Panaxadiol carbamate derivatives: Synthesis and biological evaluation as potential multifunctional anti-Alzheimer agents.

It is reported that panaxadiol has neuroprotective effects. Previous studies have found that compound with carbamate structure introduced at the 3-OH position of 20 (R) -panaxadiol showed the most effective neuroprotective activity with an EC50 of 13.17 µM. Therefore, we designed and synthesized a series of ginseng diol carbamate derivatives with ginseng diol as the lead compound, and tested their anti-AD activity. It was found that the protective effect of compound Q4 on adrenal pheochromocytoma was 80.6 ±â€¯10.85 % (15 µM), and the EC50 was 4.32 µM. According to the ELISA results, Q4 reduced the expression of Aß25-35 by decreasing ß-secretase production. Molecular docking studies revealed that the binding affinity of Q4 to ß-secretase was -49.67 kcal/mol, indicating a strong binding affinity of Q4 to ß-secretase. Western blotting showed that compound Q4 decreased IL-1ß levels, which may contribute to its anti-inflammatory effect. Furthermore, compound Q4 exhibits anti-AD activities by reducing abnormal phosphorylation of tau protein and activation of the mitogen activated protein kinase pathway. The learning and memory deficits in mice treated with Q4in vivo were significantly alleviated. Therefore, Q4 may be a promising multifunctional drug for the treatment of AD, providing a new way for anti-AD drugs.

Design, synthesis, and hypoxia-inducible factor-1α inhibitory activity evaluation of panaxadiol derivatives containing a thiazole moiety.

The hypoxia-inducible factor-1α (HIF-1α) pathway has been implicated in tumor angiogenesis, growth, and metastasis. Therefore, the inhibition of this pathway is an important therapeutic target for cancer. Here, three series of panaxadiol derivatives containing a thiazole moiety (5a-l, 7a-i, and 9a-i) were synthesized and evaluated for HIF-1α inhibitory activity using a Hep3B cell-based luciferase reporter assay. Compounds 5d and 7b showed the strongest inhibitory activity with IC50 values of 17.37 and 6.42 µM, respectively, and did not show any significant cytotoxicity. Western blot assay results indicated that these two compounds exhibited more potent inhibition, compared with panaxadiol, of the expression of HIF-1α protein in Hep3B cells at a concentration of 50 µM. Molecular docking experiments were also performed to investigate the structure-activity relationship.

Ginseng-derived panaxadiol ameliorates STZ-induced type 1 diabetes through inhibiting RORγ/IL-17A axis.

Retinoic-acid-receptor-related orphan receptor γ (RORγ) is a major transcription factor for proinflammatory IL-17A production. Here, we revealed that the RORγ deficiency protects mice from STZ-induced Type 1 diabetes (T1D) through inhibiting IL-17A production, leading to improved pancreatic islet ß cell function, thereby uncovering a potential novel therapeutic target for treating T1D. We further identified a novel RORγ inverse agonist, ginseng-derived panaxadiol, which selectively inhibits RORγ transcriptional activity with a distinct cofactor recruitment profile from known RORγ ligands. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for panaxadiol in the RORγ ligand-binding pocket. Despite its inverse agonist activity, panaxadiol induced the C-terminal AF-2 helix of RORγ to adopt a canonical active conformation. Interestingly, panaxadiol ameliorates mice from STZ-induced T1D through inhibiting IL-17A production in a RORγ-dependent manner. This study demonstrates a novel regulatory function of RORγ with linkage of the IL-17A pathway in pancreatic ß cells, and provides a valuable molecule for further investigating RORγ functions in treating T1D.

Synthesis and Antitumor Activity of Panaxadiol Pyrazole and Isooxazole Derivatives.

