Natural Xanthine Oxidase Inhibitor 5-O-Caffeoylshikimic Acid Ameliorates Kidney Injury Caused by Hyperuricemia in Mice.

Xanthine oxidase (XOD) inhibition has long been considered an effective anti-hyperuricemia strategy. To identify effective natural XOD inhibitors with little side effects, we performed a XOD inhibitory assay-coupled isolation of compounds from Smilacis Glabrae Rhizoma (SGR), a traditional Chinese medicine frequently prescribed as anti-hyperuricemia agent for centuries. Through the in vitro XOD inhibitory assay, we obtained a novel XOD inhibitor, 5-O-caffeoylshikimic acid (#1, 5OCSA) with IC50 of 13.96 µM, as well as two known XOD inhibitors, quercetin (#3) and astilbin (#6). Meanwhile, we performed in silico molecular docking and found 5OCSA could interact with the active sites of XOD (PDB ID: 3NVY) with a binding energy of -8.6 kcal/mol, suggesting 5OCSA inhibits XOD by binding with its active site. To evaluate the in vivo effects on XOD, we generated a hyperuricemia mice model by intraperitoneal injection of potassium oxonate (300 mg/kg) and oral gavage of hypoxanthine (500 mg/kg) for 7 days. 5OCSA could inhibit both hepatic and serum XOD in vivo, together with an improvement of histological and multiple serological parameters in kidney injury and HUA. Collectively, our results suggested that 5OCSA may be developed into a safe and effective XOD inhibitor based on in vitro, in silico and in vivo evidence.

[Effect of "Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma" in treatment of lung adenocarcinoma based on network pharmacology].

To explore the mechanism of Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma(HDH-SGR) in treating lung adenocarcinoma based on big data bioinformatics combined with network pharmacology analysis and molecular docking technology. The chemical components and potential therapeutic targets of HDH-SGR were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Lung adenocarcinoma-related genes were obtained from The Cancer Genome Atlas(TCGA), Therapeutic Target Database(TTD), Pharmacogenetics and Pharmacogenomics Knowledge Base(PharmGKB), Online Mendelian Inheritance in Man(OMIM), DrugBank, and GeneCards. "Drug component-target" network was constructed using Cytoscape to screen out key compounds. STRING was used to build protein-protein interaction(PPI) network and core targets were screened out by Cytoscape-CytoNCA topology analysis. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analyses of target genes were performed by R-clusterProfiler. Finally, key compounds were docked to core target genes using AutoDock. The results showed that 22 active compounds and 499 potential therapeutic targets were obtained from HDH-SGR. A total of 14 332 lung adenocarcinoma-related targets were screened out through six data platforms, including 182 common targets. Fifteen core targets were screened out from the PPI network. GO and KEGG analyses revealed significant enrichment of relevant target genes in various biological processes, cellular functions(e.g., response to lipopolysaccharide, nuclear receptor activity, and ligand-activated transcription factor activity) and close relationship between target genes and non-small cell lung cancer signaling pathways. Based on the results of molecular docking validation, diosgenin, quercetin, naringenin, taxifolin, 2-methoxy-3-methyl-9,10-anthraquinone, stigmasterol, and ß-sitosterol were able to bind tightly to the core targets. HDH-SGR can intervene in lung adenocarcinoma through multiple targets and signaling pathways, such as non-small cell lung cancer signaling pathways. The binding of active components in Chinese medicine to key targets is presumedly one of the mechanisms that produce therapeutic effects.

Rapid identification of new minor chemical constituents from Smilacis Glabrae Rhizoma by combined use of UHPLC-Q-TOF-MS, preparative HPLC and UHPLC-SPE-NMR-MS techniques.

