Quality study of animal-derived traditional Chinese medicinal materials based on spectral technology: Calculus bovis as a case.

INTRODUCTION: Calculus bovis (C. bovis) is a typical traditional Chinese medicine (TCM) derived from animals, which has a remarkable curative effect and high price. OBJECTIVES: Rapid identification of C. bovis from different types was realized based on spectral technology, and a rapid quantitative analysis method for the main quality control indicator bilirubin was established. METHODS: We conducted a supervised and unsupervised pattern recognition study on 44 batches of different types of C. bovis by five spectral pretreatment methods. Three variable selection methods were used to extract the essential information, and the partial least squares regression (PLSR) quantitative model of bilirubin by near-infrared (NIR) spectroscopy was constructed. RESULTS: The partial least squares discriminant analysis (PLS-DA) model could achieve 100% accuracy in identifying different types of C. bovis. The R2 of the NIR quantitative model was 0.979, which is close to 1, and the root mean square error of calibration (RMSEC) was 2.3515, indicating the good prediction ability of the model. CONCLUSION: The study was carried out to further improve the basic data of quality control of C. bovis and help the high-quality development of TCM derived from animals.

Identification and determination of different processed products and their extracts of Crataegi Fructus by infrared spectroscopy combined with two-dimensional correlation analysis.

The fruit of Crataegus sp. is known as "Shanzha (SZ)" in China and is widely used in the food, beverage, and traditional Chinese medicine (TCM) industries. SZ usually requires thermal processing to reduce the irritation of its acidity to the gastric mucosa. Different processed products of SZ resulting from thermal processing have different or even opposite functions in clinical applications. In addition, 5-hydroxymethylfurfural (5-HMF) intermediates produced during thermal processing are carcinogenic to humans. Therefore, the aim of this study was to explore a rapid and accurate method by Fourier transform infrared spectroscopy (FT-IR) for the identification of different processed products and the determination of 5-HMF in extracts. In qualitative identification, a three-stage infrared spectroscopy identification method (raw spectra, the second derivative spectra, and two-dimensional correlation (2DCOS) spectra) was developed to distinguish different processed products of SZ step by step. In quantitative determination, partial least squares regression combined with different variable selection methods, especially the 2DCOS method, was applied to determine the 5-HMF content. The results show that temperature-induced 2DCOS synchronous spectra can effectively identify different processed products of SZ by shape, intensity, and position of auto-peaks or cross-peaks, and the variables selected by power spectra from concentration-induced 2DCOS synchronous spectra have better prediction ability for 5-HMF compared to full variables. The above results demonstrate that 2D-COS analysis is a potential tool in qualitative and quantitative analysis, which can improve sample identification accuracy and determination capabilities. This study not only establishes a rapid and accurate method for the identification of different processed products but also provides a practical reference for food safety and the efficient use of TCM.

Metabolic profiles of Fufang Xiling Jiedu capsule in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Fufang Xiling Jiedu capsule (FXJC), a traditional Chinese medicine that evolved from "Yinqiao Powder", is widely used for the treatment of cold and influenza. However, due to a lack of in vivo metabolism research, the chemical components responsible for the therapeutic effects still remain unclear. Hence, this study aimed to describe the metabolic profiles of the FXJC in rat plasma, urine, and feces. A combined data mining strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was employed and 201 xenobiotics, including 117 prototype components and 84 metabolites were detected. Phenolic acids, flavonoids, triterpenes, and lignans were prominent ingredients absorbed in vivo, and the major metabolic pathways of the detected metabolites were glucuronidation, sulfation, methylation, and oxidation. This is the first systematic study on the metabolism of the FXJC in vivo, providing valuable information for future studies on the efficacy, toxicity, and mechanism of the FXJC.

Study on quality analysis of different species of Coptidis rhizome based on fingerprint-effect relationship.

INTRODUCTION: The quality evaluation of Coptidis rhizome (CR) is attributed to the origin and processing method, and this strategy of ignoring the bioactive components usually leads to biased quality analysis, which is difficult to indicate the clinical efficacy. OBJECTIVES: In order to evaluate the quality level of different species of CR, we collected 20 batches of CR and investigated the fingerprint-effect relationship. METHODS: High-performance liquid chromatography (HPLC) fingerprints of CR were established, and the fingerprint-effect relationship was explored using cluster analysis, principal component analysis, Pearson correlation analysis, grey relation analysis, and partial least squares regression. RESULTS: We have identified a total of 10 common peaks (1-10) with similarity scores above 0.96. The study on the relationship between spectra and potency further showed that the contents of peaks 8, 9, and 10 are potential key components. And based on a previous study, a method of one measurement and multiple evaluations of CR was established to achieve the goal of simplifying the analytical process and reducing costs. CONCLUSION: Through a combination of fingerprint analysis, antioxidant activity evaluation, fingerprint-efficacy relationship analysis, and simultaneous quantification of multiple components, a CR quality control index and method have been selected and established, which can also provide a more comprehensive quality evaluation for traditional Chinese medicine.