In this study, we designed and synthesized 19 nitrogen-containing heterocyclic derivatives of panaxadiol (PD). We first reported the antiproliferative activity of these compounds against four different tumor cells. The results of the MTT assay showed that the PD pyrazole derivative (compound 12b) had the best antitumor activity and could significantly inhibit the proliferation of four tested tumor cells. For A549 cells, the IC50 value was as low as 13.44±1.23 µM. Western blot analysis showed that the PD pyrazole derivative was a bifunctional regulator. On the one hand, it can down-regulate the expression of HIF-1α by acting on PI3 K/AKT signaling pathway in A549 cells. On the other hand, it can induce the decrease of CDKs protein family and E2F1 protein expression levels, thus playing a crucial role in cell cycle arrest. According to the results of molecular docking, we found that multiple hydrogen bonds were formed between the PD pyrazole derivative and two related proteins, and the docking score of the derivative was also significantly higher than that of the crude drug. In summary, the study of the PD pyrazole derivative laid a foundation for the development of ginsenoside as an antitumor agent.

Novel ginsenoside derivatives induce apoptosis in HepG-2 cells via the MDM2-p53 signaling pathway.

In this study, 6-aryl-5-cyanopyrimidines and quinazolinones were introduced into panaxadiol (PD) to synthesize 25 new panaxadiol derivatives. The cytotoxicity of these compounds against three cancer cells and one normal cell was examined by methylthiazoletetrazolium (MTT) cytotoxicity experiment. The findings demonstrated that PD cyanopyrimidine derivatives have superior anti-proliferative effects over quinazoline derivatives. Among them, PM14 had the strongest inhibitory activity on the proliferation of human hepatoma cell lines (HepG-2), the IC50 value was 2.13 ± 0.20 µM. 4',6-diamidino-2-phenyl-indole (DAPI) staining showed that PM14 made HepG-2 nucleus shrink and had obvious apoptosis. Mechanistic studies confirmed that the derivative PM14 activates p53 signaling pathway by reducing the expression of MDM2 protein, and further induces an increase in the intracellular Bax/Bcl-2 ratio, down-regulated the expression of Caspase-3, up-regulated Cl-Caspase-3 expression, eventually leading to cell apoptosis. This lays the foundation for subsequent development of derivatives with stronger anti-proliferative activity.

Design, Synthesis and Cytotoxicity Evaluation of Novel Indole Derivatives of Panaxadiol.

Based on the well-known cytotoxicity of indole compounds, we used the 'Fisher indole synthesis' method to introduce appropriately substituted indole rings into panaxadiol (PD), generating eighteen novel Panaxadiol indole derivatives. Six human cancer cell lines (A549, HepG-2, HCT-116, SGC-7901, MDA-MB-231, PC-3 cells) and one normal ovarian cell lines (IOSE144) were designed to evaluate the anti-proliferative activity of the PD derivatives. The results showed that the majority of PD derivatives showed enhanced anti-proliferative activity, when compared with PD, with P-Methylindolo-PD exhibiting the highest cytotoxicity. In A549 cells, IC50 value was 5.01±0.87 µM, which is roughly 12 times higher than the activity of PD and 5 times that of 5-FU. Moreover, cell morphology analysis and Annexin V-FITC/PI assays exhibited that P-Methylindolo-PD could induce A549 cell apoptosis (55.7 % of apoptotic cells at 20 µM). Moreover, molecular docking experiments were performed to explore the molecular mechanism underlining the binding of P-Methylindolo-PD to the active site of EGFR. The results support that P-Methylindolo-PD might be a promising lead compound for further studies.

(20R)-Panaxadiol as a Natural Active Component with Anti-Obesity Effects on ob/ob Mice via Modulating the Gut Microbiota.

Obesity is an important cause of diseases such as type 2 diabetes, non-alcoholic fatty liver and atherosclerosis. The use of ingredients extracted from traditional Chinese medicine for weight loss is now receiving more and more attention. Ginseng has been recorded since ancient times for the treatment of diabetes. The (20R)-Panaxadiol (PD) belongs to the ginseng diol type compounds, which are moderately bioavailable and may remain in the intestinal tract for a longer period of time. This study investigated the potential positive effect of PD in ob/ob mice and evaluated its effect against obesity. The ob/ob mice were administered PD for ten weeks. Our study showed that PD could improve obesity, glucose tolerance disorder, as well as gut dysbiosis. Panaxadiol decreased ob/ob mice's Firmicutes/Bacteroidetes (F/B). Furthermore, 16S rRNA gene sequencing of the fecal microbiota suggested that PD changed the composition of the gut microbiota in ob/ob mice and modulated specific bacteria such as lactobacillus, prevotellace and so on. Moreover, PD improved the intestinal wall integrity. In conclusion, our results suggest that (20R)-Panaxadiol, as an active ingredient of the traditional Chinese medicinal herb ginseng, may improve obesity to some extent via improving gut microbiota.