INTRODUCTION: Herbs are an important resource for new drug development. However, the conventional approach for the discovery of new compounds from herbs was time-consuming, tedious, and inefficient. OBJECTIVES: Establish a quick approach to identify new minor constituents in herbs. METHODS: The constituents in herbs were firstly analysed using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Based on the accurate masses, isotopic ions, and the characteristic fragmentation ions in the mass spectra, the molecular compositions and possible structures of compounds were first deduced. After being enriched by a preparative HPLC method, the potential new minor structures were definitely identified by an on-line UHPLC-solid phase extraction-nuclear magnetic resonance-mass spectrometry (UHPLC-SPE-NMR-MS) approach. RESULTS: By combined the use of UHPLC-Q-TOF-MS, preparative HPLC and UHPLC-SPE-NMR, three new minor compounds were definitely identified as bis-3,4-dihydroxyphenylpropanoid-substituted catechins (A2 and A3) and 4″-formyl-astilbin (B5). In addition, five isomers of bis-dihydroxyphenylpropanoid-substituted catechin (A1, A4-A7), four isomers of 4″-formyl-astilbin (B1-B4), engeletin formates and isomers (C1-C5), formyl-cinchonains (D1-D4), formyl-caffeoylshikimic acid (E1-E4) were also tentatively determined by MS and MS/MS characterisation. CONCLUSION: The combination of UHPLC-Q-TOF-MS, preparative HPLC and UHPLC-SPE-NMR-MS techniques is a quick and effective approach for finding new minor constitutes from herbs.

Advances in the chemical constituents, pharmacological activity, and clinical application of Smilacis Glabrae Rhizoma: A review and predictive analysis of quality markers (Q-markers).

Smilacis Glabrae Rhizoma (SGR) is recognized in traditional Chinese medicine for its distinctive therapeutic properties and abundant supply. Its phytochemical profile is diverse, encompassing flavonoids, steroids, saccharides, phenolic glycosides, volatile constituents, organic acids, phenylpropanoids, stilbenoids, among others. Recent pharmacological investigations reveal that SGR possesses a broad spectrum of pharmacological effects with multifaceted clinical applications. This review collates the current knowledge on SGR's chemical composition, pharmacological activities, and its clinical utility. Utilizing network pharmacology and molecular docking approaches, this study provides a preliminary identification of potential quality markers (Q-Markers) within SGR. The findings suggest that compounds such as astilbin, isoengelitin, neoisoastilbin, neoastilbin, astragaloside, diosgenin, resveratrol, stigmasterol, ß-sitosterol, and quercetin in SGR are promising candidates for Q-Markers. While flavonoids are the most extensively studied, there is a pressing need to further explore the active monomeric compounds within SGR. The introduction of Q-Markers is instrumental in developing standardized quality metrics. Specifically, astilbin has been noted for its antitumor, antidiabetic, antihypertensive, anti-hyperuricemic, and hepatoprotective potential, warranting further research for therapeutic applications.

Influence of different pretreatments and drying methods on the chemical compositions and bioactivities of Smilacis Glabrae Rhizoma.

BACKGROUND: The processing of medicinal plant materials is one of the important factors influencing the components and biological activities of TCMs. Smilax glabra Roxb. is an herbal vine widely distributed in China, and its dried rhizome (Smilacis Glabrae Rhizoma, SGR) is often used in traditional medicines and functional foods. The processing methods of fresh cutting for SGR slices have been included in ancient Chinese herbal works, some local standards of TCMs, and the current Chinese Pharmacopoeia. Nevertheless, to date, the scientific basis for the processing of fresh medicinal materials for SGR slices has not been revealed. METHODS: To optimize the processing method for preparing SGR slices from the fresh rhizomes, the chemical compositions of the un-pretreated and pretreated (boiling, steaming) samples before and after drying (sun-drying, shade-drying, oven-drying), and the contents of astilbin isomers in dried SGR were analyzed by UHPLC-Q-TOF-MS/MS and UHPLC-DAD methods, respectively. Then, the antioxidant, anti-inflammatory, xanthine oxidase and α-glucosidase inhibitory activities of the prepared SGR slices were investigated by biological assays. RESULTS: A total of fifty-two compounds were identified from the un-pretreated and pretreated samples and a total of forty-nine compounds were identified from the subsequently dried samples. After pretreated by boiling and steaming, the contents of neoastilbin, neoisoastilbin, and isoastilbin in the prepared samples all increased. As a quality marker of SGR, the content of astilbin was unchanged or decreased slightly compared with that in the un-pretreated samples. During the drying process, the contents of the four astilbin stereoisomers in the un-pretreated samples increased significantly, while those in the pretreated samples had a slight increase or decrease. The effects of different processing methods were sorted according to the bioactivities of the prepared SGR. As a result, SGR slices prepared with no pretreatment followed by a sun-drying process have a higher astilbin content, better bioactivities and more energy savings, representing the optimum processing method for SGR slices. CONCLUSIONS: This study reveals the scientific basis for the processing of fresh medicinal materials for SGR slices. The results provide scientific information for the quality control of SGR and its rational applications in herbal medicines and functional foods.

