Tetrastigma Hemsleyanum Diels Research Articles

Molecular mechanisms of Tetrastigma hemsleyanum Diels&Gilg against lung squamous cell carcinoma: From computational biology and experimental validation

Ethnopharmacological relevanceTetrastigma hemsleyanum （T. hemsleyanum）, valued in traditional medicine for its potential to boost immunity and combat tumors, contains uncharacterized active compounds and mechanisms. This represents a significant gap in our understanding of its ethnopharmacological relevance. Aim of the studyTo involve the mechanism of anti-lung cancer effect of T. hemsleyanum by means of experiment and bioinformatics analysis. Materials and methodsThe anticancer mechanism of T. hemsleyanum against lung squamous carcinoma (LUSC) in zebrafish was investigated.. The LUSC model was established by injecting NCI-H2170 cells in the zebrafish and evaluating its anti-tumor efficacy. Next, component targets and key genes were obtained by molecular complex detection (MCODE) analysis and protein-protein interaction (PPI) network analysis. Component analysis of T. hemsleyanum was performed by UPLC-Q-TOF-MS. Molecular docking was used to simulate the binding activities of key potential active components to core targets were simulated using. Prognostic and pan-cancer analyses were then performed to validate the signaling pathways involved in the prognostic genes using gene set enrichment analysis (GSEA). Subsequently, Molecular dynamics simulations were then performed for key active components and core targets. Finally, cellular experiments were used to verify the expression of glutamate metabotropic receptor 3 (GRM3) and glutamate metabotropic receptor 7 (GRM7) in the anticancer effect exerted of T. hemsleyanum. ResultsWe experimentally confirmed the inhibitory effect of T. hemsleyanum on LUSC by transplantation of NCI-H2170 cells into zebrafish. There are 20 main compounds in T. hemsleyanum, such as procyanidin B1, catechin, quercetin, and kaempferol, etc. A total of 186 component targets of T. hemsleyanum and sixteen hub genes were screened by PPI network and MCODE analyses. Molecular docking and molecular dynamics simulation results showed that Gingerglycolipid B and Rutin had higher affinity with GRM3 and GRM7, respectively. Prognostic analysis, Pan-cancer analysis and verification experiment also confirmed that GRM3 and GRM7 were targets for T. hemsleyanum to exert anti-tumor effects and to participate in immune and mutation processes. In vitro experiments suggested that the inhibitory effect of T. hemsleyanum on cancer cells was correlated with GRM3 and GRM7. ConclusionIn vivo, in vitro and in silico results confirmed the potential anticancer effects against LUSC of T. hemsleyanum, which further consolidated the claim of its traditional uses.

Kaempferol 3-O-Rutinoside, a Flavone Derived from Tetrastigma hemsleyanum Diels et Gilg, Reduces Body Temperature through Accelerating the Elimination of IL-6 and TNF-α in a Mouse Fever Model.

Fever is a serious condition that can lead to various consequences ranging from prolonged illness to death. Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) has been used for centuries to treat fever, but the specific chemicals responsible for its antipyretic effects are not well understood. This study aimed to isolate and identify the chemicals with antipyretic bioactivity in T. hemsleyanum extracts and to provide an explanation for the use of T. hemsleyanum as a Chinese herbal medicine for fever treatment. Our results demonstrate that kaempferol 3-rutinoside (K3OR) could be successfully isolated and purified from the roots of T. hemsleyanum. Furthermore, K3OR exhibited a significant reduction in rectal temperature in a mouse model of fever. Notably, a 4 μM concentration of K3OR showed more effective antipyretic effects than ibuprofen and acetaminophen. To explore the underlying mechanism, we conducted an RNA sequencing analysis, which revealed that PXN may act as a key regulator in the fever process induced by lipopolysaccharide (LPS). In the mouse model of fever, K3OR significantly promoted the secretion of IL-6 and TNF-α during the early stage in the LPS-treated group. However, during the middle to late stages, K3OR facilitated the elimination of IL-6 and TNF-α in the LPS-treated group. Overall, our study successfully identified the chemicals responsible for the antipyretic bioactivity in T. hemsleyanum extracts, and it answered the question as to why T. hemsleyanum is used as a traditional Chinese herbal medicine for treating fever. These findings contribute to a better understanding of the therapeutic potential of T. hemsleyanum in managing fever, and they provide a basis for further research and development in this field.

