An exploratory metabolomic study reveals the Dipsacus asper-Achyranthes bidentate herb pair against osteoarthritis by modulating imbalance in polyunsaturated fatty acids and energy metabolism.

Osteoarthritis (OA) is a degenerative joint disease primarily affecting the cartilage. The therapeutic potential of the Dipsacus asper-Achyranthes bidentate herb pair for OA has been acknowledged, yet its precise mechanism remains elusive. In this study, we conducted a comprehensive analysis of metabolomic changes and therapeutic outcomes in osteoarthritic rats, employing a gas chromatography-mass spectrometry-based metabolomics approach in conjunction with histopathological and biochemical assessments. The rats were divided into six groups: control, model, positive control, Dipsacus asper treated, Achyranthes bidentata treated, and herb pair treated groups. Compared to the model group, significant reductions in levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and iNOS were observed in the treated groups. Multivariate statistical analyses were employed to investigate metabolite profile changes in serum samples and identify potential biomarkers, revealing 45 differential biomarkers, with eighteen validated using standard substances. These analytes exhibited excellent linearity across a wide concentration range (R2>0.9990), with intra- and inter-day precision RSD values below 4.69% and 4.83%, respectively. Recoveries of the eighteen analytes ranged from 93.97% to 106.59%, with RSD values under 5.72%, underscoring the method's reliability. Treatment with the herbal pair effectively restored levels of unsaturated fatty acids such as linoleic acid and arachidonic acid, along with glucogenic amino acids. Additionally, levels of phosphoric acid and citric acid were reversed, indicating restoration of energy metabolism. Collectively, these findings highlight the utility of metabolomic analysis in evaluating therapeutic efficacy and elucidating the underlying molecular mechanisms of herb pairs in OA treatment.

Unraveling the potential mechanisms of the anti-osteoporotic effects of the Achyranthes bidentata-Dipsacus asper herb pair: a network pharmacology and experimental study.

Background: Osteoporosis is a prevalent bone metabolism disease characterized by a reduction in bone density, leading to several complications that significantly affect patients' quality of life. The Achyranthes bidentata-Dipsacus asper (AB-DA) herb pair is commonly used in Traditional Chinese Medicine (TCM) to treat osteoporosis. This study aimed to investigate the therapeutic compounds and potential mechanisms of AB-DA using network pharmacology, molecular docking, molecular dynamics simulation, and experimental verification. Methods: Identified compounds of AB-DA were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Traditional Chinese Medicine Information Database (TCM-ID), TCM@Taiwan Database, BATMAN-TCM, and relevant literature. The main bioactive ingredients were screened based on the criteria of "OB (oral bioavailability) ≥ 30, DL (drug-likeness) ≥ 0.18." Potential targets were predicted using the PharmMapper and SwissTargetPrediction websites, while disease (osteoporosis)-related targets were obtained from the GeneCards, DisGeNET, and OMIM databases. The PPI network and KEGG/GO enrichment analysis were utilized for core targets and pathway screening in the STRING and Metascape databases, respectively. A drug-compound-target-pathway-disease network was constructed using Cytoscape software to display core regulatory mechanisms. Molecular docking and dynamics simulation techniques explored the binding reliability and stability between core compounds and targets. In vitro and in vivo validation experiments were utilized to explore the anti-osteoporosis efficiency and mechanism of sitogluside. Results: A total of 31 compounds with 83 potential targets for AB-DA against osteoporosis were obtained. The PPI analysis revealed several hub targets, including AKT1, CASP3, EGFR, IGF1, MAPK1, MAPK8, and MAPK14. GO/KEGG analysis indicated that the MAPK cascade (ERK/JNK/p38) is the main pathway involved in treating osteoporosis. The D-C-T-P-T network demonstrated therapeutic compounds that mainly consisted of iridoids, steroids, and flavonoids, such as sitogluside, loganic acid, and ß-ecdysterone. Molecular docking and dynamics simulation analyses confirmed strong binding affinity and stability between core compounds and targets. Additionally, the validation experiments showed preliminary evidence of antiosteoporosis effects. Conclusion: This study identified iridoids, steroids, and flavonoids as the main therapeutic compounds of AB-DA in treating osteoporosis. The underlying mechanisms may involve targeting core MAPK cascade (ERK/JNK/p38) targets, such as MAPK1, MAPK8, and MAPK14. In vivo experiments preliminarily validated the anti-osteoporosis effect of sitogluside. Further in-depth experimental studies are required to validate the therapeutic value of AB-DA for treating osteoporosis in clinical practice.

