Spatial metabolome of biosynthesis and metabolism in Cyclocarya paliurus leaves.

A large number of plant metabolites were discovered, but their biosynthetic and metabolic pathways are still largely unknown. However, the spatial distribution of metabolites and their changes in metabolic pathways can be supplemented by mass spectrometry imaging (MSI) techniques. For this purpose, the combination of desorption electrospray ionization (DESI)-MSI and non-targeted metabolomics was used to obtain the spatial distribution information of metabolites in the leaves of Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus). The sample pretreatment method was optimized to have higher detection sensitivity in DESI. The changes of metabolites in C. paliurus were analyzed in depth with the integration of the spatial distribution information of metabolites. The main pathways for biosynthesis of flavonoid precursor and the effect of changes in compound structure on the spatial distribution were found. Spatial metabolomics can provide more metabolite information and a platform for the in-depth understanding of the biosynthesis and metabolism in plants.

Identification and Analysis of Protein Family Associated with RNA Interference Pathway in Juglandaceae.

BACKGROUND: One of the crucial processes for small RNA synthesis and plant disease resistance is RNA interference (RNAi). Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA binding (DRB), and Argonaute are important proteins implicated in RNAi (AGO). Numerous significant woody plants belong to the Juglandaceae; walnut is one of the four groups of woody plants on earth and one of the four groups of dried fruits. METHODS: In order to correlate walnuts and their homologues, this work integrated numerous web resources from structural analysis and transcriptome data collected from gene families in order to elucidate the evolution and functional differentiation of RNA-related proteins in the walnut (Juglans rega) genome. RESULTS: 5 DCL genes, 13 RDR genes, 15 DRB genes, and 15 AGO genes are found in the walnut genome and encode conserved protein domains and motifs with similar subcellular distribution.There are three classes and seven subclasses of walnut AGO proteins. RDRS are primarily split into four categories, whereas DRBs can be divided into six. DCLs are separated into four groups. The walnut RDR1 copy number of 9 is the exception, with 7 of those copies being dispersed in clusters on chromosome 16. Proteins are susceptible to various levels of purification selection, but in walnut, purification selection drives gene creation. These findings also indicated some resemblance in other plants belonging to the walnut family. Under various tissues and stresses, many RNA-related genes in walnut produced abundant, selective expression. CONCLUSIONS: In this study, the genome of the Juglandaceae's DCL, RDR, DRB, and AGO gene families were discovered and analysed for the first time. The evolution, structure, and expression characteristics of these families were also preliminary studied, offering a foundation for the development and breeding of the walnut RNAi pathway.

Molecular mechanism of thiamine in mitigating drought stress in Chinese wingnut (Pterocarya stenoptera): Insights from transcriptomics.

Urban garden plants are frequently affected by drought, which can hinder their growth, development, and greening effect. Previous studies have indicated that Chinese wingnut (Pterocarya stenoptera) responds to drought stress by increasing the expression of thiamine synthesis genes. In this study, it was found that exogenous thiamine can effectively alleviate the negative effects of drought stress on plants. Forward transcriptome sequencing and physiological tests were further conducted to reveal the molecular mechanism of thiamine in alleviating drought stress. Results showed that exogenous thiamine activated the expression of eight chlorophyll synthesis genes in Chinese wingnut under drought stress. Moreover, physiological indicators proved that chlorophyll content increased in leaves of Chinese wingnut with thiamine treatment under drought stress. Photosynthesis genes were also activated in Chinese wingnut treated with exogenous thiamine under drought stress, as supported by photosynthetic indicators PIabs and PItotal. Additionally, exogenous thiamine stimulated the expression of genes in the auxin-activated signaling pathway, thus attenuating the effects of drought stress. This study demonstrates the molecular mechanism of thiamine in mitigating the effects of drought stress on non-model woody plants lacking transgenic systems. This study also provides an effective method to mitigate the negative impacts of drought stress on plants.

Nitrogen addition modifies the relative gene expression level and accumulation of carbon-based bioactive substances in Cyclocarya paliurus.