Differential effects of winter cold stress on soil bacterial communities, metabolites, and physicochemical properties in two varieties of Tetrastigma hemsleyanum Diels & Gilg in reclaimed land.

Tetrastigma hemsleyanum Diels & Gilg (TDG) has been recently planted in reclaimed lands in Zhejiang Province, China, to increase reclaimed land use. Winter cold stress seriously limits the growth and development of TDG and has become the bottleneck limiting the TDG planting industry. To investigate the defense mechanisms of TDG toward winter cold stress when grown on reclaimed land, a combined analysis of soil bacterial communities, metabolites, and physicochemical properties was conducted in this study. Significant differences were observed in the composition of soil bacterial communities, metabolites, and properties in soils of a cold-tolerant variety (A201201) compared with a cold-intolerant variety (B201810). The fresh weight (75.8% of tubers) and dry weight (73.6%) of A201201 were significantly higher than those of B201810. The 16S rRNA gene amplicon sequencing of soil bacteria showed that Gp5 (25.3%), Gemmatimonas (19.6%), Subdivision3 (16.7%), Lacibacterium (11.9%), Gp4 (11.8%), Gp3 (10.4%), Gp6 (7.0%), and WPS-1 (1.2%) were less common, while Chryseolinea (10.6%) were more common in A201201 soils than B201810 soils. Furthermore, linear discriminant analysis of effect size identified 35 bacterial biomarker taxa for both treatments. Co-occurrence network analyses also showed that the structures of the bacterial communities were more complex and stable in A201201 soils compared to B201810 soils. In addition, ultra-high-performance liquid chromatography coupled to mass spectrometry analysis indicated the presence of significantly different metabolites in the two soil treatments, with 10 differentially expressed metabolites (DEMs) (8 significantly upregulated by 9.2%-391.3% and 2 significantly downregulated by 25.1%-73.4%) that belonged to lipids and lipid-like molecules, organic acids and derivatives, and benzenoids. The levels of those DEMs were significantly correlated with the relative abundances of nine bacterial genera. Also, redundancy discriminant analysis revealed that the main factors affecting changes in the bacterial community composition were available potassium (AK), microbial biomass nitrogen (MBN), microbial biomass carbon (MBC), alkaline hydrolysis nitrogen (AHN), total nitrogen (TN), available phosphorus (AP), and soil organic matter (SOM). The main factors affecting changes in the metabolite profiles were AK, MBC, MBN, AHN, pH, SOM, TN, and AP. Overall, this study provides new insights into the TDG defense mechanisms involved in winter cold stress responses when grown on reclaimed land and practical guidelines for achieving optimal TDG production.IMPORTANCEChina has been undergoing rapid urbanization, and land reclamation is regarded as a viable option to balance occupation and compensation. In general, the quality of reclaimed land cannot meet plant or even cultivation requirements due to poor soil fertility and high gravel content. However, Tetrastigma hemsleyanum Diels & Gilg (TDG), extensively used in Chinese herbal medicine, can grow well in stony soils with few nutrients. So, to increase reclaimed land use, TDG has been cultivated on reclaimed lands in Zhejiang Province, China, recently. However, the artificial cultivation of TDG is often limited by winter cold stress. The aim of this study was to find out how TDG on reclaimed land deal with winter cold stress by looking at the bacterial communities, metabolites, and physicochemical properties of the soil, thereby guiding production in practice.

Multi-omics combined with MALDI mass spectroscopy imaging reveals the mechanisms of biosynthesis of characteristic compounds in Tetrastigma hemsleyanum Diels et Gilg.

Tetrastigma hemsleyanum Diels et Gilg is recognized as a source of extracts with various desirable bioactivities. However, current knowledge regarding the mechanisms of biosynthesis of flavonoids, phenolic compounds, and other bioactive chemicals is limited. We conducted comprehensive tissue distribution studies and biosynthetic analyses of the 26 main bioactive compounds of this plant. The majority of flavonoids exhibited higher concentrations in the cortex (CT) compared to the vascular cylinder (VC). The expression levels of genes and proteins in CT and VC were quantified using mRNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ). A total of 31,700 genes were identified, among which 4921 exhibited differential expression between CT and VC. A total of 13,996 proteins were identified in the proteomes of CT and VC, with 927 showing differential expression. Co-expression network analyses of DEGs and DEPs from multiple sites demonstrated substantial pathway variations linked to flavonoid biosynthesis. Through differential enrichment analysis, a total of 32 genes involved in the flavone biosynthesis pathway were identified, with iTRAQ specifically detecting C3'H, F3H and FLS. Pearson correlation analysis revealed a strong association between the expression levels of C3'H, F3H, and FLS and the concentrations of flavonoids. The validation of multiple genes encoding pivotal enzymes was conducted using real-time fluorescence quantitative PCR (RT-qPCR). The findings provide a foundation for future investigations into the molecular mechanisms and functional characterization of T. hemsleyanum candidate genes associated with characteristic compounds.

