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Background: Buxus plants have been used in traditional medicine for a very long time. The Buxus genus has been used to cure a variety of illnesses., Objective: This review aimed to provide a literature review on the genus Buxus including its biological and phytochemical properties., Materials and Methods: The current study was conducted using several scientific databases. Correct plant names were verified from plantlist.org. The results of this search were interpreted, analyzed, and documented based on the obtained bibliographic information., Results: Within all the species of the family Buxaceae, 5 species of the genus Buxus are reported to be antibacterial, 3 species have been found to be antioxidant, 5 species are cytotoxic, 1 species is anti-inflammatory, 1 species is antidiabetic, and 4 species are antifungal. Alkaloids, terpenoids, tannins, flavonoids, peptides, and phenolic compounds are the main chemical components of this genus. The study of >11 Buxuss pecies has identified >201 compounds. Pharmacological research has demonstrated that crude extracts and some pure compounds obtained from Buxus have several pharmacological activities such as antibacterial, antioxidant, cytotoxic, anti-inflammatory, antidiabetic, and antifungal. Based on the study of the phytochemistry of Buxus species, it was concluded that all the studied plants have active compounds, among which 55 molecules showed interesting activities., Conclusions: The numerous traditional uses of Buxus species have been supported by several studies. Before Buxus plants can be fully employed clinically, further research is necessary., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 CH 3 -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 IC 50 value of 1.33 μM and 0.48 μM, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Genus Buxus plants, commonly known as "boxwood", are widely distributed in China. The stems, branches, and leaves of the plant are traditionally used for rheumatism, toothache, chest pain, abdominal gas, and other diseases. However, an overview of the genus Buxus remains to be provided., Purpose: To provide a scientific basis for the appropriate use and further research the recent advancements in the traditional usage, phytochemistry, and, pharmacology of Buxus., Study Design: Chemical composition and pharmacological correlation studies through a literature review., Methods: Between 1970 and 2023, the available data concerning Buxus was compiled from online scientific sources, such as Sci-Finder, PubMed, CNKI, Google Scholar, and the Chinese Pharmacopoeia. Plant names were verified from "The Plant List" (http://www.theplantlist.org/)., Results: To date, 266 structurally diverse chemicals have been extracted and identified from the genus Buxus. Alkaloids constitute one of its primary bioactive phytochemicals. A summary of the channels of action of Cyclovirobuxine D on the cytotoxicity of a variety of cancers has been provided., Conclusion: Numerous findings from contemporary phytochemical and pharmacological studies support the traditional use, facilitating its application. Further research is necessary to address various shortcomings, including the identification of the active ingredients and quality control of the genus Buxus., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

The scientific community has been drawn towards natural plant resources due to the rising success rates. This work aimed to explore the phytochemical, antioxidant and anti-cancer properties of an ethanolic extract derived from the leaves of Buxus wallichiana Baill. which is utilized in traditional medicine in the treatment of various disease such as respiratory disorders, GI disorders and inflammation. Chemical composition of the ethanolic extract of BW leaves was evaluated by using Gas chromatography-mass spectrometry and phytochemical analysis. Moreover, anti-oxidant and anticancer activity was carried out through 2,2-diphenyl-1-picryl hydrazyl scavenging activity method and 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay against human glioblastoma cell line. ANOVA test was used to analyze the results followed by Bonferoni's post hoc. Results were significant at P <0.05 and demonstrated that, blocking the production of free radicals also caused tumor cell proliferation on the U 87 MG. Thus, the results show the same pattern of toxicity as indicated by the American National Cancer Institute. The minimum dose for cytotoxic activity of the crude extract was less than 30μg/mL. BW leave extract is considered to have promising anticancer potential as well as antioxidant due to the presence of potent compounds and could become a source of treatment with minimum side effects.

Two new degrade cycloartane triterpenoids, named buboditones A, B ( 1 , 2 ), together with ten known alkaloids, cyclobuxoviridine ( 3 ), N -dimethylcycloxobuxovircine ( 4 ), cyclovirobuxine C ( 5 ), cyclovirobuxine A ( 6 ), cycloprotobuxine C ( 7 ), cycloprotobuxine A ( 8 ), cyclobuxoxazine ( 9 ), cyclobuxoxazine A ( 10 ), buxruguline B ( 11 ), irehine ( 12 ), were isolated from the leaves and stems of Buxus bodinieri Levl., The structures of compounds 1-2 were elucidated by 1 D and 2 D NMR spectroscopic methods including HSQC, HMBC, 1 H- 1 H COSY, NOESY, as well as HRESIMS spectroscopic analysis.