In China, lots of Cyclocarya paliurus plantations have been established for tea and functional food production on nitrogen (N)-limited land. The optimum N levels require for biosynthesis and accumulation of carbon-based bioactive substances vary among plant species. This study integrated field trial with hydroponic culture to assess impact of nitrogen addition on accumulation and relative gene expression level of carbon-based secondary metabolites in C. paliurus. N addition significantly influenced not only contents of polyphenols, flavonoids and triterpenoids and relative gene expression levels of their biosynthetic pathway in C. paliurus leaves but also leaf biomass production and the bioactive substance accumulations. An intermediate N addition induced the highest contents of polyphenols, flavonoids and triterpenoids in leaves, but the optimized accumulation of these bioactive substances in the leaves was the trade-off between their contents and leaf biomass production. Correlation analysis showed that related gene expression levels were closely correlated with contents of their leaf corresponding secondary metabolites. Compared with ratios of carbon/N (C/N) and carbon/phosphorus (C/P) in the soil, ratios of C/N and C/P in the leaves were more strongly related to the contents and accumulations of polyphenols, flavonoids and triterpenoids. To obtain higher yields of targeted phytochemicals, the threshold ratios of C/N and C/P in the leaves are recommended for N and P fertilization at similar sites. Overall, our findings would provide the theoretical basis and technical support for manipulating N fertilization in C. paliurus plantations to obtain higher accumulations of targeted bioactive substances.

Progress in Research on the Alleviation of Glucose Metabolism Disorders in Type 2 Diabetes Using Cyclocarya paliurus.

Globally, the incidence of diabetes is increasing annually, and China has the largest number of patients with diabetes. Patients with type 2 diabetes need lifelong medication, with severe cases requiring surgery. Diabetes treatment may cause complications, side-effects, and postoperative sequelae that could lead to adverse health problems and significant social and economic burdens; thus, more efficient hypoglycemic drugs have become a research hotspot. Glucose metabolism disorders can promote diabetes, a systemic metabolic disease that impairs the function of other organs, including the heart, liver, and kidneys. Cyclocarya paliurus leaves have gathered increasing interest among researchers because of their effectiveness in ameliorating glucose metabolism disorders. At present, various compounds have been isolated from C. paliurus, and the main active components include polysaccharides, triterpenes, flavonoids, and phenolic acids. C. paliurus mainly ameliorates glucose metabolism disorders by reducing glucose uptake, regulating blood lipid levels, regulating the insulin signaling pathway, reducing ß-cell apoptosis, increasing insulin synthesis and secretion, regulating abundances of intestinal microorganisms, and exhibiting α-glucosidase inhibitor activity. In this paper, the mechanism of glucose metabolism regulation by C. paliurus was reviewed to provide a reference to prevent and treat diabetes, hyperlipidaemia, obesity, and other metabolic diseases.

The Modulatory Effect of Cyclocarya paliurus Flavonoids on Intestinal Microbiota and Hypothalamus Clock Genes in a Circadian Rhythm Disorder Mouse Model.

Circadian rhythm disruption is detrimental and results in adverse health consequences. We used a multi-omics profiling approach to investigate the effects of Cyclocarya paliurus flavonoid (CPF)-enriched diets on gut microbiota, metabolites, and hypothalamus clock genes in mice with induced circadian rhythm disruption. It was observed that CPF supplementation altered the specific composition and function of gut microbiota and metabolites induced by circadian rhythm disruption. Analysis showed that the abundance of Akkermansia increased, while the abundance of Clostridiales and Ruminiclostridium displayed a significant downward trend after the CPF intervention. Correlation analysis also revealed that these gut microbes had certain correlations with the metabolites, suggesting that CPFs help the intestinal microbiota to repair the intestinal environment and modulate the release of some beneficial metabolites. Notably, single-cell RNA-seq revealed that CPF supplementation significantly regulated the expression of genes associated with circadian rhythm, myelination, and neurodegenerative diseases. Altogether, these findings highlight that CPFs may represent a promising dietary therapeutic strategy for treating circadian rhythm disruption.

Inhibition of α-glucosidase by Cyclocarya paliurus based on HPLC fingerprinting integrated with molecular docking and molecular dynamics.