Role of plant metabolites in the formation of bacterial communities in the rhizosphere of Tetrastigma hemsleyanum.

Tetrastigma hemsleyanum Diels et Gilg, commonly known as Sanyeqing (SYQ), is an important traditional Chinese medicine. The content of bioactive constituents varies in different cultivars of SYQ. In the plant growth related researches, rhizosphere microbiome has gained significant attention. However, the role of bacterial communities in the accumulation of metabolites in plants have not been investigated. Herein, the composition of bacterial communities in the rhizosphere soils and the metabolites profile of different SYQ cultivars' roots were analyzed. It was found that the composition of microbial communities varied in the rhizosphere soils of different SYQ cultivars. The high abundance of Actinomadura, Streptomyces and other bacteria was found to be associated with the metabolites profile of SYQ roots. The findings suggest that the upregulation of rutin and hesperetin may contribute to the high bioactive constituent in SYQ roots. These results provide better understanding of the metabolite accumulation pattern in SYQ, and also provide a solution for enhancing the quality of SYQ by application of suitable microbial consortia.

Kaempferol and nicotiflorin ameliorated alcohol-induced liver injury in mice by miR-138-5p/SIRT1/FXR and gut microbiota

AimsExcessive alcohol consumption can lead to alcoholic liver diseases (ALDs). Tetrastigma hemsleyanum Diels et Gilg is a rare Chinese medicinal herb. Tetrastigma hemsleyanum Diels et Gilg has been validated to be highly effective for treating hepatitis. Kaempferol and nicotiflorin are two highly representative flavonoids, which have exhibit therapeutic effects on liver disease. Therefore, the protective mechanism of kaempferol and nicotiflorin on alcohol-induced liver injury were investigated. Main methodsForty mice were used in this study. After treatment of Kaempferol and nicotiflorin, serum and liver were collected and used for determination of biochemical indicators, H&E staining, and molecular detection. The interaction of miRNAs from serum extracellular vehicles (EVs) with mRNAs and 16S rRNA sequencing of gut microbiota were also investigated. Key findingsThe results showed that kaempferol and nicotiflorins significantly ameliorated alcohol-induced liver damage and observably regulated gut microbiota. Specifically, the levels of malondialdehyde (MDA) and CYP2E1 in the liver significantly reduced, and the activity of superoxide dismutase (SOD) and glutathione (GSH) in the liver evidently increased. They also significantly relieved liver oxidative stress and lipid accumulation by suppressing miR-138-5p expression, inversely enhancing deacetylase silencing information regulator 2 related enzyme-1 (SIRT1) levels and then decreasing farnesoid X receptor (FXR) acetylation, which then modulated Nrf2 and SREBP-1c signaling pathways to regulate oxidative stress and lipid metabolism induced by alcohol. SignificanceKaempferol and nicotiflorin reduced alcohol-induced liver damage by enhancing alcohol metabolism and reducing oxidative stress and lipid metabolism. The intestinal microorganism disorder was also ameliorated after oral kaempferol and nicotiflorin.

Functional characterization of a cold related flavanone 3-hydroxylase from Tetrastigma hemsleyanum: an in vitro, in silico and in vivo study.