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 IC 50 values ranging from 6.70 to 11.00 μM, respectively.

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 Ca v 3.2 inhibitors. Compounds 2-7 exhibited potency against Ca v 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 Ca v 3.2 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Buxus sempervirens L. is a common ornamental plant in southern and central Europe, and has been used ethopharmacologically against a wide variety of diseases due to it containing nor -triterpene alkaloids of the nor -cycloartane type. Recently, we demonstrated the interesting antiprotozoal potential of some of these compounds. To characterize the temporal variability in the alkaloid profile of two different varieties and their leaves and twigs, 30 different extracts of B . sempervirens were evaluated by Ultra High Performance Liquid Chromatography/positive Mode-Electrospray Ionization Quadrupole Time-of-Flight-Tandem Mass Spectrometry (UHPLC/+ESI-QqTOF-MS/MS). The analytical profiles were thoroughly investigated by various methods of multivariate data analysis (MVDA). A principal component analysis (PCA) model elucidates the seasonal variation in the phytochemical composition of B . sempervirens var. arborescens and suffruticosa along with differences between the varieties. Analysis of a volcano plot illustrated the differences between the two organs, the leaf and twig. Eighteen compounds were highlighted by the models as constituents of the plant characteristic for a season, variety or organ. These compounds were dereplicated based on their chromatographic and +ESI-QqTOF-MS and -MS/MS data. In addition, mass spectral fragmentation pathways for already known alkaloids as well as new natural products could be postulated for the first time. In conclusion, the MVDA models give detailed information on the temporal variability in the alkaloid profile of two different varieties and their organs (leaf vs. twig) of B . sempervirens . Thus, the results of this study allow, e.g., the identification of characteristic compounds for the different varieties, plant organs, seasons, and the optimal harvesting time for the isolation of particular Buxus -alkaloids of interest for subsequent studies.

Extensive phytochemical study of the methanol extract of twigs and leaves of Buxus sempervirens resulted in the identification of 17 Buxus alkaloids, including 12 new ones, namely buxusemines A-L (1-12). Their structures were delineated by detailed analysis of the HRESIMS and NMR data, as well as quantum chemical NMR calculations. Buxusemine A (1) represents the second Buxus alkaloid with a unique spiro[4.6]undecatriene moiety, buxusemines B-C (2-3) are a rarely occurring class of Buxus alkaloids featured with an additional five-membered ring through the ether or lactone linkage between C-10 and C-23, and buxusemines D-F (4-6) are another rare type of Buxus alkaloids with an epoxy motif. In the assessment of their bioactivities, buxusemine F (6) and buxanoldine (17) displayed more potent protective effects than the positive control cyclovirobuxinum D in the doxorubicin-induced cardiac injury model., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

The increasing drug resistance of malaria parasites challenges the treatment of this life-threatening disease. Consequently, the development of innovative and effective antimalarial drugs is inevitable. O -tigloylcyclovirobuxeine-B, a nor -cycloartane alkaloid from Buxus sempervirens L., has shown promising and selective in vitro activity in previous studies against Plasmodium falciparum ( Pf ), causative agent of Malaria tropica. For further investigations, it is indispensable to develop an advanced and efficient isolation procedure of this valuable natural product. Accordingly, we used liquid-liquid chromatography including centrifugal partition chromatography (CPC) to obtain the pure alkaloid on a semi-preparative scale. Identification and characterization of the target compound was accomplished by UHPLC/+ESI-QqTOF-MS/MS, 1 H NMR and 13 C NMR. In conclusion, this work provides a new and efficient method to obtain O -tigloylcyclovirobuxeine-B, a valuable natural product, as a promising antiplasmodial lead structure for the development of innovative and safe medicinal agents.

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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 blood‑brain 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 (CVB‑D), which can cross the BBB with a novel delivery system. However, it remains unclear whether CVB‑D exerts anticancer effects against GBM and low‑grade glioma (LGG). The aim of the present study was to explore the feasibility of CVB‑D as a new effective agent in the treatment of GBM and LGG. The ability of CVB‑D 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 V‑FITC/PI assay. The expression levels of the apoptosis‑associated proteins, namely cleaved caspase‑3 and Bax/Bcl‑2, 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 CVB‑D reduced cell viability of T98G and Hs683 cells. Flow cytometry demonstrated that CVB‑D‑treated cells were arrested at the S phase of their cell cycle. The expression levels of the apoptosis‑associated proteins were increased in CVB‑D‑treated cells. Rh123 and Hoechst staining indicated morphological changes and mitochondrial membrane potential changes of the cells undergoing apoptosis. The data confirmed that CVB‑D 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 CVB‑D in the treatment of glioma.

