Sulfated Cyclocarya paliurus polysaccharides exert immunomodulatory potential on macrophages via Toll-like receptor 4 mediated MAPK/NF-κB signaling pathways

The biological activity of plant polysaccharides can be enhanced by sulfated modification.In this study,the immunomodulatory effect of sulfated Cyclocarya paliurus polysaccharides(SCP3)on macrophages RAW264.7 and its potential molecular mechanism were investigated.Results showed that SCP3 at 25-100μg/m L increased viability and improved phagocytosis of RAW264.7 cells.Meanwhile,SCP3 could activate mitogen-activated protein kinase(MAPK)and nuclear factor kappa B(NF-κB)signaling pathways,which increased the phosphorylation of Erk1/2,JNK,p38 and NF-κB p65,promoting secretion of cytokines tumor necrosis factorα(TNF-α),interleukin 6(IL-6)and nitric oxide(NO)as well as the production of reactive oxygen species(ROS).In addition,Toll-like receptor 4(TLR4)receptor inhibitors were able to block the production of NO and TNF-αby SCP3-stimulated macrophages.Based on Western blot analysis and validation using specific inhibitors against MAPK and NF-κB signaling pathways,the results demonstrated that SCP3 induced macrophages activation and enhanced TNF-αand NO production via TLR4-mediated MAPK and NF-κB pathways.In summary,SCP3 has significant immunomodulatory potential.The underlying molecular mechanism was that SCP3 activates macrophages via TLR4 receptors to promote ROS production,which in turn activates the downstream MAPK/NF-κB signaling pathway and then increases the secretion levels of cytokines and NO.

Apoptosis of colon cancer CT-26 cells induced polysaccharide from Cyclocarya paliurus and its phosphorylated derivative via intrinsic mitochondrial passway

In this study,the antitumor properties and the possible molecular mechanisms of Cyclocarya paliurus polysaccharide(CP)and its phosphorylated derivative(P-CP)on CT-26 mouse colon carcinoma cells were investigated.Results found that CP had high inhibition ratio against CT-26 cells.The flow cytometry results found that CP treatment could cause the intracellular acidification,arrest the cell cycle in the S phase and increase reactive oxygen species generation.Additionally,CP treatment triggered mitochondrial membrane potential depolarization and Ca^(2+)overloading,and broke down the balance of antioxidant system,Na^(+)/K^(+)-ATPase and Ca^(2+)-ATPase.Further analysis found CP induced cell apoptosis through improving the activities of caspase-3 and caspase-9,and increasing the level of cytochrome C.Furthermore,the comparative study of antitumor effect on CT-26 cells displayed that the phosphorylation enhanced antitumor activities of polysaccharides.These results suggest CP is a potential natural therapeutic agent for colon cancer and phosphorylation represents an effective method of enhancing the antitumor activity of CP.

Cytochemical localization of ATPase and sub-cellular variation in mesophyll cell of Cyclocarya paliurus seedlings under iso-osmotic stress and calcium regulation

The ultrastructural distribution and active location of ATPase and the ultrastructural variations were investigated in mesophyll cells of Cyclocarya paliurus seedlings after iso-osmotic salt/water treatments in combination with calcium regulation. C. paliurus seed- lings were treated with five groups （control, 85 mM NaCl, 85 mM NaCl＋ 12 mM Ca（NO3）2, PEG iso-osmotic to 85 mM NaCl and PEG iso-osmotic to 85 mM NaCl＋12 mM Ca（NO））2） in a hydroponic system in a phytotron. Results show that under normal growth conditions the ATPase activity was low and the enzyme was primarily located on the nucleus. After 12 days of iso-osmotic salt/water treatments, ATPase activity on the tonoptast increased. Osmiophilic globules for iso-osmotic water treatment were greater than that for iso-osmotie salt treatments. The ATPase activity increased and was mostly transferred onto the nucleus for calcium regulation treatment under iso- osmotic salt/water stresses, and the osmiophilic globules significantly decreased under iso-osmotic water stress with calcium regulation. The ATPase located on the nucleus indicated that the degree of salt/drought damage that seedlings suffered was slighter, while the amount of the enzyme located on the tonoplast showed that the degree of salt/drought damage there was more serious. After 4 and 20 days of iso- osmotic treatments, the injury suffered by the leaf ultrastructures of C. paliurus seedlings for iso-osmotic treatment with calcium regula- tion was lower than those without calcium regulation, especially for the iso-osmotic water treatments. Preliminary analysis suggests that the iniury suffered by C. paliurus seedlings was lower for iso-osmotic salt treatments than for iso-osmotic water treatments, while the effect of calcium regulation under iso-osmotic water stress was greater than that of the iso-osmotic salt stress.