Cyclocarya paliurus (CP) extracts have been shown to lower sugar and lipid levels in blood, but the material basis is not clear. We analyzed CP aqueous extracts using high-performance liquid chromatography "fingerprinting", checked their pharmacological parameters using virtual screening, and undertook molecular docking and molecular dynamics simulations. Also, the inhibitory effects of CP components upon α-glucosidase in vitro were evaluated. Fingerprinting and virtual screening showed that the aqueous extract of CP contained the active components protocatechuic acid, chlorogenic acid, caffeic acid and rutin, which were safe and had no side effects in vivo. Molecular docking and molecular dynamics simulations showed that chlorogenic acid and rutin might have a potent inhibitory effect on α-glucosidase. An enzyme-activity assay in vitro showed that the half-maximal inhibitory values of chlorogenic acid and rutin were 398.9 and 351.8 µg/ml, respectively. Chlorogenic acid and rutin had an inhibitory effect on α-glucosidase. Cyclocarya paliurus could be developed as a natural α-glucosidase inhibitor.

Combined RNA-seq and molecular biology technology revealed the protective effect of Cyclocarya paliurus polysaccharide on H2O2-induced oxidative damage in L02 cells thought regulating mitochondrial function, oxidative stress and PI3K/Akt and MAPK signaling pathways.

Targeting attenuates oxidative damage may serve as a promising strategy for chronic diseases therapy. Cyclocarya paliurus polysaccharide (CP) had shown promising protection effects on L02 cells in oxidative damage. However, its mechanism remains elusive. Here, the protective effect of CP against oxidative damage was demonstrated in an L02 cell line. RNA-seq analysis showed that CP regulated oxidative and mitochondrion-related signal pathways, and the results were further confirmed with experiments. Seahorse extracellular flux experiments indicated that CP recovered mitochondrion function, thereby restoring normal respiration in the cells. In addition, CP regulated the activity of phosphoinositide 3-kinase//protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPKs) and it increased the expression of antioxidant enzymes (SOD, HO-1, and NQO1). Hence, our data suggested that CP protected L02 cells by targeting attenuate oxidative stress and recovering mitochondrion function, which might be achieved through PI3K/Akt and MAPK signaling. In summary, CP may be used as a functional food and may alleviate chronic diseases involving oxidative damage.

Metabolome and Transcriptome Analyses Unravel the Molecular Regulatory Mechanisms Involved in Photosynthesis of Cyclocarya paliurus under Salt Stress.

Photosynthesis is the primary life process in nature, and how to improve photosynthetic capacity under abiotic stresses is crucial to carbon fixation and plant productivity. As a multi-functional tree species, the leaves of Cyclocarya paliurus possess antihypertensive and hypoglycemic activities. However, the regulatory mechanism involved in the photosynthetic process of C. paliurus exposed to salinity has not yet been elucidated. In this study, the photosynthetic characteristics of C. paliurus seedlings, such as photosynthetic rate (Pn), stomatal conductance (Gs), and electron transfer rate (ETR), were investigated under different salt concentrations, while the metabolome and transcriptome analyses were conducted to unravel its molecular regulatory mechanisms. Salt stress not only significantly affected photosynthetic characteristics of C. paliurus seedlings, but also severely modified the abundance of metabolites (such as fumaric acid, sedoheptulose-7-phosphate, d-fructose-1,6-bisphosphate, and 3-phospho-d-glyceroyl phosphate) involved in central carbon metabolism, and the expression of photosynthetic genes. Through the co-expression network analysis, a total of 27 transcription factors (including ERFs, IDD, DOF, MYB, RAP) were identified to regulate photosynthetic genes under salt stress. Our findings preliminarily clarify the molecular regulatory network involved in the photosynthetic process of C. paliurus under salt stress and would drive progress in improving the photosynthetic capacity and productivity of C. paliurus by molecular technology.

Transcriptome analysis of Cyclocarya paliurus flavonoids regulation of differently expressed genes in Enterococcus faecalis under low pH stress.

Enterococcus faecalis (E. faecalis) is an indigenous intestinal bacterium and has potential to be applied as probiotic supplement. Low pH is one of the main stresses that E. faecalis has to deal with to colonize in the gastrointestinal tract. Previous study indicated low concentration of flavonoids may enhance the tolerance of probiotic to environmental stress. In the present research, transcriptome analysis was employed to investigate the influence of Cyclocarya paliurus flavonoids (CPF) on E. faecalis exposed to low pH environment. The results revealed that under the stress of low pH, genes related to cell wall and membrane, transmembrane transport, metabolism process, energy production, and conversion stress proteins were significantly differentially expressed. And certain undesired changes of which (such as genes for MFS transporter were downregulated) could be partially mitigated by CPF intervention, indicating their capacity to improve the low pH tolerance of E. faecalis. Results from this study deepened our understanding of the beneficial role of CPF on the probiotic in the gastrointestinal environment.