Buxaustroines A-N ( 1 - 14 ), a series of triterpenoidal alkaloids featuring a novel 17(13→18) abeo motif, were obtained from the extract of Buxus austro-yunnanensis . Their structures were assigned based on NMR data analysis and X-ray diffraction crystallography. A putative biosynthetic pathway for one of the alkaloids from a co-isolate 15 is proposed. In the assessment of their bioactivities, some of the compounds displayed protective effects against doxorubicin-induced injury of myocardial cells. Preliminary structure-activity relationship studies of 1 - 14 , which are based on the same skeleton, were conducted.

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 IC 50 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Buxus sempervirens L. is used in Morocco as a plant with some medicinal properties including treatment of diabetes and cardiovascular diseases., Methods: The objective of the study was to assess the effect of the aqueous extract of Buxus sempervirens L. (Boxwood) leaves on the blood lipid parameters including total cholesterol, triglycerides, High-Density Lipoprotein Cholesterol (HDL-C), Low-Density Lipoprotein Cholesterol (LDL-C) and Very Low-Density Lipoprotein Cholesterol (VLDL-C) in normal and streptozotocininduced diabetic rats. In addition, antioxidant activity of aqueous Buxus sempervirens extract was evaluated by DPPH and FRAP assays., Results: The results indicated a significant decrease in serum total cholesterol and LDL-C levels, while, no change was observed on triglycerides and VLDL-C values in diabetic rats. Lipid profile of normal animals did not reveal any modification. In addition the aqueous extract of Buxus sempervirens exerted a significant antioxidant activity. Therefore, the results suggest that the aqueous extract of Buxus sempervirens exhibits lipid lowering activity which could be due partially to its antioxidant ability., Conclusion: In conclusion, Buxus sempervirens leaf extract may be helpful in controlling the development of hyperlipidemia as well as atherosclerosis in diabetic animals., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

A new flavonoid diglycoside named buxusoside (1), together with its aglycone chrysosplenol-D (2), as well as 4', 5-dihydroxy-3,6,7-trimethoxyflavone (3) and 3',4',5-tiihydroxy-3,6,7-trimethoxyflavone (4), were isolated from the 70% EtOH extract of the air-dried plant of Buxus sinica. Its structure was elucidated mainly by iD and 2D NMR spectra.

Background: The present research was carried out to investigate pharmacological properties of Buxus papillosa C.K. Schneid. (Buxaceae)., Methods: Buxus papillosa extracts of leaves (BpL), stem (BpS), roots (BpR) and BpL fractions: hexane (BpL-H), aqueous (BpL-A) also plant constituent, cyclomicrobuxine effect were studied in jejunum, atria, aorta and tracheal preparations from rabbit and guine-peg., Results: Ca ++ antagonistic effect of BpS, BpR, BpL-H, BpL-A and cyclomicrobuxine were conclusively suggested, when spontaneous contractions of rabbit jejunal preparation was relaxed along with subsequent relaxation of potassium chloride (80 mM) induced contractions. Ca ++ antagonistic effect was further confirmed, when a prominent right shift like that of verapamil was observed in Ca ++ concentration-response curves, drawn in a tissue pretreated with BpL (0.3-1.0 mg/mL). In rabbit tracheal tissues BpL, BpS, BpR, BpL-H and BpL-A produced a prominent relaxation in contractions induced by potassium chloride (80 mM) and carbachol (1 μm). When tested in rabbit aortic rings, BpL, BpS, BpR, BpL-H and BpL-A showed concentration-dependent (0.1-3.0 mg/mL) vasorelaxant effect against phenylephrine (1 μM) and high K + -induced contractions. In isolated guinea-pig right atria, BpL, BpS, BpR, BpL-H and BpL-A suppressed atrial force of spontaneous contractions, with BpL-A being most potent., Conclusions: Our results reveal that Buxus papillosa possesses gut, airways and cardiovascular inhibitory actions.