Tetrastigma hemsleyanum Diels et Gilg, a traditional Chinese medicine, frequently suffers from cold damage in the winter, leading to lower yields. There is a pressing need to improve cold resistance; however, the mechanisms underlying T. hemsleyanum responses to cold stress are still not clearly understood. Here, we explored the function of the flavanone 3-hydroxylase gene (ThF3H) in T. hemsleyanum under cold treatment. The open reading frame of ThF3H is 1092bp and encodes 363 amino acid residues. In vitro, the ThF3H enzyme was expressed in E. coli and successfully catalyzed naringenin and eriodictyol into dihydrokaempferol and dihydroquercetin, respectively. ThF3H exhibited a higher affinity for naringenin than for eriodictyol, which was in accordance with an in silico molecular docking analysis. The optimal pH and temperature for ThF3H activity were 7.0 and 30°C, respectively. In vivo, overexpression of the ThF3H gene enhanced the cold tolerance of transgenic Arabidopsis lines, which was likely due to the increase in flavonoids. Collectively, the function of a cold-related ThF3H in the flavonoid biosynthesis pathway may be helpful for improving the cold tolerance of T. hemsleyanum through molecular breeding techniques.

Fermentation-mediated variations in structure and biological activity of polysaccharides from Tetrastigma hemsleyanum Diels et Gilg

Variations in the structure and activities of polysaccharides from Tetrastigma hemsleyanum Diels et Gilg fermented by Sanghuangporus sanghuang fungi were investigated. Compare with the unfermented polysaccharide (THDP2), the major monosaccharide composition and molecular weight of polysaccharide after fermentation (F-THDP2) altered dramatically, which caused galactose-induced conversion from glucose and one-third of molecular weight. F-THDP2 had a molecular weight of 1.23 × 104 Da. Moreover, the glycosidic linkage of F-THDP2 varied significantly, a 1, 2-linked α-d-Galp and 1, 2-linked α-d-Manp backbone was established in F-THDP2, which differed from that of 1, 4-linked α-d-Glcp and 1, 4-linked β-d-Galp in THDP2. In addition, F-THDP2 showed a more flexible chain conformation than that of THDP2 in aqueous solution. Strikingly, F-THDP2 exhibited superior inhibitory effects on HeLa cells via Fas/FasL-mediated Caspase-3 signaling pathways than that of the original polysaccharide. These variations in both structure and biological activities indicated that fermentation-mediated modification by Sanghuangporus sanghuang might a promising novel method for the effective conversion of starch and other polysaccharides from Tetrastigma hemsleyanum Diels et Gilg into highly bioactive biomacromolecules, which could be developed as a potential technology for use in the food industry.

Optimization of tetrastigma hemsleyanum extraction process based on GA-BPNN model and analysis of its antioxidant effect

Tetrastigma hemsleyanum (Tetrastigma hemsleyanum Diels et Gilg) is a valuable traditional Chinese medicine with various applications. In this study, we aimed to optimize the extraction process for the total extraction yield of five flavonoid components, namely kaempferol, quercetin, rutin, kaempferol-3-O-rutinoside, and astragalin from the Tetrastigma hemsleyanum root (THR), and explore its potential molecular mechanisms in treating oxidative diseases as well as antioxidant activity. To achieve these objectives, we employed the genetic algorithm-back propagation neural network (GA-BPNN), the Box-Behnken design (BBD) with 4-factors and 3-levels to establish the optimal ethanol extraction process for the total extraction yield of the 5 components. Using public databases, the “component core targets-disease core target genes” networks were built, as well as molecular docking. Furthermore, DPPH was used to examine the antioxidant activity of the extracts obtained from THR under the optimal extraction process. The experimental value of the total extraction yield of the 5 components achieved a maximum of 788.12 mg/kg when the ethanol concentration was 73%, the solid-liquid ratio was 26 g/mL, and the ultrasonic duration was 30 min, and the ultrasonic temperature was 76 °C. When docked with protein molecules such as 6Y8I, quercetin, and other components received moderate to high scores. When the total concentration of the 5 components was 3.033 μg/mL, the DPPH radical scavenging rate was 89.81%. Compared with the BBD method, the GA-BPNN method is more efficient and reliable for optimizing the extraction process of active ingredients in THR because of its good data-fitting ability.

