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.

Achyranthes bidentata polysaccharide ameliorates type 2 diabetes mellitus by gut microbiota-derived short-chain fatty acids-induced activation of the GLP-1/GLP-1R/cAMP/PKA/CREB/INS pathway.

Gut microbiota variances reflecting the severity type 2 diabetes mellitus (T2DM). Achyranthes bidentata polysaccharide (ABP) can regulate gut microbiota. However, the hypoglycemic effect and underlying mechanism of ABP remain unclear. Herein, we characterized the structure of ABP and revealed the hypoglycemic effect of ABP in mice with T2DM. ABP repaired the intestinal barrier in T2DM mice and regulated the composition and abundance of gut microbiota, especially increasing bacteria which producing short-chain fatty acids (SCFAs), then increasing glucagon-like peptide-1 (GLP-1) level. The abundance of these bacteria was positively correlated with blood lipid and INS levels, negatively correlated with FBG levels. Colon transcriptome data and immunohistochemistry demonstrated that the alleviating T2DM effect of ABP was related to activation of the GLP-1/GLP-1 receptor (GLP-1R)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-response element binding protein (CREB)/INS pathway. Fecal microbiota transplantation (FMT) confirmed the transmissible efficacy of ABP through gut microbiota. Overall, our research shows that ABP plays a hypoglycemic role by increasing gut microbiota-derived SCFAs levels, and activating the GLP-1/GLP-1R/cAMP/PKA/CREB/INS pathway, emphasizing ABP as promising T2DM therapeutic candidates.

Achyranthes bidentata polysaccharides improve cyclophosphamide-induced adverse reactions by regulating the balance of cytokines in helper T cells.

The manuscript aimed to study the immune function maintenance effect of Achyranthes bidentata polysaccharides (ABPs). The mice were divided into the control group, cyclophosphamide-induced (CTX) group, and ABPs-treated (ABP) group. The results showed that, compared with the CTX group, ABPs could significantly improve the spleen index and alleviate the pathological changes in immune organs. Ex vivo study of whole spleen cells, the levels of interleukin-2 (IL-2), interleukin-6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were increased. The proliferation of lymphocytes and the proportion of CD3+CD4+ Th cells in peripheral blood mononuclear cells were increased. The transcription of GATA-3, Foxp3, and ROR γ t were decreased, while the transcription of T-bet was increased. The transcriptome sequencing analysis showed that the differentially expressed genes (DEGs) caused by ABPs-treated were mostly downregulated in CTX-induced mice. The Th2-related genes were significantly enriched in DEGs, with representative genes, including Il4, II13, Il9, etc., while increasing the expression of immune effector genes simultaneously, including Ccl3, Ccr5, and Il12rb2. It was suggested that ABPs possibly regulated the balance of cytokines in helper T cells to ameliorate the immune function of CTX-induced mice.

Application of microscopy and spectroscopy in investigating anti-cancer potential of Achyranthes aspera L. leaves.