Panaxadiol as a major metabolite of AD-1 can significantly inhibit the proliferation and migration of breast cancer cells: In vitro and in vivo study.

Previous studies have shown that 20 (R)-25-methoxyl-dammarane-3ß, 12ß, 20 triol (AD-1) can inhibit various cancer cell lines. This study aimed to explore the effect and mechanism of AD-1 metabolite M2 (Panaxadiol; PD) on breast cancer cells of nude mice. Five AD-1 metabolites were isolated and identified using various chromatographic techniques. PD was the main component. In vitro results showed that PD could inhibit the proliferation and migration of MDA-MB-231 cells by inducing G1-phase arrest. In addition, PD down-regulated the expression of Cyclin D1, cdk2, cdk4, cdk6, P-p38, and MMP9, and up-regulated p21 and p27. In vivo results showed that PD could effectively reduce the volume, weight, and migration of breast cancer Transcriptomics analyzed 491 differentially expressed genes by GO and KEGG enrichment. RT-PCR verification confirmed that the significant down-regulation of MMP9 was consistent with transcriptomics results. In further research showed that PD regulated the protein expression of P-p38 and MMP9 in MAPK pathway. In summary, in vivo and in vitro studies showed that PD significantly inhibit the occurrence and development of breast cancer, possibly through the MAPK pathway.

Synthesis and evaluation of HIF-1α inhibitory activities of novel panaxadiol derivatives.

Hypoxia-inducible factor 1α (HIF-1α) is a known regulator of tumor cell proliferation, migration, and angiogenesis. The presence of a high concentration of HIF-1α is positively correlated with the severity of cancer. Therefore, the inhibition of this pathway represents an important therapeutic target for the treatment of various types of cancer. Here, we designed and synthesized 30 panaxadiol (PD) derivatives and evaluated their inhibitory activities against HIF-1α transcription. Of these, compound 3l exhibited the most promising inhibitory activity (IC50 = 3.7 µM) and showed significantly decreased cytotoxicity compared with PD. Compound 9e exhibited the strongest cytotoxic effect and may be considered for further preclinical development.

Novel panaxadiol triazole derivatives induce apoptosis in HepG-2 cells through the mitochondrial pathway.

In this study, we introduced 1, 2, 4-triazole groups into panaxadiol (PD) to obtain 18 panaxadiol triazole derivatives. Five cancer cells and one normal cell were evaluated for cytotoxicity by MTT assay. The results showed that most of the derivatives could inhibit cancer cell proliferation, and the anti-proliferative activity of compound A1 was the most significant. For HepG-2 cells, the IC50 value was 4.21 ± 0.54 µM, which was nearly 15 times higher than the activity of PD. Further studies showed that compound A1 could induce apoptosis in HepG-2 cells, and could enhance the expression of Cl-caspase-3, Cl-caspase-9 and Cl-PARP. Moreover, Western blot analysis showed that after treating HepG-2 cells with compound A1, the expression of p53 protein was increased and the ratio of Bax/Bcl-2 was gradually increased. The cytoplasmic Bax is then translocated to the mitochondria, causing the release of Cyt c protein. Therefore, the results indicate that compound A1 induces apoptosis through the mitochondrial pathway and can be used the potential to develop new anti-proliferative agents.

In vivo pharmacokinetics, distribution, and excretion of an anticancer agent isolated from red ginseng, in rat.

The compound 20(S),25-epoxydammarane-3ß,12ß,24α-triol (24-hydroxy-panaxadiol or 24-OH-PD), isolated from the red Panax ginseng CA Meyer possesses anticancer activity. Our aim was to study the pharmacokinetic characteristics of 24-OH-PD, which is essential for pre-clinical research during the development of new drugs. In this study, a simple and sensitive ultra-performance liquid chromatography-mass spectrometry (LC-MS/MS) method was established and used for studying the pharmacokinetics, in vitro protein binding, tissue distribution, and elimination profiles of 24-OH-PD in rats. 24-OH-PD was characterized by linear pharmacokinetics in the dose range of 2.5-10 mg/kg and had relatively longer half-life (4.82-5.45 h) than the other ginsenosides. It had a wide tissue distribution profile in rats and was primarily distributed in the lung. Within 96 h of intravenous administration, 13.84% of 24-OH-PD was excreted out via feces and 0.02% via urine in its unchanged form. In conclusion, a simple LC-MS/MS method with high sensitivity and selectivity was established for the quantification of 24-OH-PD.