Background: Buxus sempervirens L. (Boxwood) is a medicinal plant used in Moroccan traditional medicine for diabetes treatment in Morocco. However; there is no experimental support of this ethnopharmacological use., Methods: The present investigation is aimed at studying the antidiabetic and the preliminary phytochemical of the aqueous extract of the leaves of Buxus sempervirens (AELBS). AELBS (5 mg/kg) was administered by gavage to normal and streptozotocin-induced diabetic rats and blood glucose levels were measured for 6 hours (acute study) and 15 days (chronic study). Oral glucose test tolerance (OGTT) and histopathological examination of liver and pancreas were carried out using standard methods. Furthermore, preliminary phytochemical screening and quantification of phenolic and flavonoid contents were also realized., Results: AELBS reduced the blood glucose of both healthy and diabetic rats. This extract was also able to improve oral glucose tolerance in diabetic rats and it ameliorated hepatic histology, however, no enhancement was noticed in pancreatic histology. Preliminary phytochemical study revealed that AELBS possesses many chemical compounds such as alkaloids, phenolic compounds (flavonoids, tannins, and cyanidins), carbohydrates, reducing sugars and mucilage. Additionally, this extract is rich in phenolic compounds including flavonoids., Conclusion: According to all these findings, we conclude that AELBS at a low dose demonstrated a potent antihyperglycemic effect in streptozotocin rats and further investigations should be necessary for supporting the use of this plant in the management of diabetes by the Moroccan population., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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).

Georgian flora is represented by about 4150 plant species. Many important alkaloid-containing plant species and among of them are species Buxus L. of genus in Adjara. The aims of the research were: sequential screening of the plants for the consistence of alkaloids; Study of anatomical characteristics of Buxus colchica Pojark. and revealing of specific pharmacological activity of steroidal alkaloids. The objects of research were B. colchica, B. balearika and B. sempervirens, growing in Adjara (Georgia), collected in active phase of flowering of the plants. There were revealed 370 species of alkaloid containing plants. Sum of alkaloids and crude aqueous extract have spasmolitic and antihistaminic activity. Experimental anatomical research of diagnostic characteristics of the bines showed the existence of monocyclic transient system with fiber like tracheids, dorsoventral mesophyll of the leaves; the structure for the upper part of epidermis is linear and the lower part is curved, type of stomata is paracitic.

Buxus papillosa is one of the most extensively studied species of the genus Buxus known to possess steroidal alkaloids which can be used for assessing the various pharmacological activities of this plant. This paper describes the liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (LC-ESI-QQQ-MS) method for the quantification of six steroidal alkaloids as chemical markers in the extracts of leaves, roots and stems of B. papillosa. Quantitative MS/MS analysis was carried out by optimization of the most sensitive transition for each analyte. This has yielded detection and quantification limits of 0.486-8.08 ng/mL and 1.473-24.268 ng/mL, respectively for all analytes. Linearity of response was also achieved and the regression coefficient found to be >0.99 for all analyzed compounds. The newly developed MRM (Multiple Reaction Monitoring) method showed excellent sensitivity for the quantification of steroidal alkaloids within 15 min run time. This paper describes the application of LC-QQQ-MS technique for steroidal alkaloids analysis in plant samples., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Georg Thieme Verlag KG Stuttgart · New York.)

Chemical investigation of the crude methanolic extract of Buxus macowanii resulted in the isolation of five new steroidal alkaloids, 31-hydroxybuxatrienone (1), macowanioxazine (2), 16α-hydroxymacowanitriene (3), macowanitriene (4), macowamine (5), along with five known steroidal bases, Nb-demethylpapillotrienine (6), moenjodaramine (7), irehine (8), buxbodine B (9) and buxmicrophylline C (10). Structures of compounds 1-10 were elucidated with the aid of spectroscopic methods including 1D and 2D NMR techniques and mass spectrometry. Compounds 1, 3, and 4 belong to a rare class of Buxus alkaloids having Δ(1,2) 9(10→19) abeo triene system. All isolates were evaluated for in vitro acetylcholinesterase (AChE) inhibitory activity and found to exhibit moderate to weak anti-AChE activity with IC50 values in the range of 10.8-98μM. Compounds 1 and 6 were also moderately active in BACE1 inhibitory assay., (Copyright © 2014 Elsevier Inc. All rights reserved.)