The genus Achyranthes belong to the family Amaranthaceae which constitutes an important group of herbs and shrubs with immense medicinal value. The present research work was conducted to investigate the anticancer potential of Achyranthes aspera L. leaves by focusing on the antioxidant, aniproliferative and antimitotic activities of leaf extracts. Plant extraction was carried out by soxhelt method with different solvents. Phytochemical characterization of the plants extracts using chemical methods identified the presence of cardiac glycosides, saponins, coumarins, proteins, tannins, flavonoids and triterpenes. Alkaloid was present in methanolic and ethanolic extract. High performance liquid chromatography showed presence of different concentration of myricetin, quercetin and kaempferol in different extracts with the highest concentration of myricetin (84.53 µg/mL) in n-butanolic extract. The extracts were then tested for antioxidant activity using 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging assay by spectrophotometric method. In DPPH radical scavenging assay, antioxidant activity of A. aspera ranged between 79.78 ± 0.034% and 58.63 ± 0.069%. Highest antioxidant activity was observed for methanolic extract and lowest for acetone. Antimitotic activity was determined by using Allium cepa assay in which microscopic investigation was carried out to observe normal and abnormal phases of mitosis. In this assay, n-butanolic extract had highest antimitotic activity with minimum mitotic index at 2 mg/mL (57 ± 0.0351%). The plant extracts also caused chromosomal and mitotic aberrations which were clearly observed under 40× and 100× magnification of compound microscope. Antiproliferative activity was determined by using yeast cell model in which light microscope with hemocytometer was used for cell counting. In case of Antiproliferative activity, the ethyl acetate extract of A. aspera had highest antiproliferative activity with lowest cell viability (22.14 ± 0.076%) at highest extract concentration (2 mg/mL) while methanol extract of A. aspera had highest antiproliferative activity with lower cell viability (24.24 ± 0.057%) at lowest extract concentration (0.25 mg/mL). The results of the study indicated that the leaves extract of A. aspera have strong potential to be used as a source of anti-cancer agent. RESEARCH HIGHLIGHTS: Achyranthes aspera L. leaves have various phytochemicals which contribute to its medicinal properties Various extracts of the leaves of A. aspera L. possess antioxidant, antimitotic and antiproliferative potential The results of the study indicated that the leaves extract of A. aspera have strong potential to be used as a source of anti-cancer agent.

Oligo/polysaccharides from Cyathula officinalis and Achyranthes bidentata: a review of structures and bioactivities.

OBJECTIVES: Oligo-/polysaccharides from Cyathula officinalis Kuan (COPs) and Achyranthes bidentata Blume (ABPs) have attracted researchers' attention in the fields of healthy food supplements and traditional Chinese medicine (Niúxi) due to their multiple bioactivities combined with their nontoxic and highly biocompatible nature. The purpose of this paper was to provide a systematic and comprehensive overview of the extraction, purification, and structural analysis methods, chemical characteristics, biological activities, and structure bioactivity relationship. Furthermore, the possible development trends and perspectives for future research, and traditional uses of Niúxi are also summarized. METHODS: All the information was gathered from a library search and scientific databases. KEY FINDINGS: Although COPs and ABPs are derived from different plants, they have similar structural features in type, structure, and glycosidic linkage patterns and biological activities in vivo and in vitro. However, there are differences in monosaccharide compositions, which can be used as an identification mark. CONCLUSIONS: As traditional Chinese herbal medicine, C. officinalis and A. bidentata have similar pharmacological activities. The COPs and ABP possess wide pharmacological effects such as antitumor, antioxidant, anti-osteoporosis, and anti-inflammatory. Meanwhile, the biological activity and structure-activity relationship of purified COPs and ABPs are less studied, future research should focus on them.

Isolation of Calenduloside E from Achyranthes bidentata Blume and its effects on LPS/D-GalN-induced acute liver injury in mice by regulating the AMPK-SIRT3 signaling pathway.

BACKGROUND: Acute liver injury (ALI) is a frequent fatal liver disease with a high mortality. Calenduloside E (CE) is a pentacyclic triterpenoid derived from Achyranthes bidentata Blume. It has been found that liver injury is associated with mitochondrial dysfunction, and activation of the AMPK-SIRT3 signaling pathway protects the mitochondrial function to play a role in resistance to the disease. However, whether CE is protective against ALI through the AMPK-SIRT3 signaling pathway is unclear. PURPOSE: To clarify the influences of Calenduloside E (CE) isolated from Achyranthes bidentata Blume on LPS/D-GalN-induced Acute liver injury (ALI). METHODS: A mouse model of ALI was developed, intraperitoneal injection of 10 µg/kg LPS and 700 mg/kg D-GalN, histopathological, oxidative stress, and immune inflammation of the mice were monitored. The mechanism of CE influencing liver injury was investigated by examining the gut microbiota, mitochondrial dysfunction, and the AMPK-SIRT3 signaling pathway. The antagonistic effects of specific AMPK and SIRT3 blocker, as well as AMPKα1, AMPKα2, SIRT3 transfection-mediated silencing were investigated to confirm the role of the AMPK-SIRT3 signaling pathway in this process. RESULTS: CE relieved liver pathological damage of mice and led to reduced oxidative stress and immune inflammation in mice, affected the balance of gut microbiota in mice with liver injury, as well as energy metabolism, and regulated mRNA and protein expressions of AMPK-SIRT3 signaling pathway. In addition, in vitro studies showed that CE relieved mitochondrial respiratory and protein expressions of AMPK-SIRT3 signaling pathway in LPS/D-GalN-induced AML12 and LX2 cells, and such effect was blocked by AMPK and SIRT3 inhibitors. Furthermore, silencing of AMPKα1, AMPKα2, and SIRT3 blocked the effects of CE. Overall, the influences of CE on mice with liver injury is tuned by the AMPK-SIRT3 signaling pathway. CONCLUSION: CE mediates mitochondrial function and eventually regulate energy metabolism by regulating the AMPK-SIRT3 signaling pathway. The results of this study provide molecular evidences for application of CE in treatment of ALI and provide references to the drug development for ALI.

