An oncogenic role of lncRNA SNHG1 promotes ATG7 expression and autophagy involving tumor progression and sunitinib resistance of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is a malignant tumor with high incidence in adult kidney. Long non-coding RNAs (lncRNAs) have recently been recognized as important regulators in the development of RCC. However, whether lncRNA SNHG1 is associated with RCC progression remains to be elucidated. Here, the role of SNHG1 in RCC autophagy and sunitinib resistance was evaluated. Expression of SNHG1 in RCC tissues and cells was assessed using RT-qPCR. Western blot was utilized to measure the levels of autophagy-related molecules and ATG7. RNA pull-down and RIP assays were performed to confirm the molecular axis between SNHG1/PTBP1/ATG7. Cell proliferation, migration, invasion and apoptosis were analyzed by CCK-8, EdU, transwell and flow cytometry, respectively. The subcellular localization of SNHG1 was determined by an intracellular fractionation assay. The fluorescence intensity of GFP-LC3 autophagosome in RCC cells was detected. IHC staining was performed to test ATG7 expression in tumor tissues from nude mice. Here, a positive correlation of upregulated SNHG1 with poor prognosis of RCC patients was observed in RCC tissues and cells. SNHG1 knockdown suppressed tumor growth and reversed sunitinib resistance and autophagy of RCC cells. Additionally, SNHG1 was found to directly bind to PTBP1, thereby positively regulating ATG7 expression. Furthermore, we verified that SNHG1 mediated the malignant behavior of RCC cells through the PTBP1/ATG7 axis. To sum up, SNHG1 regulates RCC cell autophagy and sunitinib resistance through the PTBP1/ATG7 axis, which highlights a promising therapeutic target for RCC treatment.

Cancer-Associated Fibroblasts Boost Tumorigenesis of Clear Cell Renal Cell Carcinoma via Exosome-Mediated Paracrine SNHG1.

Despite the dominant roles of cancer-associated fibroblasts (CAFs) have attached much attention in tumorigenesis, the CAFs-derived molecular determinants that regulate renal cell carcinoma (RCC) development remains elusive. Our previous study uncovered an oncogenic SNHG1 in the immune escape of RCC, whereas CAFs-derived exosomes could be a source accounting for increasing SNHG1 in RCC cells, this is still a mystery. The obtained CAFs and normal fibroblast (NFs) from fresh RCC and adjacent tissues were firstly identified using western blot and immunofluorescent staining. The enrichment of SNHG1 was validated by RT-qPCR. CAFs-derived exosomes were isolated from conditioned medium using ultracentrifugation method and ExoQuick-TC system. The internalization of exosomes, transfer of SNHG1, was measured by immunofluorescence. Regulation of conditioned medium or exosomal SNHG1 from CAFs on RCC biological functions was evaluated by CCK-8, EdU incorporation, colony formation, and transwell assays to assess the RCC cell proliferation, migration, and invasion. SNHG1 was significantly upregulated in CAFs isolated from RCC stroma. Exosomes derived from CAFs transferred SNHG1 to RCC cells and resulted in an increased SNHG1 expression in RCC cells. The exosomes excreted by CAFs promoted RCC cell proliferation, migration, and invasion, whereas the promotion effect of CAFs-exosomes on RCC progression was attenuated by SNHG1 knockdown. The present study revealed a new mechanism of exosomal SNHG1 extracted from CAFs enhanced RCC progression and may provide a potential target for the treatment of RCC.

Efficacy and safety of vorolanib plus everolimus in metastatic renal cell carcinoma: A three-arm, randomised, double-blind, multicentre phase III study (CONCEPT).

