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Cyclocarya paliurus has traditionally been used as medicine and functional food. This study aims at investigating the flavonoid accumulation in C. paliurus dependent on phosphate (Pi) availability and its potential association with internal carbon partitioning. One-year-old seedlings of C. paliurus were planted in four different Pi levels. Low Pi resulted in low phosphorus content within plants, while the nitrogen content increased. Further analysis revealed that the surplus carbon pool was greater and was allocated to N-metabolism and carbohydrate synthesis under low Pi conditions, as shown by the higher levels of free amino acids, starch, and soluble sugars. Low Pi availability also induced higher enzymatic activities of shikimate dehydrogenase (SDH) and flavonoid 3-hydoxylase (FHT), and higher flavonoid accumulation in leaves. Our results indicated that the surplus carbon induced by low-Pi levels can increase flavonoid synthesis in seedlings of C. paliurus. In addition, growth and biomass accumulation were increased by the elevated Pi levels. As a result, the highest flavonoid yield per plant was obtained under relative low Pi conditions. This study can provide the basis for developing new agricultural practices to maintain high yield while still keeping the quality of medicinal plants and crops.

Two dammarane glycosides (1-2) were isolated from the leaves of Cyclocarya paliurus. The structures of new compounds were established by application of spectroscopic methods, including one-dimensional and two-dimensional NMR, HRESIMS, and chemical hydrolysis. When evaluated against seven human cancer cell lines, the two compounds exhibited selective cytotoxicity to MCF-7 cells.

Flavonoids are a class of commonly occurring natural compounds in the plant kingdom with various biological activities. This study compares the content of flavonoids in Cyclocarya paliurus at different developmental stages to better inform the selection of the optimal picking period. Thus, we analyzed the transcriptome and metabolome of C. paliurus at different developmental stages. The transcriptome analysis revealed 44 genes involved in the biosynthesis of flavonoids in C. paliurus, with 10 differentially expressed genes across the four different developmental stages. The metabolites were separated and identified by a combination of chromatography and mass spectrometry, followed by multi-reaction monitoring mode analysis of triple quadrupole mass spectrometry for complete metabolite quantification. In the flavonoid synthesis pathway, a total of 137 differential flavonoids were detected. The joint transcriptome and metabolome analysis showed that the expression trends in differential metabolites and genes were significantly related. Four MYB transcription factors and two bHLH transcription factors that are closely related to flavonoid biosynthesis were identified. The regulation network of flavonoid biosynthesis in C. paliurus was thus established, providing guidance for follow-up research.

Cyclocarya paliurus (Batal.) Iljinskaja, a monotypic species in Cyclocarya of Juglandaceae, is regarded as one of important medical plants in China. In order to reveal the alterations in chloroplast (cp) genome with nuclear genome duplication, we presented the complete cp genomes of C. paliurus, and firstly analyzed on the basis of ploidy type (tetraploid and diploid C. paliurus). The total length of the cp genome of tetraploid and diploid C. paliurus is 160,938 and 161,105 bp, respectively. Both type genome consist of a large single-copy (LSC) region (90,221 and 90,391 bp), a small single-copy (SSC) region (18,593 and 18,590 bp), and an pair of invert repeats (IRs) regions (26,062 and 26,062 bp). Tetraploid and diploid plastid genome contain 132 and 137 genes, 87 and 88 protein-coding genes, 37 and 39 tRNA genes, and both eight rRNA genes, respectively. Closely phylogenetic relationship by analyzing 23 cp genomes suggests that tetraploid C. paliurus probably originated from diploid C. paliurus.