In silico Screening and Validation of Achyranthes aspera as a Potential Inhibitor of BRAF and NRAS in Controlling Thyroid Cancer.

BACKGROUND: Thyroid carcinoma (THCA) is one of the most prevalent endocrine tumors, accounting for 3.4% of all cancers diagnosed annually. Single Nucleotide Polymorphisms (SNPs) are the most prevalent genetic variation associated with thyroid cancer. Understanding thyroid cancer genetics will enhance diagnosis, prognosis, and treatment. METHODS: This TCGA-based study analyzes thyroid cancer-associated highly mutated genes through highly robust in silico techniques. Pathway, gene expression, and survival studies were performed on the top 10 highly mutated genes (BRAF, NRAS, TG, TTN, HRAS, MUC16, ZFHX3, CSMD2, EIFIAX, SPTA1). Novel natural compounds from Achyranthes aspera Linn were discovered to target two highly mutated genes. The natural compounds and synthetic drugs used to treat thyroid cancer were subjected to comparative molecular docking against BRAF and NRAS targets. The ADME characteristics of Achyranthes aspera Linn compounds were also investigated. RESULTS: The gene expression analysis revealed that the expression of ZFHX3, MCU16, EIF1AX, HRAS, and NRAS was up-regulated in tumor cells while BRAF, TTN, TG, CSMD2, and SPTA1 were down-regulated in tumor cells. In addition, the protein-protein interaction network demonstrated that HRAS, BRAF, NRAS, SPTA1, and TG proteins have strong interactions with each other as compared to other genes. The ADMET analysis shows that seven compounds have druglike properties. These compounds were further studied for molecular docking studies. The compounds MPHY012847, IMPHY005295, and IMPHY000939 show higher binding affinity with BRAF than pimasertib. In addition, IMPHY000939, IMPHY000303, IMPHY012847, and IMPHY005295 showed a better binding affinity with NRAS than Guanosine Triphosphate. CONCLUSION: The outcomes of docking experiments conducted on BRAF and NRAS provide insight into natural compounds with pharmacological characteristics. These findings indicate that natural compounds derived from plants as a more promising cancer treatment option. Thus, the results of docking investigations conducted on BRAF and NRAS substantiate the conclusions that the molecule possesses the most suited drug-like qualities. Compared to other compounds, natural compounds are superior, and they are also druggable. This demonstrates that natural plant compounds can be an excellent source of potential anti-cancer agents. The preclinical research will pave the road for a possible anti-cancer agent.

