Cardiac glycosides from Streblus asper with potential antiviral activity.

Ten undescribed cardiac glycosides, strasperosides A-J, together with twelve known analogues, were isolated from Streblus asper Lour. Their structures were elucidated on the basis of spectroscopic analysis, electronic circular dichroism data, and chemical methods. These cardiac glycosides showed diversity in steroid skeleton and sugar moiety. Strasperosides A and B are a pair of unusual stereoisomers featuring different orientation of the lactone motif. Ten cardiac glycosides demonstrated potent antiviral effects on HSV-1 in vitro with the IC50 values from 0.19 ± 0.08 to 1.03 ± 0.25 µM and the therapeutic indices from 66.61 ± 5.08 to 326.75 ± 11.75.

Bioassay-Guided Fractionation and Biological Activity of Cardenolides from Streptocaulon juventas.

The discovery that Na/K-ATPase acts as a signal transducer led us to investigate the structural diversity of cardiotonic steroids and study their ligand effects. By applying Na/K-ATPase activity assay-guided fractionation, we isolated a total of 20 cardiotonic steroids from Streptocaulon juventas, including an undescribed juventasoside B (10: ) and 19 known cardiotonic steroids. Their structures have been elucidated. Using our platform of purified Na/K-ATPase and an LLC-PK1 cell model, we found that 10: , at a concentration that induces less than 10% Na/K-ATPase inhibition, can stimulate the Na/K-ATPase/Src receptor complex and selectively activate downstream pathways, ultimately altering prostate cancer cell growth. By assessing the ligand effect of the isolated cardiotonic steroids, we found that the regulation of cell viability by the isolated cardiotonic steroids was not associated with their inhibitory potencies against Na/K-ATPase activity but reflected their ligand-binding affinity to the Na/K-ATPase receptor. Based on this discovery, we identified a unique active cardiotonic steroid, digitoxigenin (1: ), and verified that it can protect LLC-PK1 cells from hypoxic injury, implicating its potential use in ischemia/reperfusion injury and inducing collagen synthesis in primary human dermal fibroblast cells, and implicating that compound 2: is the molecular basis of the wound healing activity of S. juventas.

Investigation of cardiac glycosides from oleander in a human induced pluripotent stem cells derived cardiomyocyte model.

The ingestion of Nerium oleander and Thevetia peruviana are common causes for poisoning in Southeast Asia. All parts of the oleander shrub contain cardiac glycosides of the cardenolide type. These glycosides act via inhibition of a Na+/K+-ATPase which might cause severe arrhythmia and subsequent death in oleander-poisoned patients. The current study uses human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) in a microelectrode array (MEA) system to assess the cardiac effects of neriifolin, oleandrin, digitoxigenin, peruvoside and thevetin A from the oleander plant. Digoxin was used as established reference compound. All tested compounds showed a corrected field potential duration (FPDc) shortening and was the lowest for 600 nM digitoxigenin with -36.9 ± 1.2 %. Next to the dose-dependent pro-arrhythmic potential, a complete beat arrest of the spontaneously beating hiPSC-CM was observed at a concentration of 300 nM for neriifolin, 600 nM for oleandrin and 1000 nM for digitoxigenin and peruvoside. Thevetin A did not cause arrhythmia up to a final concentration of 1000 nM. Thus, it was possible to establish a cardiac effect rank order of the tested substances: neriifolin > oleandrin > digitoxigenin = peruvoside > digoxin > thevetin A.

Malayoside, a cardenolide glycoside extracted from Antiaris toxicaria Lesch, induces apoptosis in human non-small lung cancer cells via MAPK-Nur77 signaling pathway.