BACKGROUND: Vorolanib is a highly potent tyrosine kinase inhibitor (TKI) targeting vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor. This three-arm, randomised, registered study aimed to assess the combination of vorolanib and everolimus or vorolanib alone versus a control arm of everolimus as second-line treatment in patients with metastatic renal cell carcinoma (RCC). PATIENTS AND METHODS: Patients with advanced or metastatic RCC who had received one prior VEGFR-TKI were randomised (1:1:1) to receive the combination of vorolanib and everolimus or either monotherapy. Patients with brain metastases were excluded. The primary end-point was progression-free survival (PFS) assessed by the independent review committee per Response Evaluation Criteria in Solid Tumours v1.1. RESULTS: Between 10th March 2017 and 30th May 2019, 399 patients (133 in each group) were enrolled. By the cutoff date (30th April 2020), a significant improvement in PFS was detected in the combination group compared with the everolimus group (10.0 versus 6.4 months; hazard ratio, 0.70; P = 0.0171). PFS was similar between the vorolanib group and the everolimus group (median: 6.4 versus 6.4 months; hazard ratio, 0.94; P = 0.6856). A significantly higher objective response rate was observed in the combination group than in the everolimus group (24.8% versus 8.3%; P = 0.0003), whereas there was no significant difference between the vorolanib group and the everolimus group (10.5% versus 8.3%; P = 0.5278). The overall survival data were immature. A total of 96 (72.2%), 52 (39.1%) and 71 (53.4%) grade 3 or higher treatment-related adverse events occurred in the combination group, vorolanib group and everolimus group, respectively. CONCLUSIONS: The addition of vorolanib to everolimus as 2nd-line treatment for patients with advanced or metastatic RCC who have experienced cancer progression after VEGFR-TKI therapy provided a better objective response rate and PFS than everolimus alone with a manageable safety profile. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03095040; Chinadrugtrials, CTR20160987.

Camrelizumab plus famitinib for advanced or metastatic urothelial carcinoma after platinum-based therapy: data from a multicohort phase 2 study.

BACKGROUND: Dual blockade of immune checkpoint and angiogenesis is an effective strategy for multiple cancers. Camrelizumab is a monoclonal antibody against PD-1, and famitinib is a multitargeted receptor tyrosine kinase inhibitor with antiangiogenesis and antiproliferation activities against tumor cells. We conducted an open-label, multicenter phase 2 basket study of camrelizumab and famitinib in eight cohorts of genitourinary or gynecological cancers. Here, findings in cohort of advanced or metastatic urothelial carcinoma with platinum-progressive disease (cohort 2) are presented. METHODS: Patients who had progressed after platinum-based chemotherapy for advanced or metastatic disease or had progressed within 12 months after completion of platinum-based (neo)adjuvant therapy were given camrelizumab (200 mg intravenously every 3 weeks) plus famitinib (20 mg orally once daily). Primary endpoint was objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: Totally, 36 patients were recruited. With a median duration from enrollment to data cut-off of 11.9 months (range 6.1-28.5), ORR was 30.6% (95% CI 16.3% to 48.1%). Median duration of response (DoR) was 6.3 months (95% CI 2.1 to not reached). Median progression-free survival (PFS) was 4.1 months (95% CI 2.2 to 8.2), and median overall survival (OS) was 12.9 months (95% CI 8.8 to not reached). Patients with bladder cancer (n=18) had numerically better outcomes, with an ORR of 38.9% (95% CI 17.3% to 64.3%) and a median PFS of 8.3 months (95% CI 4.1 to not reached). Median DoR and OS in this subpopulation had not been reached with lower limit of 95% CI of 4.2 months for DoR and 11.3 months for OS, respectively. Of 36 patients, 22 (61.1%) had grade 3 or 4 treatment-related adverse events, mainly decreased platelet count and hypertension. CONCLUSIONS: Camrelizumab plus famitinib showed potent antitumor activity in advanced or metastatic urothelial carcinoma patients after platinum-based chemotherapy. Patients with bladder cancer seemed to have better response to this combination. TRIAL REGISTRATION NUMBER: NCT03827837.

Activity and safety of SHR3680, a novel antiandrogen, in patients with metastatic castration-resistant prostate cancer: a phase I/II trial.