Saponin extract from Achyranthes bidentata Blume alleviates disuse-induced muscle atrophy through PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Achyranthes bidentata Blume are one of the regularly used herbal drugs in Chinese medicine, and has been applied for strengthening the muscle and bone for a long time. However, its effect on muscle remains unclear. AIM OF THE STUDY: This paper aims to explore the anti-muscle atrophy effect of A. bidentata, and to clarify the possible signaling pathways involved. MATERIALS AND METHODS: The saponin extract of the roots of A. bidentata (ABSE) was prepared and analyzed, and its activity on myoblast differentiation was assayed with C2C12 cell culture. ABSE was then orally administered at dosage of 35, 70 and 140 mg/kg/day to disuse-induced muscle atrophy mice. The studies on mice body weight and muscle quality were conducted, and Western blot was used for exploring the possible signaling pathways involved in the muscle protective action aided with transcriptome analysis. RESULTS: The total saponin content of ABSE was 59.1%. ABSE promoted the C2C12 cells differentiation to myotube in C2C12 differentiation assay. Further study with disuse-induced muscle atrophy mice model demonstrated that ABSE significantly increased muscle fiber diameter as well as the proportion of slow muscle fibers. Possible mechanism study aided with transcriptome analysis revealed that ABSE alleviated muscle atrophy at least through activation of PI3K/Akt pathway in vivo & vitro. CONCLUSIONS: The saponin extract of the root of A. bidentata (ABSE) has a protective effect on muscle atrophy, and showed a considerable potential in prevention and treatment of muscle atrophy.

Achyranthes polysaccharide based dual-responsive nano-delivery system for treatment of rheumatoid arthritis.

Achyranthes plays the role of dredging the meridians and clearing the joints with a certain anti-inflammatory effect, peripheral analgesic activity and central analgesic activity. A novel self-assembled nanoparticles containing Celastrol (Cel) with matrix metalloproteinase (MMP)-sensitive chemotherapy-sonodynamic therapy was fabricated targeting macrophages at the inflammatory site of rheumatoid arthritis. Dextran sulfate (DS) with highly expressed SR-A receptor on the surface of macrophages is used to specifically target the site of inflammation; by introducing PVGLIG enzyme-sensitive polypeptides and ROS-responsive bonds, it can achieve the desired effect on MMP-2/9 and reactive oxygen species at the joint site. The preparation forms DS-PVGLIG-Cel&Abps-thioketal-Cur@Cel nanomicelles, referred to as D&A@Cel. The resulting micelles had an average size of 204.8 nm and the zeta potential -16.46 mV. The results show that activated macrophages can effectively capture Cel in in vivo experiments, indicating that Cel delivered by nanoparticles can significantly improve bioavailability.

[Achyranthes bidentata polysaccharide inhibits the adipogenic differentiation of rat bone marrow mesenchymal stem cells by blocking the PPARγ/TRPV4 pathway].

Objective To investigate the effect of Achyranthes bidentata polysaccharides (ABPS) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its mechanism. Methods Five SD rats were sacrificed, and the BMSCs were dissected and isolated. The BMSCs were adherently cultured to logarithmic growth phase after identification, and treated with different doses of ABPS for 48 hours. The cell survival rates were detected by MTT assay. The highest dose of ABPS without toxicity to BMSCs was selected for subsequent experiments. Cells were randomly divided into control group, ABPS group, rosiglitazone group and ABPS combined with rosiglitazone group. Cell survival rates were detected by MTT assay. Triglyceride (TG) levels in BMSCs were detected by spectrophotometry. Lipid droplet formation in BMSCs was observed by oil red O staining. The mRNA and protein expression of peroxisome proliferater-activated receptor γ (PPARγ), transient receptor potential vanilloid 4 (TRPV4) and CCAAT/enhancer binding protein α (C/EBPα) were detected by real time quantitative PCR and Western blot analysis. Results The dose of ABPS≤200 mg/L had no obvious toxic effect on the growth of BMSCs after 48 hours, and the cell survival rate of 400 mg/L ABPS group was lower. Compared with the control group, the ABPS group showed decreased levels in TG, decreased relative expression of PPARγ, TRPV4 and C/EBPα mRNA and protein, and the decreased number of cytoplasmic lipid droplets. In the rosiglitazone group, observation reported the decreased cell survival rate, increased TG level, increased relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein, along with the increased number of cytoplasmic lipid droplets. Compared with the ABPS group, the cell survival rate was decreased, TG level was increased, the relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein increased, and the number of cytoplasmic lipid droplets increased in the ABPS combined with rosiglitazone group. Compared with rosiglitazone group, the survival rate was increased, TG level was decreased, the relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein were decreased, and the number of cytoplasmic lipid droplets was decreased in the ABPS combined with rosiglitazone group. Conclusion ABPS can inhibit adipogenic differentiation of BMSCs, and the mechanism may be related to the regulation of PPARγ/TRPV4 pathway.

