Root Bark Of Pseudolarix Kaempferi Research Articles

Pseudolaric acid B induces apoptosis associated with the mitochondrial and PI3K/AKT/mTOR pathways in triple‑negative breast cancer.

Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarixkaempferi, has been shown to exert strong antitumor properties. The aim of the present study was to investigate the mechanisms underlying the proposed antitumor properties of PAB in the triple‑negative breast cancer cells, MDA‑MB‑231. The cell processes evaluated included cell proliferation by Cell Counting Kit‑8 assay, colony formation and EdU assay, apoptosis by Annexin V‑FITC/PI apoptosis assay, cell migration by Transwell migration assay and invasion by Transwell invasion assay. PAB significantly inhibited the proliferation of MDA‑MB‑231 cells through a mechanism that was considered to be associated with cell cycle arrest at the G2/M phase. There was decreased protein expression levels of CDK1 and cyclin B1 and increased protein expression levels of p53 and p21. However, there were no well‑defined inhibitory effects on the normal breast cell line MCF10A. PAB also triggered apoptosis in a concentration‑dependent manner through the mitochondrial apoptosis pathway. It caused collapse of mitochondrial membrane potential, accumulation of reactive oxygen species and release of cytochrome c, as well as upregulation of cleaved caspase‑3, cleaved caspase‑9, cleaved PARP and Bax, and downregulation of Bcl‑2 and Bcl‑xl. The migration and invasion ability of MDA‑MB‑231 cells were inhibited by decreasing the expression levels of the epithelial‑mesenchymal transition‑related markers N‑cadherin and vimentin and increasing the expression of E‑cadherin. Moreover, the expression levels of PI3K (p110β), phosphorylated (p)‑AKT (ser473) and p‑mTOR (ser2448) were downregulated and LY294002, a PI3K inhibitor, could interact additively with PAB to induce apoptosis of MDA‑MB‑231 cells. Overall, the present results demonstrated that PAB induced apoptosis via mitochondrial apoptosis and the PI3K/AKT/mTOR pathway in triple‑negative breast cancer. It also inhibited cellular proliferation, migration and invasion, suggesting that PAB may be a useful phytomedicine for the treatment of triple‑negative breast cancer.

Pseudolaric acid B triggers cell apoptosis by activating AMPK/JNK/DRP1/mitochondrial fission pathway in hepatocellular carcinoma

Pseudolaric acid B (PAB), a natural product isolated from the root bark of Pseudolarix kaempferi, has been reported to exert inhibitory effects in various cancers. However, the underlying mechanisms remain largely unclear. In the present study, we investigated the mechanism through which PAB exert its anticancer effects in hepatocellular carcinoma (HCC). PAB inhibited the viability of and induced apoptosis in Hepa1–6 cells in a dose-dependent manner. It disrupted mitochondrial membrane potential (MMP) and impaired ATP production. Furthermore, PAB induced phosphorylation of DRP1 at Ser616 and mitochondrial fission. Blocking DRP1 phosphorylation by Mdivi-1 inhibited mitochondrial fission and PAB-induced apoptosis. Moreover, c-Jun N-terminal kinase (JNK) was activated by PAB, and blocking JNK activity using SP600125 inhibited PAB-induced mitochondrial fission and cell apoptosis. Furthermore, PAB activated AMP-activated protein kinase (AMPK), and inhibiting AMPK by compound C attenuated PAB-stimulated JNK activation and blocked DRP1-dependent mitochondrial fission and apoptosis. Our in vivo data confirmed that PAB inhibited tumor growth and induced apoptosis in an HCC syngeneic mouse model by inducing the AMPK/JNK/DRP1/mitochondrial fission signaling pathway. Furthermore, a combination of PAB and sorafenib showed a synergistic effect in inhibiting tumor growth in vivo. Taken together, our findings highlight a potential therapeutic strategy for HCC.

