Uncaria Rhynchophylla and hirsuteine as TRPV1 agonists inducing channel desensitization.

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria rhynchophylla (UR) is recognized for its therapeutic applications in treating hypertension and inflammation. However, the specific molecular mechanisms how UR and its bioactive constituents modulate inflammatory pathways remain unknown. This study investigates the effects of UR extract and its constituent, hirsuteine (HST), on TRPV1 channel modulation which is related to hypertension and inflammation. MATERIALS AND METHODS: Electrophysiological recordings and calcium imaging experiments were conducted to assess TRPV1 activation by UR extract and HST in HEK293T cells and sensory neurons. RESULTS: UR extract and HST activated TRPV1 in HEK293T cells, with repeated applications causing channel desensitization. HST application on TRPV1-positive sensory neurons significantly reduced electrical activity compared to capsaicin. CONCLUSION: This study demonstrated UR extract and HST are a novel TRPV1 agonists inducing channel desensitization and a potent agent for treatment of TRPV1 dependent pain relief.

Anticancer efficacy of hirsuteine against colorectal cancer by opposite modulation of wild-type and mutant p53.

PURPOSE: The present study aimed to explore the anticancer activity of hirsuteine (HST), an indole alkaloid from the traditional Chinese herbal medicine Uncaria rhynchophylla, against colorectal cancer (CRC) and the underlining mechanism. METHODS: MTT, colony formation, flow cytometry and MDC staining were conducted to confirm the antiproliferative effect of HST on human CRC cells harboring different p53 status. Protein expressions were evaluated by the Western blot analysis. p53 protein half-life and the interaction between p53 and MDM2 were investigated using cycloheximide (CHX)-chase assay and Co-immunoprecipitation (Co-IP), respectively. Transcriptional activity of p53 was examined by qRT-PCR and Chromatin immunoprecipitation (ChIP). Xenograft tumor in nude mice was created to evaluate in vivo anticancer effect of HST against CRC. RESULTS: HST inhibited cell growth, arrested cell cycle and induced autophagy, showing efficient anticancer effects on CRC cells independent of p53 status. In HCT-8 cells, HST prolonged wtp53 half-life, and upregulated mRNA level of p21, suggesting that HST activated the p53 pathway through enhancement of wtp53 stability and transcriptional activity. Meanwhile in SW620 cells, HST induced MDM2-mediated proteasomal degradation of mutp53R273H, increased the DNA-binding ability of mutp53R273H at the p21 promoter, and upregulated mRNA levels of p21 and MDM2, demonstrating the depletion of mutp53R273H and restoration of its wild-type-like properties by HST. p53 knockdown by siRNA significantly impaired the growth inhibition of HST on HCT-8 and SW620 cells. Moreover, HST showed anticancer effects in xenograft tumors, accompanied with an opposite regulation of wtp53 and mutp53 R273H in mechanism. CONCLUSION: This study revealed the anticancer efficacy of HST against CRC via opposite modulation of wtp53 and mutp53 R273H, indicating the potential of HST to be a CRC drug candidate targeting p53 signaling.

Inhibitory effect and mechanism of hirsuteine on NCI­H1299 lung cancer cell lines.

Traditionally, the bark of Uncaria rhynchophylla (UR) has been employed for the treatment of hypertension, cancer, convulsions, haemorrhage, autoimmune disorders and other ailments. The primary aim of the present study was to explore the antiproliferative activity of hirsuteine (HTE) isolated from UR over a range of concentrations in human NSCLC NCI-H1299 cells and to explore the mechanisms underlying its therapeutic efficacy. The effects of HTE on cell viability were examined using Cell Counting Kit-8 (CCK-8) and colony formation assays, while apoptosis was assessed by flow cytometry. Cell cycle progression was additionally evaluated via propidium iodide staining, while reverse transcription-quantitative PCR and western blotting methods were employed to assess the protein levels and genes related to apoptosis and progression of the cell cycle, respectively. NCI-H1299 cell proliferation was markedly suppressed by HTE in a time- and dose-dependent manner. However, clear changes in cell morphology were also induced, resulting in G0-G1 phase cell cycle arrest, which was associated with cyclin E and CDK2 downregulation. HTE additionally induced robust NSCLC NCI-H1299 cell apoptosis, downregulation of Bcl-2 and upregulation of cytoplasmic cytochrome C, Bax, Apaf1, cleaved caspase-3 and cleaved caspase-9, which together drove the observed apoptotic cell death. HTE could effectively suppress human NSCLC NCI-H1299 cell growth by inducing apoptotic death in a dose-dependent fashion in vitro, therefore elucidating the mechanism by which this phytomedicine acts as a potent anticancer compound that warrants study as a treatment for human NSCLC patients.

Effects of hirsuteine on MDA-MB-453 breast cancer cell proliferation.

