Structurally diverse alkaloids from the Buxus sinica and their cytotoxicity.

Extensive phytochemical study of the methanol extract of twigs and leaves of Buxus sinica resulted in the identification of forty-one Buxus alkaloids, including twenty undescribed ones, namely cyclobuxusinines A-I (1-7, 16 and 20), as well as secobuxusinines A-K (8-15 and 17-19). Their structures were delineated by detailed analysis using various spectroscopic techniques. cyclobuxusinines B (2) was the first Buxus alkaloid, whose CH3-18 was oxidized, implying the presence of special oxidative enzymes in this plant. Secobuxusinines C (10), D (11), and E (12), whose C-12 or C-19 have an OH group substitution, enriched the substituent pattern in Buxus alkaloid and suggested more structurally diverse alkaloids in the Buxus spp. In the assessment of their bioactivities, some of them exhibited significant cytotoxic effects on two human tumor ovarian cancer cell lines. Notably, compound 36 displayed more potent cytotoxic effect against ES2 and A2780 cell lines with the IC50 value of 1.33 µM and 0.48 µM, respectively.

Structurally diverse triterpenoid alkaloids from Buxus rugulosa.

Four new nortriterpenoid alkaloids, namely buxrugulines E-H (1-4), along with five known ones (5-9), were isolated from the twigs and leaves of Buxus rugulosa. Their structures were identified based on extensive NMR data and MS spectroscopic analyses. Our bioassays revealed that compounds 5, 6 and 8 exhibited potent cytotoxicity in vitro against MCF-7 cell lines, with IC50 values ranging from 6.70 to 11.00 µM, respectively.

Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition.

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2-7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis.

BACKGROUND: P53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells. PURPOSE: To explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation. STUDY DESIGN AND METHODS: Different mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53. RESULTS: The triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation. CONCLUSION: Our study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.

Cyclovirobuxine D inhibits cell proliferation and migration and induces apoptosis in human glioblastoma multiforme and low­grade glioma.

Gliomas are the most common neoplasm of the human central nervous system. Glioblastoma multiforme (GBM) is one of the most serious types of gliomas. Although considerable progress has been made in the development of cancer therapeutic agents, several antineoplastic drugs fail to penetrate the blood­brain barrier (BBB), resulting in a low survival rate of glioma patients. Recent studies have revealed that the traditional Chinese medicine Buxus microphylla contains the main active component Cyclovirobuxine D (CVB­D), which can cross the BBB with a novel delivery system. However, it remains unclear whether CVB­D exerts anticancer effects against GBM and low­grade glioma (LGG). The aim of the present study was to explore the feasibility of CVB­D as a new effective agent in the treatment of GBM and LGG. The ability of CVB­D to inhibit GBM and LGG cell proliferation was detected by CCK8 assay. Flow cytometry was used to detect cell cycle progression and apoptosis induction by Annexin V­FITC/PI assay. The expression levels of the apoptosis­associated proteins, namely cleaved caspase­3 and Bax/Bcl­2, were detected by western blot analysis. The mitochondrial membrane potential (ΔΨm) was detected by Rh123 dyed fluorescence micrograph. Hoechst staining was used to observe the morphological characteristics of the apoptotic cells. The scratch test was used to evaluate the migration of GBM and LGG cells. The results indicated that CVB­D reduced cell viability of T98G and Hs683 cells. Flow cytometry demonstrated that CVB­D­treated cells were arrested at the S phase of their cell cycle. The expression levels of the apoptosis­associated proteins were increased in CVB­D­treated cells. Rh123 and Hoechst staining indicated morphological changes and mitochondrial membrane potential changes of the cells undergoing apoptosis. The data confirmed that CVB­D inhibited cell proliferation by arresting the cell cycle of GBM and LLG cells and that it promoted the induction of cell apoptosis by altering the mitochondrial membrane potential. The findings of the present study indicate the potential value of CVB­D in the treatment of glioma.

Investigations into the therapeutic effects of aerial and stem parts of Buxus papillosa C.K. Schneid.: In vitro chemical, biological and toxicological perspectives.

In this study, different solvent extracts (methanol, dichloromethane and n-hexane) from aerial and stem parts of Buxus papillosa C.K. Schneid (Buxaceae) were investigated for a panoply of bioassays. Biological profiles were established by determining antioxidant and enzyme inhibition profiles. Toxicity was tested using MTT cell viability assay on five different human cancer cell lines i.e, MCF-7, MDA-MB-231, CaSki, DU-145 and SW-480. For chemical fingerprinting, total bioactive contents and UHPLC-MS secondary metabolites profile were determined. Generally, both aerial and stem methanol extracts had highest total bioactive contents, radical scavenging and reducing power potential. DCM and n-hexane extracts were found to be most active for total antioxidant and metal chelating activity. The UHPLC-MS analysis of methanol extracts revealed the presence of several phenolic, flavonoid, alkaloid, saponin and depsipeptide derivatives. All the extracts were significantly active against butyrylcholinesterase, whereas moderate inhibition was observed for acetylcholinesterase, α-glucosidase and urease. Similarly, a considerable level of cytotoxicity was observed against all the tested cell lines with IC50 values ranging from 26 to 225.9 µg/mL. Aerial methanol and stem n-hexane extracts were found to be most cytotoxic. Principal component analysis was also performed to find any possible correlation between biological activities and total bioactive contents. On the basis of our findings, B. papillosa may be considered as promising source of bioactive molecules.