Metabolomic and microbiomic insights into color changes during the sweating process in Dipsacus asper.

Sweating is one of the most important primary processing methods of Chinese medicinal materials. Dipsacus asper is a typical representative of sweating treatment that is recommended by the Chinese Pharmacopoeia. The color change of the fracture surface of the root is the prominent feature of sweating treatment. However, few studies have focused on the mechanism of color change during sweating treatment. In this study, widely targeted metabolomics and ITS high-throughput sequencing technologies were applied to detect metabolites and microbial structure and diversity in the root of D. asper during sweating treatment. A total of 667 metabolites, including 36 downregulated and 78 upregulated metabolites, were identified in D. asper following sweating treatment. The significantly differential metabolites were divided into 12 classes, including terpenoids and phenolic acids. Moreover, all the differential terpenoids were upregulated and 20 phenolic acids showed a significant change after sweating treatment. In addition, microbial community diversity and richness increased following sweating treatment. The composition of microbial communities revealed that the relative abundances of Ascomycota and Basidiomycota significantly changed after sweating treatment. Correlation analysis revealed that Ascomycota (Fusarium sp., Macrophomina sp., Ilyonectria sp., Memnoniella sp., Penicillium sp., Cyphellophora sp., Neocosmospora sp., unclassified_f_Nectriaceae, and unclassified_o_Saccharomycetales) and Basidiomycota (Armillaria sp.) were associated with the content of terpenoids (6-deoxycatalpol and laciniatoside III) and phenolic acids (3-(4-hydroxyphenyl)-propionic acid, ethyl caffeate, 4-O-glucosyl-4-hydroxybenzoic acid, 2-acetyl-3-hydroxyphenyl-1-O-glucoside, 4-O-glucosyl-3,4-dihydroxybenzyl alcohol, 3-O-feruloylquinic acid, 3,4-O-dicaffeoylquinic acid methyl ester, O-anisic acid, and coniferyl alcohol). We speculate that the Ascomycota and Basidiomycota affect the content of terpenoids and phenolic acids, resulting in color change during sweating treatment in D. asper. This study provides a foundation for analyzing the mechanism involved in the processing of Chinese medicinal materials.

Akebia Saponin D Inhibits the Inflammatory Reaction by Inhibiting the IL-6-STAT3-DNMT3b Axis and Activating the Nrf2 Pathway.

Akebia saponin D (ASD) is derived from the Dipsacus asper Wall. ex Henry, which is a traditional Chinese medicine commonly used to treat rheumatic arthritis (RA). However, the in-depth mechanism of the anti-inflammatory effect of ASD is still unclear. This study aimed to preliminarily explore the anti-inflammatory effect of ASD and the underlying mechanisms from the perspective of DNA methylation and inflammation-related pathways. We found that ASD significantly reduced the production of multiple inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 (PGE2), in LPS-induced RAW264.7 cells. The expression of DNA methyltransferase (DNMT) 3b and inducible nitric oxide synthase (iNOS) was also obviously inhibited by the ASD treatment. The protein and mRNA levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also significantly inhibited by ASD. ASD inhibited the macrophage M1 phenotype, inhibited the high level of DNMT3b, and downregulated the signal transducer and activator of the transcription 3 (STAT3) pathway to exert its anti-inflammatory activity. Furthermore, DNMT3b siRNA and Nrf2 siRNA significantly promoted the anti-inflammatory effect of ASD. Our study demonstrates for the first time that ASD inhibits the IL-6-STAT3-DNMT3b axis and activates the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway to achieve its inhibitory effect on inflammatory reactions.

