The Alkaloid-Enriched Fraction of Pachysandra terminalis (Buxaceae) Shows Prominent Activity against Trypanosoma brucei rhodesiense.

In the course of our studies on antiprotozoal natural products and following our recent discovery that certain aminosteroids and aminocycloartanoid compounds from Holarrhena africana A. DC. (Apocynaceae) and Buxus sempervirens L. (Buxaceae), respectively, are strong and selective antitrypanosomal agents, we have extended these studies to another plant, related to the latter-namely, Pachysandra terminalis Sieb. and Zucc. (Buxaceae). This species is known to contain aminosteroids similar to those of Holarrhena and structurally related to the aminocycloartanoids of Buxus. The dicholoromethane extract obtained from aerial parts of P. terminalis and, in particular, its alkaloid fraction obtained by acid-base partitioning showed prominent activity against Trypanosoma brucei rhodesiense (Tbr). Activity-guided fractionation along with extended UHPLC-(+)ESI QTOF MS analyses coupled with partial least squares (PLS) regression modelling relating the analytical profiles of various fractions with their bioactivity against Tbr highlighted eighteen constituents likely responsible for the antitrypanosomal activity. Detailed analysis of their (+)ESI mass spectral fragmentation allowed identification of four known constituents of P. terminalis as well as structural characterization of ten further amino-/amidosteroids not previously reported from this plant.

Mechanism of Active Steroid Alkaloids from Solanum nigrum Against Non-small Cell Lung Cancer： Based on Network Pharmacology / 中国实验方剂学杂志

Objective:To explore the potential targets and pathways of steroid alkaloids<italic> </italic>from<italic> Solanum</italic> <italic>nigrum</italic> （SASN） in the treatment of non-small cell lung cancer （NSCLC） and analyze the possible mechanism. Method:The active SASN against NSCLC were searched from literature. Then potential targets of SASN were screened through SwissTargetPrediction and PharmMapper， and those of NSCLC through GeneCards. Venny was employed to yield the common targets of the two， and Cytoscape to construct the 'medicinal-component-disease-target' network. Metascape was applied to enrich the Gene Ontology （GO） terms and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways of the common targets， and STRING was used to generate the protein-protein interaction （PPI） network， followed by screening of key targets by Cytoscape. Finally， Western blot was used to verify the effects of the medicinal on key targets. Result:A total of 6 active SASN were screened out： solasonine， solamargine， solasodine， solanocapsine， solanidine， and <italic>N</italic>-methylsolasodine， which had 96 potential anti-NSCLC targets. These targets mainly involved the pathways in cancer， proteoglycans in cancer， and Forkhead box protein O （FoxO） pathway. PPI network analysis demonstrated 15 key anti-NSCLC targets of SASN， such as mitogen-activated protein kinase （MAPK）1， MAPK8， MAPK14， protein kinase B （Akt1）， signal transducer and activator of transcription 3 （STAT3）， and proto-oncogene tyrosine protein kinase （SRC）. Meanwhile， Western blot results showed that SASN could significantly down-regulate the expression of the key proteins Akt1， SRC， and STAT3. Conclusion:We predicted the potential targets and pathways of SASN against NSCLC and obtained 15 key targets， from which we selected three key proteins for validation. The validation results were consistent with the prediction results. This paper is expected to lay a scientific basis for the subsequent in-depth study of the mechanisms of SASN against NSCLC.

Quantitative determination of six steroid alkaloids by sensitive hydrophilic interaction liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic study in rats.

In this study, a sensitive hydrophilic interaction liquid chromatography (HILIC) method was developed to determine pseudojervine (PJV), veratrosine (VTS), jervine (JV), veratramine (VTM), veramarine (VA) and veratroylzygadenine (VTG) in rat plasma. Separations were carried out using LC-MS/MS with a Chrom Matrix HP amide column (5 m, 10 cm × 3.0 mm i.d.). The mobile phases were (A) 0.01 mm formic acid and (B) acetonitrile. Good linearity was found for all analytes (R2  > 0.995) in the concentration range from 5 to 1000 µg/L with LLOQ at 5 µg/L for VTM and VTS; and from 1 to 1000 µg/L with LLOQ at 1 µg/L for PJV, JV, VA and VTG. Accuracy of the assay varied from 90.5 to 108.1%. The extraction recovery and matrix effect of six analytes ranged from 72.2 to 95.5% and from 79.2 to 98.4%. According to the stability test, six analytes in rat plasma were stable during the analysis process. On the basis of validation of the assay, the pharmacokinetics of the six steroid alkaloids were investigated after oral administration of Lilu extracts to rats.

Induction of thymic stromal lymphopoietin by a steroid alkaloid derivative in mouse keratinocytes.