As one of famous shrubs in China, Buxus microphylla is considered as the important wattle. However, the constituents of Buxus microphylla stem extracts aren't used effectively. Therefore, the molecules of stem extracts in Buxus microphylla are analyzed to further utilize the resources. The results show that the optimal extraction time of ethanol/methanol extraction, petroleum ether/ benzene extraction, and benzene/alcohol extraction are 7h, 7h, and 5h, respectively. The HK-61, HK-63, HK-73, HK-81, HK-82 stem extracts are obtained 1, 9, 1, 27, and 1 components, respectively. The stem extracts of Buxus microphylla is rich in drug and biomedical activities. Buxus microphylla stem is fit to extract 1,5-hexadien-3-yne, squalene, and dibutyl phthalate.

Buxus sempervirens L. (European Box, Buxaceae) has been used in ethnomedicine to treat malaria. In the course of our screening of plant extracts for antiprotozoal activity, a CH2Cl2 extract from leaves of B. sempervirens showed selective in vitro activity against Plasmodium falciparum (IC50 = 2.79 vs. 20.2 µg/mL for cytotoxicity against L6 rat cells). Separation of the extract by acid/base extraction into a basic and a neutral non-polar fraction led to a much more active and even more selective fraction with alkaloids while the fraction of non-polar neutral constituents was markedly less active than the crude extract. Thus, the activity of the crude extract could clearly be attributed to alkaloid constituents. Identification of the main triterpene-alkaloids and characterization of the complex pattern of this alkaloid fraction was performed by UHPLC/+ESI-QTOF-MS analyses. ESI-MS/MS target-guided larger scale preparative separation of the alkaloid fraction was performed by 'spiral coil-countercurrent chromatography'. From the most active subfraction, the cycloartane alkaloid O-tigloylcyclovirobuxeine-B was isolated and evaluated for antiplasmodial activity which yielded an IC50 of 0.455 µg/mL (cytotoxicity against L6 rat cells: IC50 = 9.38 µg/mL). O-tigloylcyclovirobuxeine-B is thus most significantly responsible for the high potency of the crude extract.

In order to investigate plant physiological process of leaf senescence and aging, Fourier transform infrared (FTIR) spectroscopy was used to study the young, mature, and old yellow leaves from seven species of evergreen trees. The spectra of the leaves from different growing period are different in the region of 1 800-700 cm(-1). The absorption ratios A1 070/A2 927, A1 070/A1 160 were used to evaluate the relative changes of polysaccharides, and A1 318/A2 922 was used to estimate the change of calcium oxalate during leaf senescence. Decomposition and curve-fitting analysis was performed in the region of 1 800 -1 500 cm(-1). The sub-band absorption ratio H1 650/H1 740 was used to evaluate the relative changes of protein in the leaves. The results show that the accumulation and mobilization of polysaccharides, protein, and calcium oxalate during leaf growing period were different in different plant species. This study demonstrates the potential of mid-infrared spectroscopy for investigation of plants senescence, as well as physiological and biochemical changes of plants.

Rationale: Tandem mass spectrometric studies of natural products revealed the identification of key fragments which can be helpful for their rapid dereplication in plant extracts utilizing a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach, particularly for the thermally labile compounds. The knowledge of the collision-induced dissociation (CID) fragmentation pattern of the molecule is essentially required prior to the analysis by LC/MS/MS., Methods: The fragmentation patterns of eleven Buxus steroidal alkaloids were studied by using a positive ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Chromatographic separation of a Buxus papillosa extract was achieved using a capillary HPLC system coupled with the mass instrument., Results: ESI-QqTOF-MS (positive ion mode) showed the presence of several characteristic fragments which can be used to rapidly identify various classes of Buxus steroidal alkaloids. The presence of a cyclopropane ring in the cycloartenol skeleton and the hydroxyl group at C-10 was found to effect on the fragmentation pattern and afford characteristic peaks. This study distinguishes between different types of Buxus steroidal alkaloids based on logical fragmentation pathways. This strategy was successfully applied in LC/ESI-QqTOF-MS/MS analysis of Buxus papillosa extract to investigate and characterize Buxus steroidal alkaloids and 14 compounds were identified as steroidal alkaloids., Conclusions: The knowledge of the fragmentation pattern was used for the rapid identification of this bioactive group of biosynthetically unique steroidal alkaloids in complex plant extracts of Buxus species, especially in the absence of any reference material, by combining key fragments, exact mass measurements and relative abundances of diagnostic fragment ions., (Copyright © 2012 John Wiley & Sons, Ltd.)