HPLC analysis of bioactive diterpenoids from the root bark of Pseudolarix kaempferi

The root bark of Pseudolarix kaempferi, a famous traditional Chinese medicine, has been commonly used in China to treat skin diseases caused by fungal infections. To evaluate its pharmatherapeutic quality, a sensitive, simple and accurate high-performance liquid chromatography (HPLC) method was developed for the quantitative analysis of three major bioactive diterpenoids, pseudolaric acids A-C in P. kaempferi. A Zorbax XDB RP-C18 (4.6 mm × 250 mm, 5 μm) was used in this HPLC system, with UV detection at 260 nm. A gradient elution was effected by a mobile phase composed of acetonitrile and 1% aqueous acetic acid, where acetonitrile concentration was changed from 30% to 60% in 30 min. These three constituents were well resolved by this method with good linear relationships in the range of 0.982-98.2 μg /mL for pseudolaric acid A, 0.618-61.8 μg/mL for pseudolaric acid B, and 1.052-105.2 μg/mL for pseudolaric acid C, respectively. The correlation coefficients of the calibration curves were all over 0.999. In addition, these three compounds in six batches of the root barks of P. kaempferi were quantitatively determined to evaluate the effectiveness of this method.

Pseudolaric acid B exhibits anti-cancer activity on human hepatocellular carcinoma through inhibition of multiple carcinogenic signaling pathways

BackgroundPseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi, exhibits a potent anti-cancer activity in a variety of tumor cells. PurposeThe present study was designed to evaluate the anti-cancer effects of PAB on hepatocellular carcinoma (HCC) cell lines in vitro, and to explore the underlying mechanism. MethodsThe anti-proliferative activity of PAB on HCC cells were assessed via sulforhodamine B staining, colony formation, cell cycle analysis, respectively. Apoptosis was detected using Annexin V/propidium iodide double staining and diamidino-phenyl-indole staining, respectively. Protein expression regulated by PAB treatment was tested by western blotting. ResultsThe present results showed that PAB significantly inhibited the proliferation of HepG2, SK-Hep-1, and Huh-7 HCC cell lines in vitro with IC50 values of 1.58, 1.90, and 2.06 μM, respectively. Furthermore, PAB treatment repressed the colony formation in HepG2, SK-Hep-1, and Huh-7 HCC cell lines. Flow cytometry analysis revealed that PAB caused an obvious cell cycle arrest in G2/M phase and induced apoptosis with the induction of p21, Bax, cleaved-caspase-3, and cleaved-PARP in human HepG2 and SK-Hep-1 cells. Mechanistically, PAB treatment down-regulated the phosphorylation of STAT3, ERK1/2, and Akt. Moreover, abnormal GSK-3β/β-catenin signaling in HepG2 cells was remarkably suppressed by PAB treatment. Finally, proliferation markers including cyclin D1 and c-Myc, and anti-apoptosis proteins such as Bcl-2 and survivin were also down-regulated by PAB treatment in HepG2 cells. ConclusionTaken together, our results suggest that PAB exerts anti-cancer activity in HCC cells through inhibition of STAT3, ERK1/2, Akt, and GSK-3β/β-catenin carcinogenic signaling pathways, and may be used as a phytomedicine in the treatment of HCC.

Synthetic studies on pseudolaric acid B: Construction of the trans-fused [5–7] ring system via Dieckmann condensation

Pseudolaric acids A (1) and B (2) are diterpene acids isolated from the root bark of Pseudolarix kaempferi as the main biologically active constituents of tujinpi, a traditional Chinese medicinal herb. Studies toward the total synthesis of 2 are described. The unusual trans-fused [5–7] ring system in the core structure of 2 was successfully synthesized via Dieckmann condensation.