Polysaccharides from Tetrastigma Hemsleyanum Diels et Gilg ameliorated inflammatory bowel disease by rebuilding the intestinal mucosal barrier and inhibiting inflammation through the SCFA-GPR41/43 signaling pathway

In this study, the therapeutic effects of Tetrastigma hemsleyanum polysaccharide (THP) on inflammatory bowel disease (IBD) and its possible mechanisms were investigated based on the IBD mouse model induced by dextran sodium sulfate (DSS) and the lipopolysaccharide (LPS)-stimulated Caco-2 cell model. THP significantly alleviated the signs and symptoms of DSS-induced IBD mice, including the reduced weight, shortened colonic length, and increased colitis disease activity index. In vivo, THP significantly reduced inflammatory cell infiltration and oxidative damage, promoted intestinal mucus secretion, and restored the integrity of the intestinal epithelial barrier and mucus barrier. Furthermore, THP reversed the changes in the intestinal flora of colonized mice and restored the levels of short-chain fatty acids (SCFAs) by increasing the abundance of potentially beneficial bacteria and increasing the abundance of butyrate-producing bacteria. In addition, THP upregulated the expression of G-protein-coupled receptors (GPR41 and GPR43) both in vivo and in vitro. In summary, the current investigation showed that THP effectively protected against intestinal inflammation and impairment in the intestinal barrier in the setting of DSS-induced IBD, possibly by regulating gut microbiota structure and corresponding SCFA metabolites, and the pathway of SCFAs action may be related to SCFA-GPR41/43 signaling pathway.

Components research on Tetrastigma hemsleyanum Diels et Gilg: Identification and effect of drying methods on the content of ten main constituents by targeting metabolomics method

Tetrastigma hemsleyanum Diels et Gilg (TH) is one of the new eight Genuine Medicinal Materials of Zhejiang. It has extensive biological activities, such as anti-inflammatory, anti-tumor and analgesic activities, etc. In this study, the chemical components of TH were systematically investigated by ultra high-performance liquid chromatography-tandem quadrupole time of flight mass spectrometry (UPLC/Q-TOF-MS). Based on the MS spectrum, 39 compounds in TH extracts including 14 flavonoids, 10 fatty acids, 5 polyphenols and phenolic acids, 4 terpenes and other compounds were detected and tentatively identified. TH samples were treated under different drying methods (vacuum freeze drying, hot air drying, natural drying, light drying and vacuum drying). Besides, the effect of different drying methods on the content of 10 main chemical constituents in TH extracts including catechin, rutin, kaempferol-3-O-rutinoside and so on was also investigated by targeting metabolomics method with ultra high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-MS/MS) assisted by multivariate statistical analysis. Large differences were observed between vacuum drying and vacuum freeze drying with remarkable content changes. The contents of rutin, proanthocyanidin B1 and catechin were the most different among the various drying methods. The systematic identification of chemical constituents is helpful for the further medicinal development and application of TH. The effects of drying methods on the content of TH components were studied, which provided experimental data for the processing, storage and quality control of TH.

Screening out Biomarkers of Tetrastigma hemsleyanum for Anti-Cancer and Anti-Inflammatory Based on Spectrum-Effect Relationship Coupled with UPLC-Q-TOF-MS

Tetrastigma hemsleyanum Diels et Gilg. (T. hemsleyanum) is an economically and medicinally valuable species within the genus Tetrastigma. However, the material basis of its pharmacological action and the biomarkers associated with its anti-cancer and anti-inflammatory effects are still unclear. Additionally, the T. hemsleyanum industry cannot grow because there is a lack of a scientific, universal, and measurable quality control system. This study aimed to explore the chemical basis quality markers related to the anti-cancer and anti-inflammatory effects of T. hemsleyanum to establish an effective quality evaluation method. UPLC-Q-TOF-MSE fingerprint profiles of T. hemsleyanum from different origins were established. Pharmacodynamic studies used HepG2 and HuH-7 cells and LPS-induced RAW264.7 to evaluate the anti-tumor and anti-inflammatory effects of the active ingredients. The spectrum-effect relationships between UPLC fingerprints and anti-cancer and anti-inflammatory activities were evaluated using PCA and PLSR statistical methods. Moreover, docking analysis was performed to identify specific active biomarkers with molecular targets associated with cancer and inflammation. Chlorogenic acid, quinic acid, catechin, kaempferol 3-rutinoside, apigenin-8-C-glucoside, and linolenic acid were associated with anticancer activity, while chlorogenic acid, quercetin, quinic acid, kaempferol 3-rutinoside, rutinum, apigenin-8-C-glucoside, and linolenic acid were associated with anti-inflammatory activity. The spectrum-effect relationship of T. hemsleyanum was successfully established, and the biomarkers for anti-cancer and anti-inflammatory effects were preliminary confirmed. These findings provide a theoretical basis for the elucidation of the substance basis of T. hemsleyanum and lay the foundation for its rapid identification, quality control, industrial research, and utilization.