Lung cancer is the leading cause of cancer deaths in the world. Non-small cell lung cancer (NSCLC), with poor prognosis and resistance to chemoradiotherapy, is the most common histological type of lung cancer. Therefore, it is necessary to develop new and more effective treatment strategy for NSCLC. Nur77, an orphan member of the nuclear receptor superfamily, induces apoptosis in cancer cells including NSCLC cells, by high expression and translocation to mitochondria. Small molecules trigger expression and mitochondrial localization of Nur77 may be an ideal anti-cancer drug candidate. Here, we report malayoside, a cardiac glycoside in the extract of Antiaris toxicaria Lesch., had different sensitivities to NSCLC cells. Malayoside induced apoptosis in NCI-H460 cells. Meanwhile, malayoside induced Nur77 expression and mitochondrial localization, and its induction of apoptosis was Nur77-dependent. To investigate the molecular mechanism of malayoside inducing Nur77 and apoptosis, we found that malayoside activated MAPK signaling pathway, including both ERK and p38 phosphorylation. The suppression of MAPK signaling activation inhibited the expression of Nur77 and apoptosis induced by malayoside. Our studies in nude mice showed that malayside potently inhibited the growth of tumor cells in vivo. Furthermore, the anti-cancer effect of malayosidwas in vivo was also related to the elevated expression of Nur77, p-ERK, and p-p38 proteins. Our results suggest that malayoside possesses an anti-NSCLC activity in vitro and in vivo mainly via activation of MAPK-Nur77 signaling pathway, indicating that malayoside is a promising chemotherapeutic candidate for NSCLC.

Structural Analysis of Diastereomeric Cardiac Glycosides and Their Genins Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

Ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is an economical and indispensable tool in natural product research to investigate novel metabolites, biomarker discovery, chemical diversity exploration, and structure elucidation. In this study, the structural analysis of 38 naturally occurring cardiac glycosides (CGs) in various tissues of Nerium oleander was achieved by the extensive use of mass spectrometry. The chemical diversity of CGs was described on the basis of characteristic MS/MS fragmentation patterns, accurate mass measurement, and published scientific information on CGs from Nerium oleander. It was observed that only six genins, viz., Δ16anhydrogitoxigenin, Δ16adynerigenin, gitoxigenin, oleandrigenin, digitoxigenin, and adynerigenine, produce 38 diverse chemical structures of CGs. Among them, 20 were identified as diastereomers having a difference in a sugar (l-oleandrose, ß-d-diginose, and ß-d-sarmentose) unit. However, the differentiation of diastereomeric CGs was not possible by only MS/MS fragments. Thus, the diastereomer's chromatographic elution order was assigned on the basis of the relative retention time (RRt) of two reference standards (odoroside A and oleandrin) among their diastereomers. Besides this, the in-source fragmentation of CGs and the MS/MS of m/z 325 and 323 disaccharide daughter ions also exposed the intrinsic structure information on the sugar units. The daughter ions m/z 162, 145, 113, 95, and 85 in MS/MS spectra indicated the abundance of l-oleandrose, ß-d-diginose, and ß-d-sarmentose sugars. At the same time, m/z 161, 143, 129, and 87 product ions confirmed the presence of a ß-d-digitalose unit. As a result, the UPLC-ESI/TQD system was successfully utilized for the structure characterization of CGs in Nerium oleander tissues.

Implications for glycosylated compounds and their anti-cancer effects.

Glycosylated compounds are major secondary metabolites of plants, which have various therapeutic effects on human diseases, by acting as anti-cancer, antioxidant, and anti-inflammatory agents. Glycosylation increases stability, bioactivity, and solubility of compounds and improves their pharmacological properties. Two well-known examples of glycosylated compounds include cardiac and flavonoid, the anti-tumor activities of which have been emphasized by several studies. However, little is known about their role in the treatment or prevention of cancer. In this review, recent studies on anti-tumor properties of cardiac and flavonoid glycosides, and their mechanisms of action, have been investigated. More specifically, this review is aimed at focusing on the multifactorial properties of cardiac and flavonoid compounds as well as their correlation with signaling pathways in the treatment of cancer.

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers, leaves, and twigs of Streblus asper.

A new non-cytotoxic [(+)-17ß-hydroxystrebloside (1)] and two known cytotoxic [(+)-3'-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na+/K+-ATPase. Compound 3 docks deeply in the Na+/K+-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4' hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na+/K+-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na+/K+-ATPase. Thus, 3 was found to inhibit Na+/K+-ATPase, but 1 did not. In addition, the cytotoxic and Na+/K+-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na+/K+-ATPase but not by interacting with glucose transporters.

Therapeutic Aspects of Squill; An Evidence-Based Review.

