Fractional Deletion of Compound Kushen Injection Indicates Cytokine Signaling Pathways are Critical for its Perturbation of the Cell Cycle.

We used computational and experimental biology approaches to identify candidate mechanisms of action of aTraditional Chinese Medicine, Compound Kushen Injection (CKI), in a breast cancer cell line (MDA-MB-231). Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis. The initial fractionation separated major from minor compounds, and it showed that major compounds accounted for little of the activity of CKI. Furthermore, removal of no single major compound altered the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical with respect to CKI activity. Transcriptome analysis was used to correlate compound removal with gene expression and phenotype data. Many compounds in CKI are required to trigger apoptosis but significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.

Nonclinical safety of astilbin: A 4-week oral toxicity study in rats with genotoxicity, chromosomal aberration, and mammalian micronucleus tests.

Astilbin is an active flavonoid compound isolated from Rhizoma Smilacis Glabrae. It has been widely used as an anti-hepatic, anti-arthritic, and anti-renal injury agent. However, its safety has not yet been established. The objective of this study was to evaluate 4-week repeated oral toxicity and genotoxicity of astilbin. We examined oral toxicity in Sprague-Dawley rats after daily oral administration of astilbin at 50, 150, and 500 mg/kg for 4 weeks. Negative control animals received the same volume of the solvent. Astilbin administration did not lead to death, body weight gain, food consumption, or adverse events. There were no significant differences in toxicity between the astilbin and control group; we observed no toxic effects on hematological or urinalysis parameters, biochemical values, organ weight, or histopathological findings. We assessed the genotoxicity of astilbin with the Ames test (TA97a, TA98, TA100, TA102, and TA1535), chromosomal aberration assay (using Chinese hamster ovary cells), and mammalian micronucleus test (in mice). We found no genotoxicity in any tested strains. The no-observed-adverse-effect level (NOAEL) for astilbin in the 4-week repeated oral toxicity study in rats was greater than 500 mg/kg body weight/day, regardless of gender. Results also suggested that astilbin does not have genotoxicity potential.

[Effect of Chinese Herbs Used in Treating Multiple Sclerosis on T Subsets Using Association Rules].

OBJECTIVE: To analyze the effect of Chinese herbs used by Prof. LI Tao on peripheral blood T subsets in treating multiple sclerosis (MS) by using association rules and statistical methods, thereby providing evidence for optimizing prescriptions. METHODS: Data of MS inpatients and outpatients recorded by data collecting system, Xiyuan Hospital, China Academy of Chinese Medical Sciences were resorted. The relationship between Chinese herbs and T cell subsets were analyzed using SPSS17.0 and Aprior module in SPSS Clementine 12.0. RESULTS: Radix bupleuri, Radix Paeoniae alba, Fructus Aurantii, Atractylodes, and Radix Glycyrrhizae were most commonly used herbal combinations.Radix Aconiti lateralis preparata and Rhizoma Smilacis glabrae were often added. Radix Aconiti lateralis preparata was associated with decreased Th1 cells (confidence level 83.78%, supportive level 36.26%). Decreased Th1 cell was associated with Radix Aconiti lateralis preparata (confidence level 71.26%, supportive level 36.26%).Radix Aconiti lateralis preparata was obviously associated with decreased Th1 cells. Radix Bupleuri, Radix Paeoniae alba, bitter orange, Atractylodes , Radix glycyrrhizae, and Radix Aconiti lateralis preparata could reduce peripheral blood Th1 subsets of MS patients and elevate Th2 subsets (all P < 0.01). CONCLUSIONS: The herbal combination of Radix Bupleuri, Radix Paeoniae alba, Fructus Aurantii, Atractylodes, Radix Glycyrrhizae, Rhizoma Smilacis glabrae, and Radix Aconiti lateralis preparata could lower peripheral blood Th1 cells and elevate Th2 cells, and prevent the relapse of MS possibly by reducing Th1 cells and elevating Th2 cells. Especially Radix Aconiti lateralis preparata played important roles in aforesaid changes of Th1 and Th2.

A mixed microscopic method for differentiating seven species of "Bixie"-related Chinese Materia Medica.

