Harmonizing international quality standards for Andrographis paniculata: A comparative analysis of content determination methods across pharmacopeias.

The quality standards for Andrographis paniculata, a widely used medicinal herb, exhibited significant variations across different pharmacopeias. In this study, we compared the HPLC content determination methods and total lactone content of A. paniculata samples from different regions, as specified in the Chinese (CP), United States (USP), European (EP), Thai (TP), and Indian pharmacopeias (IP), as well as the Hong Kong Chinese Materia Medica Standards (HK). We aimed to assess the differences and similarities among these pharmacopeias and harmonized international quality standards for A. paniculata. The analysis revealed variations in sample preparation, liquid chromatographic conditions, fingerprint profiles, and total lactone content among the different pharmacopeias. Specifically, the CP and HK methods exhibited superior sample preparation and chromatographic separation. Further comparing the content of 20 A. paniculata samples with the CP, USP, EP and HK methods showed consistent determinations for the same components, indicating similar detection capabilities. The discrepancies in total lactone content primarily stemmed from differences in the number and types of detected compounds. Moreover, the acceptance criteria exhibited a stringency in the order CP > HK > EP > USP. In conclusion, this comparison analysis of content determination in CP, USP, HK, EP, TP and IP provided a scientific foundation for the international standardization and trade regulations of A. paniculata. It also served as a valuable reference for the development of international quality standards for other medicinal herbs, facilitating the harmonization of global pharmaceutical standards.

EPAS1 Promoter Hypermethylation is a Diagnostic and Prognostic Biomarker for Non-Small Cell Lung Cancer.

Background: The importance of promoter methylation in non-small cell lung cancers (NSCLC) remains to be understood. Thus, we aimed to determine the diagnostic and prognostic value of the methylation of the endothelial PAS domain containing protein-1 (EPAS1) promoter in NSCLC. Methods: EPAS1 promoter methylation levels were quantitated by methylation-specific PCR. Further, we evaluated the expression, promoter methylation, prognostic value, and impact on immune cell infiltration of EPAS1 by analyzing the TCGA database using web-based bioinformatics tools including GEPIA, UALCAN and MethSurv. Results: Our results demonstrated that promoter methylation of EPAS1 downregulated its expression in NSCLC tissues. Additionally, an AUC value of 0.772 indicated that the methylation of the EPAS1 promoter is a potential diagnostic marker for NSCLC. A Kaplan-Meier analysis demonstrated that high methylation levels of CpG sites in the EPAS1 promoter were indicative of poorer overall survival. Further, EPAS1 expression levels were highly correlated with the infiltration of several types of immune cells, including γδ T cells, T follicular helper cells, CD8+ T cells, and CD4+ T-cells. Conclusion: Collectively, our findings suggest that methylation analyses of the EPAS1 promoter could be used as a prognostic biomarker for NSCLC and that EPAS1 potentially plays an important role in immune cell infiltration in NSCLC.

PRKCDBP Methylation is a Potential and Promising Candidate Biomarker for Non-small Cell Lung Cancer.

BACKGROUND: The occurrence and development of lung cancer are closely linked to epigenetic modification. Abnormal DNA methylation in the CpG island region of genes has been found in many cancers. Protein kinase C delta binding protein (PRKCDBP) is a potential tumor suppressor and its epigenetic changes are found in many human malignancies. This study investigated the possibility of PRKCDBP methylation as a potential biomarker for non-small cell lung cancer (NSCLC). METHODS: We measured the methylation levels of PRKCDBP in the three groups of NSCLC tissues. Promoter activity was measured by the dual luciferase assay, with 5'-aza-deoxycytidine to examine the effect of demethylation on the expression level of PRKCDBP. RESULTS: The methylation levels of PRKCDBP in tumor tissues and 3 cm para-tumor were higher than those of distant (>10 cm) non-tumor tissues. Receiver operating characteristic (ROC) curve analysis between tumor tissues and distant non-tumor tissues showed that the area under the line (AUC) was 0.717. Dual luciferase experiment confirmed that the promoter region was able to promote gene expression. Meanwhile, in vitro methylation of the fragment (PRKCDBP_Me) could significantly reduce the promoter activity of the fragment. Demethylation of 5'-aza-deoxycytidine in lung cancer cell lines A549 and H1299 showed a significant up-regulation of PRKCDBP mRNA levels. CONCLUSIONS: PRKCDBP methylation is a potential and promising candidate biomarker for non-small cell lung cancer.