From ancient times, medicinal plants have been usually utilized to treat many disorders, but today, interest in these herbs is again aroused, because of their fewer side effects and low-cost. In traditional medicine, for many diseases, various medicinal herbs have been suggested so far. Drimia maritime, also named squill, is an important medicinal plant for the treatment of many diseases, especially respiratory diseases. In the current evidence-based study, we conducted a review of the general characteristics, ingredients, administration form, and side effects of squill in traditional medicine. For this purpose, traditional Persian medicine literatures and electronic databases were examined including PubMed, Scopus, and Google Scholar. Many compounds are isolated from D.maritima, including scillaren, scillirubroside, scillarenin, and bufadienolide glycosides. Oxymel is the most commonly used form of squill for various diseases, especially respiratory diseases. Besides, squill has been used in the treatment of cardiovascular, digestive, and dermatological disorders, it is also used against various cancer cells for its antioxidant and cytotoxic properties. Moreover, there is relatively reliable evidence of its benefits for bacterial and helminthic infections, rheumatism, edema, gout, abortion induction, healing of wounds and urine induction. It seems that supplementary studies are required to explore the bioactive agents and their effective mechanisms.

Identification and analysis of cardiac glycosides in Loranthaceae parasites Taxillus chinensis (DC.) Danser and Scurrula parasitica Linn. and their host Nerium indicum Mill.

To evaluate the effect of the host plant on the quality of Loranthaceae species as medicinal raw material, ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was used to identify cardiac glycosides in Nerium indicum and its parasitic plant species Taxillus chinensis and Scurrula parasitica. Samples were collected from N. indicum and these parasites, while Morus alba and its parasite T. chinensis and Osmanthus fragrans and its parasite S. parasitica were used as controls. Based on mass spectrometry data and elemental composition analysis of positive and negative ion modes, in combination with standard cardiac glycosides and relevant literature, cardiac glycosides in N. indicum and its parasites T. chinensis and S. parasitica were identified, and their correlations were analyzed. A total of 29 cardiac glycosides were identified, among which 28 were found in N. indicum parasitized by T. chinensis; 25 cardiac glycosides were identified in the same host under attack by S. parasitica; five cardiac glycosides were identified in both T. chinensis and S. parasitica, which grew parasitically on N. indicum, whereas no cardiac glycosides were identified in M. alba parasitized by T. chinensis, or in O. fragrans parasitized by S. parasitica. We conclude that UPLC-Q-TOF-MS/MS technology can identify cardiac glycosides in N. indicum and parasites T. chinensis and S. parasitica rapidly, accurately, and thoroughly. N. indicum will transfer its own cardiac glycosides to its parasites through the special host-parasite interaction. Our results provide a reference basis for evaluating the influence of the host plant on the quality of medicinal compounds obtained from Loranthaceae species.

A sensitive and selective multiple reaction monitoring mass spectrometry method for simultaneous quantification of flavonol glycoside, terpene lactones, and biflavonoids in Ginkgo biloba leaves.

In this study, an efficient and sensitive UHPLC-QQQ-MS/MS (MRM) analytical strategy was established firstly for simultaneous determination of 11 components, including 3 original flavonol glycoside, 4 terpene lactones and 4 biflavonoids in Ginkgo biloba leaves. The validated strategy exhibited proper linearity (R2 ≥0.99) in the range of 0.5-125 µg/mL, and intra and inter-day precision were lower than 4.09% and 4.80%, respectively. Limit of detection (LOD) and quantification (LOQ) were calculated, ranging from 0.2-4.6 ng/mL, with repeatability values between 1.98% and 4.48%. The average recoveries were all in the range of 98.45-106.67% with RSD (n = 3) for the related compounds. Subsequently, the proposed method was used for the analysis of Ginkgo biloba leaves during leaf senescence. Results showed the dominant flavonol glycosides were kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside, the level of terpene lactones and biflavonoids reached the highest in the latest harvest samples. Compared with conventional detection method, the present method could directly analyze original flavonol glycoside without acid hydrolysis process and terpene lactones without the ELSD in a high sensitivity. Moreover, the biflavonoids in Ginkgo biloba leaves were also simultaneously quantified. The results demonstrated that the developed method was accurate, sensitive and reliable for simultaneous quantification of multi-components in Ginkgo biloba leaves, and this study should be significant for the comprehensive utilization and development of Ginkgo biloba resources.

