospyrone, crassiflorone and plumbagin) from Diospyros canaliculata and Diospyros crassiflora as well as the crude extracts from these plants. The agar disk diffusion assay, broth microdilution method, microplate Alamar blue assay (MABA) and radiometric respiratory technique using the BACTEC 460 TB system were used. Results of the antimycobacterial assays indicated that the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations ranged from 1.22 microg/mL to 39.06 microg/mL for Mycobacterium smegmatis and all studied Mycobacterium tuberculosis strains for the crude extract from D. crassiflora, diospyrone and crassiflorone. Results of the killing rate experiment revealed that a total inhibition effect on M. tuberculosis H37Rv strain was observed at Day 18 for D. crassiflora and Day 21 for the crude extract from D. canaliculata and diospyrone at 4x MIC as determined by MABA. Results of the antigonorrhoeal assay indicated that diospyrone was able to prevent the growth of all studied strains of Neisseria gonorrhoeae. The overall results of this work provide evidence that the studied plant extracts (diospyrone, crassiflorone and plumbagin) might be potential sources of new antimicrobial drugs against tuberculosis and gonorrhoea.

A multi-locus plastid phylogenetic analysis of the pantropical genus Diospyros (Ebenaceae), with an emphasis on the radiation and biogeographic origins of the New Caledonian endemic species.

We aimed to clarify phylogenetic relationships within the pantropical genus Diospyros (Ebenaceae sensulato), and ascertain biogeographical patterns in the New Caledonian endemic species. We used DNA sequences from eight plastid regions (rbcL, atpB, matK, ndhF, trnK intron, trnL intron, trnL-trnF spacer, and trnS-trnG spacer) and included 149 accessions representing 119 Diospyros species in our analysis. Results from this study confirmed the monophyly of Diospyros with good support and provided a clearer picture of the relationships within the genus than in previous studies. Evidence from phylogenetic analyses suggests that Diospyros colonized New Caledonia multiple times. The four lineages of Diospyros in New Caledonia also differ in their degree of diversification. The molecular data indicate that one lineage is paleoendemic and derived from an ancient Australian species. The other three lineages are more closely related to several Southeast Asian species; two of them are neoendemics, and one has radiated rapidly and recently.