Natural variations in flavonoids and triterpenoids of Cyclocarya paliurus leaves

Cyclocarya paliurus is widely distributed in subtropical areas of China.Secondary metabolites in the leaves,including flavonoids and triterpenoids,provide protection against diseases such as hyperlipidemia,diabetes,hypertension,and hypoimmunity.In this study,343 C.paliurus leaves were collected from 32 distinct populations covering most of the main distribution,to deduce the response of flavonoids and triterpenoids to seven environmental factors.Principal component analysis was performed using seven flavonoid and six triterpenoid compounds.The results show that altitude,latitude,and longitude significantly contributed to variations in total flavonoid and triterpenoid contents.In addition,the interaction of these metabolites was extensively correlated with environment changes,and therefore should be considered during selection and cultivation.

Protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis

OBJECTIVE To explore the protective effects of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja on STZ-stimulated INS-1 cells through regulating of autophagy and apoptosis.METHODS INS-1cells were cultured in media containing 3n M STZ and different doses of total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja.The proliferation of cells was examined by MTT assay,ROS content were detected by fluorescence enzyme label.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),hydrogen peroxidase(CAT),malondialdehyde(MDA)were also measured by colorimetry.The activities of caspase-3,9 were also observed by Caspase colorimetric assay kit in each group.The expressions of Bcl-2 m RNA and Bax m RNA were detected by Real time PCR,protein expression of LC3-Ⅱand PARP were detected by Western blotting.RESULTS Compared with the control group,total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja could promote the proliferation of INS-1 cells no matter with STZ or not when its concentration lower than 25μg·m L-1;but when its concentration higher than 100μg·m L-(1use individually)or 50μg·m L-1(combined use),total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja might significantly inhibite the growth of the cells whether STZ existed or not.Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja(6.25,12.5,25μg·m L-1)might inhibit INS-1cell apoptosis,decrease intra-cellular ROS contents,improve the s upernatant liquid SOD,GSH-PX,CAT activities,decrease MDA level,promote INS-1 cell secreting insulin,decrease the protein expressions of autophagy protein LC3Ⅱand apoptosis regulating protein cleaved PARP protein,up-regulate the anti-apoptotic protein Bcl-2 m RNA expression,down-regulate the pro-apoptotic protein Bax m RNA expression and Bcl-2and Bax,reduce the activity of caspase-9 and caspase-3(P<0.05 or P<0.01).CONCLUSION Total triterpenoids extracts from Cyclocarya paliurus(Batal.)Iljinskaja has good protective effect on STZ-stimulated INS-1cells.It can inhibit STZ injured INS-1 cells to overproduce ROS production,enhance endogenous antioxidant enzymes(GSH-Px,SOD,CAT activities),reduce the expression of autophagy protein LC3Ⅱand apoptosisregulating protein cleaved PARP,up-reglute the antiapoptosis protein Bcl-2 expression,down-regulate the pro-apoptotic protein Bax expression,decrease the caspase-9 and caspase-3 activities,and improve the INS-1cell survival rate,and then play a protective effect on damaged INS-1 cells.

Callus culture of Cyclocarya paliurus (Batal.) Iljinskaja leaves

The hormone combinations and dark culture of the callus induction for Cyclocarya paliurus （Batal.） Iljinskaja leaves were optimized by an orthogonal experiment. The effects of hormones on callus proliferation were also studied. The results show that the optimum medium for callus induction of C. paliurus leaves was MS ＋ 1.0 mg.L-1 6-BA ＋ 0.5 mg.L-1 NAA ＋ 1.0 mg·L-1 2,4-D. The combination of 6-BA and NAA significantly affected the induction of callus, while the effect of 2,4-D was less. The maximum rate of callus induction was 95.4%. A 10-day darkness period was beneficial for callus induction. The best proliferation medium was 1/4 DKW ＋ 0.5 mg·L-1 6-BA ＋ 1.0 mg·L-1 2,4-D.