Phytochemicals from the Leaves of Cyclocarya paliurus and their 11ß-HSD1 Enzyme Inhibitory Effects.

Two new dammarane-type triterpenoid saponins, 3ß-(α-l-arabinopyranosyloxy)-24,25-dihydroxydammar-20-en-12α-yl 6-deoxy-ß-d-glucopyranoside (1) and (24R)-3ß-[(4-O-acetyl-α-l-arabinopyranosyl)oxy]-25-hydroxy-20,24-epoxydammaran-12ß-yl 6-deoxy-ß-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 11ß-HSD1 enzyme. Hederagenin (13) exhibited moderate inhibitory effect for mouse 11ß-HSD1 with an IC50 value of 0.16±0.04 µM.

Potential Role of Natural Plant Medicine Cyclocarya paliurus in the Treatment of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease that has become increasingly prevalent worldwide. It poses a serious threat to human health and places a considerable burden on global social medical work. To meet the increasing demand for T2DM treatment, research on hypoglycemic drugs is rapidly developing. Cyclocarya paliurus (Batal.) Iljinskaja is a medicinal plant that grows in China. The leaves of C. paliurus contain polysaccharides, triterpenoids, and other chemical components, which have numerous health benefits. Therefore, the use of this plant has attracted extensive attention in the medical community. Over the past few decades, contemporary pharmacological studies on C. paliurus extracts have revealed that it has abundant biological activities. Multiple in vitro and in vivo experiments have shown that C. paliurus extracts are safe and can play a therapeutic role in T2DM through anti-inflammatory and antioxidation activities, and intestinal flora regulation. Its efficacy is closely related to many factors, such as extraction, separation, purification, and modification. Based on summarizing the existing extraction methods, this article further reviews the potential mechanism of C. paliurus extracts in T2DM treatment, and we aimed to provide a reference for future research on natural plant medicine for the prevention and treatment of T2DM and its related complications.

Combined Analysis of the Metabolome and Transcriptome Identified Candidate Genes Involved in Phenolic Acid Biosynthesis in the Leaves of Cyclocarya paliurus.

To assess changes of metabolite content and regulation mechanism of the phenolic acid biosynthesis pathway at different developmental stages of leaves, this study performed a combined metabolome and transcriptome analysis of Cyclocarya paliurus leaves at different developmental stages. Metabolite and transcript profiling were conducted by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer and high-throughput RNA sequencing, respectively. Transcriptome identification showed that 58 genes were involved in the biosynthesis of phenolic acid. Among them, 10 differentially expressed genes were detected between every two developmental stages. Identification and quantification of metabolites indicated that 14 metabolites were located in the phenolic acid biosynthetic pathway. Among them, eight differentially accumulated metabolites were detected between every two developmental stages. Association analysis between metabolome and transcriptome showed that six differentially expressed structural genes were significantly positively correlated with metabolite accumulation and showed similar expression trends. A total of 128 transcription factors were identified that may be involved in the regulation of phenolic acid biosynthesis; these include 12 MYBs and 10 basic helix-loop-helix (bHLH) transcription factors. A regulatory network of the phenolic acid biosynthesis was established to visualize differentially expressed candidate genes that are involved in the accumulation of metabolites with significant differences. The results of this study contribute to the further understanding of phenolic acid biosynthesis during the development of leaves of C. paliurus.

The effects of Engelhardtia chrysolepis Hance on long-term memory and potential dopamine involvement in mice.

Engelhardtia chrysolepis Hance (ECH) is a perennial plant used in traditional medicine. A major active ingredient of ECH is astilbin (ASB), which has recently been shown to have neuroprotective effects as well as to affect catecholamine neurotransmissions in brain areas such as the prefrontal cortex. In this study, we investigated the effects of ECH and ASB on long-term memory in mice using a battery of behavioral tests. Acute ECH treatments dose-dependently facilitated nonspatial, but not spatial, memory. ECH treatments also upregulated expression of tyrosine hydroxylase, the enzyme mediating catecholamine synthesis, in neuroblastoma cell culture. Acute ASB treatments similarly improved nonspatial memory, whereas chronic ASB treatments improved both nonspatial and spatial memory. In accordance with such behavioral effects, the increased ratio of tissue concentrations of dopamine metabolites over dopamine in striatal regions was observed in mice with chronic ASB treatments. These results suggest that ECH and its active ingredient ASB may facilitate long-term memory by modulating catecholamine transmission.