Pseudolaric acid B inhibits gastric cancer cell metastasis in vitro and in haematogenous dissemination model through PI3K/AKT, ERK1/2 and mitochondria-mediated apoptosis pathways

Pseudolaric acid B (PAB) is the major bioactive constituent in the root bark of Pseudolarix kaempferi and has been reported to have cytotoxicity against tumor cells. Our in vivo experiments showed that PAB could inhibit gastric cancer cell lung metastasis in a nude mouse haematogenous dissemination model. To evaluate the anti-metastasis mechanism of PAB in gastric cancer cells, cytological experiments were performed. The results showed that PAB could inhibit the adhesion ability to matrigel, migration, invasion and colony formation ability of BGC-823 and MKN-45 cells. Western blot further confirmed that the inhibitory effects of PAB on anti-metastasis may involve regulating the expression of the metastasis-related proteins MMP-9, HIF-1α, VEGF, VEGFR2, E-Cadherin and Ezrin. We obtained further proof that PAB which could be used as a multi-targeted agent to inhibit the PI3K/AKT, ERK1/2 and mitochondria-mediated apoptosis pathways and consequently suppress tumor growth and metastasis. Our experiments suggest that PAB-induced effects may have novel therapeutic applications for the treatment of gastric cancer.

New sesquiterpene polyol esters from the root bark of Pseudolarix kaempferi

A series of sesquiterpene polyol esters, including two new β-dihydroagarofuran sesquiterpenes (1-2) and 14 known compounds (3-16), were isolated from the root bark of Pseudolarix kaempferi. The esters⿿ structures were elucidated by employing extensive spectroscopic analyses and by comparison to literature compounds. The absolute configurations of the two new compounds were determined by the CD exciton chirality method. The known compounds 3-16 were isolated from the Pseudolarix genus for the first time.

New eudesmane-type sesquiterpenoids from the root bark of Pseudolarix kaempferi

The phytochemical investigation of the root bark of Pseudolarix kaempferi yielded eight eudesmane-type sesquiterpenoids, including three new ones, 1α-hydroxyl-4(14)-en-β-dihydroagarafuran (1), 1α, 2α-diacetoxy-8β-isobutanoyloxy-9α-benzoyl oxy-15-β-(β-furancarbonyloxy)-4β, 6β-dihydroxy-β-dihydroagarofuran (7), and 1α-acetoxy-2 α-furancarbonyloxy-8β-isobutanoyloxy-9α-benzoyloxy-15β-(β-acetoxy)-4β, 6β-dihydroxy-β-dihydroagarofuran (8). Herein the new compounds 7 and 8 were reported as a mixture. The molecular structures of the isolated compounds were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, NMR, and MS, and comparison with the literature data.

Development and validation of LC–MS/MS method for quantification of pseudolaric acid B from the root bark of Pseudolarix kaempferi in rat plasma: Application to a pharmacokinetic study

Pseudolaric acid B (PAB), which is the main biologically active diterpene acid of Pseudolarix kaempferi, has presented anti-fungal, anti-tumor, anti-fertility, and anti-tubulin activities. In this study, a sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) method with multiple-reaction monitoring mode was employed for quantification of PAB in rat plasma. The calibration curve was linear over the range of 0.86–288ng/mL with correlation coefficient (r) greater than 0.995 for PAB, and the lower limit of quantification was 0.86ng/mL in rat plasma. The accuracy of PAB was between −9.1% and 7.0% relative error, and precision ranged from 1.2 to 10.6% relative standard deviation. This method was successfully applied to the pharmacokinetic studies of PAB in Sprague-Dawley rats. After single intravenous administration of 2.0, 4.0, and 8.0mg/kg PAB to rats, the t1/2 were (16.1±5.6), (30.0±13.7), and (27.4±5.3)min, respectively. The pharmacokinetics (C2min, AUC) of PAB within the used dosage range were in accordance with linear pharmacokinetic characteristics.

Pseudoferic acids A–C, three novel triterpenoids from the root bark of Pseudolarix kaempferi

Three novel triterpenoids, pseudoferic acids A and B (1 and 2) possessed a unique 16,24-cyclo-26-norlanostane skeleton, and pseudoferic acid C (3) featured a cis-fused D/E-ring system, were isolated from the root bark of Pseudolarix kaempferi. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compounds 2 and 3 exhibited weak inhibitory activities against 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1) with IC50 values of 0.44 (mouse 11β-HSD1) and 0.75μM (human 11β-HSD1), respectively.