Induction, Flavonoids Contents, and Bioactivities Analysis of Hairy Roots and True Roots of Tetrastigma hemsleyanum Diels et Gilg

The flavonoids in Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) have high medicinal value. However, because of slow growth and harsh ecological environments, T. hemsleyanum is currently an endangered species. In light of this, we present a detailed hairy root induction procedure as a promising alternative to true roots with medicinal value. The percentage of explants induced by Agrobacterium rhizogenes (A. rhizogenes) to produce hairy roots out of the total number of explants infected (induction rate 1) was 95.83 ± 7.22%, and the proportion of hairy roots that contained Rol B fragments among all the hairy roots with or without Rol B fragments (positive rate) was 96.57 ± 1.72%. The transformation was further confirmed by the expression of the GUS protein. A high-productive hairy root line was screened for the comparative profiling of six flavonoids with true roots using high-performance liquid chromatography (HPLC). The contents of (+)-catechin, (-)-epicatechin, neochlorogenic acid, luteolin-6-C-glucoside, and orientin were 692.63 ± 127.24, 163.34 ± 31.86, 45.95 ± 3.46, 209.68 ± 6.03, and 56.82 ± 4.75 μg/g dry weight (DW) of 30-day-old hairy roots, respectively, which were higher than those of 3-year-old true roots. Hairy roots have stronger antioxidant activity than true roots. Overall, the hairy roots of T. hemsleyanum could serve as promising alternative sources for the production of flavonoids with medicinal uses.

Phylogenetic Analysis of R2R3-MYB Family Genes in Tetrastigma hemsleyanum Diels et Gilg and Roles of ThMYB4 and ThMYB7 in Flavonoid Biosynthesis.

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is an extensively used Chinese folk herb with multiple bioactivities. Among these bioactivities, flavonoids are recognized as the representative active ingredients. We previously found an elevated accumulation of flavonoids in T. hemsleyanum under water stress; however, the mechanism remains unclear. R2R3-MYB transcription factors play vital roles in the plant response to environmental stress and the regulation of secondary metabolites. Herein, a systematic transcriptome identification of R2R3-MYB family genes under water stress in T. hemsleyanum was performed to explore their potential function in the biosynthesis of flavonoids. A total of 26 R2R3-MYB genes were identified, most of which were clustered into functional branches of abiotic stress. ThMYB4 and ThMYB7 were then screened out to be associated with the biosynthesis of flavonoids through a protein-protein interaction prediction. An expression correlation analysis based on RNA-seq further confirmed that ThMYB4 and ThMYB7 were positively related to the flavonoid biosynthetic pathway genes of T. hemsleyanum. In ThMYB4- and ThMYB7-overexpression hairy roots, it was found that the expression of ThCHS and ThCHI was significantly increased, suggesting that ThMYB4 and ThMYB7 may act as regulators in flavonoid biosynthesis. This will shed new light on the promotion of flavonoid production and the medicinal value of T. hemsleyanum by manipulating transcription factors.

Tetrastigma hemsleyanum Diels et Gilg Roots from Different Origins: Pharmacognostical Identification, Content Determination of Functional Composition and in vitro Antioxidant Activity

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is a unique and rare Chinese medicinal herb mainly distributed in the south of the Yangtze River in China. In the present study, T. hemsleyanum roots from seven different sites in the south of the Yangtze River (Guangxi [Baise, Guilin, and Hechi], Hunan [Fenghuang], and Zhejiang [Hangzhou, Taizhou, and Wenzhou] provinces) were collected, and differences in the macroscopic and microscopic appearance of the roots, content of their active ingredients, and antioxidant activity of the root extracts were analyzed. The Pearson correlation coefficient was used to analyze the correlation between the antioxidant activity and the total flavonoids and total polyphenols contents of T. hemsleyanum roots. The results of macroscopic and microscopic root appearance indicated that T. hemsleyanum roots from different sites could be clearly differentiated based on size and epidermal characteristics, the color of dry root powder, and the shape and arrangement of the root canal. The results of content determination showed no significant differences (P>0.05) between the contents of flavonoids, polyphenols, rutin, and catechin in T. hemsleyanum roots from Guilin, Guangxi, and those from Zhejiang and Hunan. However, the contents of quercetin and kaempferol showed a significant difference (P<0.01) between T. hemsleyanum roots from Guilin, Guangxi, and those from Zhejiang, Hunan, and Guangxi (Baise and Hechi). Furthermore, the root extracts of T. hemsleyanum showed a high antioxidant activity ranging from 2.254±0.240 to 14.655±4.528 mg ascorbic acid equivalent/g sample. Pearson correlation analysis showed that the antioxidant activity of T. hemsleyanum roots had a higher correlation with the total flavonoids content than with the total polyphenols content. The results of this study provided a reference for the quality evaluation of T. hemsleyanum from different origins and provided technical support for subsequent resource development.