Separation and purification of quinolyridine alkaloids from seeds of Thermopsis lanceolata R. Br. by conventional and pH-zone-refining counter-current chromatography.

In this work, the preparative separation of quinolyridine alkaloids from seeds of T. lanceolata by conventional and pH-zone-refining counter-current chromatography. Traditional counter-current chromatography separation was performed by a flow-rate changing strategy with a solvent system of ethyl acetate-n-butanol-water (1:9:10, v/v) and 200 mg sample loading. Meanwhile, the pH-zone-refining mode was adopted for separating 2.0 g crude alkaloid extracts with the chloroform-methanol-water (4:3:3, v/v) solvent system using the stationary and mobile phases of 40 mM hydrochloric acid and 10 mM triethylamine. Finally, six compounds, including N-formylcytisine (two conformers) (1), N-acetycytisine (two conformers) (2), (-)-cytisine (3), 13-ß-hydroxylthermopsine (4), N-methylcytisine (5), and thermopsine (6) were successfully obtained in the two counter-current chromatography modes with the purities over 96.5%. Moreover, we adopted nuclear magnetic resonance and mass spectrometry for structural characterization. Based on the obtained findings, the pH-zone-refining mode was the efficient method to separate quinolyridine alkaloids relative to the traditional mode.

Qualitative and quantitative determination of chemical constituents in Jinbei oral liquid, a modern Chinese medicine for coronavirus disease 2019, by ultra-performance liquid chromatography coupled with mass spectrometry.

Introduction: Traditional Chinese medicine (TCM) has the advantages of syndrome differentiation and rapid determination of etiology, and many TCM prescriptions have been applied to the clinical treatment of coronavirus disease 2019 (COVID-19). Among them, Jinbei Oral Liquid (Jb.L) has also shown an obvious curative effect in the clinic, but the related material basic research is relatively limited. Methods: Therefore, in this process, a systematic data acquisition and mining strategy was established using ultra-high- performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Results and Discussion: With the optimized conditions, a total of 118 peaks were tentatively characterized, including 43 flavonoids, 26 phenylpropanoids, 14 glycosides, 9 phthalides, 8 alkaloids and others. To determine the content of relevant pharmacological ingredients, we firstly exploited the ultra-performance liquid chromatography method coupled with triple-quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method for simultaneous detection of 31 active ingredients within 17 min, and the validation of methodology showed that this method has good precision and accuracy. Moreover, analyzing the pharmacology of 31 individual of the medicinal material preliminarily confirmed the efficacy of Jb.L and laid a foundation for an in-depth study of network pharmacology.

Cardioprotective Effects of Tetrahydropalmatine on Acute Myocardial Infarction in Rats.

Tetrahydropalmatine (THP) is an active component of Corydalis yanhusuo W. T. Wang. The current study investigates the possible cardioprotective effects of tetrahydropalmatine in acute myocardial ischemia (AMI) rats. The anterior descending coronary artery of SD rats was ligated to establish an AMI model. After two weeks of gavage of THP, cardiac function was determined by echocardiography. The organ index and the infarct size were assessed after the experiment, and the histopathological myocardial tissue changes were observed. In addition, the apoptosis index of myocardial cells was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The levels of SOD, MDA, CAT, GSH-Px, BNP, and cTn-I were measured by enzyme-linked immunosorbent assay. To determine relevant proteins, the Western blot and molecular docking were applied. Compared with the model group, THP could enhance rat cardiac ejection function to improve cardiac function, drastically lessen the infarct size, reduce myocardial cell damage and inflammatory cell infiltration. THP might also prevent ischemic myocardial damage by inhibiting myocardial cell apoptosis and efficiently reducing oxidative stress. Specifically, THP could decrease MDA, BNP, c-TnI activities, as well as the expression of Bax and Caspase-3 protein, while increasing SOD, GSH-Px, CAT activities, and Bcl-2 level. Furthermore, THP could significantly promote the phosphorylation of PI3K and Akt proteins. The involved pathways and proteins have also been verified through molecular docking. According to these findings, THP may preserve the myocardium due to its anti-oxidant and anti-apoptotic properties.