While providing either NH4+ or NO3− to Cyclocarya paliurus, a mixture of NO3− and NH4+ at a ratio of 7/3 results in the best combination of plant growth and leaf secondary compound production. Cyclocarya paliurus is a well-known multiple function tree species with leaves that have been used as medicine and nutraceutical foods based on their high content of polyphenolic secondary compounds. Understanding C. paliurus growth and phytochemical accumulation in response to different NO3−/NH4+ ratios is essential to optimize plant growth and secondary compound production. Seedlings of C. paliurus were grown in a growth chamber, and five NO3−/NH4+ ratio treatments were applied. There were significant differences in seedling growth, measured secondary metabolites, and relative gene expression among different NO3−/NH4+ ratio treatments. The highest seedling growth was achieved at the NO3−/NH4+ ratio of 50/50, while the highest induced polyphenol and flavonoid content in plants occurred at NO3−/NH4+ ratios of 100/0 and 0/100 (i.e., when a single nitrogen source was used). C. paliurus leaves were the main sites of the polyphenol and flavonoid accumulation, and the highest yields of measured phytochemicals in leaves were obtained at the NO3−/NH4+ ratio of 70/30. A sole NH4+ or NO3− nutrition promoted related gene expression of the polyphenol and flavonoid biosynthesis, while a significantly positive correlation was detected between the phytochemical contents and related gene expression levels. This study provides a basis for manipulating NO3−/NH4+ ratio to achieve the highest yields of phytochemicals in C. paliurus plantations.

Context Cyclocarya paliurus (CP) (Batal.) Iljinsk (Cyclocaryaceae), a plant native to China, is the sole species in the genus Cyclocarya. Its leaves have been widely used as a remedy for hyperlipidaemia in traditional folk medicine. However, the mechanism underlying CP-induced lipolysis, especially in the liver, has not been entirely elucidated. Objective This study investigates the effect of CP ethanol extract (CPE) on hepatic steatosis and the underlying molecular mechanisms involved. Materials and methods The effect of CPE at concentrations of 0, 6.25, 12.5, 25, 50, and 100 μg/mL on the viability of HepG2 cells was examined using the cell counting kit-8 (CCK-8) assay after incubation for 24 h. CPE-induced changes in intracellular lipid content were assessed by measuring the absorbance of oil red O staining at 520 nm, and the possible underlying mechanisms were further studied using quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, immunofluorescence studies and transmission electron microscopy. Results The half-maximal inhibitory concentration (IC50) of CPE in HepG2 cells was 97.27 μg/mL. Treatment with 50 μg/mL CPE increased lipid clearance, which was associated with increased autophagy in HepG2 cells. CPE-induced autophagy involved downregulation of phosphorylation level of mammalian target of rapamycin (0.87 ± 0.08 vs. 1.31 ± 0.10). Fluorescent double staining and electron microscopy images showed lipid deposits within autolysosomes, thereby confirming the abovementioned findings. Discussion and conclusions CPE can induce hepatic fat clearance through the autophagy-lysosome pathway known as lipophagy. CPE has potential as a functional food.

Soil water capacity (SWC) is a very important factor for the artificial cultivation and production of seedling in Cyclocarya paliurus. To understand SWC requirement for seedling cultivation and to investigate morphological and physiological changes under different SWCs, a 100-day SWC treatment was conducted during artificial cultivation; four treatments were 10–20 wt% SWC (W1), 30–40 wt% SWC (W2), 50–60 wt% SWC (W3), and 70–80 wt% SWC (W4). The result showed that W3 was suitable for seedling cultivation. Compared with W3, growth biomass decreased and water content increased at W1, W2 and W4; K, Ca, and Mg content increased under W1, while Na content increased under W4; SOD, PPO, POD, and CAT activity in leaf significantly increased under W1 and W4, of which SOD activity was the highest, and MDA content reached its maximum under W1. W1 and W4 had negative effects on seedling growth, and seedlings adapt to unfavorable water condition by morphological and physiological responses. Our research would be useful for artificial cultivation and management of Cyclocarya species.

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.

Extracts of Cyclocarya paliurus (CP) leaves, a popular sweet tea, inhibit pancreatic β cell apoptosis and have potent hypoglycemic effects, but the identities of the anti-apoptotic bioactive components are still unknown. In the present study, a method using UPLC-Q-TOF/MS based on serum pharmacochemistry combined with target cell extraction was established to rapidly identify direct-acting pancreatic protectants from CP. After orally administering a set amount of CP extract to rats, blood samples were collected to characterize the components that can be absorbed into the blood using UPLC-Q-TOF/MS. Also, target cells (pancreatic β NIT-1 cells) were incubated with CP extract for 24 hours, and cells were collected to identify the components that can bind to the cells using UPLC-Q-TOF/MS. Finally, to evaluate the protective effect of the bioactive components of CP, MTT and TUNEL assays were performed on treated NIT-1 cell induced by streptozotocin (STZ). Three potential direct-acting pancreatic protectants -- kaempferol, quercetin, quadranoside IV -- were identified, and anti-apoptotic effects of kaempferol and quercetin were confirmed in STZ-induced NIT-1 cells. The findings indicate that this combined approach is a feasible, rapid, and expedient tool for capturing potential direct-acting components from natural products such as those from CP leaves.