Total flavonoids of Tetrastigma hemsleyanum Diels et Gilg inhibits colorectal tumor growth by modulating gut microbiota and metabolites

Tetrastigma hemsleyanum Diels et Gilg is a dietary supplement in southern China. The total flavonoids of T. hemsleyanum (THTF) can be used for gastrointestinal disease treatment. Colorectal cancer (CRC) is associated with gut microbiota dysbiosis. This study was designed to investigate the effect of THTF on CRC from gut microbiota and fecal metabolomics. THTF (120 mg/kg) oral gavage reduced tumor growth and protected intestinal function (p-p65/p65, ZO-1) in HCT116 xenografts. THTF increased probiotics Bifidobacteriales, Bifidobacteriaceae, Bifidobacterium, Bifidobacterium pseudolongum, and decreased “harmful” bacteria Bacteroidota, Firmicutes, Bacteroidia, Rikenellaceae, Odoribacter, Alistipes richness. Furthermore, THTF restored abnormal fecal metabolite levels. It showed a strong correlation among gut microbiota, metabolites, and tumor weight. Finally, THTF promoted Bifidobacterium pseudolongum growth in vitro, whose cell-free supernatant further inhibited HCT116 cell proliferation and clonogenicity. Together, THTF delays CRC tumor growth by maintaining microbiota homeostasis, restoring fecal metabolites, and protecting intestinal function.

Flavonoid Metabolism in Tetrastigma hemsleyanum Diels et Gilg Based on Metabolome Analysis and Transcriptome Sequencing

Tetrastigma hemsleyanum Diels et Gilg, known as a "plant antibiotic", possesses several attractive properties including anti-inflammatory, anti-tumor, and antioxidant effects, with its efficacy being attributed to flavonoids. However, the flavonoid biosynthesis of T. hemsleyanum has rarely been studied. In this study, we investigated the flavonoid metabolism of T. hemsleyanum through metabolome analysis and transcriptome sequencing. The metabolomic results showed differences in the flavonoids of the leaves and root tubers of T. hemsleyanum. A total of 22 flavonoids was detected, and the concentrations of most flavonoids in the leaves were higher than those in the root tubers. Transcriptome analysis revealed that differentially expressed genes (DEGs) in the leaves and root tubers were enriched in photosynthesis-antenna proteins. Pearson correlation analysis indicated that the expression levels of chalcone isomerase (CHI) and UDP-glycose flavonoid glycosyltransferase (UFGT) were highly correlated with the concentrations of most flavonoids. Further, this study found that the photosynthesis-antenna proteins essentially contributed to the difference in the flavonoids in T. hemsleyanum. The gene expressions and concentrations of the total flavonoids of leaves and root tubers in Hangzhou, Jinhua, Lishui, and Taizhou in Zhejiang Province, China, showed that CHI (CL6715.Contig1_All, Unigene19431_All, CL921.Contig4_All) and UFGT (CL11556.Contig3_All, CL11775.Contig1_All) were the potential key genes of accumulation of most flavonoids in T. hemsleyanum.

Antitumor effects of polysaccharides from Tetrastigma hemsleyanum Diels et Gilg via regulation of intestinal flora and enhancing immunomodulatory effects in vivo.