Characterization of Silver Nanoparticles Synthesized by Leaves of Lonicera japonica Thunb.

Background: The leaves of L. japonica (LLJ) are widely used as medicine in China. It is rich in caffeoylquinic acids, flavonoids and iridoid glycosides and has strong reducing capacities. Therefore, it can be used as a green material to synthesize silver nanoparticles. Methods: LLJ was used as a reducing agent to produce the LLJ-mediated silver nanoparticles (LLJ-AgNPs). The structure and physicochemical properties of LLJ-AgNPs were characterized by ultraviolet spectroscopy (UV-Vis), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and x-ray powder diffraction (XRD). Antioxidant activity of LLJ-AgNPs was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging. Antibacterial activity was determined by 96 well plates (AGAR) gradient dilution, while the anticancer potential was determined by MTT assay. Results: The results showed LLJ-AgNPs had a spherical structure with the maximum UV-Vis absorption at 400 nm. In addition, LLJ-AgNPs exhibited excellent antioxidant properties, where the free radical scavenging rate of LLJ-AgNPs was increased from 39% to 92% at concentrations from 0.25 to 1.0 mg/mL. Moreover, LLJ-AgNPs displayed excellent antibacterial properties against E. coli and Salmonella at room temperature, with minimum inhibitory values of 10-6 and 10-5 g/L, respectively. In addition, the synthetic LLJ-AgNPs exhibited a better inhibition effect in the proliferation of cancer cells (HepG2, MDA-MB -231, and Hela cells). Conclusion: The present study provides a green approach to synthesize LLJ-AgNPs. All those findings illustrated that the produced LLJ-AgNPs can be used as an economical and efficient functional material for further applications in food and pharmaceutical fields.

An efficient high-speed counter-current chromatography method for the preparative separation of potential antioxidants from Paeonia lactiflora Pall. combination of in vitro evaluation and molecular docking.

Paeonia lactiflora Pall., one of the most famous classical herbal medicine, has been used to treat diseases for over 1200 years. In this research, the functional ingredients were purified by online-switch two-dimensional high-speed counter-current chromatography combined with inner-recycling and continuous injection mode. The antioxidant activity was evaluated by investigating the 2,2'-azobis (2-amidinopropane) dihydrochloride-induced oxidant damage in vitro and confirmed through molecular docking. n-Butanol/ethyl acetate/water (2:3:5, v/v) solvent system was used for the first-dimensional separation and optimized the sample loading. Two pure compounds and a polyphenol-enriched fraction were separated. The polyphenol-enriched fraction was separated with a solvent system n-hexane/ethyl acetate/methanol/water (2:8:4:6, v/v) with continuous injection mode. Five compounds were successfully separated, including gallic acid (1), methyl gallate (2), albiflorin (3), paeoniflorin (4), and ethyl gallate (5). Their structures were identified by mass spectrometry and NMR spectroscopy. The results from the antioxidant effect showed that albiflorin had stronger antioxidant activity. Molecular docking results indicated that the affinity energy of the identified compounds ranged from -3.79 to -8.22 kcal/mol and albiflorin showed the lowest affinity energy. Overall, all those findings suggested that the strong antioxidant capacity of albiflorin can be potentially used for the treatment of diseases caused by oxidation.

Anti-pandemic influenza A (H1N1) virus potential of Xilingjiedu capsule in vitro.

This study is aimed to investigate the effect of Xilingjiedu capsule (XLC), one of a preparation of traditional Chinese medicine, on influenza A (H1N1) virus as well as its preliminary mechanism. The median cell mortality (TC50) to A549 cells and half effective inhibition concentration (IC50) of influenza A (H1N1) virus of XLC were determined by MTT assay. Reed-Muench method was used to calculated the 50% tissue culture infective dose (TCID50) of H1N1 virus to A549 cells. In mechanism research, the mRNA expression levels of MyD88, TLR4, TLR7 and TRAF6 and the protein expression level of MyD88 were detected by using RT-PCR and Western blot, respectively. The results suggested that XLC showed good anti influenza A (H1N1) virus activity. The antiviral mechanism of XLC was related to the Toll-like signaling pathway. It could drown regulate the mRNA expression level of MyD88 and TLR4 and the protein level of MyD88. This research provides reference for the application of XLC in anti influenza virus.