Synthesis and Cytotoxicity Evaluation of Panaxadiol Derivatives.

In this study, 13 panaxadiol (PD) derivatives were synthesized via reactions with aromatic compounds and amino acids. Following this, the cytotoxicity of these compounds was evaluated against four cancer cell lines (human hepatoma cells HepG-2, human lung cancer cells A549, human breast cancer cells MCF-7, and human colon cancer cells HCT-116) and one normal cell lines (human gastric epithelial cells GES-1). The results showed that the panaxadiol derivatives 3, 12, and 13 showed significant inhibition of cellular proliferation against cancer cells compared with PD, and the panaxadiol derivative 12 had the lowest IC50 value for A549 (IC50 =18.91±1.03 µm). For MCF-7 cells, most compounds exhibited good inhibition of cellular proliferation, and the panaxadiol derivative 13 showed the strongest inhibitory effect (IC50 =8.62±0.23 µm), which significantly increased the cytotoxicity of PD and was stronger than the positive control (mitomycin). For normal cells, all compounds exhibited low or no toxic effects; thus, these derivatives can be used to develop novel antiproliferative agents.

Esterified Derivatives of Panaxadiol and Their Inhibitory Effect on HL-60, THP-1, and PC-3 Cell Lines.

Panaxadiol is a dammarane-type ginsenoside having high ginseng content. The 3-hydroxy group of panaxadiol (PD) was modified by fatty acids and diacids. The modified panax glycol had enhanced anticancer activity. Twelve PD derivatives were evaluated and purified by chemical synthesis, column chromatography, co-synthesis, and identification. The human leukemia cells THP-1, HL-60, and human prostate cancer cell lines PC-3 were evaluated; PD derivatives were tested and evaluated in vitro by MTT assay. The results showed that the antitumor activities of some derivatives on three tumor cell lines were better than those of PD.

Metabonomics Approach To Comparing the Antistress Effects of Four Panax ginseng Components in Rats.

Different components of Panax ginseng have different properties and medicinal effects. Metabonomics was a prospective approach to analyze the global response of endogenous metabolites to physiological and pathological processes. In this study, an untargeted metabonomics method using GC/TOFMS combined with multivariate statistical techniques was applied to compare entire metabolite differences and the antistress variations among four components of P. ginseng, namely, total ginsenosides (TG), panaxadiol (PD), panaxatriol (PT), and ginseng polysaccharide (PS), in Wistar rats. The results of metabolite analysis showed that numerous urine metabolites involving neurotransmitters, amino acids, organic acids, and gut microbiota metabolites were changed after administration of the four components of P. ginseng, with TG having the least impact on urinary metabolites. The urinary metabolite profiling of these rats exposed to acute combined stress (forced swimming and behavior restriction) demonstrated that the four ginseng components attenuated urine metabolite changes involving gut microbiota metabolites, tricarboxylic acid (TCA) cycle and energy metabolites, and organic acids to different degrees, with TG improving most of the metabolites altered by stress.

(R)-panaxadiol by whole cells of filamentous fungus Absidia coerulea AS 3.3382.

The microbial transformation of 20(R)-panaxadiol (PD) by the fungus Absidia coerulea AS 3.3382 afforded three new and three known metabolites. The structures of the metabolites were characterized as 3-oxo-20(R)-panaxadiol (1), 3-oxo-7ß- hydroxyl-20(R)-panaxadiol (2), 3-oxo-22ß-hydroxyl-20(R)-panaxadiol (3), 3-oxo- 7ß,22ß-dihydroxyl-20(R)-panaxadiol (4), 3-oxo-7ß,24ß-dihydroxyl-20(R)-panaxadiol (5), and 3-oxo-7ß,24α-dihydroxyl-20(R)-panaxadiol (6). Among them, 2-4 were new compounds. In addition, compounds 3 and 4 exhibited significant anti-hepatic fibrosis activity.

