Activation of autophagy by Citri Reticulatae Semen extract ameliorates amyloid-beta-induced cell death and cognition deficits in Alzheimer’s disease

Amyloid-beta-induced neuronal cell death contributes to cognitive decline in Alzheimer’s disease.Citri Reticulatae Semen has diverse beneficial effects on neurodegenerative diseases,including Parkinson’s and Huntington’s diseases,however,the effect of Citri Reticulatae Semen on Alzheimer’s disease remains unelucidated.In the current study,the anti-apoptotic and autophagic roles of Citri Reticulatae Semen extract on amyloid-beta-induced apoptosis in PC12 cells were first investigated.Citri Reticulatae Semen extract protected PC12 cells from amyloid-beta-induced apoptosis by attenuating the Bax/Bcl-2 ratio via activation of autophagy.In addition,Citri Reticulatae Semen extract was confirmed to bind amyloid-beta as revealed by biolayer interferometry in vitro,and suppress amyloid-beta-induced pathology such as paralysis,in a transgenic Caenorhabditis elegans in vivo model.Moreover,genetically defective Caenorhabditis elegans further confirmed that the neuroprotective effect of Citri Reticulatae Semen extract was autophagy-dependent.Most importantly,Citri Reticulatae Semen extract was confirmed to improve cognitive impairment,neuronal injury and amyloid-beta burden in 3×Tg Alzheimer’s disease mice.As revealed by both in vitro and in vivo models,these results suggest that Citri Reticulatae Semen extract is a potential natural therapeutic agent for Alzheimer’s disease via its neuroprotective autophagic effects.

A Network Pharmacology Study on Active Components and Targets of Citri Reticulatae Pericarpium for Treating Keloids

[Objectives]To investigate the mechanisms and pharmacologic effects of Citri Reticulatae Pericarpium against keloids by network pharmacology systematically.[Methods]TCMSP,Uniprot and BATMAN-TCM databases were used to obtain the active constituents and targets of Citri Reticulatae Pericarpium."Keloid"was used as key word to search for related therapeutic targets from Drug Bank,OMIM,TTD,and GEO databases.The Chinese medicine compound-target network was constructed by Cytoscape software.Besides,gene ontology(GO)and Kyoto Encyclopedia of genes and genome enrichment analysis were also performed.Afterward,Discovery Studio software was used to assess the interaction of key components and genes.[Results]Five active components of Citri Reticulatae Pericarpium,773 compound targets and 676 keloid treatment targets were obtained in the databases.After the intersection,there are 47 targets of Citri Reticulatae Pericarpium for treating keloids.Hub genes were identified such as MMP9,IL6,TNF,TP53,and VEGFA,which were enriched in tumor necrosis factor-α,nuclear factor kappa-B,and other signaling pathways.The molecular docking stimulation confirmed the interaction between the MMP9 and three components of Citri Reticulatae Pericarpium.[Conclusions]Citri Reticulatae Pericarpium may play an important role in treating keloids through modulating genes and signaling pathways.The present study sheds light on the mechanisms of active compounds of Citri Reticulatae Pericarpium for the treatment of keloids.

Establishment of HPLC Fingerprint, Cluster Analysis and Principle Component Analysis of Citri Reticulatae Pericarpium Viride