Thymic stromal lymphopoietin (TSLP) plays critical roles in inducing and exacerbating allergic diseases. Chemical compounds that induce TSLP production can enhance sensitization to antigens and exacerbate allergic inflammation. Hence, identifying such chemicals will be important to prevent an increase in allergic diseases. In the present study, we found, for the first time, that a steroid alkaloid derivative, code no. 02F04, concentration and time dependently induced mRNA expression and production of TSLP in a mouse keratinocyte cell line, PAM212. In particular, the activity of 02F04 was selective to TSLP. As an analogue of the liver X receptor (LXR) endogenous ligand, 02F04 rapidly increased ATP-binding cassette transporter A1 (ABCA1) expression by regulating the nuclear receptor of LXR. However, instead of being inhibited by the LXR antagonist, 02F04-induced TSLP production was delayed and markedly suppressed by inhibitors of phospholipase C (PLC), pan-protein kinase C (PKC), PKCδ, Rho-associated protein kinase (ROCK), extracellular signal-regulated kinase (ERK) 1/2, and IκΒ kinase 2 (IKK2). Treatment with 02F04 caused the formation of F-actin filaments surrounding the nucleus of PAM212 cells, which then disappeared following addition of ROCK inhibitor. 02F04 also induced phosphorylation of ERK1/2 from 2h after treatment, with a maximum at 24h, and increased nuclear factor-κB (NF-κB) promoter activity by 1.3-fold. Taken together, these results indicate that 02F04-induced TSLP production is regulated via distinct signal transduction pathways, including PLC, PKC, ROCK, ERK1/2, and NF-κB but not nuclear receptors. 02F04, with a unique skeletal structure in inducing TSLP production, can represent a potential new tool for investigating the role of TSLP in allergic diseases.

Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense.

In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, "sleeping sickness"), we have investigated extracts from the leaves and bark of the West African Holarrhenaafricana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM) against Tbr were recorded for 3ß-holaphyllamine (0.40 ± 0.28), 3α-holaphyllamine (0.37 ± 0.16), 3ß-dihydroholaphyllamine (0.67 ± 0.03), N-methylholaphyllamine (0.08 ± 0.01), conessimine (0.17 ± 0.08), conessine (0.42 ± 0.09), isoconessimine (0.17 ± 0.11) and holarrhesine (0.12 ± 0.08) with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs) could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆5,6 unsaturation slightly increased the activity while hydrolysis of C-12ß ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

Quality differences between Mongolian and traditional Chinese medicine Veratri Nigri Radix et Rhizoma by HPLC fingerprint and acute toxicity / 中草药

Objective: To evaluate the quality differences between Mongolian medicine and traditional Chinese medicine (TCM) Veratri Nigri Radix et Rhizoma in HPLC fingerprint and acute toxicity, as a fundamental research for quality standazation. Methods: HPLC fingerprint methods were used to analyze the steroid alkaloids and stilbenes of Veratri Nigri Radix et Rhizoma from different origins. A total of 14 batches of crude drugs, including eight Mongolian and six TCM samples, were examined, and their common pattern were established by authoritative software "fingerprint similarity evaluation system". Characteristic peaks in these fingerprints, including 32 steroid alkaloids and 16 stilbenes, were selected to performing cluster analysis, using SPSS 20.0 software with relative peak area as variables. Through the experiments of acute toxicity in mice, their toxicities were examined. Results: HPLC fingerprint methods of steroid alkaloids and stilbenes were established for the quality evaluation of Veratri Nigri Radix et Rhizoma, and their common patterns of Mongolian medicines (RMJ, RMZ) and TCMs (RZJ, RZZ) with high similarities between groups were obtained. In clustering analysis, 14 batches of Veratri Nigri Radix et Rhizoma were divided into two categories distinctly, as Mongolian medicine and TCM groups. Mongolian medicine showed stronger toxicity than TCM with the LD50 values as 0.503 0 and 17.934 3 g/kg, respectively. Conclusion: The quality of Mongolian medicine and TCM Veratri Nigri Radix et Rhizoma in the aspects of efficacy and idications, producing area, chemical composition and acute toxicity were of significant differences. It is necessary and emergency to distinguish the two crude drugs, and establish quality standard of Mongolian medicine Veratri Nigri Radix et Rhizoma.

Unraveling the structure-activity relationship of tomatidine, a steroid alkaloid with unique antibiotic properties against persistent forms of Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is responsible for difficult-to-treat and relapsing infections and constitutes one of the most problematic pathogens due to its multiple resistances to clinically available antibiotics. Additionally, the ability of S. aureus to develop small-colony variants is associated with a reduced susceptibility to aminoglycoside antibiotics and in vivo persistence. We have recently demonstrated that tomatidine, a steroid alkaloid isolated from tomato plants, possesses anti-virulence activity against normal strains of S. aureus as well as the ability to potentiate the effect of aminoglycoside antibiotics. In addition, tomatidine has shown antibiotic activity against small-colony variants of S. aureus. We herein report the first study of the structure-activity relationship of tomatidine against S. aureus.

Derivative Synthesis of Wanpeinine A, a Major Steroidal Alkaloid from Fritillaria shuchengensis / 中草药·英文版

Objective To design and synthesize derivatives of wanpeinine A, the main steroidal alkaloid isolated from the plant Fritillaria shuchengensis, and further study on the structure-activity relationship of the steroidal alkaloid. Methods Acylation and alkylation were used to synthesize the derivatives and their structures were identified via NMR and MS.Results The acylation of wanpeinine A (1) produced 3β,6α-diacetylwanpeinine A (2), 3β,6α-dipropionylwanpeinine A (3), 3β,6α-dichloracetylwanpeinine A (4), 3β,6α-dibenzoylwanpeinine A (5), and 3β-methoxyacylwanpeinine A (6). The alkylation of wanpeinine A formed 3β,6α-dimethoxymethylwanpeinine A (7). Conclusion All compounds are new except for 3β,6α-diacetylwanpeinine A.