Pseudolaric Acid B, a Novel Microtubule-Destabilizing Agent That Circumvents Multidrug Resistance Phenotype and Exhibits Antitumor Activity In vivo

Pseudolaric acid B (PAB) is the major bioactive constituent in the root bark of Pseudolarix kaempferi that has been used as an antifungal remedy in traditional Chinese medicine. Previous studies showed that PAB exhibited substantial cytotoxicity. The aims of this study were to elucidate the molecular target of PAB, to examine its mechanism of action, and to evaluate the efficacy of this compound in vivo. The effect of PAB on cell growth inhibition toward a panel of cancer cell lines was assayed. Cell cycle analysis, Western blotting, immunocytochemistry, and apoptosis analysis were carried out to examine the mechanism of action. Tubulin polymerization assays were conducted to examine the interaction between PAB and tubulin. A P-glycoprotein-overexpressing cell line was used to evaluate the efficacy of PAB toward multidrug-resistant phenotypes. In vivo efficacy of PAB was evaluated by the murine xenograft model. PAB induces cell cycle arrest at G2-M transition, leading to apoptosis. The drug disrupts cellular microtubule networks and inhibits the formation of mitotic spindles. Polymerization of purified bovine brain tubulin was dose-dependently inhibited by PAB. Furthermore, PAB circumvents the multidrug resistance mechanism, displaying notable potency also in P-glycoprotein-overexpressing cells. Finally, we showed that PAB is effective in inhibiting tumor growth in vivo. We identified the microtubules as the molecular target of PAB. Furthermore, we showed that PAB circumvents P-glycoprotein overexpression-induced drug resistance and is effective in inhibiting tumor growth in vivo. Our work will facilitate the future development of PAB as a cancer therapeutic.

Chemical constituents from Pseudolarix Kaempferi

A novel degraded triterpenoid isopseudolaritone A (1) and one new oligosaccharide, 1-O-isopropyl-6-O-[2-O-methyl-α-l-rhamnopyranosyl(1 → 6)]-β-d-glucopyranose (2), two new artefacts, 9-O-formacyl cedrusin (5) and 9,9′-O-diformacyl cedrusin (6), as well as 12 known phenolic Compounds (3–4, 7–16), were isolated from the root bark of Pseudolarix kaempferi. Their structures were elucidated mainly by spectroscopic methods.

Antifungal Diterpenoids of Pseudolarix kaempferi, and Their Structure—Activity Relationship Study.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Antifungal diterpenoids of Pseudolarix kaempferi, and their structure–activity relationship study

The in vitro antifungal activities of 19 structurally diversified analogues of pseudolaric acids tested against the major pathogenic fungus Candida albicans has led to the establishment of a very clear structure–activity relationship of pseudolaric acids derivatives. Pseudolaric acid A was first found to be a potent antifungal component comparable with pseudolaric acid B. Among the tested 19 diterpenoids, pseudolaric acids A 2 ( 1), B 2 ( 3), B 3 ( 4) and methyl pseudolarate A 2 ( 2) are new isolates of the root bark of Pseudolarix kaempferi, and their structures were elucidated mainly by 2D-NMR techniques and chemical methods. Compounds 12– 19 were first semi-synthesized by efficient routines from pseudolaric acid B.

Five New Diterpenoids from Pseudolarix kaempferi

Five new diterpenoids, pseudolaric acids F (1), G (2), and H (3), 2',3'-dihydroxy-1'-propoxypseudolarate B (4), and 6'-O-acetylpseudolaric acid B O-beta-D-glucopyranoside (5), along with nine known diterpenoids, pseudolaric acids A, B, and C(1), deacetylpseudolaric acid C(2), deacetylpseudolaric acid A, methyl pseudolarate A, methyl pseudolarate B, pseudolaric acid A-O-beta-D-glucopyranoside, and pseudolaric acid B-O-beta-D-glucopyranoside, were isolated from the root bark of Pseudolarix kaempferi. Their structures and stereochemistry were elucidated mainly by spectral data, especially 2D NMR techniques.