Development of Kompetitive Allele Specific PCR markers for identification of persimmon varieties using genotyping-by-sequencing

BACKGROUND: Persimmon (Diospyros kaki Thunb.) is the most widely cultivated species of the genus Diospyros. In this study, genetic diversity and variations in persimmon genotypes were investigated using single nucleotide polymorphism (SNP) markers identified by genotyping-by-sequencing (GBS) analysis. RESULTS: Ninety-five persimmon accessions grown in the Pear Research Institute, National Institute Horticultural and Herbal Science, were sequenced using the Illumina Hiseq2500 platform and polymorphic SNPs were detected to develop molecular markers. These reliable SNPs were analyzed using the Kompetitive Allele Specific PCR (KASP) assay to discriminate among persimmon genotypes. GBS generated a total of 447,495,724 trimmed reads, of which 89.7% were raw reads. After demultiplexing and sequence quality trimming, 108,876,644 clean reads were mapped to the reference transcriptome. An average of 1,146,070 genotype reads were mapped. Filtering of raw SNPs in each sample led to selection of a total of 1,725,401 high-quality SNPs. The number of homozygous and heterozygous SNPs ranged from 1,933 to 6,834 and from 846 to 5,927, respectively. CONCLUSIONS: Of the 49 SNPs selected for development of an identification system for persimmons, 15 SNPs were used in the KASP assay to analyze 32 persimmon accessions. These KASP markers discriminated among all accessions.

The Effects of Shiteito for Hiccups / 日本東洋医学雑誌

Persistent hiccups impair the quality of life of patients, and are sometimes difficult to treat. In our hospital, we usually use a shiteito decoction to treat hiccups, however, there are no published data about the effect of shiteito on hiccups. We retrospectively investigated patients with hiccups treated with a shiteito decoction as a first-line drug from February 1, 2014 to February 28, 2016. Twenty-seven patients were included (three women), and their ages ranged from 24 to 89 years (mean age : 65.4 ± 15.1 years). The shiteito decoction was completely or partially effective in 66.7% of all patients, and 59.3% of all patients completely recovered. In most cases, the hiccups disappeared within two days, and 88.9% of the patients with complete or partial recovery found the decoction to be effective within four days. Shiteito was an effective hiccup treatment, and since it could be easily administered at bedside, it would be useful regardless of patient pattern.

An overview of dermatological and cosmeceutical benefits of Diospyros kaki and its phytoconstituents

Abstract Diospyros kaki L.f. belonging to family Ebenaceae, commonly known as persimmon is used as a medicinal plant in Chinese traditional medicine since many years for different ailments including cosmetics and dermatologic applications. Traditionally this plant is used to treat different skin conditions including pimples, skin eruptions and eczema. Present interest has been focused toward use of natural bioactive compounds in various curative and beautifying applications in dermatological and cosmeceutical disciplines. The objective of this article is to present cumulative data on potential use of D. kaki for its possible role in dermatologic and cosmetic applications. Scientific data has revealed an excellent position of D. kaki in both dermatology and cosmetic discipline making it a valuable choice in respective field. Active principles from different plant parts have shown to possess anti-inflammatory, antiallergic, photo-protective, and anti-wrinkle effects with appreciable activities against tyrosinase, elastase, and collagenase enzymes. Promising antioxidant activity and skin whitening potential, augmented by reduction in sebum contents, and reduction in size and number of skin pores make it a suitable choice as cosmetic ingredient. Data has been summarized and presented on available molecular mechanism that can contribute toward phytoconstituents usage in cosmetics and dermatology mediated by different cellular pathways. Crude extracts and some of phytochemical obtained from this plant such as isoquercitrin and hyperin have better reported activities than well-known cosmetic ingredients viz., arbutin, kojic acid and hydroquinone with possibility of having no side effects. Photo protection against degenerative effects of UVA, UVB and gamma radiation can help skin to fight well against oxidative stress and reactive oxygen species. Further investigation need to be directed toward human subjects for evaluation of these reported activities for obtaining optimum commercial and industrial benefits from this valuable plant.

Characterization and influence of subtropical persimmon cultivars on juice and jelly characteristics

ABSTRACT To increase the availability to consumers and add more value to persimmon (Diospyros kaki L.), which is a very perishable and seasonal fruit and in order to identify which cultivars grown in subtropical regions are more suitable for processing in the form of juice and jelly, as well as understand what the consumer profile is for these products, the objective of this study was to evaluate the influence of different persimmon cultivars (Rama Forte, Mel, Guiombo and Taubaté) grown in subtropical regions of Brazil on the physicochemical characteristics, rheological properties and sensory acceptance of the resulting juice and jelly in order to identify cultivars with the greatest potential for industrial use. The different studied persimmon cultivars had different physical and physicochemical characteristics which resulted in juices and jellies with different physicochemical, rheological and sensory characteristics. Based on sensory acceptance and productivity/adaptability of persimmon crop in Brazil, the most suitable persimmon cultivars for processing are Rama Forte and Guiombo. In this study it was found that the consumer prefers a more acidic persimmon juice and a less firm or softer, less sweet, clearer and more intense yellow color persimmon jelly.