Cytotoxicity of Triterpenoid Alkaloids from Buxus microphylla against Human Tumor Cell Lines.

Three new triterpenoid alkaloids, namely buxmicrophyllines P-R (1-3), were isolated from the twigs and leaves of Buxus microphylla. Their structures were elucidated on the basis of NMR and MS spectroscopic analyses. Structurally, compounds 1-3 belong to the 9,10-cycloartane type alkaloids. In addition, compound 3 exhibited moderate cytotoxic activities in vitro against HL-60, SMMC-7221, A-549, MCF-7, and SW480 cell lines (with IC50 values ranging from 4.51 to 15.58 µM).

New Bioactive Lupane Triterpene Coumaroyl Esters Isolated from Buxus cochinchinensis.

Five new lupane triterpene coumaroyl esters (1-5), together with betulin (6) and a known Buxus alkaloid, N-3-benzoyldihydrocyclomicrophylline F (7), were isolated from a CHCl3-soluble partition of a methanol extract of Buxus cochinchinensis Pierre ex Gagnep. (Buxaceae) collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29). The structures of the new compounds (1-5) were determined on the basis of spectroscopic data interpretation. In addition to their cytotoxicity against HT-29 cells and nuclear factor-kappa B (p65) inhibitory activity in an enzyme-linked immunosorbent assay, all isolates as well as two semisynthetic compounds derived from betulin and 5, respectively, were also evaluated for their in vitro antiplasmodial activities against the drug-resistant Dd2 strain of Plasmodium falciparum and antifungal effects on the growth of the pathogenic yeast Candida albicans. The new lupane triterpene coumaroyl esters (1-5), along with a betulin derivative and the known Buxus alkaloid, were found to show significant in vitro antimalarial activities, with IC50 values ranging from 0.26 to 2.07 µM.

Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells.

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC(50) ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC(50) of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC(50) did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.

Triterpenoid alkaloids from Buxus microphylla.

Two new triterpenoid alkaloids, buxmicrophyllines J and K (1 and 2, resp.), together with four analogues, 3-6, were isolated from the leaves and stems of Buxus microphylla. The structures of the new compounds were elucidated by NMR and MS spectroscopic analyses. The partial assignments of the NMR spectra of 3 were also revised. Compounds 1 and 3-6 were evaluated for their growth inhibitory activity against human cell lines HL-60, SMMC-7721, A-549, SK-BR-3, and PANC-1. Compound 6 showed significant cytotoxicity against HL-60, SK-BR-3, and PANC-1 cell lines, with IC(50) values of 6.46, 19.61, and 28.57 microM, respectively.

[Study on the constituents of petroleum ether fraction of Buxus microphylla].

OBJECTIVE: To study the chemical constituents from the petroleum ether fraction of Buxus microphylla. METHODS: The petroleum ether fraction of Buxus microphylla was isolated and identified by silica gel column chromatography. And the anticancer activity of different chemical constituents was measured by MTT reduction test. RESULTS: Eight compounds were isolated and identified as lupeol (1), butulin (3), beta-sitosterol (4), stigmasterol (5), dibutyl phthalate (6), 3beta, 30-dihydroxy-lup-20 (29) ene (7), daucosterol (8). Compound 7 inhibited KB cells' proliferation in a dose-dependent manner. CONCLUSION: Compounds 2 - 5, 7, 8 are isolated from this genus for the first time. Compound 7 has certainly anticancer effects.

Cytotoxic triterpenoid alkaloids from Buxus microphylla.

Five new triterpenoid alkaloids, buxmicrophyllines E-I (1-5), and six known ones (6-11) were isolated from the leaves and stems of Buxus microphylla. The structures of compounds 1-5 were elucidated by NMR and MS spectroscopic analysis, and the relative stereochemistry of 5 was determined by single-crystal X-ray crystallography. Compounds 3 and 9 were cytotoxic against HepG2 cells, with IC(50) values of 0.89 and 0.78 microM, and compounds 2, 3, 7, 8, and 9 were cytotoxic against K562 cells, with IC(50) values of 2.95, 4.44, 1.70, 5.61, and 0.37 microM, respectively.

[Structural modification of cyclovirobuxine D and their inhibition activity on lipid peroxidation].

AIM: To search for compounds through structural modification of cyclovirobuxine D, using 20 or 21-aminosteroids as lead compound for the treatment of cerebrovascular diseases with better lipid peroxidation inhibitory activity. METHODS: According to rational drug design principle, a series of cyclovirobuxine D analogues were prepared, and their bioactivities were tested. RESULTS: Four new compounds were obtained and confirmed by spectra. CONCLUSION: Lipid peroxidation inhibitory effects of cyclovirobuxine D analogues were tested by using TBA method. Some compounds showed better activity than that of cyclovirobuxine D.