[Cloning and bioinformatics analysis of ß-amyrin synthase in Dipsacus asper].

Dipsaci Radix is one of the commonly used Chinese medicinal materials in China, with a long history. It has the medicinal activities of nourishing liver and kidney, recovering from broken sinews, and treating bone fracture. Triterpenoid saponins are the main functional ingredients of Dipsacus asper. ß-Amyrin synthases(ß-AS) as a superfamily of oxidosqualene cyclases(OSCs) can catalyze the construction of the skeleton structure of oleanane-type triterpenoid saponins. There are only a few studies about the ß-AS in D. asper, and the catalytic mechanism of this enzyme remains to be explored. To enrich the information of ß-AS, according to the transcriptome sequencing results, we cloned DaWß-AS gene from D. asper into a specific vector for heterologous expression in Escherichia coli. In the meantime, real-time PCR was performed to analyze the relative expression of DaWß-AS in four different tissues of D. asper. The results of RT-qPCR showed DaWß-AS had the highest expression level in leaves. Bioinformatics results indicated that DaWß-AS had a conserved domain of PLN03012 superfamily, belonging to the cl31551 superfamily. There was no transmembrane domain or signal peptide in DaWß-AS. This study provides a scientific basis for revealing the biological pathways of triterpenoid saponins in D. asper, which will facilitate the biosynthesis of the associated saponins and afford reference for the cultivation and development of high-quality resources of D. asper.

Simultaneous determination of 7 components in Dipsacus asper by HPLC / 中国药房

OBJECTIVE To establis h the method for the simultaneous determination of six iridoids （loganic acid ，loganin， sweroside，dipsanoside B ，dipsanoside A ，sylvestroside Ⅰ）and one triterpene saponin （asperosaponin Ⅵ）in Dipsacus asper . METHODS High performance liquid chromatography （HPLC） method was adopted. The determination was performed on Symmetry® C18 column with mobile phase consisted of acetonitrile- 0.1％ phosphoric acid solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 212 nm（asperosaponin Ⅵ）and 237 nm（dipsanoside B ，dipsanoside A ， sweroside，loganic acid ，sylvestroside Ⅰ，loganin）. The column temperature was set at 30 ℃，and sample size was 20 μL. RESULTS The linear range of loganic acid ， loganin， sweroside， sylvestroside Ⅰ ， dipsanoside B ， dipsanoside A and asperosaponin Ⅵ were 399.24-931.56，50.30-150.90，48.24-168.84，27.00-70.20，12.93-38.80，40.64-121.92，42.08-147.28 µg/mL （all r＞0.999 0）. RSDs of precision ，reproducibility and stability tests （24 h）were all less than 2％. Average recoveries were 104.43％（RSD＝0.63％，n＝6），101.74％（RSD＝1.11％，n＝6），100.76％（RSD＝1.06％，n＝6），98.00％（RSD＝1.58％，n＝6）， 99.03％（RSD＝2.31％，n＝6），102.93％（RSD＝2.26％，n＝6），102.31％（RSD＝1.00％，n＝6），respectively，The contents were 142.5-280.6，5.5-49.0，28.0-112.9，7.2-35.8，4.4-16.9，17.2-79.3，0.8-54.5 mg/g，respectively. CONCLUSIONS Established method is accurate and reliable ，and can be used for the content determination of 7 components in D. asper .

[Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis].

The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.

Polycaprolactone/Polyethylene Glycol Blended with Dipsacus asper Wall Extract Nanofibers Promote Osteogenic Differentiation of Periodontal Ligament Stem Cells.