Hirsuteine is extracted from Uncaria rhynchophylla, the bark of which has traditionally been used to treat hypertension, cancer, convulsions, hemorrhage, auto-immune disorders, and other ailments. The anticancer properties of hirsuteine are of significant importance to the research community; however, its underlying mechanism of action is not well understood. The aim of the present study was to examine the antiproliferative ability of hirsuteine using human breast cancer MDA-MB-453 cells and to determine the underlying molecular mechanism involved in its therapeutic efficacy. The effects of hirsuteine on cell viability were determined using CCK-8 and colony formation assays, while apoptosis was assessed using flow cytometry. Cell cycle distribution was assessed using flow cytometry, and apoptotic cell quantification was performed using via Annexin V-FITC/PI staining and flow cytometry. Reverse transcription-quantitative PCR and western blotting were used to assess the expression of cell cycle progression and apoptosis associated genes and proteins. MDA-MB-453 cell proliferation was significantly reduced by hirsuteine in a concentration and time-dependent manner. Hirsuteine-treated cells exhibited G2/M phase arrest, as evidenced by the increase in G2/M phase cells and a decrease in the G0/G1 phase cells, and this was related to cyclin B1 and CDK1 downregulation. Furthermore, hirsuteine accelerated MDA-MB-453 cell apoptosis by downregulating Bcl-2 while upregulating cytoplasmic cytochrome c, Bax, Apaf1, cleaved caspase-3, and cleaved caspase-9 levels, which together drove apoptotic cell death. Thus, hirsuteine suppressed MDA-MB-453 cancer cell proliferation by inducing cell cycle arrest and promoting apoptosis.

Metabolites identification and pharmacokinetic profile of hirsuteine, a bioactive component in Uncaria in rats by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Hirsuteine is one of the major bioactive tetracyclic indole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks, possessing a wide range of pharmacological activities including neuroprotective, anticonvulsant, antihypertensive, sedative and hypnotic, and so forth. The present study was undertaken to assess the metabolism and plasma pharmacokinetics of hirsuteine in rats. After oral administration of hirsuteine at the dose of 30 mg/kg, 13, 21, and 8 metabolites were detected in rat plasma, urine, and bile by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, respectively. Furthermore, plasma concentrations of hirsuteine and its four metabolites, 4-hirsuteine N-oxide, 3,4-dehydrohirsuteine, 11-hydroxyhirsuteine, and 11-hydroxyhirsuteine-11-O-glucuronide were simultaneously quantified in rat plasma, using carbamazepine as the internal standard. The linear calibration curve of hirsuteine was in the concentration range of 0.005-5.0 µg/ml. The lower limit of quantitation in the rat plasma was 5 ng/ml for hirsuteine. This study is the first to comprehensively investigate the metabolism process of hirsuteine and the pharmacokinetic profiles of hirsuteine and its major metabolite, and will provide a scientific basis to further elucidate the pharmacodynamic material basis and therapeutic mechanism of Uncaria prescriptions.

Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1.

Chronic myeloid leukemia (CML) is a slowly progressing hematopoietic cell disorder. Sphingosine kinase 1 (SPHK1) plays established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers, including leukemia. However, small-molecule inhibitors targeting SPHK1 in CML still need to be developed. This study revealed the role of SPHK1 in CML and investigated the potential anti-leukemic activity of hirsuteine (HST), an indole alkaloid obtained from the oriental plant Uncaria rhynchophylla, in CML cells. These results suggest that SPHK1 is highly expressed in CML cells and that overexpression of SPHK1 represents poor clinical outcomes in CML patients. HST exposure led to G2/M phase arrest, cellular apoptosis, and downregulation of Cyclin B1 and CDC2 and cleavage of Caspase 3 and PARP in CML cells. HST shifted sphingolipid rheostat from sphingosine 1-phosphate (S1P) towards the ceramide coupled with a marked inhibition of SPHK1. Mechanistically, HST significantly blocked SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways. In addition, HST can be docked with residues of SPHK1 and shifts the SPHK1 melting curve, indicating the potential protein-ligand interactions between SPHK1 and HST in both CML cells. SPHK1 overexpression impaired apoptosis and proliferation of CML cells induced by HST alone. These results suggest that HST, which may serve as a novel and specific SPHK1 inhibitor, exerts anti-leukemic activity by inhibiting the SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways in CML cells, thus conferring HST as a promising anti-leukemic drug for CML therapy in the future.

Simultaneous separation and determination of hirsutine and hirsuteine by cyclodextrin-modified micellar electrokinetic capillary chromatography.

INTRODUCTION: Hirsutine and hirsuteine are the main pharmacological activity ingredients of Uncaria rhynchophylla (UR), playing an important role in treating mental and cardiovascular diseases, such as Alzheimer's disease, hypertension, Parkinson's disease, potential anti-cancer activities and so on. OBJECTIVE: To develop a cyclodextrin-modified micellar electrokinetic capillary chromatography (CD-MEKC) method for the simultaneous separation and determination of hirsutine and hirsuteine from UR and its formulations. METHODOLOGY: The optimal method was developed by investigating influences of significant factors on the separation, and this method was successfully applied for the determination of hirsutine and hirsuteine in UR and its formulations. RESULTS: The optimal background electrolyte (BGE) consisted of 40 mM sodium dihydrogen phosphate (pH 7.0), 150 mM 2,6-dimethyl-ß-cyclodextrin (DM-ß-CD), 3 mM mono-(6-ethylenediamine-6-deoxy)-ß-cyclodextrin (ED-ß-CD), and 30 mM sodium cholate (SC). Under these conditions, hirsutine and hirsuteine were successfully separated within 13 min at the separation voltage of 15 kV, temperature of 25°C and the detection wavelength of 224 nm. For the analytes, linear calibration curves were performed within the range 5.0-160.0 µg/mL. The limit of detection (LOD, S/N = 3) and the limit of quantitation (LOQ, S/N = 10) were 0.41, 1.42 µg/mL for hirsutine and 0.60, 2.17 µg/mL for hirsuteine, respectively. The recoveries of three samples were from 97.9% to 102.3%. CONCLUSION: The method was successfully applied to the determination of hirsutine and hirsuteine in UR and its formulations. Meanwhile, it provides an effective reference of the quality control of UR and its formulations.