Chloroform extract from Sophora Tonkinensis Gagnep. inhibit proliferation, migration, invasion and promote apoptosis of nasopharyngeal carcinoma cells by silencing the PI3K/AKT/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophora Tonkinensis Gagnep. (STG) has been used as a folk medicine for the treatment of different cancers, especially for nasopharyngeal carcinoma, cervical cancer, liver cancer, stomach cancer, lung cancer and leukemia in China. However, the main chemical composition and anticancer mechanism of chloroform extract of STG (CESTG) were still not very clear. AIM OF STUDY: This work was carried out to investigate the anticancer effects and mechanisms of chloroform extract of STG (CESTG) on NPC. METHODS: Cultured NPC CNE1, CNE2 and Np69 cells were treated with CESTG. Cells were subjected to cell proliferation, colony-forming, migration and invasion assays. Cell cycle and apoptosis were measured by flow cytometry. Western blotting and morphological analysis were also performed. Tumor xenografts and drug treatments were made in BALB/c nude mice. The main compounds of CESTG was separated by HPLC. RESULTS: CESTG inhibited cell viability, clonal growth and induced cell apoptosis in a dose-dependent manner by silencing the PI3K/AKT/mTOR signaling pathway, which is associated with upregulation of cleaved PARP, caspase 3/7/8/9, cleaved caspase 3/7/8/9, Bax and downregulation of PARP, P-PI3K, PI3K, P-AKT, AKT, P-mTOR, mTOR and Bcl-2. In addition, CESTG arrested cell cycle in the G1/S phase, correlating with decreased levels of cyclin D1/B1, CDK 4 and 6. CESTG decreased cell migration and invasion which correlated with decreased expression of ß-catenin, vimentin and snail. CESTG significantly inhibited the tumor growth without toxicity. CONCLUSION: The results presented here suggest that CESTG could be use as a potential source of NPC therapeutic drug.

Oligosaccharides from Polygonatum Cyrtonema Hua: Structural characterization and treatment of LPS-induced peritonitis in mice.

Fructooligosaccharide was isolated from Polygonatum Cyrtonema Hua (PFOS) for the first time. Structure characterized using FT-IR, MALDI-TOF-MS, NMR, AFM, and TEM, indicated that PFOS was graminan-type fructan with a degree of polymerization ranging from 5 to 10. A murine model of lipopolysaccharide (LPS)-induced peritonitis was used to evaluate the in vivo anti-inflammatory and lung protective efficacy of PFOS. The result shown that pretreatment with PFOS (1.0 mg/mL) in peritonitis-induced mice could significantly inhibit the level of pro-inflammatory cytokines (TNF-α, IL-1ß) in serum (P < 0.001), increase mice survival rate from 12.5 % to 54 % (P < 0.05), and alleviated lung injury through ameliorating the damage of the pulmonary cellular architecture and reducing inflammatory monocyte accumulation in lung tissue. This effect of oligosaccharides could explain the traditional usage of P. cyrtonema as a tonic medicine for respiratory problems and it could be used as a potential natural ingredient with anti-inflammatory activity.

Aromatin D-J: Seven previously undescribed labdane diterpenoids isolated from Blumea aromatica.

Blumea aromatica is a traditional Chinese medicine used for treating various diseases such as rheumatoid arthritis, eczema, and pruritus. Previous studies on B. aromatica used a mass defect-filtering strategy via the ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and reported the presence of several labdane diterpenoids (LADs). To determine the actual structures of these LADs and investigate their biological activities, seven previously undescribed LADs (aromatin D-J) were isolated from the whole B. aromatica herb. The structures of these isolated compounds were characterized using high-resolution mass spectrometry and extensive 1D and 2D NMR analyses. In addition, the absolute configurations of these compounds were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra as well as using X-ray crystallographic analysis. All isolated compounds were evaluated for their ability to activate adenylate cyclase by measuring the levels of cyclic adenosine 3',5'-monophosphate (cAMP) in rat ventricular tissue. Aromatin E, F, and J showed moderate activities with an increase in cAMP levels by 67%, 69%, and 64%, respectively, compared with the control group.