Ferruginol prevents degeneration of dopaminergic neurons by enhancing clearance of α-synuclein in neuronal cells.

Lewy bodies are characteristic spherical inclusions in Parkinson's disease (PD) that are formed by α-synuclein fibrils. Ferruginol (Fer) is an amonomeric compound isolated from a traditional Chinese herb. Here, we show that Fer exerted potent neuroprotective effects in both in vitro and in vivo PD models. Neuronal cells transfected with A53T mutant (A53T) α-synuclein plasmids and treated with Fer exhibited attenuated the cytotoxicity induced by pathogenic A53T α-synuclein overexpression. Further, when we transfected neuronal cells with siRNA-SNCA (alpha-synuclein) plasmids and incubated them with Fer, the protective role of Fer decreased. We also found that Fer was a potent α-synuclein inhibitor in neuronal cells, which promotes the clearance of αsynuclein in dopaminergic neurons exposed to 1-Methyl-4-phenylpyridinium (MPP +). Fer could inhibit abnormal α-synuclein aggregation and dopaminergic neuron depletion in A53T-Tg mice, suggesting that a role for Fer in α-synuclein accumulation and nigrostriatal pathway injury. Our study revealed that Fer strongly alleviated neurodegeneration by promoting α-synuclein clearance, indicating a neuroprotective role against α-synuclein oligomer-induced neurodegeneration, which makes it a promising candidate for the treatment of PD and other neurodegenerative diseases.

The rapid identification of chemical constituents in Fufang Xiling Jiedu capsule, a modern Chinese medicine, by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry and data mining strategy.

Fufang Xiling Jiedu capsule is an effective Chinese medicine widely used for the treatment of cold and influenza. However, its chemical constituents had not been determined, which entailed a huge obstacle to further pharmacological studies, clinical-safe medication administration, and quality evaluation. To identify the chemical constituents in Fufang Xiling Jiedu capsule, an efficient and systematic approach using ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry in conjunction with a data mining strategy was adopted in this study. As a result, 145 compounds were qualitatively identified, including 26 phenolic acids, 46 flavonoids, 39 triterpenes, and 34 other compounds, among which 6 were potentially new and 144 were being reported from Fufang Xiling Jiedu capsule for the first time. This research not only provides useful information for quality control of Fufang Xiling Jiedu capsule and its involved single herbs but also serve as basis data for further study of Fufang Xiling Jiedu capsule in vivo. Moreover, it provides a reference for the characterization of the chemical constituents of other Chinese medicine preparations.

Chinese Medicine She-Xiang-Xin-Tong-Ning, Containing Moschus, Corydalis and Ginseng, Protects from Myocardial Ischemia Injury via Angiogenesis.

The Chinese patent medicine She-Xiang-Xin-Tong-Ning (SXXTN) is a clinical medication for coronary heart disease (CHD) and angina pectoris. This study aimed to investigate pharmacological effects of SXXTN and elucidate the role in angiogenesis on human umbilical vein endothelial cells (HUVECs) and acute myocardial ischemia (AMI) rats. We prepared SXXTN to treat the cells to reveal their effects on oxidative stress-damaged cell viability, as well as cell proliferation, migration, and tube formation processes. SXXTN was also used to treat coronary artery ligation-induced acute myocardial ischemia rats to confirm whether it had positive effect on myocardial issues by hematoxylin and eosin (HE), 2,3,5-triphenyltetrazolium chloride (TTC) staining and immunohistochemical staining. We measured the levels of peroxidative damage-related enzymes in cytoplasm and serum by biochemical kits and detected vascular endothelial growth factor (VEGF), angiotensin II (Ang II), thromboxane B2 (TXB2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1α) levels in cells and rats by enzyme-linked immunosorbent assay (ELISA) kits. The results showed that SXXTN protects HUVECs against oxidative stress damage and reversed the decrease of superoxide dismutase (SOD), glutathione (GSH) and increase of creatine kinase (CK), lactate dehydrogenase (LDH) caused by oxidative stress. SXXTN promoted angiogenesis through stimulating cell migration, tube formation, and activating VEGF/VEGFR2 and ERK1/2 pathways. Furthermore, SXXTN reduced infarct size and inhibited PGI2/TXA2 imbalance, preventing atherosclerosis plaque rupture leading to worsening coronary heart disease. Taken together, we report the first in vivo and in vitro evidence that SXXTN reduced oxidative stress-mediated damage and enhanced angiogenesis, which might be useful in treatment of myocardial infarction.