BACKGROUND: Antagonizing the androgen-receptor (AR) pathway is an effective treatment strategy for patients with metastatic castration-resistant prostate cancer (CRPC). Here, we report the results of a first-in-human phase 1/2 study which assessed the safety, pharmacokinetics, and activity of SHR3680 (a novel AR antagonist) in patients with metastatic CRPC. METHODS: This phase 1/2 study enrolled patients with progressive metastatic CRPC who had not been previously treated with novel AR-targeted agents. In the phase 1 dose-escalation portion, patients received oral SHR3680 at a starting daily dose of 40 mg, which was subsequently escalated to 80 mg, 160 mg, 240 mg, 360 mg, and 480 mg per day. In phase 2 dose-expansion portion, patients were randomized to receive daily dose of 80 mg, 160 mg, or 240 mg of SHR3680. The primary endpoint in phase 1 was safety and tolerability and in phase 2 was the proportion of patients with a prostate-specific antigen (PSA) response (≥ 50% decrease of PSA level) at week 12. RESULTS: A total of 197 eligible patients were enrolled and received SHR3680 treatment, including 18 patients in phase 1 and 179 patients in phase 2. No dose-limiting toxicities were reported and the maximum tolerated dose was not reached. Treatment-related adverse events (TRAEs) occurred in 116 (58.9%) patients, with the most common one being proteinuria (13.7%). TRAEs of grade ≥ 3 occurred in only 23 (11.7%) patients, and no treatment-related deaths occurred. Antitumor activities were evident at all doses, including PSA response at week 12 in 134 (68.0%; 95% CI, 61.0-74.5) patients, stabilized bone disease at week 12 in 174 (88.3%; 95% CI, 87.2-95.5) patients, and responses in soft tissue lesions in 21 (34.4%, 95% CI, 22.7-47.7) of 61 patients. CONCLUSION: SHR3680 was well tolerated and safe, with promising anti-tumor activity across all doses tested in patients with metastatic CRPC. The dose of 240 mg daily was recommended for further phase 3 study. TRIAL REGISTRATION: Clinical trials.gov NCT02691975; registered February 25, 2016.

Continuous Synthesis of Uniformly Dispersed Mesoporous SBA-15 Supported Silver Nanoparticles in a Coiled Flow Inverter Reactor.

Mesoporous silica supported nanocatalysts have shown great potential in industrial processes due to their unique properties, such as high surface area, large pore volume, good chemomechanical stability and so on. Controllable and tunable synthesis of supported nanocatalysts is a crucial problem. Continuous synthesis of supported nanoparticles has been reported to get uniformly dispersed nanomaterials. Here, a method for continuous synthesis of uniformly dispersed mesoporous SBA-15 supported silver nanoparticles in a coiled flow inverter (CFI) microreactor is described. Compared to Ag/SBA-15 synthesized in the conventional batch reactor and Ag synthesized in continuous flow, mesoporous silica nanocatalysts synthesized in continuous flow are found to have smaller average size (7-11 nm) and narrower size distribution. The addition of capping agents can effectively change the characteristic of catalysts. Moreover, two kinds of support with different surface area and pore size have been added into the continuous synthesis. This method can provide further understandings for the synthesis of uniformly dispersed supported nanocatalysts in continuous flow, especially for mesoporous nanomaterials, which provides the possibilities of large-scale yield process of supported nanocatalysts in industry.

HMMR is a downstream target of FOXM1 in enhancing proliferation and partial epithelial-to-mesenchymal transition of bladder cancer cells.

Our previous that HMMR upregulation independently predicts poor survival in patients with papillary muscle-invasive bladder cancer (MIBC). In this study, we explored its downstream regulations and the potential transcriptional factors activating its expression. MIBC derived T24 cells, and non-MIBC (NMIBC) derived RT4 cells were used for in vitro and in vivo studies. HMMR expression enhanced cell proliferation, the expression of mesenchymal markers, and cell invasion. It induced the nuclear entry of ß-catenin, increased its active form in the nuclear part, and elevated the relative TOP/FOP activity. The promoter region of HMMR has a canonical FKH motif. FOXM1 bound to this site and activated HMMR transcription. HMMR knockdown significantly weakened FOXM1 overexpression induced bladder cancer growth, invasion, partial epithelial-to-mesenchymal transition (pEMT), as well as the activation of the Wnt/ß-catenin signaling pathway. In conclusion, the findings in this study expanded our understanding of the mechanisms underlying HMMR dysregulation and the functional role of the FOXM1-HMMR axis in bladder cancer.