Dipsacus asper wall (DA) is an ancient Chinese medicinal material that has long been used to maintain the health of human bones. The present study aimed to evaluate the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) of Dipsacus asper wall extracts (DAE). Microwave-assisted alcohol extraction of 100 mesh DA powder under optimal conditions can obtain 58.66% (w/w) yield of the crude extract. PDLSCs have excellent differentiation potential. PDLSCs treated with DA extract (DAE) underwent osteogenesis, exhibiting a higher expression of the Col-1, ALP, Runx2, and OCN genes, and had a 1.4-fold increase in mineralization, demonstrating the potential of DAE to promote osteogenic differentiation. After the addition of PI3K inhibitor LY294002, the expression of osteogenic genes was significantly inhibited, confirming that PI3K is an important pathway for DAE to induce osteogenesis. Mix DAE with polycaprolactone/polyethylene glycol (PCL/PEO) to obtain nanofibers with a diameter of 488 nm under optimal electrospinning conditions. The physical property analysis of nanofibers with and without DAE includes FTIR, mechanical strength, biodegradability, swelling ratio and porosity, and cell compatibility. When cells induced by nanofibers with or without DAE, the mineralization of PDLSCs cultured on PCL/PEO/DAE was 2.6-fold higher than that of PCL/PEO. The results of the study confirm that both DAE and PCL/PEO nanofibers have the effect of promoting osteogenic differentiation. In order to obtain the best induction effect, the optimal amount of DAE can be discussed in future research.

Identification and expression analysis of the DaNAC transcription factor family in Dipsacus asper / 药学学报

italic>NAC transcription factor genes play an important role in regulating plant adversity stress tolerance and secondary metabolism. To explore DaNAC transcription factor participation in the synthesis of asperosaponin Ⅵ in Dipsacus asper, we analyzed the expression of DaNAC genes based on full-length transcriptome data from different tissues (root, stem, leaf, flower, seed) to provide a theoretical foundation for regulating the metabolism of D. asper. RNA-seq data was used to identify open reading frames. Bioinformatic methods were used to identify the conserved domain motifs and construct an evolutionary tree. qRT-PCR was carried out to analyze tissue-specific and adversity-stressed expression. Twenty-nine DaNAC sequences were identified, all of which contain the conserved NAM domain and conserved motif 1 and motif 2 at the N terminal. Five DaNAC genes are closely related to the NAC genes in Arabidopsis thaliana and rice that are involved in adversity stress and are clustered in the Group Ⅰ subfamily. qRT-PCR revealed that DaNAC genes are differentially expressed between tissues. The expression levels were highest in leaves, followed by roots, stems and petioles, and the lowest in flowers and seeds. Compared with normal growth conditions, the expression of four NAC genes was up-regulated by treatment with low temperature (15 ℃). The expression of three genes (34564NAC2, 33883NAC48, 6727NAC14) was up-regulated and one gene (34480NAC22) was down-regulated by 150 μmol·L-1 MeJA. The results illustrate that the expression of NAC genes is induced by adversity stress, which provides a foundation for further study on the role of NAC family members in adversity stress in D. asper.

Methyl jasmonate stimulates the synthesis and accumulation of asperosaponin VI in the roots of Dipsacus asper / 药学学报

We investigated the effect of methyl jasmonate (MeJA) on the content of asperosaponin VI and the expression of genes involved in its synthesis. Dipsacus aspero seedlings were treated with MeJA at different concentrations of 50, 100, 150, 200 and 300 μmol·L-1, and leaves and roots were sampled following treatment for 1, 3 and 5 days. The content of asperosaponin VI and superoxide anion in the roots, malondialdehyde (MDA) content in leaves and superoxide dismutase were determined. The results show that 150 μmol·L-1 MeJA significantly increased the accumulation of asperosaponin VI in roots. The content of asperosaponin VI was greatest after treatment for 3 days, and was 2.16 times higher than the control. After MeJA treatment, SOD activity decreased and MDA content increased in leaves. Moreover, superoxide anion content in roots increased. The expression of squalene epoxidase (DaSE1) and geranyl diphosphate synthase (DaGPS), key enzymes in the synthesis of asperosaponin VI, were up-regulated compared with the control group. These results indicate that an optimal concentration of 150 μmol·L-1 MeJA increases the accumulation of asperosaponin VI by up-regulating the expression of key enzymes involved in the synthesis of asperosaponin VI, which facilitates resistance to adversity stress stimulated by MeJA.

Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis / 中国中药杂志

The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.

Traditional uses, processing methods, phytochemistry, pharmacology and quality control of Dipsacus asper Wall. ex C.B. Clarke: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipsacus asper Wall. ex C.B. Clarke, a traditional Chinese herbal medicine, has long been used in China for the therapy of bone diseases (e.g. bone fracture, osteoporosis, rheumatic arthritis), traumatic hematoma, uterine bleeding and those caused by the deficiency of liver and kidney. AIM OF THE STUDY: This work aims to evaluate current research progress on chemical constituents, pharmacological activities, quality control, and pharmacokinetic of Dipsacus asper Wall. ex C.B. Clarke, pinpoint the shortcomings of existing studies, and provide meaningful guidelines for our future investigations. METHODS: Extensive database retrieval, such as PubMed, SciFinder and CNKI, was carried out by using keywords such as "Dipsacus asper", "Radix Dipsaci", and "Xuduan". Furthermore, relevant textbooks, patents, reviews, and digital documents were consulted to collate all available scientific literature and to provide a complete science-based survey of the topic. RESULTS: More than 100 compounds have been isolated and identified from Dipsacus asper Wall. ex C.B. Clarke, a substantial proportion of which were reported to be triterpenoids and iridoids. Biological effects such as protective effects against bone fracture, anti-osteoporosis, neuroprotective, cardioprotective, anti-aging and protection of reproductive system activities were also evaluated in vitro and in vivo. CONCLUSIONS: Diaphoretic processing resulted in the drop in the content of asperosaponin VI, which was highly associated with bone protective effect of DA. Therefore, diaphoretic processing was not a suitable processing method for DA. Although Dipsacus asper Wall. ex C.B. Clarke was traditionally used for therapy of osteoarthritis, the in-depth study of the underlying mechanism was very rare. Much endeavor had been made on the effect of DA on bone fracture. Notably, high-dose of Dipsacus asper administration may cause adverse impacts in maternal healthy and embryo-fetal development. It's not suitable for further development in those bioactivities, such as anti-inflammatory and free radical scavenging, which are shared in many other plant species. Pharmacological effects of individual component of DA is not equivalent to its traditional usage. Attention should be paid to the traditional effect of extract of DA.

Cloning and expression stability analysis of Actin, Tubulin and GAPDH genes in Dipsacus asper / 中草药

Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.

Absolute structure assignment of an iridoid-monoterpenoid indole alkaloid hybrid from Dipsacus asper.

Iridoid-monoterpenoid indole alkaloid hybrids (IMIAHs) represent a rare class of natural products reported from only several plants of Rubiaceae and Dipsacaceae families, while their structural assignments remain a very challenging work due to complexity and flexibility. In the current study, a new IMIAH (1) was isolated from the roots of Dipsacus asper and its structure with absolute configuration was unambiguously established by a combination of spectroscopic analyses, chemical degradation and ECD calculation. A new oleanane-type triterpenoid saponin (2) and 15 known co-metabolites were also obtained and structurally characterized. Our biological evaluations showed that compound 2 exhibited moderate inhibition against acetylcholine esterase (AChE) with an IC50 value of 15.8 ±â€¯0.56 µM, and compound 15 displayed potent cytotoxicity selectively against human A549 and H157 lung cancer cells with IC50 values of 6.94 ±â€¯0.24 and 9.06 ±â€¯0.12 µM, respectively.

Triterpenoids and triterpenoid saponins from Dipsacus asper and their cytotoxic and antibacterial activities.