Synthesis and anti-tumor evaluation of panaxadiol halogen-derivatives.

In the current work, 13 novel panaxadiol (PD) derivatives were synthesized by reacting with chloroacetyl chloride and bromoacetyl bromide. Their in vitro antitumor activities were evaluated on three human tumor cell lines (HCT-116, BGC-823, SW-480) and three normal cells (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2) by MTT assay. Compared with PD, the results demonstrated that compound 1e, 2d, 2e showed significant anti-tumor activity against three tumor cell lines, the IC50 value of compound 2d against HCT-116 was the lowest (3.836µM). The anti-tumor activity of open-ring compounds are significantly better than the compounds of C-25 cyclization. Compound 1f, 2f, 2g showed the strong anti-tumor activity. The IC50 value of compound 2g against BGC-823 and SW-480 were the lowest (0.6µM and 0.1µM, respectively). Combined with cytotoxicity test, the IC50 value of compound 1e, 2d, 2e are greater than 100. the open-ring compounds (1f, 2f, 2g) showed a strong toxicity. The toxicity of 1f is lower than 2f and 2g. These compounds may be useful for the development of novel antiproliferative agents.

Synthesis and biological evaluation of panaxadiol ester derivatives possessing pyrazole and pyrrole moiety as HIF-1α inibitors.

Hypoxia-inducing factor-1α (HIF-1α) is overexpressed in variety of tumor patients and plays an important role in the regulation of hypoxia response in tumor cells. Therefore, its inhibitors have become one of the targets for the treatment of a variety of cancers. Two series of panaxadiol (PD) ester derivatives containing pyrazole (18a-j) and pyrrole (19a-n) moiety were synthesized and their HIF-1α inhibitory activities were evaluated. Among all the target compouds, compounds 18c, 19d, and 19n (IC50 = 8.70-10.44 µM) showed better HIF-1α inhibitory activity than PD (IC50 = 13.35 µM). None of these compounds showed cytotoxicity above 100 µM and inhibited HIF-1α transcription in a dose-dependent manner. These compounds showed good antitumor activity and provide lead compounds for further design and activity study of PD ester derivatives.

Inhalation of panaxadiol alleviates lung inflammation via inhibiting TNFA/TNFAR and IL7/IL7R signaling between macrophages and epithelial cells.

Background: Lung inflammation occurs in many lung diseases, but has limited effective therapeutics. Ginseng and its derivatives have anti-inflammatory effects, but their unstable physicochemical and metabolic properties hinder their application in the treatment. Panaxadiol (PD) is a stable saponin among ginsenosides. Inhalation administration may solve these issues, and the specific mechanism of action needs to be studied. Methods: A mouse model of lung inflammation induced by lipopolysaccharide (LPS), an in vitro macrophage inflammation model, and a coculture model of epithelial cells and macrophages were used to study the effects and mechanisms of inhalation delivery of PD. Pathology and molecular assessments were used to evaluate efficacy. Transcriptome sequencing was used to screen the mechanism and target. Finally, the efficacy and mechanism were verified in a human BALF cell model. Results: Inhaled PD reduced LPS-induced lung inflammation in mice in a dose-dependent manner, including inflammatory cell infiltration, lung tissue pathology, and inflammatory factor expression. Meanwhile, the dose of inhalation was much lower than that of intragastric administration under the same therapeutic effect, which may be related to its higher bioavailability and superior pharmacokinetic parameters. Using transcriptome analysis and verification by a coculture model of macrophage and epithelial cells, we found that PD may act by inhibiting TNFA/TNFAR and IL7/IL7R signaling to reduce macrophage inflammatory factor-induced epithelial apoptosis and promote proliferation. Conclusion: PD inhalation alleviates lung inflammation and pathology by inhibiting TNFA/TNFAR and IL7/IL7R signaling between macrophages and epithelial cells. PD may be a novel drug for the clinical treatment of lung inflammation.

Panaxadiol saponin ameliorates ferroptosis in iron-overload aplastic anemia mice and Meg-01 cells by activating Nrf2/HO-1 and PI3K/AKT/mTOR signaling pathway.