[Objectives] This study aimed to establish HPLC fingerprint and conduct cluster analysis and principle component analysis for Citri Reticulatae Pericarpium Viride. [Methods] Using the HPLC method, the determination was performed on XSelect~® HSS T3-C_(18) column with mobile phase of acetonitrile-0.5% acetic acid solution(gradient elution) at the flow rate of 1.0 mL/min. The detection wavelength was 360 nm. The column temperature was 25℃. The sample size was 10 μL. With peak of hesperidin as the reference, HPLC fingerprints of 10 batches of Citri Reticulatae Pericarpium Viride were determined. The similarity of the 10 batches of samples was evaluated by Similarity Evaluation System for Chromatographic Fingerprint of TCM(2012 edition) to determine the common peaks. Cluster analysis and principal component analysis were performed by using SPSS 17.0 statistical software. [Results] The HPLC fingerprints of the 10 batches of medicinal materials had total 11 common peaks, and the similarity was 0.919-1.000, indicating that the chemical composition of the 10 batches of medicinal materials was consistent. There were 11 common components in the 10 batches of medicinal materials, but their contents were different. When the Euclidean distance was 20, the 10 batches of samples were divided into two categories, S4 in the first category, and the others in the second one. When the Euclidean distance was 5, the second category could be further divided into two sub-categories, S1 and S10 in one sub-category, and S2, S3, S5, S6, S7, S8 and S9 in the other one. The principle component analysis showed that cumulative contribution rate of the two main component factors was 92.797%, and the comprehensive score of S7 was the highest with the best quality. [Conclusions] The results of HPLC fingerprinting, cluster analysis and principle component analysis can provide reference for the quality control of Citri Reticulatae Pericarpium Viride.

Evaluation of bioactive flavonoids in Citri Reticulatae Pericarpium from different regions and its association with antioxidant andα-glucosidase inhibitory activities

Background:To explore the differences of 14 batches of Citri Reticulatae Pericarpium from different regions.Methods:The main aim of this study was to develop a high-performance liquid chromatography method-photodiode array detection for determining the contents of five flavonoids and the chromatographic fingerprints of Citri Reticulatae Pericarpium from different regions.Theα-glucosidase inhibitory activities and antioxidant properties of Citri Reticulatae Pericarpium,based on free-radical scavenging assays against 1,1-diphenyl-2-picrylhydrazyl,2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),and hydroxyl radicals,were estimated and compared.Results:Among the tested compounds,the content of hesperidin(13.386-68.235 mg/g)was the highest and that of hesperitin(0.045-0.277 mg/g)was the lowest.In comparing different sources of Citri Reticulatae Pericarpium,the contents of narirutin in Citri Reticulatae Pericarpium from Guangdong(0.824-0.851 mg/g)and Sichuan(1.069-1.204 mg/g)provinces of China were lower than in other provinces.In contrast,nobiletin(8.429-12.237 mg/g)and tangeretin(3.947-4.613 mg/g)were most abundant in Guangdong sources of Citri Reticulatae Pericarpium,followed by samples from Sichuan Province(nobiletin:6.761-7.658 mg/g;tangeretin:3.422-3.933 mg/g).Correlation analysis showed that nobiletin and tangeretin were the main contributors to the antioxidant capacity,and narirutin was the main active component inhibiting theα-glucosidase activity of Citri Reticulatae Pericarpium.Conclusion:This work revealed that the intrinsic quality of Citri Reticulatae Pericarpium was affected by different growing regions,which provides a scientific basis for controlling the quality of Citri Reticulatae Pericarpium and rationally developing and utilizing Citri Reticulatae Pericarpium.

Clinical Study on Dark Tea with Pericarpium Citri Reticulatae Combined with Metformin for Type 2 Diabetes Mellitus

[Objectives]To observe the clinical effect of dark tea with pericarpium citri reticulateae combined with metformin for type 2 diabetes mellitus.[Methods]A total of 80 cases of patients with type 2 diabetes mellitus were randomly divided into the control group and the observation group with 40 cases in each group.The control group was given metform for treatment,and the observation group was given dark tea with pericarpium citri reticulateae combined with metformin for treatment.The two groups were continuously treated for 12 weeks.The indexes of patients were observed and recorded,such as the body mass index(BMI),abdominal circumference(AC),hemoglobin A1c(HbA1c),fasting blood glucose(FBG),postprandial 2 h blood glucose(P2hBG),uric acid(UA),total cholesterol(TC),and triglyceride(TG).The incidence of adverse reactions during the treatment period and the patient satisfaction for treatment in the two groups were counted.[Results]After treatment,the FBG,P2hBG and HbA1c in the two groups were decreased when compared with those before treatment(P<0.05)and the decrease of the above indexes in the observation group was more significant than that in the control group(P<0.05).After treatment,the BMI,AC,UA,TC and TG in the observation group were decreased more significantly when compared with those before treatment and those in the control group after treatment,differences were statistically significant(P<0.05).The above indexes in the control group did not change significantly before and after treatment(P>0.05).The incidence of adverse reactions was 2.5%in the observation group and 5.0%in the control group,the difference was not statistically significant(P>0.05).The patient satisfaction was 97.5%in the observation group and 55.0%in the control group,the difference was significant(P<0.05).[Conclusions]The curative effect of dark tea with pericarpium citri reticulateae combined with metformin for type 2 diabetes mellitus is better than that of western medicine alone and has no obvious adverse reactions,thus it is worthy of clinical promotion and application.