Chemical profiles, antioxidant activity and acute toxicity of raw and sulfur-fumigated Smilacis Glabrae Rhizoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilacis Glabrae Rhizoma (SGR), known as Tu-fu-ling in the China, Japan and Korea, is an herb that has been used for clearing damp and detoxification in traditional Chinese medicine for many years. The post-harvest drying of SGR has traditionally been done by the sun, but sometimes sulfur fumigation is used instead due to its low cost and high efficiency. Recent reports show that sulfur fumigation can change the chemical constitution of herbal medicines and decrease their biology activity. AIM OF THE STUDY: This study will investigate the changes to the chemical constitution, acute toxicity and antioxidant potential of SGR that occur after sulfur fumigation. To date, no studies have investigated these aspects simultaneously. MATERIALS AND METHODS: An ultra-performance liquid chromatography fingerprint method was developed for analysing changes to SGR's chemical constitution caused by sulfur fumigation. The chromatography conditions were as follows: all samples were analysed on a Waters Acquity UHPLC HT3 C18 column; the linear gradient elution was conducted with a mobile phase prepared from acetonitrile and water. All calibration curves showed good linear regression (R > 0.9991) within the tested range. The method was validated for precision, accuracy, limit of detection and quantification. Total flavonoids of the raw and sulfur-fumigated samples were also determined by ultraviolet spectrophotometry. The antioxidant properties of the extracts were evaluated using both DPPH and ABTS radical scavenging assays. The acute toxicities of the raw and sulfur-fumigated samples were investigated. RESULTS: The results demonstrate that the amounts of astilbin, neoastilbin, neoisoastilbin, isoastilbin, resveratrol and total flavonoids were lower in sulfur-fumigated samples than in raw samples. The antioxidant activity of the sulfur-fumigated samples was also significantly lower. Therefore, sulfur fumigation may cause chemical transformation, alter the chemical constitution, and decrease the bioactivity of SGR. Orally-administered doses did not cause mortality or changes in the general behaviour of tested mice. The LD50 was > 5000 mg/kg DW. However, the high-dose S-SGR mice had significant liver damage and high levels of plasma biochemical parameters (ALT, AST, DBIL, TBIL). CONCLUSIONS: The results of the current study suggest that sulfur fumigation can decrease antioxidant activity in vitro; and that orally-administrated S-SGR is unsafe at doses > 3000 mg/kg dried materia medica. Therefore, sulfur-fumigation processing should be forbidden for SGR until its efficacy and safety has been demonstrated. An alternative method of sulfur fumigation for the post-harvest processing of SGR should also be developed.

Assessing the quality of Smilacis Glabrae Rhizoma (Tufuling) by colormetrics and UPLC-Q-TOF-MS.

BACKGROUND: The quality of the materials used in Chinese medicine (CM) is generally assessed based on an analysis of their chemical components (e.g., chromatographic fingerprint analysis). However, there is a growing interest in the use of color metrics as an indicator of quality in CM. The aim of this study was to investigate the accuracy and feasibility of using color metrics and chemical fingerprint analysis to determine the quality of Smilacis Glabrae Rhizoma (Tufuling) (SGR). The SGR samples were divided into two categories based on their cross-sectional coloration, including red SGR (R-SGR) and white SGR (W-SGR). METHODS: Forty-three samples of SGR were collected and their colors were quantized based on an RGB color model using the Photoshop software. An ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/QTOF MS) system was used for chromatographic fingerprint analysis to evaluate the quality of the different SGR samples. Hierarchical cluster analysis and dimensional reduction were used to evaluate the data generated from the different samples. Pearson correlation coefficient was used to evaluate the relationship between the color metrics and the chemical compositions of R-SGR and W-SGR. RESULTS: The SGR samples were divided into two different groups based on their cross-sectional color, including color A (CLA) and B (CLB), as well as being into two separate classes based on their chemical composition, including chemical A (CHA) and B (CHB). Standard fingerprint chromatograms were for CHA and CHB. Statistical analysis revealed a significant correlation (Pearson's r = -0.769, P < 0.001) between the color metrics and the results of the chemical fingerprint analysis. CONCLUSIONS: The SGR samples were divided into two major clusters, and the variations in the colors of these samples reflected differences in the quality of the SGR material. Furthermore, we observed a statistically significant correlation between the color metrics and the quality of the SGR material.