Cyclocarya paliurus, an important endangered plant in China, has considerable medicinal, timber, and horticultural value. However, little is known about diseases that affect its health. In recent years, stem canker diseases on C. paliurus have been observed frequently in newly established nurseries in Jiangsu Province, China. Symptomatic trees showed elliptical, sunken lesions on the bark, with internal discoloration, leading to enlarging cankers with delineated margins. Pathogenicity tests with fungi isolated from symptomatic samples reproduced typical canker symptoms on both detached branches and potted plants of C. paliurus. Moreover, conidia from pycnidia of isolate ZB-23 could also cause stem canker on C. paliurus. Through combined morphological observation and DNA sequences of ITS region, β-tubulin, and translation elongation factor 1-α genes, the pathogen was identified as Botryosphaeria dothidea. Multigene maximum likelihood and maximum parsimony phylogenetic analyses further supported the identification of the pathogen. To our knowledge, this is the first report of B. dothidea causing stem canker on C. paliurus in China.

Cyclocarya paliurus is commonly used for the prevention and treatment of hypertension, diabetes, and inflammation in South China. Although research on the anti-inflammatory effects of C. paliurus leaves has been reported, no active anti-inflammatory compounds have been identified. In the present study, RAW 264.7 cells were used to establish a bioactivity-guided identification model to verify the inhibitory effects of C. paliurus leaves on inflammation and identify the anti-inflammatory constituents. The active fraction was isolated to yield 18 dammarane triterpenoid saponins, including 11 new 3,4-seco-dammarane triterpenoid saponins (1-11), the structures of which were identified on the basis of analyses of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) and comparison with literature data. Compounds 7, 8, 10, and 11 showed strong inhibition on nitric oxide (NO) productions, with IC50 values ranging from 8.23 to 11.23 μM. These four compounds significantly decreased the secretion of tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), and interleukin 6 (IL-6) in lipopolysaccharide-activated RAW 264.7 cells. Furthermore, compound 7 decreased the expression of the proteins cyclooxygenase-2 (COX-2), inducible nitric-oxide synthase (iNOS), and nuclear factor kappa-B (NF-κB/p65). In addition, the structure-activity relationships of the isolates were investigated. The results suggest that 3,4-seco-dammarane triterpenoid saponins may be used as potential anti-inflammatory drugs and warrant further investigation.

Obesity is a global epidemic. Recent studies have shown that Cyclocarya paliurus (C. paliurus) leaves have the potential to alleviate fat deposits. However, the fat-reducing mechanism of it remains unclear. Using Caenorhabditis elegans (C. elegans) as a model, we found that C. paliurus polysaccharide (CPP) significantly decreased fat storage in both normal and high-fat worms without affecting the movement. Moreover, the size and number of lipid droplets were reduced in CPP-treated ZXW618 worms. In energy metabolism, CPP decreased Escherichia coli (E. coli) OP50 growth and pharyngeal pumping and increased the expression of vit-2. In lipid metabolism, CPP down-regulated the expression of the sbp-1 and nhr-49 genes by modulating mdt-15 to prevent the expression of the Δ9-desaturase genes (fat-5, fat-6 and fat-7). Meanwhile, the expression of the acs-2 genes, the downstream of nhr-49, was suppressed by CPP. These findings provided insights into the CPP-induced anti-fat mechanisms, which contributed to the application of CPP in anti-obesity drugs.