Antioxidant effect of persimmon leaf extract to HEK293-APPswe transgenic cells and effect to Nrf2/HO-1 pathway / 中国免疫学杂志

Objective:To investigate the effect of persimmon leaf extract (PLE) on HEK293-APPswe transgenic cells (20E2).Methods:To determine whether the 20E2 cells model was successfully established,the level of Aβ1-40 in SH-SY5Y was detected and 20E2 cells(HEK293 cells stably expressing Swedish mutant APP)cultured in vivo by ELISA kit,and the expression of APP protein level was detected by Western blot.Cell viability was assayed by CCK-8 method and then selected the best concentration.Set groups:SH-SY5Y as normal control group (NC group),20E2 as model group (20E2 group),treating with PLE as treating group (20E2+PLE group).Reactive oxygen species (ROS) levels of each group were detected with DCFH-DA fluorescent probe.The extracellular level of Aβ1-42 were detected by ELISA kit.Cytoplasmic Nrf2,Nuclear Nri2,Whole-cell HO-1 were detected by Western blot.Results:Compared with model group,the expressions of ROS,Aβ1-42 were down-regulated and the Nuclear Nrf2 and Whole-cell HO-1 were up-regulated in 20E2+PLE group.Conclusion:PLE can reduce the level of oxidative stress of model group effectively,it possibly reduce the aggregation of Aβ1-42 and prevent oxidizing via activating Nrf2/HO-1 pathway.

Screening and Optimization of the Extraction Technology of Total Flavonoids in Persimmon Leaves by Semi-bionic-enzyme Method / 中国药房

OBJECTIVE:To screen the method for extracting total flavonoids in persimmon leaves and optimize extraction tech-nology. METHODS:Using extract quality and flavonoids content as indexes,the effects of extracting total flavonoids in persim-mon leaves by ethanol refluxing method,enzyme method(cellulase,β-glucanase and complex enzyme mixed by equal amounts of both),semi-bionic method and semi-bionic-enzyme method (the same enzymes) were compared. Using flavonoids content as in-dex,solid-liquid radio,reflux temperature,reflux time as factors,orthogonal test was designed to optimize the extraction technolo-gy conditions of flavonoids in persimmon leaves by semi-bionic-enzyme method,and the verification test was conducted. RE-SULTS:The extract quantity and flavonoids content by semi-bionic-enzyme method was the highest among the 4 extraction meth-ods,and the complex enzyme was the most suitable;the optimized extracting condition of semi-bionic-enzyme method were as fol-lows as solid-liquid radio of 1:14,reflux temperature of 50 ℃,reflux time of 2.0 h;extraction rates of flavonoids in 3 verification tests were 5.9%,5.8%,5.9%(RSD=0.98%,n=3). CONCLUSIONS:The optimized semi-bionic-enzyme method is efficient and stable in extracting flavonoids in persimmon leaves.

Molecular Phylogeny and Morphology of Mycosphaerella nawae, the Causal Agent of Circular Leaf Spot on Persimmon

In this study, the phylogeny and morphology of Mycosphaerella nawae (Dothideomycetes, Ascomycota) were examined using Korean and Japanese isolates, to establish the phylogenetic relationship between M. nawae and its allied species. Korean and Japanese isolates of M. nawae were collected from circular leaf spot-diseased leaves and were confirmed based on internal transcribed spacer (ITS) sequence data. Phylogenetic analysis was conducted using multiple genes, including the ITS region, 28S rDNA, β-tubulin, translation elongation factor-1α, and actin genes. Our results revealed that M. nawae is closely related to members of the genus Phaeophleospora but are distant from the Ramularia spp. In addition, microscopic analysis revealed pseudothecia on the adaxial and abaxial surface of overwintered diseased leaves (ODL) and only on the abaxial surface of diseased leaves. Ascospores are oval to fusiform, one-septate, tapered at both ends, 1.7~3.1 × 8.1~14.1 µm, and were observed in ODL. Conidia are oval, guttulate, one-septate, 3.5~4.9 × 12.8~19.8 µm, and barely discernable on 30-day cultures. To our knowledge, this is the first report on the phylogeny of M. nawae, which is closely related to the genus Phaeophleospora, especially P. scytalidii.