Camrelizumab plus Famitinib in Patients with Advanced or Metastatic Renal Cell Carcinoma: Data from an Open-label, Multicenter Phase II Basket Study.

PURPOSE: Blockade of immune checkpoint and angiogenesis is an effective treatment strategy for advanced or metastatic renal cell carcinoma (RCC). We report the results of camrelizumab plus famitinib in the RCC cohort of an open-label, multicenter, phase II basket study. PATIENTS AND METHODS: Eligible patients were enrolled to receive camrelizumab (200 mg i.v. every 3 weeks) and famitinib (20 mg orally once daily). Primary endpoint was objective response rate (ORR) per RECIST version 1.1. RESULTS: Totally, 38 patients were recruited, including 13 (34.2%) treatment-naïve and 25 (65.8%) previously treated patients. With a median duration from enrollment to data cutoff of 16.5 months (range, 6.1-20.4), 23 patients achieved a confirmed objective response, and ORR was 60.5% [95% confidence interval (CI), 43.4-76.0]. Responses in 18 (78.3%) responders were still ongoing, and Kaplan-Meier estimated median duration of response had not been reached yet (range, 1.0+-14.8+ months). Median progression-free survival (PFS) was 14.6 months (95% CI, 6.2-not reached). ORR was 84.6% (95% CI, 54.6-98.1) in treatment-naïve patients and 48.0% (95% CI, 27.8-68.7) in pretreated patients; median PFS had not been reached and was 13.4 months (95% CI, 4.1-not reached), respectively. Most common grade 3 or 4 treatment-related adverse events included proteinuria (18.4%), hypertension (18.4%), decreased neutrophil count (13.2%), palmar-plantar erythrodysesthesia syndrome (10.5%), and hypertriglyceridemia (10.5%). No treatment-related deaths occurred, and no new safety signals were observed. CONCLUSIONS: Camrelizumab plus famitinib showed potent and enduring antitumor activity in patients with advanced or metastatic RCC, both in treatment-naïve and previously treated population.

A multi-institutional retrospective study of hyperthermic plus intravesical chemotherapy versus intravesical chemotherapy treatment alone in intermediate and high risk nonmuscle-invasive bladder cancer.

Objective: To compare the efficacy and safety of hyperthermic intravesical chemotherapy (HIVEC) and intravesical chemotherapy (IVEC) in patients with intermediate and high risk nonmuscle-invasive bladder cancer (NMIBC) after transurethral resection. Methods: We included 560 patients diagnosed with primary or recurrent NMIBC between April 2009 and December 2015 at 1 of 6 tertiary centers. We matched 364 intermediate or high risk cases and divided them into 2 groups: the HIVEC+IVEC group [chemohyperthermia (CHT) composed of 3 consecutive sessions followed by intravesical instillation without hyperthermia] and the IVEC group (intravesical instillation without hyperthermia). The data were recorded in the database. The primary endpoint was 2-year recurrence-free survival (RFS) in all NMIBC patients (n = 364), whereas the secondary endpoints were the assessment of radical cystectomy (RC) and 5-year overall survival (OS). Results: There was a significant difference in the 2-year RFS between the two groups in all patients (n = 364; HIVEC+IVEC: 82.42% vs. IVEC: 74.18%, P = 0.038). Compared with the IVEC group, the HIVEC+IVEC group had a lower incidence of RC (P = 0.0274). However, the 5-year OS was the same between the 2 groups (P = 0.1434). Adverse events (AEs) occurred in 32.7% of all patients, but none of the events was serious (grades 3-4). No difference in the incidence or severity of AEs between each treatment modality was observed. Conclusions: This retrospective study showed that HIVEC+IVEC had a higher 2-year RFS and a lower incidence of RC than IVEC therapy in intermediate and high risk NMIBC patients. Both treatments were well-tolerated in a similar manner.

Exploiting equilibrium-kinetic synergetic effect for separation of ethylene and ethane in a microporous metal-organic framework.

