From a Medicinal Mushroom Blend a Direct Anticancer Effect on Triple-Negative Breast Cancer: A Preclinical Study on Lung Metastases

Bioactive metabolites isolated from medicinal mushrooms (MM) used as supportive treatment in conventional oncology have recently gained interest. Acting as anticancer agents, they interfere with tumor cells and microenvironment (TME), disturbing cancer development/progression. Nonetheless, their action mechanisms still need to be elucidated. Recently, using a 4T1 triple-negative mouse BC model, we demonstrated that supplementation with Micotherapy U-Care, a MM blend, produced a striking reduction of lung metastases density/number, paralleled by decreased inflammation and oxidative stress both in TME and metastases, together with QoL amelioration. We hypothesized that these effects could be due to either a direct anticancer effect and/or to a secondary/indirect impact of Micotherapy U-Care on systemic inflammation/immunomodulation. To address this question, we presently focused on apoptosis/proliferation, investigating specific molecules, i.e., PARP1, p53, BAX, Bcl2, and PCNA, whose critical role in BC is well recognized. We revealed that Micotherapy U-Care is effective to influence balance between cell death and proliferation, which appeared strictly interconnected and inversely related (p53/Bax vs. Bcl2/PARP1/PCNA expression trends). MM blend displayed a direct effect, with different efficacy extent on cancer cells and TME, forcing tumor cells to apoptosis. Yet again, this study supports the potential of MM extracts, as adjuvant supplement in the TNBC management.     

Chemical profiling and marker characterization of Huangqin decoction prepared with three types of peony root by liquid chromatography with electrospray ionization mass spectrometry

Clarification of the quality and biological effect equivalence of traditional Chinese medicines containing multi‐origin species is essential to improve their current quality standards, and also is the core problem to clarify the origins of single herbs with multi‐species in Chinese formulas that will guarantee their clinical application. Huangqin decoction is the typical one of multi‐origin formulas frequently used in traditional Chinese medicine and Kampo medicine. An ultra high performance liquid chromatography with electrospray ionization–tandem mass spectrometry was developed for chemical profiling and marker quantification of Huangqin decoction prepared with two different original types of peony root, white and red peony root. Forty‐seven main peaks in chemical profiling of Huangqin decoction prepared with white and red peony root were identified: nine were from peony root, 20 from baical skullcap root, 17 from licorice root, and one from jujubae fruit. The markers characteristics of the respective types of peony root in Huangqin decoction differ from that in single herbs, especially in terms of monoterpenoids and hydrolysable tannins. Subsequently, 17 representative markers in Huangqin decoction prepared with three types of peony root and their chemical characteristics and content distribution were carried out.     

Comparison of the active components of Aster tataricus from different regions and related processed products by ultra‐high performance liquid chromatography with tandem mass spectrometry

We investigated crude Aster tataricus, vinegar‐processed Aster tataricus, honey‐processed Aster tataricus, and steamed Aster tataricus as a case study and developed a comprehensive strategy integrating quantitative analysis and chemical pattern recognition methods for the evaluation and differentiation of Aster tataricus from different regions, as well as related processed products. In the study, 15 batches of raw Aster tataricus collected from seven provinces were analyzed. A sensitive and rapid ultra‐high performance liquid chromatography with tandem mass spectrometry method for simultaneous determination of 15 compounds was established to evaluate the quality of raw and processed Aster tataricus. Furthermore, multivariate statistical techniques were applied to compare the differences among Aster tataricus samples. As a result, the herbs collected from seven provinces were divided into two categories, and chlorogenic acid was the most important component distinguishing between the regions. Moreover, all of the raw and processed samples were classified by partial least squares discriminant analysis based on the 15 analyzed compounds. Results showed that raw Aster tataricus, vinegar‐processed Aster tataricus, honey‐processed Aster tataricus, and steamed Aster tataricus were clustered in four different areas. Shionone, chlorogenic acid and kaempferol were the significant constituents differentiating the raw and differently processed Aster tataricus samples.     

Untargeted metabolomic study on the insomnia effect of Suan‐Zao‐Ren decoction in the rat serum and brain using ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry combined with data processing analysis

Insomnia is a common clinical disease that can seriously damage the normal lives of sufferers. Suan‐Zao‐Ren decoction has been used to treat insomnia for a long time. However, the underlying molecular mechanism of Suan‐Zao‐Ren decoction is still not clear. In this study, the nontargeted metabolomics based on high‐resolution mass spectrometry and multiple statistical approaches were initially used to investigate the changes of potential serum and brain biomarkers and metabolic pathways in the insomnia model rat. Principal component analysis‐discriminate analysis indicated that the Suan‐Zao‐Ren decoction treatment improved the metabolic phenotype insomnia. Moreover, the heatmap analysis identified the most important biomarkers involved in insomnia. According to the pathway analysis, phenylalanine metabolism, tryptophan metabolism, and so on were recognized as the most affected metabolic pathways associated with insomnia disease. These findings provided a comprehensive understanding of the regulative effects of Suan‐Zao‐Ren decoction on the host metabolic phenotype of the insomnia rats. Our work demonstrated that the metabolomics approach is a promising tool that could help us to conduct the exploration of the therapeutic effects and mechanism of traditional Chinese medicines.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

Pseudotargeted screening and determination of constituents in Qishen granule based on compound biosynthetic correlation using UHPLC coupled with high‐resolution MS