Rapid characterization of 96 chemical constituents in Citri Reticulatae Folium(leaves of ‘Fuju') using HPLC-DAD-ESI-MS^n

In order to systematically investigate the chemical constituents of Citri Reticulatae Folium （leaves of ＇Fuju＇）, an analytical method that included high-performance liquid chromatography, diode array detection, electrospray ionization, and ion-trap time-of-flight mass spectrometry （HPLC-DAD-ESI-MSn） was used to separate and identify the individual chemical components of Citri Reticulatae Folium. As a result, 96 compounds were tentatively identified in this study： including 31 phenolic acids, 4 flavonoid aglycones, 6 flavonoid mono-O-glycosides, 10 flavonoid-O-diglycosides, 5 flavonoid mono-C-glycosides, 5 flavonoid di-C-glycosides, 6 flavonoid O,C-glycosides, 5 （3-hydroxy-3-methylglutaryl） glycosyl flavonoids, 1 flavan-3-ol, and 2 alkaloids. In addition, 21 polymethoxy flavonoids （PMFs） were identified in this paper. Among these compounds, 52 compounds, which were previously found in other Citrus plants, have been identified for the first time in Citri Reticulatae Folium. 15 compounds have not been previously found in the Citrus genus were identified. Moreover, 9 potentially new compounds have also been detected in this paper. This is the first report of the full characterization of chemical components of Citri Reticulatae Folium （leaves of＇Fuju＇） by HPLC-DAD-ESI-MSn.

Chemical Constituents from Pericarpium Citri Reticulatae

Objective To study the chemical constituents from Pericarpium Citri Reticulatae （Citrus reticulata）. Methods The chemical constituents were isolated and purified by silica gel column, Sephadex LH-20, and ODS column chromatography. The structures were identified by spectral data. Results Nineteen compounds were isolated and identified as 4＇,5,6,7-tetramethoxyflavone （1）, 3,3＇,4＇,5,6,7,8-heptmethoxyflavone （2）, sinensetin （3）, 5-O-demethylnobiletin （4）, tangeretin （5）, nobiletin （6）, apigenin （7）, 5-O- desmethyltangeretin （8）, 5,7-dihydroxy-3,3＇,4＇,6-tetramethoxyflavone （9）, pachypodol （10）, 4＇,5,6,7-tetramethoxyflavanone （11）, 3＇,4＇,5,7,8-pentamethoxyflavanone （12）, agestricin C （13）, scoparone （14）, isoscopoletin （15）, hesperidin （16）, didymin （17）, methylhesperidine （18）, and naringin （19）. Conclusion Compounds 9-15 are obtained from this plant for the first time.

Crc1,6RhaT is involved in the synthesis of hesperidin of the main bioactive substance in the Citrus reticulata ‘Chachi' fruit