Clinical observation of persimmon leaf extract on secondary prevention of ischemic cerebrovascular disease / 中国生化药物杂志

Objective To investigate persimmon leaf extract on secondary prevention of ischemic cerebrovascular disease, and the impact on blood pressure, blood sugar, blood lipid, hemorheology, blood rheology indexes and blood homocysteine levels in patients with ischemic cerebrovascular disease.Methods 200 cases of patients with ischemic cerebral vascular disease were chosen, which were randomly divided into observation group and control group with 100 cases in each group.Both of the groups were given conventional therapy.And the observation group received persimmon leaf extract intervention.Then the rate of recurrence of ischemic cerebrovascular disease was observed after 3 months and 6 months.At the same time, the indexes of blood pressure and blood sugar, blood lipid, hemorheology, blood flow and blood homocysteine were recorded and compared in the second morning after being selected and after 3 months and 6 months treatment.Results After treatment for 3 months, the recurrence rate of observation group was 4.40%, 3.70% of control group; after treatment for 6 months, 8.24% of observation group, 13.75% of control group, and there were no significant differences between two groups of recurrence of ischemic cerebrovascular disease.After 6 months, systolic and diastolic blood pressure in both groups were lower than before treatment.and they were lower in observation group than control group (all P<0.05).After 3, 6 months, TC, TG, LDLC levels decreased and HDLC increased compared with before treatment, and the above indexes improved more in observation group than control group (all P<0.05).After 3, 6 months, FBG, 2hPG, HbA1C levels decreased compared with before treatment, and the above indexes decreased more at the same time point in observation group than control group (all P<0.05).After 3, 6 months, HCY levels decreased compared with before treatment, and HCY decreased more at the same time point in observation group than control group ( all P<0.05 ).After 6 months, blood rheology indexes levels improved more compared with before treatment, and they improved more at the same time point in observation group than control group (all P<0.05).After 6 months, main artery blood flow velocity levels increased compared with before treatment, and they increased more at the same time point in observation group than control group ( all P<0.05 ) .Conclusion Persimmon leaf extract intervention therapy can effectively improve blood pressure , sugar, lipid, hemorheology, homocysteine and prevent platelet aggregation and thrombosis.

Effects of Kaki (Persimmon) Extract-containing Diet on Levels of Volatile Sulfur Compounds in Oral Gas and Feces, as well as on Subjective Fecal Odor, in Healthy Adults / 日本補完代替医療学会誌

<b>Objectives:</b> To examine in two tests the potential of kaki (persimmon) extract-containing diet (KE diet) to reduce malodorous volatile sulfur compounds (VSC), such as hydrogen sulfide (H₂S), methyl mercaptan (CH₃SH) and dimethyl sulfide (CH₃SCH₃), as well as on subjective fecal odor on healthy adults.<br> <b>Methods:</b> In the first test, 11 subjects were given garlic-containing soup. For a period of time, they were given a single dose of KE diet (150 mg as kaki extract) with water, and only water for the rest of the study period. Two hours after the administration, oral gas samples were collected from individual subjects and analyzed for VSC. In the second test, 14 subjects were given a single dose of KE diet for 7 days. Fecal samples were collected from individual subjects before and after the 7-day KE diet intervention. Levels of VSC were determined and the magnitude of subjective fecal odor was estimated based on ratings in the self-administered questionnaire.<br> <b>Results:</b> Levels of CH₃SCH₃ in oral gas were significantly lowered when subjects were on a KE diet. On the other hand, although decreases in the level of any VSC in feces before and after the 7-day KE diet intake did not reach a statistical significance, subjective fecal odor significantly improved by the KE diet intake.<br> <b>Conclusion:</b> KE diet appears to have a beneficial effect on VSC-associated oral malodor and subjective fecal odor.<br>

First Report of Black Spot Disease Caused by Alternaria alternata on Sweet Persimmon Fruits

Black spot of sweet persimmon, caused by Alternaria alternata, occurred in an orchard in Gyeongnam province, Korea in 2012. The symptom was appearance of 0.5 to 4 cm black spots on the surface of fruit. The pathogen was isolated from flesh of disease lesions. The causal agent was identified as A. alternata by morphological characteristics and sequencers of the internal transcribed spacer (ITS) 1 and ITS4 regions of rRNA. Artificial inoculation of the pathogen resulted in development of disease symptoms and the re-isolated pathogen showed characteristics of A. alternata.

Antioxidative and Antidiarrheal Effects of Persimmon Extracts / 한국실험동물학회지

Since oxidative stresses are involved in gastroenteritis and diarrhea, we investigated antioxidative and antidiarrheal activities of persimmon flesh extract (PFE) and persimmon calyx extract (PCE) in vitro and in vivo, respectively. PCE significantly scavenged 1,1-diphenyl-2-picrylhydrazyl hydrate and 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) from 500 microg/mL, although PFE was ineffective. In addition, PFE and PCE exhibited strong nitric oxide-scavenging effects from 1 microg/mL, in which PCE was superior to ascorbic acid (50 microM). Furthermore, PFE and PCE significantly inhibited FeCl3-induced lipid peroxidation as well as Cu2+/H2O2-induced protein oxidation from 10 microg/mL. In vivo charcoal-propulsion assay, in contrast to a negligible effect of PFE, treatment with PCE (160-500 mg/kg) markedly inhibited intestinal motility. The results indicate that extracts of persimmon, especially PCE, possess antioxidative, antiinflammatory and antidiarrheal activities. Therefore, it is suggested that persimmon extracts could be used for the relief of gastroenteritis and diarrhea.