Diabetes is a complex illness requiring long-term therapy. Cyclocarya paliurus, a recently confirmed new food resource, shows significant hypoglycemic and hypolipidemic effects in type II diabetes. Triterpenoid saponins are considered as the effective medicinal components of C. paliurus and are useful for the treatment of diabetes mellitus. However, little is known regarding their specific mechanism of actions. In this study, we used active ingredient screening and target prediction techniques to determine the components of C. paliurus responsible for its anti-diabetic effects as well as their targets. In addition, we used bioinformatics technology and molecular docking analysis to determine the mechanisms underlying their anti-diabetic effects. A total of 39 triterpenes were identified through a literature search and 1 triterpene compound by experiments. In all, 33 potential target proteins associated with 36 pathways were predicted to be related to diabetes. Finally, 7 compounds, 15 target proteins, and 15 signaling pathways were found to play important roles in the therapeutic effects of C. paliurus against diabetes. These results provide a theoretical framework for the use of C. paliurus against diabetes. Moreover, molecular docking verification showed that more than 90% of the active ingredients had binding activity when tested against key target proteins, and a literature search showed that the active ingredients identified had anti-diabetic effects, indicating that the results were highly reliable.

MADS-box transcription factors (TFs) have fundamental roles in regulating floral organ formation and flowering time in flowering plants. In order to understand the function of MIKC-type MADS-box family genes in Cyclocarya paliurus (Batal.) Iljinskaja, we first implemented a genome-wide analysis of MIKC-type MADS-box genes in C. paliurus. Here, the phylogenetic relationships, chromosome location, conserved motif, gene structure, promoter region, and gene expression profile were analyzed. The results showed that 45 MIKC-type MADS-box were divided into 14 subfamilies: BS (3), AGL12 (1), AP3-PI (3), MIKC* (3), AGL15 (3), SVP (5), AGL17 (2), AG (3), TM8 (1), AGL6 (2), SEP (5), AP1-FUL (6), SOC1 (7), and FLC (1). The 43 MIKC-type MADS-box genes were distributed unevenly in 14 chromosomes, but two members were mapped on unanchored scaffolds. Gene structures were varied in the same gene family or subfamily, but conserved motifs shared similar distributions and sequences. The element analysis in promoters’ regions revealed that MIKC-type MADS-box family genes were associated with light, phytohormone, and temperature responsiveness, which may play important roles in floral development and differentiation. The expression profile showed that most MIKC-type MADS-box genes were differentially expressed in six tissues (specifically expressed in floral buds), and the expression patterns were also visibly varied in the same subfamily. CpaF1st24796 and CpaF1st23405, belonging to AP3-PI and SEP subfamilies, exhibited the high expression levels in PA-M and PG-F, respectively, indicating their functions in presenting heterodichogamy. We further verified the MIKC-type MADS-box gene expression levels on the basis of transcriptome and qRT-PCR analysis. This study would provide a theoretical basis for classification, cloning, and regulation of flowering mechanism of MIKC-type MADS-box genes in C. paliurus.

Cyclocarya paliurus is commonly used to treat diabetes in China. However, the natural habitats of C. paliurus are typically affected by salt stress. Hydrogen sulfide (H2S) is a growth regulator that is widely used to enhance plant stress tolerance, but the possible mechanism underlying H2S-alleviated salt stress in C. paliurus remains unclear. C. paliurus seedlings pretreated with NaHS (an H2S donor) were exposed to salt stress, and then, the leaf and total biomass, chlorophyll fluorescence parameters, nitric oxide (NO) content, oxidative damage, and proline and phenolic content were investigated to test the hypothesis that H2S and NO were involved in the salt tolerance of C. paliurus. The results showed that H2S pretreatment maintained chlorophyll fluorescence and attenuated the loss of plant biomass. We also found that H2S pretreatment further increased the endogenous NO content and nitrate reductase activity compared with salt treatment. Moreover, H2S pretreatment alleviated salt-induced oxidative damage, as indicated by lowered lipid peroxidation, through an enhanced antioxidant system including more proline and phenolic accumulation and increased antioxidant enzyme activities. However, C. paliurus leaves treated with the NO scavenger significantly diminished H2S-mediated NO production and alleviation of membrane lipid peroxidation. Thus, we concluded that H2S-induced NO was involved in C. paliurus salt tolerance.