Physisorption is a promising technology to cut cost for separating ethylene (C2H4) from ethane (C2H6), the most energy-intensive separation process in the petrochemical industry. However, traditional thermodynamically selective adsorbents exhibit limited C2H4/C2H6 selectivity due to their similar physiochemical properties, and the performance enhancement is typically at the expense of elevated adsorption heat. Here, we report highly-efficient C2H4/C2H6 adsorption separation in a phosphate-anion pillared metal-organic framework ZnAtzPO4 exploiting the equilibrium-kinetic synergetic effect. The periodically expanded and contracted aperture decorated with electronegative groups within ZnAtzPO4 enables effective trapping of C2H4 and impedes the diffusion of C2H6, offering an extraordinary equilibrium-kinetic combined selectivity of 32.4. The adsorption heat of C2H4 on ZnAtzPO4 (17.3 to 30.0 kJ mol-1) is substantially lower than many thermodynamically selective adsorbents because its separation capability only partially relies on thermodynamics. The separation mechanism was explored by computational simulations, and breakthrough experiments confirmed the excellent C2H4/C2H6 separation performance of ZnAtzPO4.

Enantioseparation of racemic amlodipine using immobilized ionic liquid by solid-phase extraction.

In this paper, a novel l-glutamate based immobilized chiral ionic liquid (SBA-IL (Glu)) was prepared by chemical bonding method and applied as a solid sorbent for chiral separation of amlodipine. The performance of SBA-IL (Glu) was investigated for the absorption of (S)-amlodipine and separation of amlodipine enantiomer. The static experiment showed that equilibrium adsorption was achieved within 80 minutes, and the saturation adsorptions capacity was 12 mg/g. The complex was then packed in a glass chromatographic column for the separation of amlodipine and the enantiomeric excess (%ee) of (S)-amlodipine reached 24.67%. The immobilized ionic liquids exhibit good reusability, and the separation efficiency remains 18.24% after reused five times, which allows potential scale-up for the chiral separation of amlodipine.

Systematic screening of protein-coding gene expression identified HMMR as a potential independent indicator of unfavorable survival in patients with papillary muscle-invasive bladder cancer.

Papillary and non-papillary are two histological patterns of bladder carcinogenesis and are considered as dual-track oncogenic pathways, which have different genetic alterations. The TCGA-bladder cancer (BLCA) database contains clinicopathological, genomic and survival data from over 400 muscle-invasive bladder cancer patients. In this study, using data from this database, we performed a systematic screening of gene expression to identify the protein-coding gene that might have prognostic value in papillary and non-papillary muscle-invasive bladder cancer (MIBC). The data of patients with primary MIBC in TCGA-BLCA was acquired from the UCSC Xena project (http://xena.ucsc.edu) for re-analysis. By setting |log2 fold change|≥2 and adjusted p value <0.01 as the screening criteria, we found 751 significantly dysregulated genes, including 183 overexpressed and 568 downregulated genes. HMMR was identified as a potential prognostic marker with unique expression. Multivariate analysis showed that its expression was an independent prognostic indicator of shorter progression-free survival (PFS) (HR: 1.400, 95%CI: 1.021-1.920, p = 0.037) in the papillary subtype. ENST00000393915.8 and ENST00000358715.3, two transcripts that contain all 18 exons and encode the full length of HMMR, were significantly upregulated in cancer tissues compared with normal bladder tissues. None of the 17 CpG sites in its DNA locus was relevant to HMMR expression. 26/403 (6.5%) MIBC cases had HMMR gene-level amplification, which was associated with upregulated HMMR expression compared with the copy-neutral and deletion groups. Gene set enrichment analysis (GSEA) in papillary MIBC found that the high HMMR expression group was associated with upregulated genes enriched in multiple gene sets with well-established role in BC development, including G2M checkpoint, E2 F Targets, Myc Targets V1, Myc Targets V2 and Glycolysis. Based on these findings, we infer that HMMR expression might be a specific prognostic marker in terms of PFS in papillary MIBC. DNA amplification might be an important mechanism of its elevation.

Enantioseparation of flurbiprofen enantiomers using chiral ionic liquids by liquid-liquid extraction.