Characterization, quantitation, similarity evaluation and combination with Na+,K+-ATPase of cardiac glycosides from Streblus asper.

Streblus asper Lour. (Moraceae) is a medicinal plant in Asian countries including India and Thailand, possessing activities of anti-tumor, anti-allergy, anti-parasitic and anti-bacterial. In this paper, characterization, quantitation and similarity evaluation of cardiac glycosides in different parts of S. asper were investigated by HPLC-Q-TOF-MS and chemometric methods. Then, the inhibition of Na+,K+-ATPase activity by the compounds isolated from S. asper was measured. Meanwhile, enzyme kinetics and molecular docking were determined to exhibit the combination modes between cardiac glycosides and Na+,K+-ATPase. As a result, twenty peaks of cardiac glycosides were assigned. Strophanthidin-3-O-α-l-rhamnopyranosyl-(1 → 4)-6-deoxy-ß-d-allopyranoside (1), glucostrebloside (2), strebloside (4) and mansonin (8) with a significant activity of inhibiting Na+,K+-ATPase (IC50 7.55-13.60 µM) were chosen for the determination of enzyme kinetics, exhibiting anticompetitive inhibitory characteristics towards Na+,K+-ATPase. Compound 4 could reasonably bind to the active sites of Na+,K+-ATPase, proved by molecular docking. Furthermore, the contents of the major compounds in four different parts of S. asper were extremely different, analyzed by chemometric methods, similarity analysis and principle compounds analysis. All these findings indicated that the contents of major compounds in different parts of S. asper were extremely different with a significant activity of inhibiting Na+,K+-ATPase, providing a reference for determination of effective part and administered dosage. The combination modes between cardiac glycosides and Na+,K+-ATPase were also revealed by enzyme kinetics and molecular docking, which provided a basis for further study of pharmacological activity.

Identification and screening of cardiac glycosides in Streptocaulon griffithii using an integrated data mining strategy based on high resolution mass spectrometry.

The present study was designed to develop a practical strategy to tackle the problem of lacking standard compounds and limited references for identifying structure-related compounds in Streptocaulon griffithii Hook. f., especially those in trace concentrations, with a focus on antitumor activity. The cardiac glycosides (CGs)-enriched part was determined using in vitro bioactive assays in three cancer cell lines and then isolated using macroporous resins. The MS and MS/MS data were acquired using a high performance liquid chromatography coupled with hybrid quadrupole-time of flight (HPLC-Q-TOF-MS) system. To acquire data of trace compound in the extract, a multiple segment program was applied to modify the HPLC-Q-TOF-MS method. A mass defect filter (MDF) approach was employed to make a primary MS data filtration. Utilizing a MATLAB program, the redundant peaks obtained by imprecise MDF template calculated with limited references were excluded by fragment ion classification, which was based on the ion occurrence number in the MDF-filtered total ion chromatograms (TIC). Additionally, the complete cleavage pathways of CG aglycones were proposed to assist the structural identification of 29 common fragment ions (CFIs, ion occurrence number ≥ 5) and diagnostic fragment ions (DFIs, ion occurrence number < 5). As a result, 30 CGs were filtered out from the MDF results, among which 23 were identified. This newly developed strategy may provide a rapid and effective tool for identifying structure-related compounds in herbal medicines.

The Foxgloves (Digitalis) Revisited.

This review provides a renewed look at the genus Digitalis. Emphasis will be put on those issues that attracted the most attention or even went through paradigmatic changes since the turn of the millennium. PubMed and Google Scholar were used ("Digitalis" and "Foxglove" were the key words) to identify research from 2000 till 2017 containing data relevant enough to be presented here. Intriguing new results emerged from studies related to the phylogeny and taxonomy of the genus as well as to the biosynthesis and potential medicinal uses of the key active compounds, the cardiac glycosides. Several Eastern and Western Foxgloves were studied with respect to their propagation in vitro. In this context, molecular biology tools were applied and phytochemical analyses were conducted. Structure elucidation and analytical methods, which have experienced less exciting progress, will not be considered here in great detail.

Na⁺i,K⁺i-Dependent and -Independent Signaling Triggered by Cardiotonic Steroids: Facts and Artifacts.