Study on Response Surface Methodology Optimization for Extraction Process of Cyclocarya paliurus Extract and Its Blood Sugar-lowering Effect

[Objectives]This study was conducted to discuss the mechanism of the blood sugar-lowering effect of Cyclocarya paliurus extract.[Methods]With C.paliurus leaves as the raw material,the ethyl acetate-butyl acetate microwave extraction process of C.paliurus was studied.With the yield of C.paliurus extract as an evaluation index,single-factor experiments were carried out on such 6 factors as the dry C.paliurus leaf powder size,solid-to-liquid ratio,extraction time,microwave frequency,microwave power,and microwave extraction time,and the extraction and purification process was further optimized by response surface analysis.The normal mice and the hyperglycemic mice modeled by alloxan were injected with C.paliurus extract to study the blood sugar-lowering effects of different groups of C.paliurus extract.[Results]The optimal process combination for the extraction of C.paliurus leaves was obtained:dry C.paliurus powder size 90-100 mesh,solid-to-liquid ratio 1∶20(g/ml),extraction time 192 min,microwave frequency 2500 MHz,microwave power 490 W,and extraction time 248 s.Under these conditions,the yield of C.paliurus extract reached the maximum,95.10%,reaching 99.83%of the predicted value.The blood sugar-lowering test on mice showed that the C.paliurus extract had a good effect on lowering blood sugar,indicating that C.paliurus extract can improve the free radical scavenging capacity to a certain extent.[Conclusions]This study provides certain reference for the in-depth study of the physiological effects of C.paliurus extract and the comprehensive development and utilization of C.paliurus extract.

Whole-genome Duplication Reshaped Adaptive Evolution in A Relict Plant Species,Cyclocarya paliurus

Cyclocarya paliurus is a relict plant species that survived the last glacial period and shows a population expansion recently.Its leaves have been traditionally used to treat obesity and diabetes with the well-known active ingredient cyclocaric acid B.Here,we presented three C.paliurus genomes from two diploids with different flower morphs and one haplotype-resolved tetraploid assembly.Comparative genomic analysis revealed two rounds of recent whole-genome duplication events and identified 691 genes with dosage effects that likely contribute to adaptive evolution through enhanced photosynthesis and increased accumulation of triterpenoids.Resequencing analysis of 45 C.paliurus individuals uncovered two bottlenecks,consistent with the known events of environmental changes,and many selectively swept genes involved in critical biological functions,including plant defense and secondary metabolite biosynthesis.We also proposed the biosynthesis pathway of cyclocaric acid B based on multi-omics data and identified key genes,in particular gibberellinrelated genes,associated with the heterodichogamy in C.paliurus species.Our study sheds light on evolutionary history of C.paliurus and provides genomic resources to study the medicinal herbs.

Geographical discrimination of Cyclocarya paliurus tea for origin traceability based on multielement analysis by ICP-OES and chemometrics multivariate

Objective: This paper focused on the geographical discrimination of Cyclocarya paliurus tea for origin traceability based on multielement analysis by ICP-OES and chemometrics multivariate.Methods: In this study, eleven trace element concentrations were determined by ICP-OES and processed by multivariate statistical analysis.Results: Based on ANOVA, the mean concentrations of 10 elements except Co differed significantly among six origins. Pearson’s correlation analysis showed that 11 pairs of elements have a positive significant correlation and 12 pairs have a negative significant correlation. The geographical origins were effectively differentiated using the eleven elements combined with PCA. And the S-LDA model offered a 100%differentiation rate.Conclusion: The overall results suggested that the combination of multielement analysis by ICP-OES and chemometrics multivariate could trace the geographical origins of tea. And the paper can provide reference for quality control and quality evaluation of C. paliurus in the future.