Hesperidin is a dihydroflavonoids, accounting for more than 50% of the total flavonoids in Citri Reticulatae Pericarpium(CRP) of traditional Chinese medicine. It is an effective antioxidant and free radical scavenger that has anti-inflammatory, antiviral and hypoglycemic properties.The latest studies reported that hesperidin has a potential for novel coronavirus resistance. However, little is known about the synthesis regulation and accumulation site of hesperidin in plants. In this study, hesperidin synthase gene Crc1,6RhaT was cloned, and the protein can be completely transformed flavanone-7-O-glucoside into hesperidin in vitro and in vivo. Studies on biological characteristics of ovary walls and exocarps showed that the relative expression levels of the Crc1,6RhaT gene and protein decreased gradually with the development of citrus fruits, and the relative content of hesperidin firstly increased, then sequentially decreased. In situ hybridization results further revealed that Crc1,6RhaT transcription was mainly concentrated in the secretory cavity cells, which are revealed to be the site of flavonoid synthesis.Immunocytochemistry localization results showed that the Crc1,6RhaT was mainly located in the endoplasmic reticulum, nucleus and vacuole of secretory cells. We inferred that the Crc1,6RhaT was synthesized in the endoplasmic reticulum, then was transported into the vacuoles through enlarged vesicles at the end of the endoplasmic reticulum. Our results not only revealed that Crc1,6RhaT may be involved in the synthesis of hesperidin of the main bioactive substance in the medicinal plant Citrus reticulata ‘Chachi' fruit, but also provided novel insights into the main subcellular sites of hesperidin biosynthesis in vacuoles.

基于模糊建模解析不同产地沃柑果实感官品质差异

【目的】果实感官品质是决定果品市场竞争力最重要的因素,建立科学的柑橘果实综合感官品质评价方法,明确不同产地柑橘果实感官品质差异,探究消费者偏好性,筛选与柑橘感官属性密切相关的代谢产物,为柑橘育种、实际生产及市场销售提供理论支撑和数字化方法。【方法】以具有品质差异的9个产区沃柑果实为试验材料,首先组织评价员对样品各感官属性及权重进行评价,利用模糊数学得出不同感官属性的权重排名,并对不同产地样品的综合感官品质进行排序及分组;同时,检测常规理化指标,并通过气相色谱质谱联用仪(gas chromatography-massspectrometry,GC-MS)和高效液相色谱(high performance liquid chromatography,HPLC)分析与果实风味相关的可溶性糖、有机酸、氨基酸、类黄酮等代谢产物;进而利用OPLS-DA分析不同组间样品的主要差异代谢物,并通过皮尔逊相关性分析将样品各感官属性与相关代谢物质数据进行关联;最终获得可反映沃柑风味品质的重要代谢标志物。【结果】沃柑感官属性权重排名为果肉甜度>果肉酸度>果肉化渣性>果实色泽>果实香气>果实外形;常规指标固酸比不适合用于沃柑果实样品内在品质优劣的精准分级。因此,基于综合感官品质差异,9个产地沃柑样品被划分为两组,高感官品质组沃柑样品综合得分均高于60分,低感官品质组综合得分均低于60分。OPLS-DA分析表明,3种主要差异代谢产物可在风味上显著区分高、低感官品质组沃柑样品,这些物质分别为L-谷氨酸、橙皮苷和L-缬氨酸。皮尔逊相关性分析表明,α-酮戊二酸、L-谷氨酸和橙皮苷含量与沃柑综合感官品质、甜度和酸度得分多呈显著负相关。【结论】模糊数学可用于沃柑果实综合品质评价,消费者对沃柑的果肉甜度和酸度更为关注,L-谷氨酸和橙皮苷对沃柑果实品质差异的形成具有重要作用。

Effects of inorganic nutrients and environmental factors on the removal of n-propylbenzene and isopropylbenzene in seawater by cryptophytes Rhinomonas reticulata S6A