High irradiance and relatively low temperature, which characterize Mediterranean winters, cause chilling stress in plants. Downregulation of photosynthetic efficiency is a mechanism that allows plants to survive these conditions. This study aims to address whether this process shows a regular spatial pattern across leaf surface or not. Three species (Buxus sempervirens, Cistus albidus and Arctostaphylos uva-ursi) with contrasting responses to winter stress were studied. During 7 days, macro and micro Fv/Fm spatial patterns were monitored by the use of chlorophyll fluorescence imaging techniques. In the field, the strongest photoinhibition was found in B. sempervirens, while there was almost no chronic photoinhibition in C. albidus. In leaves of the first species, Fv/Fm decreased from base to tip while in C. albidus it was uniform over the leaf lamina. An intermediate behavior is shown by A. uva-ursi leaves. Spatial heterogeneity distribution of Fv/Fm was found inside the leaves, resulting in greater Fv/Fm values in the inner layers than in the outer ones. Neither xanthophyll-linked downregulation of Fv/Fm nor protein remobilization were the reasons for such spatial patterns since pigment composition and nitrogen content did not reveal tip-base differences. During recovery from winter, photoinhibition changes occurred in Fv/Fm, pigments and chloroplast ultrastructure. This work shows for the first time that irrespective of physiological mechanisms responsible for development of winter photoinhibition, there is an acclimation response with strong spatio-temporal variability at leaf level in some species. This observation should be taken into account when modeling or scaling up photosynthetic responses., (Copyright © Physiologia Plantarum 2011.)

Objective: To investigate hemodynamic in anaesthetized dogs after the intravenous injection of cyclovirobuxine D (CVB-D)., Methods: The hemodynamic of anaesthetized dogs were observed after intravenous injection of CVB-D at doses of 0.1, 0.2, 0.4 mg/kg., Results: CVB-D of 0.2 and 0.4 mg/kg could decrease HR, TPVR and increase CBF. In addition, CVB-D of 0.4 mg/kg could increase SAP and SV. Yet MAP and CO of dogs showed no remarkable changes with the treatment of CVB-D., Conclusion: CVB-D has effect of improving cardiac function, which may be the mechanism of anti-myocardial ischemia effect of CVB-D.

The effect of Cyclovirobuxine D, an active ingredient from Buxus microphylla, was investigated in the potential prevention of cardiac dysfunction in rats with congestive heart failure. Heart failure was induced by left coronary artery occlusion and verified using echocardiography. Cyclovirobuxine D was administered for 30 days (0.5, 1.0 and 2.0mg/kg, ig) and mortality, cardiac function, hemodynamics, microcirculation, histology and ultrastructure assessments were observed. Results from the present study suggest that Cyclovirobuxine D is beneficial for heart failure induced by myocardial infarction and supports the potential for Cyclovirobuxine D as a new therapy for heart failure., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Acetylcholinesterase (AChE) inhibition-directed phytochemical studies on the methanolic extract of Buxus natalensis, collected in South Africa, resulted in the isolation of 12 compounds: O(2)-natafuranamine (1), O(10)-natafuranamine (2), cyclonataminol (3), 31-demethylbuxaminol A (4), buxaminol A (5), buxafuranamide (6), buxalongifolamidine (7), buxamine A (8), cyclobuxophylline K (9), buxaminol C (10), methyl syringate (11), and p-coumaroylputrescine (12). Compounds 1-4 were new alkaloids, and compound 5 was isolated for the first time as a natural product. Their structures were elucidated with the aid of extensive NMR and mass spectroscopic studies. Compounds 1 and 2 are members of a rarely occurring class of Buxus alkaloids, having a tetrahydrofuran ring incorporated in their structures. Compounds 1-12 exhibited strong to moderate AChE inhibitory activity.

From the aerial parts of Buxus hyrcana, three triterpenoidal alkaloids, 17-oxo-3-benzoylbuxadine (1), buxhyrcamine (2), and 31-demethylcyclobuxoviridine (3), along with 16 known compounds, cyclobuxoviridine (4), N(b)-dimethylcyclobuxoviricine (5), E-buxenone (6), Z-buxenone (7), moenjodaramine (8), homomoenjodarmine (9), buxamine A (10), buxamine B (11), 31-hydroxybuxamine B (12), N(20)-formylbuxaminol E (13), papillozine C (14), buxmicrophylline F (15), buxrugulosamine (16), cyclobuxophylline O (17), spirofornabuxine (18) and arbora-1,9(11)-dien-3-one (19) were isolated. Their structures were elucidated by using NMR spectroscopic methods. All of the compounds exhibited moderate to weak acetylcholinesterase, butyrylcholinesterase and glutathione S-transferase inhibitory activities. Compounds 1-19 also exhibited modest anti-fungal activities against Candidaalbicans. Compounds 1, 2, 8, 9 and 18 also exhibited weak anti-leishmanial activity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