UHPLC-QTOF-MS based metabolite profiling analysis and the correlation with biological properties of wild and artificial agarwood.

To date, the agarwood has been over exploited worldwide in the wild due to high demand. As an alternative, the agarwood obtained through artificial methods has greatly resolved the shortage of agarwood supply in the global market. However, little information about the difference in chemical constituents and bioactivities of the wild agarwood and the artificial agarwood is available. This study aims to systematically compare the chemical composition and the bioactivities between wild and artificial agarwood on the basis of the integrated method of ultra-high-performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-TOF-MS) and multivariate statistical analysis. The invitro antioxidant activity was determined using the 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical scavenging activity assays. The cytotoxic activity of agarwood from different origin against three human cancer cell lines (i.e., A375, U251, and Skov3) were compared using the MTT assay. Fifty metabolites from UPLC-QTOF-MS spectra were identified and included in the multivariate analysis. Among these metabolites, 2-(2-phenylethyl) chromone derivatives (PECs), bi-2-(2-phenylethyl) chromone derivatives (BPECs) and sesquiterpene-2-(2-phenylethyl) chromone conjugates (SPECs) were found to be the major metabolites and acted as discriminant compounds in agarwood from different origin. The antioxidant activity study showed that the wild agarwood displayed significant antioxidant capacity compared with the artificial agarwood. Particularly, the content of secondary metabolites of SPEC analogs shown a positive effect on the radical scavenging activities, whereas the PECs were negatively correlated. Interestingly, no significant difference was observed between wild and artificial agarwood in terms of cytotoxic activity against three human cancer cell lines (i.e., A375, U251, and Skov3). To the best of our knowledge, this research is the first to study the metabolite profiles and bioactivities of the wild and the artificial agarwood in a holistic approach, and is expected to provide a rational basis for the quality assessment of artificial agarwood as a substitute for wild agarwood.

Hypermethylation of tumor necrosis factor decoy receptor gene in non-small cell lung cancer.

Abnormal methylation of the TNFRSF10C and TNFRSF10D genes has been observed in numerous types of cancer; however, no studies have investigated the methylation of these genes in non-small cell lung cancer (NSCLC). The aim of the present study was to investigate the association between TNFRSF10C and TNFRSF10D methylation and NSCLC. Methylation levels of 44 pairs of NSCLC tumor tissues and distant non-tumor tissues were analyzed using quantitative methylation specific PCR and methylation reference percentage values (PMR). The methylation levels of the TNFRSF10C gene in NSCLC tumor tissue samples were significantly higher compared with those in the distant non-tumor tissues (median PMR, 2.73% vs. 0.75%; P=0.013). Subgroup analysis demonstrated that the methylation levels of TNFRSF10C in tumor tissues from male patients were significantly higher compared with those in distant non-tumor tissues (median PMR, 2.73% vs. 0.75%; P=0.041). The levels of TNFRSF10C methylation were also higher in the tumor tissues of patients who were non-smokers compared with their distant non-tumor tissues (median PMR, 2.50% vs. 0.63%; P=0.013). TNFRSF10C methylation levels were higher in the tumor tissues from male patients compared with those from female patients (median PMR, 2.50% vs. 0.63%; P=0.031). However, no significant differences in the methylation levels of the TNFRSF10D gene were observed between the sexes. Using the cBioPortal and The Cancer Genome Atlas lung cancer data, it was demonstrated that TNFRSF10C methylation levels were inversely correlated with TNFRSF10C mRNA expression levels (r=-0.379; P=0.008). In addition, demethylation of lung cancer cell lines A549 and NCI-H1299 using 5'-aza-deoxycytidine further confirmed that TNFRSF10C hypomethylation was associated with significant upregulation of TNFRSF10C mRNA expression levels [A549 fold-change (FC)=8; P=1.0×10-4; NCI-H1299 FC=3.163; P=1.143×10-5]. A dual luciferase reporter gene assay was also performed with the insert of TNFRSF10C promoter region, and the results revealed that the TNFRSF10C gene fragment significantly enhanced the transcriptional activity of the reporter gene compared with that in the control group (FC=1.570; P=0.032). Overall, the results of the present study demonstrated that hypermethylation of TNFRSF10C was associated with NSCLC.