Flurbiprofen is a kind of nonsteroidal anti-inflammatory drug, which has been widely used in clinic for treatment of rheumatoid arthritis and osteoarthritis. It has been reported that S-flurbiprofen shows good performance on clinic anti-inflammatory treatment, while R-enantiomer almost has no pharmacological activities. It has important practical values to obtain optically pure S-flurbiprofen. In this work, chiral ionic liquids, which have good structural designability and chiral recognize ability, were selected as the extraction selector by the assistance of quantum chemistry calculations. The distribution behaviors of flurbiprofen enantiomers were investigated in the extraction system, which was composed of organic solvent and aqueous phase containing chiral ionic liquid. The results show that maximum enantioselectivity up to 1.20 was attained at pH 2.0, 25°C using 1,2-dichloroethane as organic solvent, 1-butyl-3-methylimidazole L-tryptophan ([Bmim][L-trp]) as chiral selector. The racemic flurbiprofen initial concentration was 0.2 mmol L-1 , and [Bmim][L-trp] concentration was 0.02 mol L-1 . Furthermore, the recycle of chiral ionic liquids has been achieved by reverse extraction process of the aqueous phase with chiral selector, which is significant for industrial application of chiral ionic liquids and scale-up of the extraction process.

Combining Protein and miRNA Quantification for Bladder Cancer Analysis.

We combine the telomerase extension reaction and microRNA (miRNA)-induced rolling circle amplification, followed by graphene oxide (GO) and nicking enzyme-assisted signal amplification as a method to analyze telomerase and miRNA-21 in urine samples with the following merits. First, it is a binary assay and can simultaneously output double signals that correspond to the quantities of telomerase and miRNA, respectively. Second, telomerase activity is enhanced by using a DNA molecular beacon probe to inhibit the formation of G-quadruplex. Third, background noise is decreased significantly via introduction of GO. Fourth, performance tests on about 258 urine samples demonstrate that this binary assay can distinguish between urine from bladder cancer patients, those with cystitis, and normal individuals. Finally, this strategy also shows great potential in distinguishing between muscle-invasive bladder cancers and non-muscle-invasive bladder cancers. The proposed strategy will greatly contribute to clinical decision-making and individualized treatments.

Association of myeloid-derived suppressor cells and efficacy of cytokine-induced killer cell immunotherapy in metastatic renal cell carcinoma patients.

Metastatic renal cell carcinoma (MRCC) is one of the malignancies that are sensitive to immunotherapy. However, the underlying immune inhibitory factors such as myeloid-derived suppressor cells (MDSCs) might restrain the efficacy of immunotherapy. The present study investigates the clinical efficacy of cytokine-induced killer (CIK) cell therapy in patients with MRCC and explores whether the levels of peripheral MDSCs are associated with the prognosis of patients receiving this therapy. Twenty-nine patients with measurable MRCC were treated with an adoptive transfer of autologous CIK cells, followed by 5 consecutive days of interleukin-2 administration. The tumor response and 1-year survival were observed. The proportion of MDSCs in the peripheral blood was detected, and the correlation of MDSCs with prognosis was analyzed. Of 29 evaluable patients, no complete responses were seen; 4 patients exhibited a partial response (13.8%), 18 patients displayed stable disease (62.1%), and 7 patients showed progressive disease (24.1%). Twenty patients (69.0%) were alive 14.8-41.4 months at the time of the last follow-up (median follow-up=20.2 mo). The 1-year survival was 82.8% (24/29). Peripheral blood MDSCs were elevated in almost all MRCC patients and decreased after CIK-cell infusion. Subgroup analysis indicated that patients with a relatively low proportion of MDSCs exhibited prolonged survival. In conclusion, our data suggest that transfusion of autologous CIK cells can induce regression of MRCC, and MDSCs can serve as a potential marker for the prognosis of patients receiving a CIK-based therapy.

Axial dispersion coefficient in high-speed counter-current chromatography.