Cyclocarya paliurus (Batal.) Iljinskaja, as a woody tree species, has various health-promoting phytochemicals and is commonly used in tea production in China. A field trial for C. paliurus was conducted at two typical sites to evaluate genotype × environmental interaction (GEI) effects and identify the stability and adaptability of 13 families. The genotype main effects and genotype × environment interaction effects (GGE) were used to analyze the GEI effects for tree height, diameter at breast height (DBH), and leaf flavonoid and triterpenoid contents. Two-way analysis of variance indicated that the tree growth and leaf phytochemical contents were mainly affected by genotype and GEI. Based on the results of GGE, the JH1 family had high mean values and high stability in total flavonoid and total triterpenoid accumulations, while the KM4 family exhibited high means and high stability in tree height and DBH growth, as well as high means and medium stability in total triterpenoid content. Pearson’s correlation analysis indicated that the annual mean DBH was significantly and positively correlated with leaf phytochemical content in C. paliurus when all data for the 13 families at the two sites were used. Our results suggest that JH1 should be mainly extended as a superior genotype for the production of flavonoids and triterpenoids in the leaves, while KM4 could be spread for timber production or leaf triterpenoid production. This study provides useful information on the selection of C. paliurus genotypes in target sites and cultivation strategies for establishing oriented plantations.

Background Cyclocarya paliurus (Batal.) Iljinskaja is a common endemic tree species and used as a Chinese medicine. The main active components in the leaves of this plant are polysaccharides. However, the temporal patterns of gene expression underlying the synthesis of polysaccharides in C. paliurus at different leaf developmental stages and its relationship with the polysaccharide content and antioxidant activities has not been reported to date. Methods RNA-seq was used to investigate the biosynthesis pathway of polysaccharides at the four developmental stages of C. paliurus leaves. The content and the antioxidant activities of polysaccharides were measured with typical biochemical methods and the identified correlations were statistically evaluated. Results Sixty-nine differentially expressed genes were found in the leaves during different developmental stages of C. paliurus. These are associated with glycosyltransferases and belong to 18 families. During different developmental stages of C. paliurus, the polysaccharide content first increased and then decreased, and the UDP-glucose 4-epimerase gene was found to be significantly positively correlated with the polysaccharide content. The clearance rates of DPPH radicals, superoxide anion radicals, hydroxyl radicals, and the reducing power of polysaccharides in the leaves of C. paliurus at different developmental stages showed a dose-dependent relationship with the concentration of polysaccharides. Conclusions The smallest fully expanded leaves are suitable for high-quality tea, and leaves with sizes below the largest fully expanded leaves are suitable for industrial production of polysaccharides.

As a multiple function tree species, Cyclocarya paliurus is planted and managed for timber production and medicinal use. However, it remains unclear to what extent geographical gradients in growth and wood quality are shaped by the environment. We used data of 197 trees from 22 natural populations to assess geographic variation patterns in radial growth and wood density, and their correlations with environmental factors. Significant differences in mean annual DBH and wood basic density were observed among the 22 populations (p

Two novel nortriterpenoids, cyclopalitins A and B (1 and 2), were isolated from aerial parts of Cyclocarya paliurus. Their structures were elucidated on the basis of extensive spectroscopic analyses including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D NMR and 2D NMR. Compounds 1 and 2 were tested for their cytotoxicity towards B16F10. The results revealed that they didn’t show cytotoxic activity against B16F10 with 10 ug/mL treatment for 48 h.

Rhamnaceae was widespread over the Northern Hemisphere during most of the Cenozoic. Abundant fossil materials play an important role in evaluating divergence times, which aids in understanding the morphological evolution and biogeographic histories within the family. Two occurrences, Paliurus hirsuta J.L. Dong et B.N. Sun sp. nov., and Ventilago lincangensis K.N. Liu et S.P. Xie, are presented here from the middle Miocene of Zhangpu County, South Fujian Province, based on diagnostic winged fruits. The morphological and anatomical features of the two species are described in detail. The fossil records of Ventilago are rarely documented, and our discovery adds new information to understand the history of this genus. Paliurus hirsuta sp. nov. fruits are pubescent which show a closer affinity to the P. ramosissimus group. As a result, we deduce that Paliurus probably had differentiated into two groups in low latitude South Fujian by the middle Miocene. Fossil assemblages presented in the Fotan flora indicate that a diverse forest flora surrounding a lake or wetland in Zhangpu County, Fujian Province, during the middle Miocene.