Detection and determination of many known/unknown compounds in traditional Chinese medicines have always been challenging. To comprehensively identify compounds in Qishen granule, which is a widely prescribed herbal formula for treating chronic heart failure, a pseudotargeted screening method was proposed based on compound biosynthetic correlation using ultra high‐performance liquid chromatography coupled with high‐resolution mass spectrometry. Firstly, all possible compounds of Qishen granule were classified into nine types according to their core skeletons, and potential analogue molecular formulas were predicted according to core compound‐related biosynthetic correlations, such as methylation, hydroxylation, and glucosidation. Secondly, nine pseudocompound databases consisting of core compounds, deduced biosynthetic correlations, and predicted analogue molecular formulas were established. Then, compounds of interest were directly located by pseudotargeted screening of high resolution mass spectrometry data and further verified by target tandem mass spectrometry. As a result, 213 constituents were identified and 21 of them were determined as potential new compounds. This demonstrated that pseudotargeted screening based on compound biosynthetic correlations significantly facilitated the processing of extremely large information data and improved the efficiency of compound identification. This research provided essential data for exploration of effective substances in Qishen granule and enriched the methodology for comprehensive characterization of constituents in complex traditional Chinese medicines.     

Comprehensive separation of iridoid glycosides and triterpenoid saponins from Dipsacus asper with salt‐containing solvent by high‐speed countercurrent chromatography coupled with recycling mode

The roots of Dipsacus asper Wall as a commonly used traditional Chinese medicine are used for tonifying liver and kidney and strengthening bones and muscles. However, an effective separation strategy for comprehensive and rapid separation of the main active compounds from the roots of D. asper is nonexistent. This investigation provided an effective separation method based on AB‐8 macroporous resin column chromatography using different ratios of ethanol in water and two different modes of high‐speed countercurrent chromatography with salt‐containing solvent system for rapid enrichment and separation from the roots of D. asper. The macroporous resin column chromatography was performed on AB‐8 resin using ethanol in water ratios of 10, 30, 40, 50, and 80% as the optimized enrichment conditions for iridoid glycosides and triterpenoid saponins with different polarities. For high‐speed countercurrent chromatography separation, the conventional and recycling modes were combined together to develop a strategy for 12 compounds ( 1 – 12 ) from the enriched parts of 30, 40, and 80% ethanol, including six high‐polarity iridoid glycosides ( 1 – 6 ) using inorganic salt‐containing solvent system and six triterpenoid saponins ( 7 – 12 ). Recycling high‐speed countercurrent chromatography separation was successfully applied to separate two isomers ( 9 and 10 ) after 11 cycles.     

Magnetic molecularly imprinted specific solid‐phase extraction for determination of dihydroquercetin from Larix griffithiana using HPLC

The naturally occurring quercetin flavonoid, dihydroquercetin, is widely distributed in plant tissues and has a variety of biological activities. Herein, a magnetic molecularly imprinted solid‐phase extraction was tailor made for selective determination of dihydroquercetin in Larix griffithiana using high‐performance liquid chromatography. Amino‐functionalized core‐shell magnetic nanoparticles were prepared and characterized using scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry, and infrared spectroscopy. The polymer had an average diameter of 250 ± 2.56 nm and exhibited good stability and adsorption for template molecule, which is enriched by hydrogen bonding interaction. Multiple factors for extraction, including loading, washing, elution solvents, and extraction time, were optimized. The limit of detection was 1.23 μg/g. The precision determined at various concentration of dihydroquercetin was less than 4% and the mean recovery was between 74.64 and 101.80%. It has therefore been shown that this protocol can be used as an alternative extraction to quantify dihydroquercetin in L. griffithiana and purify quercetin flavonoid from other complex matrices.     

中药微量元素的主成分分析

基于主成分分析的方法,研究了中药的相似性。主成分综合得分越近,中药就越相似。     

Determination of Polycyclic Aromatic Hydrocarbons in Traditional Chinese Medicine Raw Material,Extracts, and Health Food Products

The four polycyclic aromatic hydrocarbon markers (PAH4) of benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), and benzo[a]pyrene (BaP) are indicators showing polycyclic aromatic hydrocarbon (PAH) contamination levels in Chinese medicine raw materials (CMRMs), extracts and health food products; Samples of herbal medicine, herbal extracts, and food supplements were extracted with n-hexane, then cleaned up sequentially on Florisil and EUPAH solid-phase extraction (SPE) columns. A gas chromatography–mass spectrometry method for the determination of four polycyclic aromatic hydrocarbon markers in Chinese medicine raw material, extracts, and health food products was established; In spiked-recovery experiments, the average recovery was about 78.6–107.6% with a precision of 2.3–10.5%. The limit of quantification (LOQ) and limit of detection (LOD) of the PAH4 markers in this method were 2.0 μg/kg and 0.7 μg/kg, respectively. When the developed method was utilized to determine PAH4 contents in 12 locally available health food products, 3 samples contained over 10.0 μg/kg BaP, and 5 samples contained over 50.0 μg/kg PAH4. The European Union (EU) limits for BaP and PAH4 are 10 and 50.0 μg/kg, respectively; therefore, more attention must be drawn to the exposure risk of BaP and PAH4 in CMRMs, their extracts, and health food products. According to the risk assessment based on the Margin of Exposure (MOE) method, it is recognized that the products mentioned in this study pose a low risk.