Buxidin (1) and E-Buxenone (2), steroidal alkaloids from Buxus hyrcana, are found to possess potent immunosuppressive properties. The activity was tested in vitro on oxidative burst, chemotaxis, T-cell proliferation, and cytokine production. Both compounds showed a significant immunomodulatory activity with clear suppressive effect on oxidative burst and chemotaxis in a dose-dependent manner. They also exhibited suppressive effect on the phytohemagglutinin-stimulated T-cell proliferation. The immunomodulatory activity was further confirmed by the suppression of IL-2 and IL-4 production. Furthermore, molecular docking studies were performed to investigate the binding mode of Buxidin (1) and E-Buxenone (2) with IL-2. Despite the structural differences between Buxidin (1) and E-Buxenone (2), docking results revealed that they adopt a similar binding pattern at the active site of the IL-2. A good agreement between practical and theoretic results indicates that the current docking study could provide an alternate tool for the structural optimization of recently identified ligand as more potent IL-2 inhibitors.

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.

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.

Objectives: To study the cardiac protection of Cyclovirobuxinum D (Cvb-D) in rats model., Methods: The rats were subjected to left main coronary artery occlusion. The change about S-T segment, the area of myocardial injury (necrotic and ischemic areas), the amount of cardiac tissue malondialdehyde (MDA), the cardiactissue creatine phosphokinase (CPK) and the cardiac tissue superoxide dismutase (SOD) activation were measured., Results: Compared to the model group, Cvb-D (1.1 mg/kg, 2.2 mg/kg dos-age) significantly reduce myocardial damage, reduce myocardial ischemia mode rats' sigma s-t of ECG, significantly reduce cardiac tissue CPK activation and MDA content, raise the cardiac tissue SOD activation in the rats with myocardial ischemia., Conclusion: Cvb-D is effective in treatment of myocardial ischemia in rats.

Objective: To observe the effect of cyclovirobuxinum-D (CVB-D) on cerebral ischemia-reperfusion injury in rats and explore its mechanisms., Method: One hundred and twenty rats were randomly divided into three CVB-D groups (2, 1, 0.5 mg x kg(-1)), Nimodipine group (2 mg x kg(-1)), model group and sham operated group, 20 rats each group. Rat cerebral ischemia-reperfusion injury model was induced by middle cerebral artery occlusion, the nerve injury symptoms was evaluated, the level of SOD and MDA in brain tissue were determined, the concentration of intracellar Ca2+ of brain was measured, and the pathological change of brain was also observed., Result: CVB-D could improve the nerve injury symptoms, reduce the infarction area of brain, the concentration of intracellar Ca2+ and the level of MDA, increase the activity of SOD, and decrease the pathological change of brain., Conclusion: CVB-D has protective effect on cerebral ischemia-reperfusion injury in rats.

Buxus alkaloids display a wide range of interesting pharmacological activities. Here, we present a short and efficient approach towards one of the alkaloids, buxozine-C. The first synthesis has been achieved with 91% yield starting from cyclovirobuxine-D by forming a tetrahydro-oxazine ring with formaldehyde at room temperature.

Phytochemical studies on the crude methanolic extract of Buxus hyrcana, collected from Iran, resulted in the isolation of two new steroidal alkaloids, (+)-O6-buxafurandiene (1) and (+)-7-deoxy-O6-buxafurandiene (2) along with four known steroidal bases, (+)-benzoylbuxidienine (3), (+)-buxapapillinine (4), (+)-buxaquamarine (5) and (+)-irehine (6). The structures of these new and known compounds were established with the aid of extensive NMR spectroscopic studies. Compounds 1 and 2 belong to the rarely occurring class of Buxus alkaloids having a tetrahydrofuran ring incorporated in their structures. Compounds 1-6 exhibited acetylcholinesterase enzyme inhibitory activity.