Identification of a UDP-Glucosyltransferase favouring substrate- and regio-specific biosynthesis of flavonoid glucosides in Cyclocarya paliurus.

Cyclocarya paliurus (Batalin) Iljinsk is a medicinal plant belonging to the Juglandaceae family, and its leaves are used for a traditional sweet herbal tea with bioactivity against obesity and hyperglycaemia in China. It contains various bioactive specialised metabolites, such as flavonoids, triterpenes and their glucosides, while no glycosyltransferases (GTs) have been reported in C. paliurus to date. Herein, we identified and cloned the first glucosyltransferase C. paliurus GT1. The expression profiles of C. paliurus GT1 showed very high expression in young leaves, callus and branches, but relatively low expression in old leaves and bark and no expression in root. The recombinant C. paliurus GT1 protein was heterologously expressed in Escherichia coli and exhibited catalytic activity towards multiple flavonoids favouring substrate- and regio-specific biosynthesis. Further enzyme assays indicated a preference for certain hydroxyl group glucosylation by C. paliurus GT1. C. paliurus GT1 actively catalysed the glucosylation of flavones and flavonols, but it was less active towards isoflavones, flavanones or triterpenes. C. paliurus GT1 was also able to catalyse the attachment of sugars to the thiol (S-) or amine (N-) sites on aromatic compounds but not on aliphatic compounds. Molecular docking and site-directed mutagenesis analyses indicated that A43F, V84P, and M201Y dramatically altered the regio-selectivity and activity, and the W283M mutation and deletion of the V309-D320 region enhanced the activity and the formation of disaccharides. Herein, we present the identification and characterization of the first multi-functional glucosyltransferase in C. paliurus and provide a basis for understanding the biosynthesis of flavonoid glucosides. C. paliurus GT1 could be utilized as a synthetic biology tool for the synthesis of O-, N-, or S-glucosylated natural/unnatural products.

Nitrogen availability alters flavonoid accumulation in Cyclocarya paliurus via the effects on the internal carbon/nitrogen balance.

Cyclocarya paliurus has traditionally been used in medicines and nutraceutical foods. The aims of this study were to determine whether flavonoid accumulation in C. paliurus is dependent on nitrogen (N) availability and to investigate the internal C (carbon)/N balance under controlled conditions. One-year-old seedlings were grown under five increasing available N level treatments (N1-5) and were harvested throughout the 15-d experimental period. The greatest total chlorophyll amount and photosynthetic rate were achieved during the intermediate N treatments (N3 and N4). The greatest starch level was detected in N3. The total C level was relatively stable, but the total N and free amino acid levels increased, which resulted in a decreased C:N ratio. The flavonoid contents in roots and stalks decreased, while leaves showed a different pattern (peaking in N3). The flavonoid level was closely correlated with flavanone-3-hydroxylase activity, which displayed a similar variation pattern, and their levels were significantly positively correlated with those of total C and starch. Thus, the partitioning of C among primary and secondary metabolisms could be responsible for flavonoid biosynthesis and provide the basis for maintaining high yields, which increases the nutritional values of crops and medicinal plants.

Effects of nitrogen availability on mineral nutrient balance and flavonoid accumulation in Cyclocarya paliurus.

Cyclocarya paliurus has traditionally been used as medicine or nutraceutical foods. This study aims at investigating flavonoid accumulation in C. paliurus dependent on nitrogen availability and the following internal mineral nutrient balance under controlled condition. The 1-year-old seedlings of C. paliurus were grown in five different nitrogen levels. Along with the N gradient, C/N was significantly decreased, and the concentrations of phosphorus, potassium, calcium and magnesium were changed within plant. In the leaves, the main accumulation organ in C. paliurus, the highest flavonoid accumulation was achieved in intermediate N level (N3), which was closely related to flavanone-3-hydroxylase (FHT) activity as they had the similar variation patterns. Correlation analysis suggested that internal mineral nutrient balance can significantly affect flavonoid accumulation, especially for Mg within plant. These data revealed that nitrogen availability and the following altered internal mineral balance can significantly affect flavonoid accumulation. This study can provide the basis for developing new agricultural practices to maintain high yield while still keeping the nutritional value of crop or medicinal plants.