Effect of Cyclocarya paliurus polysaccharides on streptozotocin-induced diabetic nephropathy in rats

OBJEVTIVE:To investigate the efficacy of Cyclocarya paliurus(C.paliurus)polysaccharides on streptozotocin-induced diabetic nephropathy in rats.METHODS:Rats were divided into 6 groups,including group of normal control,group of diabetic control,group of metformin treatment,low-dose group of C.paliurus polysaccharides treatment,middle-dose group of C.paliurus polysaccharides treatment and high-dose group of C.paliurus polysaccharides treatment.Histological analysis of kidney was analyzed using hematoxilin and eosin.Levels of blood glucose,creatinine,urea,uric acid were determined by spectrophotometry.Anti-oxidative enzymes were measured by real-time polymerase chain reaction(PCR)and enzyme-linked immunosorbent assay(ELISA).Advanced glycation end products(AGEs)and transforming growth factor-β1(TGF-β1)level was measured by ELISA.RESULTS:Abnormal changes were observed in the group of diabetic control characterized by atrophy of the renal glomeruli with hypercellularity,congestion of glomerular tufts,dilation of the renal spaces,and degeneration of renal tubule.Compared with that of normal group,blood glucose,creatinine,urea,uric acid level was significantly increased in the group of diabetic control.Superoxide dismutase,catalase,glutathione peroxidase,glutathione reductase level was significantly decreased,but AGEs and TGF-β1 level was significantly increased.By contrast,administration of C.paliurus polysaccharides and metformin could reverse the above-mentioned results of the group of diabetic control,especially in the high-dose group of C.paliurus polysaccharides.CONCLUSION:Our findings suggest that C.paliurus polysaccharides may play a protecting role for nephropathy of diabetic rats by lowering glucose,creatinine,urea,uric acid level,enhancing the antioxidative ability,and reducing AGEs and TGF-β1 expression.

Antioxidative and immunological effects of Cyclocarya paliurus polysaccharides on the spleen injury of diabetic rats

OBJEVTIVE:To investigate the effects of Cyclocarya paliurus(C.paliurus)polysaccharides on the spleen injury of diabetic rats.METHODS:Animals were divided into 6 groups,including normal group,model group,control group,low-dose group of C.paliurus polysaccharides treatment,middle-dose group of C.paliurus polysaccharides treatment and high-dose group of C.paliurus polysaccharides treatment.Histological analysis of spleen was analyzed using hematoxilin and eosin.Levels of biological parameters and anti-oxidative enzymes were determined by spectrophotometry.Interleukin-7(IL-7)and IL-10 were measured by enzyme-linked immunosorbent assay.RESULTS:Compared with that of model group,superoxide dismutase,catalase,glutathione peroxidase,glutathione reductase level increased 78.63%(P<0.05),51.76%(P<0.05),2.95 times(P<0.01)and 41.11%(P<0.05)in the high-dose group of C.paliurus polysaccharides treatment,respectively.IL-7 and IL-10 increase 1.66(P<0.01)and 1.21 times(P<0.01)in the high-dose group of C.paliurus polysaccharides treatment,respectively.CONCLUSION:It is suggested that C.paliurus polysaccharides may play a protecting role for spleen injury of diabetic rats by enhancing the antioxidative ability and evaluating the immunity.

New triterpenoid saponins from the leaves of Cyclocarya paliurus

Two new secodammarane triterpenoid saponins, cyclocariosides 1 and J （1 and 2）, and a new epoxydammarane triterpenoid saponin, cyclocarioside K （3） were isolated from the ethanol extracts of the leaves of Cyclocarya paliurus. The structures of these compounds were elucidated by spectroscopic techniques.

Cyclocarysaponins A–J,dammarane-type triterpenoid glycosides from the leaves of Cyclocarya paliurus

Ten previously undescribed dammarane-type triterpenoid glycosides,cyclocarysaponins A–J(1–10),were isolated from the leaves of Cyclocarya paliurus(Batal.)Iljinskaja.The structures of these compounds were characterized through detailed spectroscopic analysis,including 1D and 2D nuclear magnetic resonance(NMR)and high-resolution electrospray ionization mass spectrometry(HR-ESI-MS).The cytotoxic activities of all isolates were assessed against five human cancer cell lines(Bel-7402,Caski,BGC-823,A2780,and HCT-116).Of the tested compounds,compounds 1,7,and 9 exhibited selective cytotoxicity against one or more human cancer cell lines.