Effect of "Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma" in treatment of lung adenocarcinoma based on network pharmacology / 中国中药杂志

To explore the mechanism of Hedyotis Diffusae Herba-Smilacis Glabrae Rhizoma(HDH-SGR) in treating lung adenocarcinoma based on big data bioinformatics combined with network pharmacology analysis and molecular docking technology. The chemical components and potential therapeutic targets of HDH-SGR were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Lung adenocarcinoma-related genes were obtained from The Cancer Genome Atlas(TCGA), Therapeutic Target Database(TTD), Pharmacogenetics and Pharmacogenomics Knowledge Base(PharmGKB), Online Mendelian Inheritance in Man(OMIM), DrugBank, and GeneCards. "Drug component-target" network was constructed using Cytoscape to screen out key compounds. STRING was used to build protein-protein interaction(PPI) network and core targets were screened out by Cytoscape-CytoNCA topology analysis. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) analyses of target genes were performed by R-clusterProfiler. Finally, key compounds were docked to core target genes using AutoDock. The results showed that 22 active compounds and 499 potential therapeutic targets were obtained from HDH-SGR. A total of 14 332 lung adenocarcinoma-related targets were screened out through six data platforms, including 182 common targets. Fifteen core targets were screened out from the PPI network. GO and KEGG analyses revealed significant enrichment of relevant target genes in various biological processes, cellular functions(e.g., response to lipopolysaccharide, nuclear receptor activity, and ligand-activated transcription factor activity) and close relationship between target genes and non-small cell lung cancer signaling pathways. Based on the results of molecular docking validation, diosgenin, quercetin, naringenin, taxifolin, 2-methoxy-3-methyl-9,10-anthraquinone, stigmasterol, and β-sitosterol were able to bind tightly to the core targets. HDH-SGR can intervene in lung adenocarcinoma through multiple targets and signaling pathways, such as non-small cell lung cancer signaling pathways. The binding of active components in Chinese medicine to key targets is presumedly one of the mechanisms that produce therapeutic effects.

Simultaneous Determination of Twelve Constituents in Yigan Jiedu Capsules by HPLC / 中国药学杂志

OBJECTIVE: To develop a method for determining 12 constituents in Yigan Jiedu Capsules by HPLC. METHODS: The quantitative analysis was carried out on a Waters Atlantic T3 C18column maintained at 40 ℃, using mobile phase consisting of acetonitrile(A)and 0.1% phosphoric acid solution(B)by a gradient elution program(0-10 min, 4%→13%A; 10-20 min, 13%→15%A; 20-25 min, 15%→19%A; 25-35 min, 19%A; 35-36 min, 19%→23%A; 36-45 min, 23%→27%A; 45-56 min, 27%→43%A; 56-65 min, 43%→65%A) at a flow rate of 1 mL•min-1. The detection wavelength was set at 270 nm in the first 22 min, and then changed to 290 nm between 22 and 44 min, and last changed to 270 nm. RESULTS: The linear ranges of gallic acid, vanillic acid, picroside Ⅱ, astilbin, engeletin, picroside, baicalin, berberine hydrochloride, wogonoside, emodin, chrysophanol and wogonin fell with in the range of 0.012 19-0.243 8 μg, 0.036 70-0.734 0 μg, 0.033 77-0.675 4 μg, 0.057 38-1.147 6 μg, 0.006 580-0.131 6 μg, 0.015 38-0.307 6 μg, 0.067 11-1.342 2 μg, 0.028 04-0.560 8 μg, 0.041 48-0.829 6 μg, 0.006 310-0.126 2 μg, 0.013 64-0.272 8 μg and 0.014 22-0.284 4 μg. The recoveries were 98.7%, 98.9%, 99.4%, 99.2%, 98.9%, 99.1%, 98.9%, 99.6%, 99.3%, 98.9%, 98.6% and 99.0%, respectively. The relative standard deviations were 1.02%, 0.46%, 0.64%, 0.86%, 1.1%, 1.3%, 0.60%, 0.92%, 0.61%, 1.5%, 0.93%and 0.98%, respectively(n=6). CONCLUSION: This method is simple, accurate, reproducible and convenient for the quality control over Yigan Jiedu Capsules.