Confusion in the species associated with the name of "Bixie" in Chinese Materia Medica began centuries ago. In recent decades, diverse medicinal plants from the genera Dioscorea and Smilax, and even minor species from the genus Heterosmilax, have been documented under the name Bixie or a very similar name as folk medicines in different areas of China. However, the traditional efficacies and chemical profiles of these herbs are not exactly the same and even vary wildly. Comprehensive authentication of multiple Bixie herbs has not yet been attempted. To differentiate and ensure the correct use of these Bixie-related herbs, in this study, seven sorts of representative Bixie herbs (Dioscorea collettii, D. zingiberensis, D. nipponica, D. septemloba, Smilax china, S. glabra, and Heterosmilax japonica) were characterized based on the microscopic examination of their powders and cryotomed transverse sections. This is not only the first attempt to distinguish Bixie herbs by a comprehensive microscopic techniques, including common light microscopy, fluorescence microscopy, and polarized light microscopy, but also it is the first research to observe characteristics of transections of crude drugs under polarized lighting for the purpose of authentication. Polarized light has been found to provide a number of unique characteristics. The results indicate that starch granules, stone cells, vascular bundles, and other significant tissue features can be used to authenticate "Bixie" herbs. The method was proven to be quick, handy, specific, and simple. It should be widely applicable to other herbal materials.

[Rapid determination of the multi-marker ingredients in Heterosmilacis Japonicae Rhizoma and Sophorae Flavescentis Radix with near-infrared diffused reflection spectroscopy].

A rapid NIRS method for determination of macrozamin in Heterosmilacis japonicae rhizoma (HJR), and the total content of oxymatrine and matrine (OMT + MT) as well as the total content of oxysophocarpine and sophocarpine (OSC + SC) in sophorae flavescens radix (SFR) was developed to explore the application feasibility of NIRS for the quality assurance system of Chinese patent drugs. The contents of macrozamin in HJR samples, and OMT + MT and OSC + SC in SFR samples were determined by HPLC as reference values. The NIR spectra of the samples were measured in a diffused reflection mode. The different characteristic wavebands and pretreatment methods were optimized. The quantitative calibration models between the NIR spectra and the content reference values of marker components in HJR and SFR samples, were established with partial least square method, and further optimized through the cross validation and external validation. The contents of macrozamin in 88 batches of HJR samples were over the range of 0.36-12.88 mg · g(-1). The total contents of OMT + MT and OSC + SC in 75 batches of SFR samples were over the range of 8.87-66.31 and 2.30-15.11 mg · g(-1), respectively. The performance of the final models for macrozamin, OMT + MT and OSC + SC was evaluated well according to correlation coefficients (r), root mean square error of cross-validation (RMSECV) and root mean square error of prediction (RMSEP). The R2 values of the cross-validation for macrozamin, OMT + MT and OSC + SC were 0.9025, 0.9491 and 0.9137, and those of RMSECV were 0.961, 2.45 and 0.724 mg · g(-1) respectively. The R2 values of external validation for the three models were 0.9817, 0.9826 and 0.9609, and those of RMSEP were 0.693, 2.27 and 0.658 mg · g(-1), respectively. This is the first report on rapid determination of macrozamin in Heterosmilacis japonicae rhizoma, and oxymatrine, matrine, oxysophocarpine and sophocarpine in sophorae flavescens radix by NIRS method. The presented method can fulfill the requirement of rapid acquirement of chemical information of raw medicinal materials prior the manufacturing of compound Kushen injection.

Chemical profiling of constituents of Smilacis glabrae using ultra-high pressure liquid chromatography coupled with LTQ Orbitrap mass spectrometry.

A reliable LC-MS method has been applied for the separation and identification of major constituents of the rhizome of Smilacis glabrae. Identification of the constituents was carried out by interpretation of their retention time, and MS and MS/MS data, especially by comparing these with Sarcandra glabra under the same LC-MS conditions, as well as the data provided by the literature. Thirty-three compounds, including catechin derivatives, flavanonols, phenolic acid derivatives and phenylpropanoid glycosides were either identified or tentatively characterized. Among them, compound 12 was deduced to be a new phenylpropanoid-substituted catechin. Fragmentation behaviors of the three major categories of compounds were also investigated. This UPLC-PDA/ESI-MS(n) method was effective for the separation and identification of the constituents and could be the basis for the comprehensive quality control of Smilacis glabrae.

Estrogenic and anti-estrogenic compounds from the Thai medicinal plant, Smilax corbularia (Smilacaceae).