Panaxadiol saponin (PND) is a latent targeted drug for the treatment of aplastic anemia (AA). In this study, we examined the effects of PND on ferroptosis in iron-overload AA and Meg-01 cells. We utilized RNA-seq to analyze differentially expressed genes in iron-induced Meg-01 cells treated with PND. The effects of PND or combined with deferasirox (DFS) on iron deposition, labile iron pool (LIP), several ferroptosis events, apoptosis, mitochondrial structure, as well as ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR pathway-related markers in iron-induced Meg-01 cells were examined by Prussian-blue staining, flow cytometer, ELISA, Hoechst 33342 staining, transmission electron microscope, and Western blot assays, respectively. Additionally, an AA mice model with iron overload was established. Then, the blood routine was assessed, and the number of bone marrow-derived mononuclear cells (BMMNCs) in mice was counted. Also, serum iron, ferroptosis events, apoptosis, histology, T lymphocyte percentage, ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR signaling-related targets in primary megakaryocytes of AA mice with iron overload were assessed by commercial kits, TUNEL staining, hematoxylin and eosin (H&E) staining, Prussian blue staining, flow cytometer, and qRT-PCR analysis, respectively. PND suppressed iron-triggered iron overload, and apoptosis, and ameliorated mitochondrial morphology in Meg-01 cells. Importantly, PND ameliorated ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR signaling-related marker expressions in iron-induced Meg-01 cells or primary megakaryocytes of AA mice with iron overload. Moreover, PND ameliorated body weight, peripheral blood cell counts, the number of BMMNCs, and histological injury in the iron-overload AA mice. Also, PND improved the percentage of T lymphocytes in the iron-overload AA mice. PND attenuates ferroptosis against iron-overload AA mice and Meg-01 cells via activating Nrf2/HO-1 and PI3K/AKT/mTOR pathway and is a promising novel therapeutic candidate for AA.

(20R)-panaxadiol improves obesity by promoting white fat beigeing.

Introduction: Obesity is an important cause of a range of metabolic diseases. However, the complex mechanisms of obesity and its related diseases make some weight loss methods ineffective or have safety issues. Ginseng, a specialty of Jilin Province in China with both edible and medicinal value, contains mainly ginsenosides and other components. In order to study the anti-obesity effect of ginseng, network pharmacology was used to predict and screen the active ingredients, action targets and signaling pathways of ginseng. We found (20R)-panaxadiol (PD) is a more desirable active ingredient due to its high drug-like properties and high bioavailability. Moreover, it is closely related to cAMP pathway which is more important in metabolism regulation. The corresponding pharmacodynamic targets of PD include ADRB2 (the gene encoding the ß2-adrenoceptor receptor). Our study aimed to investigate whether Panaxadiol can promote white adipocyte beigeing and increase thermogenesis through modulating the ß2/cAMP pathway to exert anti-obesity effects. Methods: In vivo, we established high-fat feeding obesity model, genotypically obese mice (ob/ob) model, and administered PD (10 mg/kg). PD treatment in ob/ob mice along with ß2 receptor inhibitor ICI118551. In vitro, differentiated mature 3T3-L1 cells were given palmitate (PA) to induce hypertrophy model along with PD (20 µM). Results: The results of this study demonstrated that PD significantly reduced body weight, improved glucose tolerance and lipid levels in high-fat-induced obese mice and ob/ob mice, and also reduced lipid droplet size in PA-treated hypertrophic adipocytes in vitro. Molecular biology assays confirmed that cAMP response element binding protein (CREB) phosphorylation was increased after PD administration, and the expression of thermogenesis-related proteins UCP1, PRDM16 and mitochondrial biosynthesis-related proteins PGC-1α, TFAM and NRF1 were increased. Molecular docking results showed a low binding energy between ß2 receptors and PD, indicating an affinity between the ß2 receptor and PD. In addition, the ß2 receptor inhibition, reversed the anti-obesity effect of PD on the body weight, lipid droplets, the expression of thermogenesis-related proteins and CREB phosphorylation in ob/ob mice. Discussion: These results suggest that PD may promote the expression of thermogenic proteins through phosphorylation of CREB via ß2 receptor activation, and thus exert anti-obesity effects.