Light quality affects flavonoid production and related gene expression in Cyclocarya paliurus.

Understanding the responses of plant growth and secondary metabolites to differential light conditions is very important to optimize cultivation conditions of medicinal woody plants. As a highly valued and multiple function tree species, Cyclocarya paliurus is planted and managed for timber production and medical use. In this study, LED-based light including white light (WL), blue light (BL), red light (RL), and green light (GL) were used to affect leaf biomass production, flavonoid accumulation and related gene expression of one-year C. paliurus seedlings in controlled environments. After the treatments of 60 days, the highest leaf biomass appeared in the treatment of WL, while the lowest leaf biomass was found under GL. Compared to WL, the total flavonoid contents of C. paliurus leaves were significantly higher in BL, RL, and GL, but the highest values of selected flavonoids (kaempferol, isoquercitrin and quercetin) were observed under BL. Furthermore, the greatest yields of total and selected flavonoids in C. paliurus leaves per seedling were also achieved under BL, indicating that blue light was effective for inducing the production of flavonoids in C. paliurus leaves. Pearson's correlation analysis showed that there were significantly positive correlations between leaf flavonoid content and relative gene expression of key enzymes (phenylalanine ammonia lyase, PAL; 4-coumaroyl CoA-ligase, 4CL; and chalcone synthase, CHS) in the upstream, which converting phenylalanine into the flavonoid skeleton of tetrahydroxy chalcone. It is concluded that manipulating light quality may be potential mean to achieve the highest yields of flavonoids in C. paliurus cultivation, however this needs to be further verified by more field trials.

De Novo Transcriptome Assembly and Annotation of the Leaves and Callus of Cyclocarya Paliurus (Bata1) Iljinskaja.

Cyclocarya Paliurus (Bata1) Iljinskaja contains various bioactive secondary metabolites especially in leaves, such as triterpenes, flavonoids, polysaccharides and alkaloids, and its leaves are widely used as an hyperglycemic tea in China. In the present paper, we sequenced the transcriptome of the leaves and callus of Cyclocarya Paliurus using Illumina Hiseq 4000 platform. After sequencing and de novo assembly, a total of 65,654 unigenes were generated with an N50 length of 1,244bp. Among them, 35,041 (53.37%) unigenes were annotated in NCBI Non-Redundant database, 19,453 (29.63%) unigenes were classified into Gene Ontology (GO) database, and 7,259 (11.06%) unigenes were assigned to Clusters of Orthologous Group (COG) categories. Furthermore, 11,697 (17.81%) unigenes were mapped onto 335 pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG), among which 1,312 unigenes were identified to be involved in biosynthesis of secondary metabolites. In addition, a total of 11,247 putative simple sequence repeats (SSRs) were detected. This transcriptome dataset provides a comprehensive sequence resource for gene expression profiling, genetic diversity, evolution and further molecular genetics research on Cyclocarya Paliurus.

Separating four diastereomeric pairs of dihydroflavonol glycosides from Engelhardia roxburghiana using high performance counter-current chromatography.

Four pairs of diastereomers were successfully isolated and separated from the water extract of Engelhardia roxburghiana by high performance counter-current chromatography (HPCCC) using a two-step procedure. The diastereomers were initially separated by a two-phase solvent system composed of n-hexane-n-butanol-0.1% trifluoroacetic acid (1:2:3, v/v/v) and followed by the same solvent system using hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as an additive. The chromatographic conditions, elution mode, and concentrations of the additive were refined. The two-step HPCCC isolation yielded 43.7mg (2S, 3S)-astilbin, 27.6mg (2R, 3R)-astilbin, 5.9mg (2S, 3R)-astilbin, 4.8mg (2R, 3S)-astilbin, 6.9mg (2S, 3S)-engelitin, 3.1mg (2R, 3R)-engelitin, 8.2mg (2S, 3R)-engelitin, and 6.0mg (2R, 3S)-engelitin from 384mg crude extract in four runs with purities of 99.3%, 96.2%, 99.8%, 99.9%, 97.0%, 96.5%, 96.1%, and 96.8%, respectively. The present study revealed that HP-ß-CD can be used as an additive in HPCCC to effectively improve the resolution of the diastereomers. The established HPCCC method may serve as an approach to obtain high purity diastereomers on a large scale.