[Current quality standards of Andrographis Herba of different countries and regions].

Andrographis Herba is a commonly used plant medicine, and has been recorded in pharmacopeias of different countries. However, there are some differences in the quality standards. Based on this, this paper compare the quality standards of Andrographis Herba between Chinese Pharmacopoeia, Hong Kong Chinese Materia Medica Standards, United States Pharmacopoeia, European Pharmacopoeia and Indian Pharmacopoeia, including origin, botanical characteristics, identification(microscopic identification and chromatographic identification), content determination, specific test(such as impurities, loss on drying, extractives, pesticides, heavy metals, mycotoxins, and other items) and storage requirements, so as to provide a reference for studying international quality standards of Andrographis.

Rapid Screening of Forskolin-Type Diterpenoids of Blumea aromatica DC Using Ultra-High-Performance Liquid Chromatography Tandem Quadrupole Time-of-Flight Mass Spectrometry Based on the Mass Defect Filtering Approach.

The discovery of new active compounds of natural products tends to be increasingly more challenging due to chemical complexity and unpredictable matrices. Forskolin is an active natural labdane-type diterpenoid ingredient widely used worldwide for the treatment of glaucoma, heart failure, hypertension, diabetes, and asthma, and is expected to be a promising anticancer, anti-inflammation, and anti-HIV agent. In recent years, demand for forskolin in the medicine market has increased dramatically. However, natural forskolin originates exclusively from traditional Indian herb medicine Coleus forskohlii (Willd.) Briq. In a previous study, we isolated a series of diterpenoids including an 8,13-epoxy-14ene labdane carbon skeleton from Blumea aromatica DC. In order to identify alternative plant resources, a novel and effective strategy was proposed for the screening of potential forskolin-type diterpenoids (FSKD) compounds obtained from B. aromatica, using the mass defect filtering (MDF) strategy via ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) approach. Within a narrow, well-defined mass defect range, the strategy developed could significantly improve the detection efficiency of selected FSKD compounds by filtering out certain major or moderate interference compounds. Additionally, the MS/MS cleavage behavior and the characteristic diagnostic ions of the FSKD compounds were proposed to be used in aiding structural identification of the filtration compounds. As a result, a total of 38 FSKD of B. aromatica were filtered out and tentatively identified. To the best of our knowledge, it was the first time that these forskolin-type diterpenoids were identified in B. aromatica, which significantly expands our understanding of the chemical constituents of Blumea species, and allows B. aromatica to be used as a potential alternative plant resource that contains these forskolin-type active compounds. The strategy proposed was proven efficient and reliable for the discovery of novel compounds of herbal extracts.

Cytotoxic Prenylated Xanthones from the Leaves of Garcinia bracteata.

Six new prenylated xanthones (1: -6: ) and seventeen known xanthones were isolated from extracts of Garcinia bracteata leaves. Their structures were determined by extensive NMR and MS spectroscopic data analysis. The inhibitory activities of the isolates were assayed on HeLa, A549, PC-3, HT-29, and WPMY-1 cell lines. Compounds 1: and 15: -17: showed moderate inhibitory effects on tumor cell growth, with IC50s ranging from 3.7 to 14.7 µM.

Bioactive scalemic caged xanthones from the leaves of Garcinia bracteata.

Four pairs of previously undescribed caged xanthones (1-4) and twelve known caged xanthones (5-16) were isolated from the leaf extract of Garcinia bracteata. Their structures were unambiguously elucidated on the basis of spectroscopic methods. The planar structure and relative configuration of 1 was confirmed by X-ray crystallographic analysis. The enantiomers of compounds 1, 2, 4 were further resolved by semi-preparative chiral HPLC, and the absolute configurations of enantiomers of compounds 1 and 4 were determined by measurement and calculation of electronic circular dichroism (ECD) spectra and specific rotations. The inhibitory activities of the isolated compounds against human HeLa, A549, PC-3, HT-29, and WPMY-1 cell lines were assayed, and garcibractatin A (4) showed the most potent inhibitory activities in vitro with IC50 values from 1.11 to 2.93 µM. A preliminary structure-activity relationship has been discussed, and some helpful conclusions have been drawn.