Tetrastigma hemsleyanum Diels et Gilg is a traditional Chinese herbal medicine with high medicinal value, and antitumor, antioxidant and anti-inflammatory biological activities. However, while several studies have focused on flavonoids in Tetrastigma hemsleyanum tubers, there are few studies on the enhanced immune effect of Tetrastigma hemsleyanum polysaccharides (THP). In this study, we evaluated the antitumor effect of THP in a lung tumor model and explored the mechanism of antitumor activity through intestinal flora. In addition, a cyclophosphamide (CTX)-induced immunosuppression model was used to declare the immunomodulatory effect of THP in the immunosuppressive state induced by antitumor drugs. The results showed that THP increased the content of ileum secreted immunoglobulin A (SIgA) and cecum short-chain fatty acids (SCFAs) and improved microbial community diversity, regulating the relative abundance of dominant microbiota flora from the phylum level to the genus level, and recovering the intestinal microflora disorder caused by tumors. Additionally, THP can increase the organ indices and improve immune organ atrophy. THP can upregulate routine blood counts and stimulate the production of the serum cytokines. THP also promoted the macrophage phagocytic index, NK-cell activation, and complement and immunoglobulin (IgG, IgA, IgM) levels. The detection of Splenic lymphocyte proliferation and T lymphocyte subsets also sideways reflects that THP can restore CTX-induced immune inhibition in mice. In conclusion, this study suggests that THP can effectively achieve the enhanced antitumor effects, regulate gut microbiota and improve the immunosuppression induced by antitumor drugs. Therefore, THP can enhance the immune capacity and provide novel immunomodulatory and antineoplastic adjuvant agents.

Determination of the geographical origin of Tetrastigma hemsleyanum Diels & Gilg using an electronic nose technique with multiple algorithms

Tetrastigma hemsleyanum Diels & Gilg, an herbal medicinal plant, is planted widely in bamboo forests in southern China to promote economic benefits. Volatile compounds (VOCs) of T. hemsleyanum from different geographical regions are difficult to identify in field forests. In this study, VOCs from leaf samples of different geographical origins were analyzed using an electronic nose with 10 different sensors. Principal component analysis (PCA), partial least-squares regression (PLS), hierarchical cluster analysis (HCA), and radial basis function (RBF) neural networks were used to determine differences among different local samples. The results demonstrated that PCA achieved an accurate discrimination percentage of 91.31% for different samples and HCA separated the samples into different groups. The RBF neural network was successfully applied to predict samples with no specified localities. T. hemsleyanum samples from geographically close regions tended to group together, whereas those from distant geographical regions showed obvious differences. These results indicate that an electronic nose is an effective tool for detecting VOCs and discriminating the geographical origins of T. hemsleyanum. This study provides insights for further studies on the fast detection of VOCs from plants and effect of forests and plant herbal medicines on improving air quality.

Polysaccharides from Tetrastigma Hemsleyanum Diels et Gilg attenuate LPS-induced acute lung injury by modulating TLR4/COX-2/NF-κB signaling pathway

Tetrastigma Hemsleyanum Diels et Gilg (Sanyeqing, SYQ), a traditional herb native to China, has been widely used in the treatment of inflammatory diseases, febrile convulsions and dysentery. Previous studies have demonstrated the hypoglycemic, anti-inflammatory and anti-pyretic effects of polysaccharides from SYQ (SYQP). However, the role of SYQP in acute lung injury (ALI) remained unknown. Our present study aimed to explore the protective effects of SYQP in ALI and explained its underlying mechanism. In vivo, Balb/c mice were intragastrically administered with SYQP and dexamethasone (DXMS) for 14 days which were intratracheally instilled (IT) with LPS at the last day. In vitro, A549 cells were pretreated with SYQP, DXMS or TAK-242, following by LPS stimulation for 24 h. The histopathological results showed that SYQP remarkably alleviated pulmonary macrophage infiltration and myeloperoxidase (MPO) levels. Additionally, SYQP evidently suppressed wet/dry (W/D) ratio of lung and white blood cells (WBC) in bronchoalveolar lavage fluid (BALF). Moreover, SYQP markedly reduced TNF-α, IL-6, IL-1β and COX-2 levels both in vivo and in vitro. Meanwhile, SYQP significantly attenuated oxidative stress through upregulating the levels of SOD and GSH-Px and downregulating the expressions of MDA and LDH in vivo, which was confirmed by in vitro results that SYQP could increase SOD activity and decrease MDA and NO contents. Importantly, a decline in protein expressions of TLR4, COX-2, NF-κB p50, phospho-NF-κB p65 and phospho-IκB-α was detected in vivo and in vitro. Taken together, our results demonstrated that SYQP ameliorated LPS-induced ALI by alleviating inflammation and oxidative stress via attenuating TLR4/COX-2/NF-κB signaling pathway.