Fruit Soft Rot of Sweet Persimmon Caused by Mucor piriformis in Korea

A fruit soft rot caused by Mucor piriformis occurred on sweet persimmon storages in Jinju, Changwon and Gimhae, Gyeongnam province, Korea, 2003. The disease infection usually started from wounding after cracking of fruits. At first, the lesions started with water soaked and rapidly softened and diseased lesion gradually expanded. Colonies on potato dextrose agar at 20degrees C were whitish to olivaceous-buff Sporangia were globose, black and 96~153microm in size. Sporangiophores were 26~42microm in width. Sporangiospores were ellipsoid and 5.8~10.6x4.3~7.6microm in size. Columella was obovoid, cylindrical-ellipsoidal, pyriform, subglobose and 80~125microm in size. Optimal temperature for mycelial growth was 20degrees C on PDA. The causal organism was identified as M. piriformis. This is the first report of fruit soft rot on sweet persimmon caused by M. piriformis in Korea.

Anticancer Effect of Persimmon Leaf Extracts on Korean Gastric Cancer Cell

This study was performed to investigate the in vitro and in vivo anticancer effects of persimmon leaf extracts on human gastric cancer cells. In vitro anticancer effects of persimmon leaf extracts (water extract at 80 degrees C for 3 hours, water extract at room temperature for 48 hours, 50% ethanol extract at 80 degrees C for 3 hours, 50% ethanol extract at room temperature for 48 hours, 75% ethanol extract at 80 degrees C for 3 hours and 75% ethanol extract at room temperature for 48 hours) on SNU16 (Korean gastric cancer cell) were investigated by MTT assay. Persimmon leaf extracts exhibited strong in vitro anticancer effects. We found that the higher the ethanol content of the solvent, the stronger the in vitro anticancer effects. Extraction yields, contents of flavonoids, vitamin A, vitamin C and vitamin E were measured. We found that the higher the ethanol content of the solvent, the higher the extraction yields and the contents of flavonoids, vitamin A and vitamin E. Among persimmon leaf extracts, 75% ethanol 80 degrees C extract showed the highest extraction yield, the highest contents of flavonoids, vitamin A and vitamin E and exhibitied the strongest in vitro anticancer effect on SNU16. Therefore, 75% ethanol 80 degrees C extract was chosen as the material to investigate in vivo anticancer effects. In vivo anticancer effect of persimmon leaf 75% ethanol 80 degrees C extract was investigated in SNU16 transplanted nude mice. Twenty five female nude mice (BALB/c) were blocked into five groups according to body weight and raised for 4 weeks with diets containing 4% (w/w), 8% (w/w) persimmon leaf 75% ethanol 80 degrees C extract, with IT (intratumoral) injection treatment with 1.65 mg/100 microliter, 3.3 mg/100 microliter concentration every other day 3 weeks after SNU16 was transplanted. Persimmon 75% ethanol 80 degrees C extract significantly lowered tumor weight and tumor volume in SNU16 transplanted nude mice. Tumor weight and tumor volume in all experimental groups were significantly lower than those in the control group. Helper T cell (CD4) levels of mice injected with 3.3 mg/100 microliter extract significantly increased. Cytotoxic T cell (CD8) levels in all experimental groups significantly increased and helper/cytotoxic T cell ratios in all experimental groups significantly decreased. Natural killer cell and MHC class II molecule in all experimental groups significantly increased. In conclusion, persimmon leaf 75% ethanol 80 degrees C extract exhibited strong in vitro and in vivo anticancer effects against SNU16 cells and it increased cytotoxic T cell, natural killer cell and MHC classII molecule in experimental groups in SNU16 transplanted nude mice.

THE ANTI-OXIDATIVE EFFECT OF PERSIMMON LEAF FLAVONOID IN VITRO / 营养学报

Objective: To study the anti oxidative effects of PLF. Methods: The ?OH scavenging effect was measured by salicylic acid method, the content of MDA in liver mitochondria and microsome was measured by TBA assay; hemolysis of RBC and the swelling of mitochondria were detected by spectrophotometric methods. Results: PLF can scavenge ?OH, inhibit production of MDA, swelling of mitochondria and hemolysis of RBC. Conclusion: PLF has anti oxidation effects invitro.