Phytochemical investigation of the ethyl acetate soluble part, generated from the ethanol extract of the roots of Dipsacus asper, led to the separation and identification of three undescribed triterpenoids including one arborinane type, one ursane type and one oleanane type, two unreported oleanane type triterpenoid arabinoglycosides, and 18 known analogues. Structures of these compounds were determined by comprehensive spectroscopic analyses, with the absolute configurations of 25-acetoxy-28-dehydroxyrubiarbonone E and 2α,3ß-dihydroxy-23-norurs-4(24),11,13(18)-trien-28-oic acid being established by evaluation of their experimental and calculated ECD spectra. 25-Acetoxy-28-dehydroxyrubiarbonone E features an oxygenated C-25 that is the first case among arborinane type triterpenoids, while 2α,3ß,24-trihydroxy-23-norurs-12-en-28-oic acid incorporates a sp3 C-24 that is a rare structural feature of 23-norursane type triterpenoids. Of these isolates, 2',4'-O-diacetyl-3-O-α-l-arabinopyranosyl-23-hydroxyolea-12-en-28-oic acid and hederagonic acid exhibited moderate antibacterial activity against Staphylococcus aureus with IC50 values of 12.3 and 10.3 µM, respectively, while those with either a feruloyloxy group or an arabinosyl moiety at C-3 displayed potent cytotoxic activities against four tumor cell lines A549, H157, HepG2 and MCF-7.

Protective effect of a polysaccharide from Dipsacus asper Wall on streptozotocin (STZ)-induced diabetic nephropathy in rat.

The effects of a polysaccharide (DAP) from the roots of Dipsacus asper on renal complication in streptozotocin (STZ)-induced diabetic rats were investigated in the present study. Administration of DAP (100 and 300 mg/kg) for 4 weeks significantly decreased the fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) levels, but increase the body weight of STZ-induced diabetic rats. Increased kidney weight index, serum creatinine (Scr), blood urea nitrogen (BUN), urine protein, and urinary albumin excretion (UAE) levels along with decreased creatinine clearance (Ccr) were nearly reversed to the normal levels by DAP. Moreover, DAP was able to normalize hyperlipidemia, improve oxidative stress, and down-regulate the formation of advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE). In conclusion, our work demonstrates the preventive effect of DAP was achieved via antihyperglycemic, hypolipidemic, and antioxidant activity along with inhibition of AGE accumulation and RAGE expression in STZ-treated diabetic rats.

Protective effects of Dipsacus asper polysaccharide on osteoporosis in vivo by regulating RANKL/RANK/OPG/VEGF and PI3K/Akt/eNOS pathway.

A homogenous polysaccharide (DAP), with a molecular weight of 2.61 × 104 Da, was isolated from the roots of Dipsacus asper Wall. Gas chromatography (GC) indicated that DAP was composed of galactose and mannose with a molar ratio of 1:1. The purpose of this study was to evaluate the effect of DAP on the progress of bone loss in the ovariectomized (OVX) rat model of osteoporosis. Administration of DAP (50 and 200 mg/kg/body wt. day) for 12 weeks significantly prevented OVX-induced bone loss, biomechanical reduction, the body weight gain, the loss of the uterus weight, as well as increased U-Ca/Cr, U-P/Cr, ALP, TRAP, OC and DPD/Cr levels in rats, which was further supported by the histopathological examinations. Furthermore, we found that the mechanism by which DAP elicited anti-osteoporotic effects was mediated by up-regulation of VEGF and OPG, but down-regulation of RANK and RANKL in both protein and mRNA expression in OVX rats, as well as the activation of PI3K/Akt/eNOS signaling pathway, indicating that DAP can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

Qualitative and quantitative analysis of furofuran lignans, iridoid glycosides, and phenolic acids in Radix Dipsaci by UHPLC-Q-TOF/MS and UHPLC-PDA.