The current phytochemical investigation on Buxus hyrcana Pojark. has resulted in the isolation of the triterpenoid alkaloids 1-10. The structures of five new alkaloids, hyrcanone (1), hyrcanol (2), hyrcatrienine (3), N(b)-dimethylcycloxobuxoviricine (4), and hyrcamine (5), were elucidated by means of modern spectroscopic techniques, while the known alkaloids, buxidin (6), buxandrine (7), buxabenzacinine (8), buxippine-K (9) and E-buxenone (10), were identified by comparing their spectral data with those reported earlier. Compounds 1 and 3-9 were found to be acetyl- and butyrylcholinesterase inhibitors. The IC50 values were estimated to be in the range of 83.0-468.0 microM against AChE and 1.12-350.0 microM against BChE. The structure-activity relationship studies suggested that the presence of dimethylamino moieties at C(3) and C(20) is the most important factor influencing the activity of these compounds against the cholinesterase enzymes. All compounds were also evaluated for cytotoxicity on a fibroblast cell line with incubation of 24 h. No cytotoxic effects were exerted by any compound.

Objective: To establish a new method for the determination of cyclovirobuxine-D in huangyangning tablets., Method: Agilent Zorbax Extend C18 (4.6 mm x 250 mm, 5 microm) column with temperature 40 degrees C was used. The mobile phase was 0.3% diethylamine methanol solution and 0.3% diethylamine water solution, with a linear gradient of 0.3% diethylamine methanol solution from 78% to 95% in 9 minutes, and then maintained for 6 minutes. The flow rate was 0.8 mL x min(-1). Evaporative light scattering detector was used., Result: The calibration curve was linear at a range of 0.57-11.44 microg for the cyclovirobuxine D (r = 0.9996). The average recovery was 97.2% and RSD was 1.1% (n = 9)., Conclusion: This method is rapid, simple, and reproducible, and can be used as a quality control method for this preparation.

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.

Aim: To search for compounds for the treatment of cardiovascular diseases through prodrug structural modifications of cyclovirobuxine D, a single efficient composition distilled from Box plant in China, which was used to treat angina and myocardial infarction., Methods: According to prodrug design principle, a series of cyclovirobuxine D analogues were prepared, suc as succinate, phosphate and amino acid ester, and their biological activities were tested., Results: Seven new compounds were obtained and confirmed with 1H NMR, MS, and element analysis., Conclusion: In pharmacology experiment, for treating arrhythmia induced by aconitine, succinate and amino acid ester of cyclovirobuxine D (I and VII) showed better activities than that of cyclovirobuxine D. The normal rhythm of the heart duration of I and VII were ( 11.53 +/- 7.62) min and (12.68 +/- 9.25) min, compared with 0.9% NaCl solution and cyclovirobuxine D, (2.36 +/- 1.68) min and (10.25 +/- 6.59) min (P < 0.01), respectively. Another pharmacology experiment, for treating arrhythmia induced by chloroform, the negative ratio of I and VII were 80% and 82%, compared with 0.9% NaCl solution and cyclovirobuxine D, 43% and 52% (P < 0.05), respectively. The difference between new compounds and cyclovirobuxine D was distinct.

Aim: To determine the effects of cyclovirobuxine D (CD) on intracellular Ca2+ mobilization and L-type Ca2+ current (I(Ca-L)) in isolated rat cardiomyocytes., Methods: The effects of CD on the amplitude of I(Ca-L) and intracellular Ca2+ mobilization induced by KCl and caffeine were studied with the method of patch-clamp technique and laser scanning confocal microscopy in rat ventricular myocytes., Results: CD decreased the amplitude of I(Ca-L) in a concentration-dependent manner. At 10 mV, 1 and 10 micromol x L(-1) CD decreased I(Ca-L) density from (- 9.9 +/- 1.8) pA/pF to (-6.4 +/- 1.4) and (-4.2 +/- 0.6) pA/pF, respectively. Confocal experiments showed that intracellular fluorescent intensity (FI) value of [Ca2+] in control resting level was not changed by 1 and 10 micromol x L(-1) CD. [Ca2+] increase in response to KCl could not be reduced by CD. The rise of [Ca2+]i in response to caffeine was further enhanced by pretreatment with CD., Conclusion: CD decreased I(Ca,L) in a concentration-dependent manner and increased [Ca2+]i release induced by caffeine in rat ventricular cardiomyocytes.

Aim: To search for new compounds for the treatment of cardiovascular diseases by structural modification of cyclovirobuxine D., Methods: According to rational drug design principle, a series of cyclovirobuxine D analogues were prepared, and their bioactivities were tested., Results: Ten new compounds were syntheized and confirmed by spectra., Conclusion: Endurance lacking oxygen activity and antiarrhythmia effects of some analogues of cyclovirobuxine D were tested. Some compounds showed better activity than cyclovirobuxine D.