Experimental measurements of axial dispersion coefficients in high-speed counter-current chromatography have been carried out in the single-phase and two-phase modes. Axial dispersion coefficients were calculated from the residence time distribution curve (or the elution profile). The experimental data obtained were used to develop a model involving Peclet number Pe, Reynolds number and the ratio of flow velocity u to linear angular velocity u(theta) for predicting the axial dispersion coefficient. Furthermore, the models obtained from the single-phase and two-phase modes were compared, and a counterintuitive phenomenon was found in that the effects of the flow rate and the rotation speed on the axial dispersion coefficients are inconsistent: the axial dispersion coefficient decreases with the rotation speed and increases with the flow rate in the single-phase mode, but increases with rotation speed and decreases slightly with the flow rate in the two-phase mode.

Simultaneous determination of four sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. extracts by high-performance liquid chromatography.

Wilfortrine, wilfordine, wilforgine and wilforine are four major bioactive sesquiterpene alkaloids in Tripterygium wilfordii Hook. F. The first analytical determination of the four major bioactive alkaloids is described. The four alkaloids are well-resolved within 15 min using the developed HPLC method. The identity of the analytes was confirmed by an HPLC-MS experiment, with all compounds being clearly assignable by atmospheric pressure chemical ionization (APCI) positive mode analysis. The method was validated for limit of qualification, linearity and inter-day variation of precision and accuracy. Seven T. wilfordii samples (extracts and commercial product) were successfully analysed.

Sample capacity in preparative high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) is a versatile technique in preparative separation and purification of pure compounds from complex matrices. As a preparative chromatography, there is a need to maximize the column production. Based on the plate theory of Van Deemter, the effect of the sample load on the separation was investigated in a preparative HSCCC with a 1000 ml column capacity. The test samples of hydroquinone, pyrocatechol and phenol were separated using a two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (1:1:1:1, v/v/v/v) at different sample loads. The results showed that for the case of HSCCC, the agreement of the effect of sample load on peak height and peak width between the Van Deemter's theory and the experiments is excellent. Furthermore, the factors limiting the mass load, including the resolution between the peaks, the partition isotherm and the solute solubility were also discussed.

Preparative isolation and purification of atractylon and atractylenolide III from the Chinese medicinal plant atractylodes macrocephala by high-speed counter-current chromatography.

The medicinal plant Atractylodes macrocephala (Baizhu in Chinese) has been widely used in traditional Chinese medicine for energy and stomach complaints, treatment of dyspepsia and anorexia, anti-inflammation, anticancer and for increasing assimilation. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of two main bioactive components, namely, atractylon and atractylenolide III from A. macrocephala by using light petroleum (60-90 degrees C)-ethyl acetate-ethanol-water (4:1:4:1 v/v) as the two-phase solvent system in dual-mode elution. Compared with the separation using the normal-mode elution, the dual-mode HSCCC can be achieved with shorter elution time. Atractylenolide III (32.1 mg) at 99.0% purity and 319.6 mg atractylon at 97.8% purity could be obtained from 1000 mg crude sample in a single run. The recoveries of atractylenolide III and atractylon were 95.4 and 92.6%, respectively.

Simultaneous quantification of three major bioactive triterpene acids in the leaves of Diospyros kaki by high-performance liquid chromatography method.

The leaf of Diospyros kaki, which is a traditional Chinese medicine, has been used for the treatment of various diseases. In order to improve the quality assurance of the leaves of D. kaki, derived extracts and phytomedicines, a simple, rapid and accurate high-performance liquid chromatography (HPLC) method was developed to simultaneously assess the three bioactive triterpene acids: barbinervic acid (BA) and its epimer, rotungenic acid (RA), along with 24-hydroxy ursolic acid (HA). This HPLC assay was performed on a reversed-phase C18 column with methanol and aqueous H3PO4 as the mobile phase and using a monitoring wavelength at 210 nm. This method was successfully applied to quantify these three bioactive triterpene acids in five different solvent extracts of the leaves of D. kaki and in the leaves from six different locations in China. The results demonstrated the total content and quantity of each of the main bioactive compounds were strongly dependent on the extraction solvents and locations, indicating that the quality control of the bioactive ingredients in the leaves of D. kaki, derived extracts and phytomedicines is critical to ensure its clinical benefits. The content of the total triterpenoids was also determined by the less selective colorimetric method, and the comparison with the HPLC method was given.