Discovery of a cysteine-rich peptide with glycation modification from Achyranthes bidentata Blume.

Cysteine-rich peptides (CRPs) are stable molecules that contain multiple disulphide bonds. Various CRPs are found in plants and animals, representing potential compounds for drug development with diverse activities. Modification of CRPs, such as glycation, has attracted increased attention due to its special structural and functional properties. Hence, this study explored a CRP isolated from the Chinese herb Achyranthes bidentata Blume, which contains a glycation modification. Herein, a reverse phase high-performance liquid chromatography system with mobile phases was used to extract and purify the peptide. The eluted peptide was detected using high resolution mass spectrometry and structurally identified using high resolution mass spectrometry and nuclear magnetic resonance. The effect of the peptide on the viability of N-methyl-D-aspartic acid (NMDA)-induced HT22 cells was determined using a cell assay. Here, a new cysteine-rich glycation peptide, termed glycation-bidentatide (Gly-BTP), with three pairs of disulphide bonds and a glycation modification at the N-terminus linked to cysteine, was discovered. Cell bioactivity assay results suggested that Gly-BTP might be a potential therapeutic and provide a neuroprotective effect in NMDA-induced HT22 murine hippocampal neuronal cells. The discovery of Gly-BTP will promote the understanding of the role of CRPs in neuroprotection.

Dietary inclusion of Achyranthes japonica extract to corn-soybean meal-wheat-based diet on the growth performance, nutrient digestibility, cecal microflora, excreta noxious gas emission, and meat quality of broiler chickens.

This research was conducted to determine the effects Achyranthes japonica extract (AJE) supplementation to corn-soybean meal-wheat-based diet on the growth performance, nutrient digestibility, cecal microflora, excreta noxious gas emission, and meat quality of broiler chickens. A total of 432 one-day-old male Ross 308 broiler chickens, having initial body weight (IBW) of 41.11 ± 1.65 g were randomly allotted to 4 dietary treatments. Each treatment had 6 replicates cages with 18 broilers per cage. Dietary treatments composed of corn-wheat-soybean meal-based diets along with the addition of 0, 0.025, 0.05, and 0.1% of AJE. Bodyweight gain (BWG) and average daily feed intake (ADFI) were linearly influenced by the supplementation of AJE duringphase1 and 2 and the overall trial period. Inclusion of increasing levels of AJE linearly improved the digestibility of dry matter (DM) on d 35. Dietary supplementation of increasing levels of AJE failed to show significant effects on cecal Lactobacillus, coliform, and Salmonella counts, excreta noxious gas ammonia, hydrogen sulfide, total mercaptans, carbon dioxide, acetic acid and propionic acid emission, meat quality, and relative organ weight. Therefore, we concluded that supplementation of 0.1% of AJE in diets could improve BWG and ADFI, dry matter digestibility in broilers.

Anti-Osteoporosis Effects of the Eleutherococcus senticosus, Achyranthes japonica, and Atractylodes japonica Mixed Extract Fermented with Nuruk.