Cyclocarya paliurus is an extremely valuable and multifunctional tree species whose leaves have traditionally been used in used in medicine or as a medicinal tea in China. In recent years, anthracnose has been frequently observed on young leaves of C. paliurus in several nurseries located in Jiangsu Province, resulting in great yield and quality losses. To date, no information is available about the prevalence of C. paliurus anthracnose in China. The main purpose of the present study was to characterize the etiology of C. paliurus anthracnose. Phylogenetic analysis of the eight-loci concatenated dataset revealed that all 44 single-spore Colletotrichum isolates belonged to three species in the Colletotrichum gloeosporioides species complex, namely, Colletotrichum aenigma, Colletotrichum fructicola, and C. gloeosporioides sensu stricto. Phenotypic features, including the colony appearance and the morphology of conidia, appressoria, and ascospores, were consistent with the phylogenetic grouping. Virulence tests validated that the three Colletotrichum species could cause typical symptoms of anthracnose on C. paliurus leaves, similar to those observed in the field. The optimum mycelial growth temperature ranged from 25 to 30°C for all representative isolates, while C. gloeosporioides s. s. isolates exhibited greater tolerance to high temperature (40°C). Fungicide sensitivity assays indicated that all three Colletotrichum species were sensitive to tetramycin, which may be a potential alternative for the management of C. paliurus anthracnose. To our knowledge, this study provides the first report of C. aenigma, C. fructicola, and C. gloeosporioides s. s. causing C. paliurus anthracnose in China as well as in the world.

Cyclocarya paliurus seedlings were cultivated in three types of lead (Pb)-contaminated soils with Pb concentration of 305 ± 17 mg/kg (T1), 1964 ± 59 mg/kg (T2) and 3502 ± 107 mg/kg (T3), respectively. The results showed that after 180 days of cultivation, the contents of exchangeable and carbonate-bound Pb fractions significantly decreased in T1 and T2, but increased in T3. The growth indices of C. paliurus seedlings decreased with increasing Pb concentration; however, no difference was found between that in T1 and in Pb-free soil. The Pb concentration in the roots was an order of magnitude higher than that in the stems and in the leaves. The bioconcentration factor (BCF) of the leaves was the lowest among the three tissues investigated, and decreased with the higher concentration of Pb in the soils. These results suggest that C. paliurus can be used as a sustainable and profitable plant for the phytomanagement of Pb-contaminated soil.

Cyclocarya paliurus (Batal.) Iljinskaja, a woody medicinal species in the Juglandaceae, grows extensively in subtropical areas of China. Triterpenoids in the leaves have health-promoting effects, including hypoglycemic and hypolipidemic activities. To understand triterpenoid biosynthesis, transport and accumulation in C. paliurus during the growing season, gene cloning, gene expression and RNA in situ hybridization of related genes were used, and accumulation was examined in various organs. The complete coding sequences (CDSs) of three genes, CpHMGR, CpDXR and CpSQS, were obtained from GenBank and RACE. RNA in situ hybridization signals of the three genes mainly occurred in the epidermis, palisade tissue, phloem and xylem of leaf, shoot and root, with the signals generally consistent with the accumulation of metabolites in tissues, except in the xylem. Both gene expression and triterpenoid accumulations showed seasonal variations in all organs. However, total triterpenoid content in the leaves was significantly higher than that in the shoots, with the maximum in shoots in August and in leaves in October. According to Pearson correlation analysis, triterpenoid accumulation in the leaves was significantly positively related with the relative expression of CpSQS. However, the relation between gene expression and accumulation was dependent on the role of the gene in the pathway as well as on the plant organ. The results suggested that most of the intermediates catalyzed by CpHMGR and CpDXR in young shoots and roots were used in growth and flowering in the spring, whereas subsequent triterpenoid biosynthesis in the downstream catalyzed by CpSQS mainly occurred in the leaves by using transferred and in situ intermediates as substrates. Thus, this study provides a reference to improve triterpenoid accumulation in future C. paliurus plantations.