Na⁺,K⁺-ATPase is the only known receptor of cardiotonic steroids (CTS) whose interaction with catalytic α-subunits leads to inhibition of this enzyme. As predicted, CTS affect numerous cellular functions related to the maintenance of the transmembrane gradient of monovalent cations, such as electrical membrane potential, cell volume, transepithelial movement of salt and osmotically-obliged water, symport of Na⁺ with inorganic phosphate, glucose, amino acids, nucleotides, etc. During the last two decades, it was shown that side-by-side with these canonical Na⁺i/K⁺i-dependent cellular responses, long-term exposure to CTS affects transcription, translation, tight junction, cell adhesion and exhibits tissue-specific impact on cell survival and death. It was also shown that CTS trigger diverse signaling cascades via conformational transitions of the Na⁺,K⁺-ATPase α-subunit that, in turn, results in the activation of membrane-associated non-receptor tyrosine kinase Src, phosphatidylinositol 3-kinase and the inositol 1,4,5-triphosphate receptor. These findings allowed researchers to propose that endogenous CTS might be considered as a novel class of steroid hormones. We focus our review on the analysis of the relative impact Na⁺i,K⁺i-mediated and -independent pathways in cellular responses evoked by CTS.

New Knowledge About Old Drugs: The Anti-Inflammatory Properties of Cardiac Glycosides.

In the 19th century, cardio-active steroid glycosides, shortly cardiac glycosides, were scientifically established as drugs against heart failure. Their in vivo, cellular, and molecular actions as well as their predominant target, Na+-K+-ATPase, have been comprehensively investigated in the 20th century and the discovery of endogenous cardiac glycosides has fostered this research field. In the last years, however, results from clinical trials and meta-analyses have questioned their therapeutic value due to efficacy and safety issues. This has led to a considerable decline of their usage. Beyond the cardiovascular system, cardiac glycosides have been increasingly recognized as antitumor compounds and Na+-K+-ATPase has evolved into a promising drug target in oncology. A wealth of review articles exists that intensively discuss these topics. Surprisingly, the anti-inflammatory actions of cardiac glycosides, which were discovered in the 1960s, have so far hardly been perceived and have not yet been summarized. This review provides an overview of the in vivo and in vitro actions of cardiac glycosides on inflammatory processes and of the signaling mechanisms responsible for these effects: cardiac glycosides have been found to decrease inflammatory symptoms in different animal models of acute and chronic inflammation. Regarding the underlying mechanisms most research has focused on leukocytes. In these cells, cardiac glycosides primarily inhibit cell proliferation and the secretion of proinflammatory cytokines.

Cardiac Glycoside Constituents of Streblus asper with Potential Antineoplastic Activity.

Three new (1-3) and two known (4 and 5) cytotoxic cardiac glycosides were isolated and characterized from a medicinal plant, Streblus asper Lour. (Moraceae), collected in Vietnam, with six new analogues and one known derivative (5a-g) synthesized from (+)-strebloside (5). A preliminary structure-activity relationship study indicated that the C-10 formyl and C-5 and C-14 hydroxy groups and C-3 sugar unit play important roles in the mediation of the cytotoxicity of (+)-strebloside (5) against HT-29 human colon cancer cells. When evaluated in NCr nu/nu mice implanted intraperitoneally with hollow fibers facilitated with either MDA-MB-231 human breast or OVCAR3 human ovarian cancer cells, (+)-strebloside (5) showed significant cell growth inhibitory activity in both cases, in the dose range 5-30 mg/kg.

A novel alkaloid from Portulaca oleracea L.

A novel alkaloid named oleraciamide C (1), with six known compounds, hydroxydihydrobovolide (2), uracil (3), catechol (4), 4-aminophenol (5), vanillic acid (6) as well as 3-hydroxypyridine (7), were isolated from Portulaca oleracea L. Additionally, hydroxydihydrobovolide (2), 4-aminophenol (5), 3-hydroxypyridine (7) were obtained from the plant for the first time. Structure of the new compound was determined using spectroscopic methods including HR-ESI-TOF-MS, 1D and 2D NMR. Others were elucidated through 1H NMR, 13C NMR spectra and comparison with literature data. Notably, Compound 1 possessed an unusual bis-substituted eight-membered ring linked with the ß-glucopyranose moiety. The cytotoxicity of compound 1 was evaluated against human adipose-derived stem cells (hADSCs) by CCK-8 method.