Vigeo is a mixture of fermented extracts of Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK) manufactured using the traditional Korean nuruk fermentation method. Although the bioactive effects of ESM, AJN, and AJK have already been reported, the pharmacological effects of Vigeo have not been proven. Therefore, in this study, we investigated whether Vigeo had inhivitory effects on lipopolysaccharide (LPS)-induced inflammatory bone loss in vivo and receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclastogenesis and the related mechanism in vitro. Vigeo administration conferred effective protection against bone loss induced by excessive inflammatory response and activity of osteoclasts in LPS-induced inflammatory osteoporosis mouse model. In addition, Vigeo significantly suppressed the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by RANKL and inhibited F-actin formation and bone resorbing activity without any cytotoxicity. Moreover, Vigeo significantly inhibited RANKL-induced phosphorylation of p38, ERK, JNK, IκB, and AKT and degradation of IkB. Additionally, Vigeo strongly inhibited the mRNA and protein expression of c-FOS and NFATc1 and subsequently attenuated the expression of osteoclast specific marker genes induced by RANKL. We demonstrated for the first time the anti-osteoporosis effect of Vigeo, suggesting that it could be a potential therapeutic candidate for the treatment of osteoclast-mediated inflammatory bone diseases.

Bidentatide, a Novel Plant Peptide Derived from Achyranthes bidentata Blume: Isolation, Characterization, and Neuroprotection through Inhibition of NR2B-Containing NMDA Receptors.

Increasing attention is being focused on the use of polypeptide-based N-methyl-d-aspartate (NMDA) receptor antagonists for the treatment of nervous system disorders. In our study on Achyranthes bidentata Blume, we identified an NMDA receptor subtype 2B (NR2B) antagonist that exerts distinct neuroprotective actions. This antagonist is a 33 amino acid peptide, named bidentatide, which contains three disulfide bridges that form a cysteine knot motif. We determined the neuroactive potential of bidentatide by evaluating its in vitro effects against NMDA-mediated excitotoxicity. The results showed that pretreating primary cultured hippocampal neurons with bidentatide prevented NMDA-induced cell death and apoptosis via multiple mechanisms that involved intracellular Ca2+ inhibition, NMDA current inhibition, and apoptosis-related protein expression regulation. These mechanisms were all dependent on bidentatide-induced inhibitory regulation of NR2B-containing NMDA receptors; thus, bidentatide may contribute to the development of neuroprotective agents that would likely possess the high selectivity and safety profiles inherent in peptide drugs.

Structural characterization and in vitro osteogenic activity of ABPB-4, a heteropolysaccharide from the rhizome of Achyranthes bidentata.

Achyranthes bidentata is a species of flowering plant that is mainly distributed in China. The A. bidentata rhizome is a famous traditional Chinese medicine that has been widely used to treat lumbago, arthritis, and bone hyperplasia. In this work, A. bidentata rhizome was isolated and purified to obtain a pectic polysaccharide (ABPB-4). Chemical and spectral analyses showed that ABPB-4 had a main chain of →4)-α-d-GalpA-(1→ and →2,4)-α-l-Rhap-(1→, and the branch chains included →4)-ß-d-Galp-(1→, →6)-ß-d-Galp-(1→, →3,6)-ß-d-Galp-(1→, →5)-α-l-Araf-(1→ and →3,5)-α-l-Araf-(1→, and it was terminated with α-l-Araf-(1→ and ß-d-Galp-(1→. At concentrations of 0.01, 0.02, and 0.04 µmol/L, ABPB-4 significantly promotes the proliferation, differentiation, and mineralization of MC3T3-E1 cells in vitro, and it appreciably enhances the mRNA expression levels of osteogenic-related genes in these cells. Overall, the results reported herein indicate that ABPB-4 has outstanding osteogenic activity, and that it may be used as an anti-osteoporosis agent in the future.