Radix Dipsaci (RD), the dried root of Dipsacus asper, is used in traditional Chinese medicine as a remedy for bone fractures, traumatic hematoma, threatened abortion, and uterine bleeding. A novel ultra high-performance liquid chromatography coupled with quadrupole-time-of-flight tanderm mass spectrometry (UHPLC-Q-TOF/MS) approach was performed to rapidly characterize the chemical constituents of RD. Consequently, 21 compounds, including 12 iridoid glycosides (IGs), 4 furofuran lignans (FLs), and 5 phenolic acids (PAs) were discovered and identified from RD. Among these compounds, 3 IGs were previously unreported. Furthermore, a rapid and reliable UHPLC-DAD-based method was developed. The linearity (R2 > 0.999), repeatability (RSDs < 3.0%), intra-day and inter-day precision (RSDs < 1.6%), recovery (98.9%-102.5%), limits of detection (0.2-2.75 ng), and limits of quantification (0.75-8.5 ng) of the method was validated. The method was successfully applied for the simultaneous determination of 11 compounds in 20 batches of RD collected from various geographical regions in China. Different RD samples exhibited significantly varied contents of 11 analytes, among which IGs and PAs are abundant compounds that could be used as suitable quality markers for RD. The present study provided a useful approach for comprehensively evaluating the chemical composition and quality of RD.

Study of Transfer Rate of Standard Decoction of Dipsacus asper Decoction Pieces / 中国药房

OBJECTIVE:To study transfer rate of asperosaponinⅥ in standard decoction of Dipsacus asper decoction pieces. METHODS:The content of asperosaponin Ⅵ in Dipsacus asper decoction pieces and its standard decoction was determined by HPLC. The determination was performed on SinoChrom ODS-AP with mobile phase consisted of acetonitrile-water(30∶70,V/V)at the flow rate of 1.0 mL/min. The detection wavelength was set at 212 nm,and column temperature was 30℃. The sample size was 10 μL. By the ingredients content obtained,the transfer rate of asperosaponin Ⅵ was calculated during decoction piece to standard decoction. RESULTS:The linear range of asperosaponin Ⅵ was 0.484-4.84 μ g(r＝0.999 9). RSDs of precision,stability and reproducibility tests were all lower than 2%. The limits of quantification and detection were 0.3 and 0.1 μ g,respectively. The average recoveries in D. asper decoction pieces and standard decoction were 95.13% -100.22%(RSD＝1.78%,n＝6), 97.07%-100.08%(RSD＝0.98%,n＝6). RSD of durability test was lower than 1%.The transfer rate of asperosaponin Ⅵ in standard decoction of D. asper decoction pieces ranged 26.3%-49.5%. CONCLUSIONS:The method is simple,accurate,precise, stable,reproducible and durable,and can be used for transfer rate of asperosaponin Ⅵ in standard decoction of D. asper decoction pieces.

Antifungal activity of sterols and dipsacus saponins isolated from Dipsacus asper roots against phytopathogenic fungi.

The in vivo antifungal activity of crude extracts of Dipsacus asper roots was evaluated against the phytopathogenic fungi Botrytis cinerea, Colletotrichum coccodes, Blumeria graminis f. sp. hordei, Magnaporthe grisea, Phytophthora infestans, Puccinia recondita and Rhizoctonia solani using a whole-plant assay method. Ethyl acetate and acetone extracts, at 1000µg/mL, suppressed the development of tomato gray mold (TGM) and tomato late blight (TLB) by 90%. Through bioassay-guided isolation, five antifungal substances were isolated from the D. asper roots and identified as ß-sitosterol (1), campesterol (2), stigmasterol (3), cauloside A (4) and a novel dipsacus saponin, named colchiside (3-O-ß-d-xylopyranosyl-23-O-ß-d-glucopyranosyl-28-O-ß-d-(6-O-acetyl)-glucopyranosyl hederagenin) (5). Of those, cauloside A (4) displayed the greatest antifungal efficacy against rice blast, TGM and TLB. Colchiside (5) moderately suppressed the development of TLB, but exhibited little effect against the other diseases. The synergistic effects of the isolated compounds against TLB were also assessed. Synergistic and additive interactions were observed between several of the sterol compounds. This study indicated that the crude extracts of, and bioactive substances from, the roots of D. asper suppress TGM and TLB. In addition, cauloside A (4) and colchiside (5) could be used as antifungal lead compounds.