To effectively remove n-propylbenzene(n-PBZ)and isopropylbenzene(i-PBZ)leaked into seawater using Rhinomonas reticulata S6A(a newly isolated marine microalga),the effects of three inorganic nutrients and four environmental factors on their degradation were determined after 7 d of inoculation.Results show that NaNO_(3) at 300 mg/L caused a higher removal efficiency of both n-PBZ and i-PBZ(44.79%and 39.26%),while for NaH_(2) PO_(4)·H_(2) O,greater removal rates of two PBZs(47.30%and 42.23%)were achieved at 30 and 20 mg/L,respectively.NaHCO_(3) supplementation(500-750 mg/L)resulted in a large reduction(43.67%-45.04%)in i-PBZ concentration.The change in seawater pH(from 6 to 9)did not affect the elimination of n-PBZ and i-PBZ.The most suitable salinity and temperature were 30 and 25-30℃,respectively,leading to the PBZs removal of~40%.Light intensity exhibited significant influence on elimination of PBZs,and the maximum removal efficiencies of 56.07%(n-PBZ)and 55.00%(i-PBZ)were recorded under 200 and 600μmol/(m^(2)·s),respectively.In addition,the microalga could still remove PBZs when it failed to grow well due to darkness,strong light,low temperature,or low salinity,which might mean that good growth of alga is not always a necessary condition for PBZs removal.Therefore,attention should be paid to the suitability of nutrient levels and environmental conditions(excluding pH)in seawater when using microalgae for bioremediating PBZs-contaminated seawater.

柑橘皮多糖抗氧化性研究

[目的]研究柑橘皮多糖的抗氧化活性。[方法]利用水提醇沉法,提取柑橘皮多糖并进行初步纯化,采用苯酚-硫酸法测定柑橘皮多糖的提取率并采用水杨酸法研究柑橘皮多糖清除.OH的效果。[结果]柑橘皮中多糖的含量为6.32%;不同浓度的柑橘皮多糖对H2O2/Fe2+体系通过Fenton法反应产生的.OH都具有清除作用,且随浓度的增加,清除率逐渐升高,即清除率与多糖的用量存在一定的量效关系。[结论]柑橘皮多糖具有明显地抗氧化性,对.OH具有清除作用。

橘园生草栽培的研究进展

柑橘(Citrus reticulata Blanco)生草栽培技术作为一种土壤管理技术,具有提高土壤肥力,改善土壤生态环境,提高果实产量及品质的功能。综述了生草对橘园病虫害、生态环境、土壤物理性质、土壤pH、土壤养分、土壤有机质、土壤微生物和土壤酶等果树不同生长环境因素的影响,并探讨了生草促进果树生长,提高果实产量和品质的作用,进而提高柑橘的生产性能和经济效益的作用方式。

柑橘组织中橙皮苷的分析

采用超高效液相色谱法对陕西汉中柑橘(Citrus reticulata)的皮、肉、橘络、橘子4个组织中的橙皮苷含量进行测定。结果表明,柑橘中的橙皮苷与其他成分均能达到有效分离,橘皮、橘肉、橘络、橘子中橙皮苷含量分别为332.21、50.85、581.34和641.73 mg/kg。该方法能够用于柑橘及相关产品中橙皮苷的分析检测。

湖北部分地区柑橘农药残留情况调查与分析

采用超高效液相色谱-串联质谱联用仪和气相色谱-串联质谱联用仪对湖北省宜昌市区、丹江口市、秭归县和兴山县的柑橘(Citrus reticulata Blanco)样品农药残留情况进行定量检测和分析。结果表明,柑橘样品农药残留的检出率高达91.9%,其中,禁用农药检出率为1.6%;限用农药检出率为9.5%;允许使用农药检出率为68.3%;未登记使用农药检出率为15.9%。农药使用情况较为普遍,不过由于禁、限用农药控制程度较好,因此柑橘质量整体安全性较高。

春季防虫网温湿度变化对砂糖橘春梢生长的影响

探讨了防虫网覆盖栽培技术对网室温湿度和砂糖橘(Citrus reticulata Blanco cv.Shatangju)春梢生长的影响。结果显示,40目透光防虫网对1-4月份(春季)砂糖橘春梢的生长具有促进作用;同时比较了防虫网内外温湿度变化对粉虱类、红蜘蛛类、潜叶蛾类数量的影响,认为防虫网对砂糖橘春梢生长的促进作用可能是通过对柑橘害虫和网室温湿度的调控这两个方面来实现的。