From the rhizomes of Smilax corbularia Kunth. (Smilacaceae), 11 compounds, (2R,3R)-2″-acetyl astilbin, (2R,3R)-3″-acetyl astilbin, (2R,3R)-4″-acetyl astilbin, (2R,3R)-3″-acetyl engeletin, (2R,3S)-4″-acetyl isoastilbin, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10R)-2H,8H-benzo [1,2-b:3,4-b'] dipyran-8-one, 2-(4-hydroxyphenyl)-3,4,9,10-tetrahydro-3,5-dihydroxy-10-(3,4-dihydroxyphenyl)-(2R,3R,10S)-2H, 8H-benzo [1,2-b:3,4-b'] dipyran-8-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(3,4-dihydroxy-phenyl)-5-[(1E)-2-(3,4-dihydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, 3,4-dihydro-7-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-5-[(1E)-2-(4-hydroxyphenyl) ethenyl]-2H-1-benzopyran-2-one, and 5,7,3',4'-tetrahydroxy-3-phenylcoumarin along with 34 known compounds were isolated and characterized as 19 flavonoids, 14 catechin derivatives, 6 stilbene derivatives, and 6 miscellaneous substances. All isolates had their estrogenic and anti-estrogenic activities determined using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D. The major constituents were recognized as flavanonol rhamnosides by the suppressive effect on estradiol induced cell proliferation at a concentration of 1µM. Meanwhile, flavanonol rhamnoside acetates demonstrated estrogenic activity in both MCF-7 and T47D cells at a concentration of 100µM, and they enhanced the effects of co-treated E2 on T47D cell proliferation at concentrations of more than 0.1µM.

Phenylpropanoid glycosides from Heterosmilax erythrantha and their antioxidant activity.

By various chromatographic methods, one new phenylpropanoid glycoside, heterosmilaside (1), two known phenylpropanoid glycosides, helonioside B (2), and 2',6'-diacetyl-3,6-diferuloyl sucrose (3), and three known flavonoids, isoquercetin (4), quercetin-3-O-beta-D-glucuronopyranoside (5), and quercetin-3-O-(2''-alpha-L-rhamnopyranosyl)-beta-D-glucuronopyranoside (6) were isolated from the methanolic extract of the aerial part of Heterosmilax erythrantha Baill. Their structures were elucidated on the basis of spectroscopic analyses. All the isolated compounds were tested for antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Among them, compounds 5 and 6 showed significant antioxidant activity with SC(50) values of 3.7 and 6.5 microg/mL, respectively.

[Studies on chemical constituents of Heterosmilax yunnanensis].

OBJECTIVE: To study the chemical constituents of Heterosmilax yunianensis. METHODS: The compounds were isolated and repeatedly purified with chromatograph and the structures were elucidated by physico-chemical properties and spectral analysis. RESULTS: Eight compounds were obtained and elucidated as beta-sitosterol (I), glycerol monopalmitate (II), daucosterol (IIl), hexacosanoic acid (IV), 5-hydroxymethyl furaldehyde (V), hergapen (VI), ursolic acid (VII), liquiritigenin (VIII). CONCLUSION: They have been isolated from this plant for the first time, and IV - VIII are obtained from the plants of Heterosmilax for the first time.

[Study on the chemical constituents of Heterosmilax japonica].

OBJECTIVE: To analyze the chemical constituents of Heterosmilax japonica Kunth. METHODS: The sample was separated and analyzed by GC-MS. RESULTS: 62 ingredients were elucidated. The major components were steroids and fatty acids. CONCLUSION: The method is reliable, and the result provides a reference for further study of Heterosmilax japonica Kunth.

Neuroprotective effect of oxyresveratrol from smilacis chinae rhizome on amyloid Beta protein (25-35)-induced neurotoxicity in cultured rat cortical neurons.

We previously reported that Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. The present study evaluated the neuroprotective effect of oxyresveratrol isolated from Smilacis chinae rhizome against Abeta (25-35)-induced neurotoxicity on cultured rat cortical neurons. Oxyresveratrol over the concentration range of 1-10 microM significantly inhibited 10 microM Abeta (25-35)-induced neuronal cell death, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Oxyresveratrol (10 microM) inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Oxyresveratrol (1, 10 microM) also inhibited glutamate release into medium and reactive oxygen species (ROS) generation induced by 10 microM Abeta (25-35). These results suggest that oxyresveratrol prevents Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release and ROS generation. Furthermore, these effects of oxyresveratrol may be associated with the neuroprotective effect of Smilacis chinae rhizome.