Evaluation of oral toxicity and genotoxicity of Achyranthis Radix extract.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Achyranthes bidentata Blume, Achyranthis Radix (AR), is used as a traditional medicine ingredient in East Asia. It has anti-inflammatory, anti-oxidative, and anti-diabetic activities. AIM OF THE STUDY: In the present study, we aimed to evaluate the oral toxicity and genotoxicity of single-dose and 4-week repeated-doses of AR hot water extract (ARE), under the good laboratory practice principles. MATERIALS AND METHODS: For oral toxicity studies, SD rats (n = 5 per sex and group) were administered ARE at concentrations of 500, 1000, and 2000 mg/kg/day once (single dose) or once per day for 4 weeks (repeated dose). The non-clinical genotoxicity study consisted of bacterial reverse mutation using Escherichia coli (WP2 uvrA) and Salmonella typhimurium (TA98, TA100, TA1535, and TA1537), in vitro chromosomal aberration test with Chinese hamster lung cells (CHL/IU), and in vivo mouse bone marrow micronucleus test using bone marrow cells collected from male ICR mice (n = 5) that were orally administered ARE. RESULTS: In the single-dose oral toxicity study, mortality and treatment-related changes in body weight were not observed throughout the study, and the lethal dose was estimated to be > 2000 mg/kg in rats. In the 4-week repeated-dose oral toxicity study, ARE did not induce significant changes in body weight, organ weight, food intake, or hematological and serum biochemical parameters in any group. In the bacterial reverse mutation test, ARE did not induce gene mutations in any tested strain. In the chromosomal aberration test, ARE did not cause chromosomal aberrations. The micronucleus test showed no significant increase in the number of micronucleated polychromatic erythrocytes or the mean ratio of polychromatic to total erythrocytes. CONCLUSIONS: These results showed that ARE does not induce oral toxicity and genotoxicity in the in vivo and in vitro test systems.

Achyranthes aspera L. leaf extract induced anticancer effects on Dalton's Lymphoma via regulation of PKCα signaling pathway and mitochondrial apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Epidemiological studies promote the inclusion of natural-products in diet due to their inhibitory effects on various types of cancer. Among them, Achyranthes aspera L. (Family Amaranthaceae) is a medicinal plant in Ayurvedic pharmacopeia, found in India, Southeast Asia, America, and Sub-Saharan Africa. It is endowed with anti-inflammatory, anti-oxidant, and anti-cancer activities. However, its potential effect on Non-Hodgkin lymphomas (NHLs), has not yet been clarified. AIM OF THE STUDY: In the present study, we aimed to investigate the effect of Achyranthes aspera L. leaf extracts on highly aggressive murine NHL called Dalton's Lymphoma (DL) in vitro and in vivo. MATERIAL AND METHODS: GC-HRMS analysis was carried out for the identification of compounds present in A. aspera leaf extract. The cytotoxicity of various A. aspera leaf extracts was evaluated on DL cells by MTT assay. Chromatin condensation, nuclear fragmentation, and morphological changes were observed by microscopy technique. Flow cytometry was used to measure the changes in mitochondrial membrane potential (ΔΨm) and apoptosis. In addition, the expressions of apoptosis-related proteins were detected by western blotting. Meanwhile, the in vivo anti-tumor effect of leaf extract was tested in DL induced Balb/c mice. RESULT: GC-HRMS analysis of A. aspera methanolic leaf extract (AAML) revealed the presence of ten pharmacologically active compounds. The results showed that AAML suppressed cell proliferation, decreased mitochondrial membrane potential, changed the morphological structure, and induced apoptosis. Moreover, AAML could promote the release of cytochrome c by regulating Bcl-2 family proteins and then activated caspase-9/ -3 to triggered cell apoptosis. At the same time in DL cells treated with AAML, the protein kinase Cα (PKCα) pathway was inhibited in a concentration-dependent manner. Remarkably, in vivo, AAML mediated suppression of DL growth in Balb/c mice was accompanied by attenuation of the PKCα pathway and induction of apoptosis. Our result suggested that AAML promotes mitochondrial apoptotic cascade in DL cells by suppressing the PKCα signaling pathway. CONCLUSION: The study suggests that AAML could potently suppress DL progression by promoting apoptosis via mitochondrial-cascade and attenuation of the PKCα signaling pathway.