鄂柑一号椪柑少核芽变的AFLP分析

利用AFLP技术,对鄂柑一号椪柑(Citrus reticulata Blanc.cv.ponkan)的3个少核芽变及其母株进行分子鉴定。筛选了64对引物,有60对引物可扩增出清晰的条带,共获得2 281条清晰的扩增谱带,其中有9对引物可扩增出明显多态性条带,多态性位点共15个,占总数的0.66%。结果表明,鄂柑一号椪柑的少核芽变由遗传物质改变引起,通过AFLP技术可以有效鉴定。

湖北柑橘主产区主要病虫害种类及发生期调查

为了解湖北省柑橘(Citrus reticulata Blanco)主产区主要病虫害种类及发生特点,2017—2018年连续两年采用实地调查,并采集标本进行种类鉴定。结果表明,主要病害有11种,以柑橘炭疽病、沙皮病、绿斑病(青苔病)、根腐病为害最严重;主要害虫有19种,以柑橘红蜘蛛、潜叶蛾、柑橘大实蝇为害最严重。

1961～2009年湖北省柑橘冻害等级及其特征分析

利用湖北省70个气象站点1961～2009年冬季气温、降水资料,分析了湖北省区域柑橘(Citrus reticulata Blanco)越冬期极端低温、低温最长持续时间、降水最长持续时间和降水量的时空变化特征,并以此为基础提出柑橘综合冻害指数和冻害等级的对应关系。结果表明,自1961年以来,湖北省冬季极端最低气温以每10年0.62℃的速率升高,全省一致上升,且鄂东南高于鄂西北;柑橘越冬期间低温最长持续时间每10年下降0.8 d,20世纪80年代后减少明显,但2008年出现历史次高值,属于反常年份;柑橘越冬期降水量每10年增加4.6 mm,而降水最长持续时间变化不大。49年来湖北省柑橘的综合冻害指数呈下降趋势,每10年缩短0.36;以20世纪60年代的综合冻害指数最高,至21世纪的头9年,综合冻害指数下降至最低值。在气候变暖的背景下,柑橘发生重度冻害以上的几率明显下降,但轻度、中度冻害的几率增加。

锌胁迫对不知火杂柑和椪柑叶片光合特性及超微结构的影响

采用砂基培养法,运用CI-340便携式光合测定仪、H-7650透射电子显微镜研究了不同锌浓度如0 mg/L(锌缺乏)、0.05 mg/L(对照)和0.5 mg/L(锌过量)处理下不知火杂柑(简称不知火)和椪柑叶片叶绿体色素含量、净光合速率及超微结构的变化。结果表明:(1)锌缺乏处理的不知火及锌过量处理的椪柑叶片叶绿素(a+b)和类胡萝卜素含量、锌缺乏与锌过量处理的两者叶片净光合速率均相对低于对照。(2)锌缺乏处理下,两者叶片叶绿体出现变形或空室化,其中椪柑叶绿体内淀粉粒和质体小球增多,细胞核及线粒体正常,而不知火淀粉粒少,细胞核中出现许多黑色小颗粒物质。锌过量处理下,两者叶片叶绿体出现空室化或叶绿体膜模糊,线粒体解体,其中椪柑叶绿体内质体小球增多,淀粉粒少;而不知火淀粉粒明显增多、增大,基粒片层膨胀、松弛。可见,锌胁迫对两者叶片叶绿体色素、净光合速率及超微结构有着明显的影响,其影响程度因品种而异。

孔雀鱼HSC70基因的电子克隆与序列分析

为了研究热激同源蛋白70(70 kDa heat shock cognate,HSC70)在孔雀鱼(Poecilia reticulata Peters)对环境适应中的作用,采用电子克隆与传统实验克隆相结合的技术,分析了孔雀鱼的一个HSC70基因(命名为PrHSC70)。PrHSC70基因包含一个长1941 bp的完整编码区,含有8个内含子。PrHSC70蛋白具有HSP70蛋白家族的特征序列,且与其他动物HSC70和HSP70都高度相似。在进化树上PrHSC70与所有鱼类HSC70聚为一簇,与它们的氨基酸序列一致性为93.2%～98.9%。