A novel acylated quercetin glycoside and compounds of inhibitory effects on α-glucosidase from Panax ginseng flower buds.

A novel acylated quercetin glycoside, floralpanasenoside A (1) and five known flavonoid glycosides, panasenoside (2), quercetin 3-O-(2''-ß-D-glucopyranosyl)-ß-D- galactopyranoside (3), trifolin (4) kaempferol 7-O-α-L-rhamnoside (5), and afzelin (6) were isolated from the flower buds of Panax ginseng. Their structures were established by spectroscopic data and comparison with the literature values. Four of the six isolated compounds including 1 (IC50 = 62.4) exhibited α-glucosidase inhibitory activity with IC50 values lower than acarbose (385.2 µM). The molecular docking study indicated that 1 bound to the active site of α-glucosidase with numerous hydrogen bond interactions.

Cytotoxic epimeric ginsenosides from the flower buds of Panax ginseng.

Three pairs of ginsenoside epimers, including three new compounds (2, 3 and 5), were isolated from the flower buds of Panax ginseng. The structures of the isolated compounds were elucidated on the basis of considerable spectroscopic analyses and comparison with the reported data. All six compounds were evaluated for their cytotoxicties against three human cancer cell lines, HL-60, MGC80-3 and Hep-G2. Compounds 1, 3, and 6 with S configurations at C-24 or C-20 showed moderate inhibitory activities with IC50 values of 25.32, 18.76, and 38.64 µM in HL-60 cells, respectively. Our findings showed that different configurations of these isolated ginsenosides had a significant impact on the antitumor activity, and S epimers were higher than R.

[Chemical constituents of liquid culture of symbiotic Chaetomium globosum ML-4 of oyster and their in vitro antitumor activity].

Isolation and purification of chemical constituents of liquid culture of symbiotic Chaetomium globosum ML-4 of oyster was performed through silica gel column chromatography, gel filtration over Sephadex LH-20, preparative TLC and HPLC. Five compounds were obtained and their structures were determined as chaetoglobosin V(1), chaetoglobosin Vb(2), tyrosol(3), 5-methyluracil(4)and uracil(5), respectively, based on HR-MS and NMR data and comparison with literatures. In vitro cytotoxicity of compounds against human hepatocellular carcinoma cell line SMMC-7721 were measured byMTT method, and results showed that compound 1 could obviously inhibit the proliferation of SMMC-7721 cells with an IC50 value of 60.5 mg•L⁻¹, while the IC50 value of positive control cisplatin was 19.96 mg•L⁻¹. Further studies discovered that compound 1 could lead to G2 phase arrest in SMMC-7721 cells and induce SMMC-7721 cells apoptosis. The ratio of Bcl-2/Bax in SMMC-7721 cells was decreased. The expression of protein Caspases-3,-8,-9 was improved and the expression and phosphorylation level of Akt were reduced. Aforementioned results revealed that in vitro antitumor activity of compound 1 against SMMC-7721 cells were related to G2 phase cell cycle arrest and induced-apoptosis. The induced-apoptosis was involved in both the mitochondrial pathway and the death receptor pathway and connected with activity decline of PI3K/Akt signaling pathway.

New inhibitors of nitric oxide production from the seeds of Myristica fragrans.

Six dihydrobenzofuran type neolignans were isolated from the dried ripe seeds of Myristica fragrans Houtt. (family: Myristicaceae) and their chemical structures were identified as licarin B (1), 3'-methoxylicarin B (2), myrisfrageal A (3), isodihydrocainatidin (4), dehydrodiisoeugenol (5), and myrisfrageal B (6), respectively, on the basis of spectroscopic data analyses. Among them, compounds 3 and 6 are new compounds. Compounds 1-6 showed inhibition of nitric oxide production in lipopolysaccharide-activated murine monocyte-macrophage RAW264.7 with IC50 values of 53.6, 48.7, 76.0, 36.0, 33.6, and 45.0 µM, respectively. These values were compared to those of the positive controls, indomethacin and L-N(6)-(1-iminoethyl)-lysine, which have IC50 values of 65.3 and 27.1 µM, respectively. Further compounds 3, 5 and 6 suppressed LPS-induced iNOS mRNA expression in a does-dependent manner in RAW 264.7 cells assayed by real-time RT-PCR. Compounds 3, 5 and 6 may inhibit NO overproduction via inhibition of iNOS mRNA expression. The results provided valuable information for further investigation of compounds 1-6 as anti-inflammatory and chemopreventive agents.