HPLC测定青钱柳叶片中槲皮素、山奈酚的含量

研究青钱柳[Cyclocarya paliurus(Bata1.)Iljinskaja]叶片中2种黄酮苷元(槲皮素、山奈酚)的水解提取条件及其含量的高效液相色谱(HPLC)测定方法,为青钱柳叶片质量控制提供参考。以乙醇-盐酸混合溶液为溶剂,采用单因素试验和正交试验结合的方法考察提取水解条件;含量测定以Agilent C18色谱柱(250 mm×4.6 mm,5μm)为分离柱,乙腈-0.1%甲酸水溶液(体积比为35∶65)为流动相,检测波长370 nm,柱温30℃,流速1.0 mL/min。结果表明,优化的提取水解条件为以乙醇-盐酸(体积比为8∶1)为溶剂,每次按料液比为1∶20的量加入,90℃恒温水浴回流60 min。槲皮素和山奈酚的进样量分别在0.0808~0.8087μg(R^(2)=0.9997)、0.0964~0.9646μg(R^(2)=0.9999)范围内呈良好的线性关系;槲皮素的平均加样回收率为100.97%、相对标准偏差(RSD)为2.93%,山奈酚的平均加样回收率为97.25%、相对标准偏差为3.29%。提取水解工艺可明显提高青钱柳叶片黄酮苷元含量;建立的检测方法快速、准确、重复性好,可为青钱柳叶片药材的质量评价提供技术参考。

青钱柳叶提取物耦合茉莉酸甲酯对猕猴桃涂膜的保鲜效果

为研究能有效延长猕猴桃(Actinidia chinensis Planch)保质期的方法,选取壳聚糖为基质,添加一定浓度的茉莉酸甲酯和青钱柳(Cyclocarya paliurus(Batal.)Iljinsk)叶提取物对猕猴桃进行涂膜处理,分别采用单因素试验和正交试验,分析贮藏期间猕猴桃品质的变化规律,筛选和优化猕猴桃复合涂膜保鲜液的最佳配比组合。结果显示,壳聚糖0.8g/100mL、青钱柳叶提取物1.5g/100mL和茉莉酸甲酯0.12mmol/L的复合保鲜液对猕猴桃涂膜保鲜效果最佳;贮藏保鲜第16天时,最佳复合涂膜处理组的腐烂指数是对照组的1/4,固酸比始终低于对照组(P<0.05)。结果表明该复合涂膜保鲜液可供猕猴桃常温环境贮藏保鲜应用参考。

豫南青钱柳人工林叶片干物质含量时空格局

为了提高豫南地区青钱柳[Cyclocarya paliurus(Batal.)Ijinsk.]叶用林管理和利用的水平,调查该区青钱柳人工林叶片在不同年度、月份,不同树冠位置的干物质含量,以及叶绿素相对含量、比叶面积。结果表明,豫南地区青钱柳人工林5月叶片干物质率最低,为38.38%,8月叶片干物质率在不同年度间变化幅度最大,极差达38.27%,9—11月叶片干物质率则趋于稳定,在全年中位水平。不同年度平均干物质率不同,但相对稳定。在空间格局上,叶片干物质含量随树冠高度自下而上逐渐升高,均值分别为34.30%、46.41%、50.49%,上部叶片干物质率显著高于下部叶片,叶片干物质率在树上部各方位上无显著差异。叶绿素相对含量(SPAD)均值自下而上分别为2.11、2.51、2.63,比叶面积均值自下而上分别为33.43、21.58、18.63 cm^(2)/g。根据结论得出,青钱柳中、上部叶片在10—11月干物质含量高,叶片密度、厚度较大,易于烘干或炒制,更适于加工茶品;林地管理上通过树体修剪矮化,多采用青钱柳上部叶片作为加工原料,能提高青钱柳叶茶的品质。

Cyclocarya cf. paliurus （Batal.） Iljinskaja （Juglandaceae） from the Hunchun Formation （Eocene）, Jilin Province, China

The leaflet architecture of Cyclocarya cf. paliurus （Batal.） Iljinskaja from the Hunchun Formation （Middle Eocene） shows similarity to that of modem C. paliurus （Batal.） Iljinskaja and the specimen is the oldest fossil record in Europe and Asia. The distributions of C. cf. paliurus and other fossil records, such as Glyptostrobus, Metasequoia, Nyssa, and Liquidambar, in Hunchun flora show that it would have been a warmer-temperature to subtropical climate in Hunchun District during the Eocene period.