High-Performance Liquid Chromatography Fingerprint Analysis of Smilacis Glabrae Rhizoma and Its Adulterants / 中国药学杂志

OBJECTIVE: To investigate the chemical differences between Smilacis Glabrae Rhizoma and its adulterants, and provide a basis for the identification and quality evaluation of Tufuling samples purchased from the markets. METHODS: The method of HPLC fingerprinting was used to analyze Smilacis Glabrae Rhizoma, Heterosmilacis Chinensis Rhizoma, Heterosmilacis Yunnanensis Rhizoma, and 102 Tufuling samples which were collected around China. The fingerprints were analyzed by the methods of similarity and principal component analysis (PCA). RESULTS: The analytical method of HPLC fingerprinting was established. Eleven, fifteen and eight common peaks were selected in the fingerprints of Smilacis Glabrae Rhizoma, Heterosmilacis Chinensis Rhizoma, and Heterosmilacis Yunnanensis Rhizoma, respectively. Only five common peaks were found in the fingerprints of the three species, which were No. 1, 2, 3, 13, and 14 peaks. A total of twelve peaks were characterized in the three fingerprints. Nine peaks were characterized in the fingerprint of Smilacis Glabrae Rhizoma, among which, four constituents were characterized for the first time. Six and two constituents were for the first time characterized in the fingerprints of Heterosmilacis Chinensis Rhizoma and Heterosmilacis Yunnanensis Rhizoma, respectively. The result of PCA analysis showed that the chemical differences between the three species were quite obvious and they could be distinguished from each other. The established method was used for the analysis of Tufuling samples purchased from the markets. Sixty-five samples were identified as Smilacis Glabrae Rhizoma, seventeen samples were identified as Heterosmilacis Yunnanensis Rhizoma, and twenty samples were identified as Heterosmilacis Chinensis Rhizoma. CONCLUSION: The established method is simple and reliable, and the development of fingerprint and its chemical pattern recognition provide the way and basis for identification of Smilacis Glabrae Rhizoma and its adulterants.

Optimization of the Extraction Technology of Astilbin in Puling Penyankang Capsules by Orthogonal Test / 中国药房

OBJECTIVE:To optimize the extraction technology of astilbin from medicinal herbs in Puling penyankang cap-sules. METHODS:The extraction technology of astilbin from ingredients(Smilacis glabrae rhizoma,chuanxiong rhizoma,Eucom-miae cortex,notoginseng radix et rhizoma,Plantaginis semen)of Puling penyankang capsules was optimized with concentration of ethanol,immersion time and percolation speed as factors,and using the yield of extractum and the extraction amount of astilbin as index. RESULTS:The optimized extraction technology was as follows as 2-fold 70% ethanol,immersed for 24 h,percolated with 70% ethanol with percolation speed of 3 ml/min,10-fold percolate volume was colleted. In verification test,the yield of extractum were 6.79%,6.92% and 6.84%,respectively,with average value of (6.85 ± 0.96)%(n=3);the extraction amounts of astilbin were 39.23,39.67 and 39.69 mg,with average value of (39.53 ± 0.66) mg (n=3). CONCLUSIONS:The optimized extraction technology of astilbin in Puling penyankang capsules is stable and practical.

Development and comparative evaluation of the UPLC and HPLC methods for the determination of dihydroflavonoids in Smilacis glabrae Rhizoma / 中国药科大学学报

Aim: To develop and compare the ultra-performance liquid chromatography( UPLC) and HPLC methods for the determination of dihydroflavonoids in Smilacis glabrae Rhizoma, and establish the quality evaluation system of the above-mentioned crude drug. Methods: Four dihydroflavonoids in the crude drugs collected from 15 localities were determined using the UPLC and HPLC methods, respectively. The resolution, sensitivity, precision, accuracy and the content determination results of the four compounds were compared between the two methods. Results: The UPLC method was more fast and sensitive than the HPLC method with no significant differences among the linearity range, precision, accuracy and the content determination results between the two methods. Conclusion: The developed HPLC method was proved practicable and reliable for the quality control of Smilacis glabrae Rhizoma. The UPLC method was provided to be a more sensitive, fast and solvent-saving method compared to HPLC and can be applied in the quality evaluation of Chinese medicines.