Leaves of Cyclocarya paliurus are a sweet tea traditionally used to treat obesity and diabetes in China. However, its protective mechanisms against hyperglycemia remains unclear. Here, we demonstrate that the extract of C. paliurus leaves significantly decreased body loss, food intake and blood glucose level, and increased blood insulin level, β-cell number and insulin-producing β cells in high-fat diet-low dose STZ-induced diabetic mice. In vivo and in vitro studies also showed the extract of C. paliurus leaves significantly inhibited pancreatic β cell apoptosis by suppressing the expression of caspase 8, caspase 9 and cleaved caspase-3, as well as Bax/Bcl-2 ratio, down-regulating p38, ERK and JNK phosphorylation, and up-regulating Akt phosphorylation. These effects were significantly enhanced by inhibitor p-38 or ERK or JNK, and counteracted by inhibitor of PI3K. In addition, the extract of C. paliurus leaves also significantly improved hepatic steatosis, nephropathy and cardiac hypertrophy of diabetic mice. Taken together, these results provide the insight into the effects of C. paliurus leaves on pancreatic β cell preservation in standing glucolipotoxicity. Therefore, C. paliurus tea leaves may be used as a new remedy for diabetes through enhancing pancreatic β cell preservation by inhibiting β cell apoptosis.

Thirteen undescribed dammarane triterpenoid saponins (cypaliurusides A–M), including eleven seco-dammarane type triterpenoids, were isolated from Cyclocarya paliurus. Each of these compounds has the unique feature of having a monosaccharide attached to C-11, rather than C-12, compared to the same type of saponins found in this plant. The structures of them were determined by comprehensive analysis of 1D, 2D NMR and HRESIMS data. Cypaliuruside J showed significant α-glucosidase inhibitory effect with IC50 value of 2.22 ± 0.13 μM. In addition, Cypaliurusides F and K exhibited modest cytotoxic activities against selected human cancer cell lines in vitro, with IC50 values ranging from 4.61 ± 0.13 to 15.23 ± 3.88 μM.

An original way of brewing Cyclocarya paliurus leaves, especially in China, is to steep leaves in hot water before people drink it directly. Recently, infusing tea leaves with cold water, a creative way in Taiwan, has become a popular way of making tea. This study was designed to investigate the differences in metabolites among three brewing methods (cold-brewing, steep-brewing, and boil-brewing) based on UPLC-QTOF-MS metabolomics experiments and the feasibility of cold-brewing methods for C. paliurus leaves. Unsupervised analysis (PCA) explained 54.6% (positive ion mode) and 57.4% (negative ion mode) of the total variance, whereas supervised analysis (OPLS-DA) with cross-validated R2Y and Q2 values > 0.5, could reveal potential metabolites with better discrimination among the three brewing methods. Fifteen potential differential metabolites were chosen and identified, and nine of them were further confirmed with reference standards. This study suggested that the cold-brewing method without an increase in temperature protected the phenol aromatic ring, thereby obtaining more phenolic acid compounds from C. paliurus leaves. These results provided a basis for making cold tea and promoting the development of cold tea with C. paliurus leaves as raw materials.

Two new dammarane-type triterpenoid saponins, 3β-(α-l-arabinopyranosyloxy)-24,25-dihydroxydammar-20-en-12α-yl 6-deoxy-β-d-glucopyranoside (1) and (24R)-3β-[(4-O-acetyl-α-l-arabinopyranosyl)oxy]-25-hydroxy-20,24-epoxydammaran-12β-yl 6-deoxy-β-d-glucopyranoside (2), and fourteen known triterpenoids were isolated from the 70 % MeOH extract of the leaves of Cyclocarya paliurus. Their structures were established based on analyses of spectroscopic data. All compounds were tested for their inhibitory activities against the 11β-HSD1 enzyme. Hederagenin (13) exhibited moderate inhibitory effect for mouse 11β-HSD1 with an IC50 value of 0.16±0.04 μM.