Deciphering the metabolic profile and pharmacological mechanisms of Achyranthes bidentata blume saponins using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry coupled with network pharmacology-based investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Achyranthes bidentata Blume (AB) is a traditional Chinese medicine (TCM) widely used as a dietary supplement and anti-arthritis drug. Pharmacological studies have shown that Achyranthes bidentata Blume saponins (ABS) are the main bioactive ingredient. However, the metabolic profile and mechanisms of action of ABS against rheumatic arthritis (RA) remain to be established. AIM OF THE STUDY: Our main objective was to investigate the metabolic profile and pharmacological activities of ABS against RA. MATERIALS AND METHODS: In this study, an analytical method based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) coupled with a metabolism platform was developed for metabolic profiling of ABS in rat liver microsomes and plasma. Then, the in vivo metabolites of ABS and their targets associated with RA were used to construct the network pharmacological analysis. Gene ontology (GO) enrichment, KEGG signaling pathway analyses and pathway network analyses were performed. The therapeutic effect of ABS on RA was further evaluated using an adjuvant arthritis (AA) model and network pharmacology results validated via Western blot. RESULTS: Overall, 26 and 21 metabolites of ABS were tentatively characterized in rat liver microsomes and plasma, respectively. The metabolic pathways of ABS mainly included M+O, M+O-H2, M+O2, and M+O2-H2. Data form network pharmacology analysis suggested that MAPK, apoptosis, PI3K-AKT and p53 signaling pathways contribute significantly to the therapeutic effects of ABS on RA. In pharmacodynamics experiments, ABS ameliorated the symptoms in AA rats in a dose-dependent manner and restored the homeostasis of pro/anti-inflammatory factors. Western blot results further demonstrated a significant ABS-induced decrease in phosphorylation of ERK in the MAPK pathway (P < 0.01). CONCLUSION: Application of an analytical method based on UPLC-QTOF/MS, network pharmacology and validation experiments offers novel insights into the components and mechanisms of ABS that contribute to its therapeutic effects against RA, providing useful directions for further research.

Achyranthes bidentata polypeptide k enhances the survival, growth and axonal regeneration of spinal cord motor neurons in vitro.

Achyranthes bidentata polypeptide k (ABPPk), a powerful active component from a traditional Chinese medicinal herb-Achyranthes bidentata Bl., has exhibited promising neuroprotective activity due to its multiple-targeting capability. However, the effect of ABPPk on the survival, growth and axonal regeneration of spinal cord motor neurons remains unclear. Here, a modified method, which is more optimized for embryonic cells in ambient carbon dioxide levels, was used for acquisition of rat embryonic spinal cord motor neurons with high survival and purity. ABPPk concentration-dependently enhanced the neuronal viability and promoted the neurite outgrowth. Co-culture of motor neurons and skeletal myocytes model indicated that ABPPk enhanced the neuromuscular junction development and maturation. A microfluidic axotomy model was further established for the axonal disconnection, and ABPPk significantly accelerated the axonal regeneration of motor neurons. Furthermore, we demonstrated that the upregulation of three neurofilament protein subunits in motor neurons might be relevant to the mechanisms of the growth-promoting effect of ABPPk. Our findings provide an experimental and theoretical basis for the development of ABPPk as a potential application in the development of treatment strategy for nerve injury diseases.

Isolation of triterpenoids and phytosterones from Achyranthes bidentata Bl. to treat breast cancer based on network pharmacology.

Breast cancer (BC) is a cancer with a high incidence and mortality of female, threatening women's physical and mental health. Achyranthes bidentata Blume, a traditional Chinese medicine, has been popular in folk due to its effective pharmacology activities including anti-inflammatory and anti-tumour effects. In order to identify the active ingredients from the roots of A. bidentata for treating breast cancer, the separation guided by network pharmacology analysis was employed which led to the isolation of 7 triterpenoids and 10 phytosterones. According to the in vitro experiments, the isolated compounds showed significant inhibitory activities on MCF-7 cells as well as anti-inflammatory activities by attenuating the production of NO and TNF-α in LPS-induced macrophage cells. In conclusion, this study successfully predicted and confirmed the positive impact of triterpenoids and phytosterones on breast cancer, which provided a foundation for further in vivo studies.