Cytotoxic cardiac glycosides from the fruit (pods) of Adenium obesum (Forssk.) Roem. & Schult.

Phytochemical investigation of methanolic extract of Adenium obesum led to the isolation of 42 (1-42) compounds belongs to cardiac glycosides, triterpenoids and steroids. The chemical structures of isolated compounds were elucidated by spectral techniques UV, IR, NMR and FAB MS. The cardiac glycosides were tested against three human cell lines, 3T3 (normal cells), HeLa (Human cervical cancer cell lines) and PC-3 (Human prostate cancer cell lines). The cardiac glycoside, honghelin (4), obeside B (5) and obeside C (6) showed significant effects against cell lines Hela, 3T3 and PC-3 compared to standard drug doxorubicin. Compounds 4, 5 and 6 exhibited very low IC50 (µM) against the PC3 human cell line. 4 and 6 also showed least IC50 against the HeLa human cell lines as compared to the standard drug doxorubicin whereas these three compounds showed effect on 3T3 cell line with high IC50 values compared to drug cycloheximide.

Cardiac glycosides from the seeds of Thevetia peruviana and their pro-apoptotic activity toward cancer cells.

Phytochemical investigation of the seeds of Thevetia peruviana resulted in the isolation of seven cardiac glycosides (1-7), including two new compounds (1 and 2). Cytotoxicity of them toward cancer cell lines P15 (human lung cancer cell), MGC-803 (human gastric cancer cells), SW1990 (human pancreatic cancer cells), and normal hepatocyte cell LO2 suggested that compound 1 could selectively inhibit the proliferation of cancer cell lines with IC50 from 0.05 to 0.15 µM. Pro-apoptotic activity revealed that it induced the apoptosis of MGC-803 cancer cells in a dose-dependent manner. Meanwhile, treatment of MGC-803 cancer cells with 1 resulted in diminution of pro-caspases 3 and 9 and activation of caspases 3 and 9, while it increased the Bax/Bcl-2 ratio in a dose-dependent manner. These meant that 1 induced the apoptosis of cancer cells by involving the intrinsic apoptotic pathway. In addition, the cell cycle distribution of MGC-803 cancer cells treated by 1 revealed that it could lead to cell cycle arrest at the G2/M phase. Altogether, this study suggested that compound 1 may exhibit anticancer activity by its capability of induction of intrinsic apoptosis and cell cycle arrest at G2/M phase.

Compound Library Screening Identified Cardiac Glycoside Digitoxin as an Effective Growth Inhibitor of Gefitinib-Resistant Non-Small Cell Lung Cancer via Downregulation of α-Tubulin and Inhibition of Microtubule Formation.

Non-small-cell lung cancer (NSCLC) dominates over 85% of all lung cancer cases. Epidermal growth factor receptor (EGFR) activating mutation is a common situation in NSCLC. In the clinic, molecular-targeting with Gefitinib as a tyrosine kinase inhibitor (TKI) for EGFR downstream signaling is initially effective. However, drug resistance frequently happens due to additional mutation on EGFR, such as substitution from threonine to methionine at amino acid position 790 (T790M). In this study, we screened a traditional Chinese medicine (TCM) compound library consisting of 800 single compounds in TKI-resistance NSCLC H1975 cells, which contains substitutions from leucine to arginine at amino acid 858 (L858R) and T790M mutation on EGFR. Attractively, among these compounds there are 24 compounds CC50 of which was less than 2.5 µM were identified. We have further investigated the mechanism of the most effective one, Digitoxin. It showed a significantly cytotoxic effect in H1975 cells by causing G2 phase arrest, also remarkably activated 5' adenosine monophosphate-activated protein kinase (AMPK). Moreover, we first proved that Digitoxin suppressed microtubule formation through decreasing α-tubulin. Therefore, it confirmed that Digitoxin effectively depressed the growth of TKI-resistance NSCLC H1975 cells by inhibiting microtubule polymerization and inducing cell cycle arrest.