Background: Cyclocarya paliurus (C. paliurus) leaves contain multiple health benefical metabolites, and therefore are often used in functional foods and Chinese herbal medicines. However, breeding difficulties confines its utilization. Consequently, cell suspension culture was developed to produce the bioactive secondary metabolites of C. paliurus leaves, but the content was comparatively low. In the present paper, Aspergillus Niger Elicitor (ANE) was used to stimulate the synthesis of triterpenoids in the suspension cultured C. paliurus cells, and the signal molecules crosstalk involved in this elicitation was further studied to interpret the underlying mechanism. Results: Total triterpenoids accumulation of the cultured C. paliurus cells elicited by 200 µg/mL ANE was 9.17 times higher than that of the untreated cells. Nitric oxide (NO), hydrogen peroxide (H2O2) and jasmonic acid (JA) played important roles in the elicitation as signal molecules. Under the ANE stimulation, the concentrations of NO, H2O2 and JA all increased significantly, but presented different change profiles and peaked at different times. Based on series experiments of NO quenching by C-PTIO, H2O2 blocking by DMTU, and JA synthesis inhibition by IBU and NDGA, together with exogenous NO, H2O2 and JA addition experiments, it was deduced that ANE improved triterpenoids synthesis in the suspension cultured C. paliurus cells via a complex signal transduction network, in which three deduced and three hypothetical signal transduction pathways might be involved. JA was not only the junction of NO and H2O2 signal pathways, but also the critical point in the whole signal network. RNA-seq analysis showed that a total of 3 candidate JA synthesis pathway genes including 1 LOX and 2 OPR were found to be significantly up-regulated under the ANE stimulation, along with 5 down-regulated JAZs and 1 up-regulated JAR1 regulating to JA signal transduction.Conclusions: ANE can significantly increase the triterpenoids synthesis in the suspension cultured C. paliurus cells. RNA-seq analysis validated the pivotal role of JA in this ANE elicitation. Our results provided references for the further studies on triterpenoids synthesis in C. paliurus under ANE treatment.

Eight phenolic compounds including five glucuronides were isolated from the leaves of Cyclocarya paliurus (Batal.) Ijinskaja, which is used as a source of tea. Their structures were characterized by spectroscopic methods. The results of biological assay showed that compounds 1 and 5 had good activities against HIV-1 induced cytopathic effects in C8166 lymphocyte at non-cytotoxic concentrations, with EC50 values of 31.74 and 10.76 μM, and therapeutic index (TI) values of ＞13.02 and 25.46, respectively. Relationship between molecular structures and their bioactivities was discussed.

Cyclocarya paliurus (Batalin) Iljinsk. is a multiple function tree species distributed in subtropical areas, and its leaves have been used in medicine and nutraceutical foods in China. However, little information on the effects of nitrogen (N) forms and ratios on growth and secondary metabolite accumulation is available for C. paliurus. The impact of five NO3&minus, /NH4+ ratios on biomass production, triterpenoid accumulation and related gene expression in C. paliurus seedlings was evaluated at the middle N nutrition supply. Significant differences in seedling growth, triterpenoid accumulation and relative gene expression were observed among the different NO3&minus, /NH4+ ratio treatments. The highest triterpenoid content was achieved in a sole NO3&minus, or NH4+ nutrition, while the mixed N nutrition with equal ratio of NO3&minus, to NH4+ produced the highest biomass production in the seedlings. However, the highest triterpenoid accumulation was achieved at the treatment with the ratio of NO3&minus, /NH4+ = 2.33. Therefore, the mixed N nutrition of NO3&minus, and NH4+ was beneficial to the triterpenoid accumulation per plant. The relative expression of seven genes that are involved in triterpenoid biosynthesis were all up-regulated under the sole NH4+ or NO3&minus, nutrition conditions, and significantly positive correlations between triterpenoid content and relative gene expression of key enzymes were detected in the leaves. Our results indicated that NO3&minus, is the N nutrition preferred by C. paliurus, but the mixture of NO3&minus, and NH4+ at an appropriate ratio would improve the leaf triterpenoid yield per area.