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

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

Cytotoxic androstane derivatives from Sarcococca ruscifolia.

Sarcorusones A-D (1-4), four new androstane (C19-steroid) derivatives were characterized from Sarcococca ruscifolia along with five known compounds. Their structures were elucidated on the basis of extensive MS and NMR spectroscopic analysis. All the new structures share common 14-hydroxyl and 17-ketone functional groups, and compounds 2-4 feature a seneciamide group connecting to C-3 position. The inhibitory activities of all the isolates against melanoma cell B16F10 and lung cancer cell H1299 were evaluated, and compounds 2, 3, 5, and 6 exhibited moderate cytotoxic activities against B16F10 and H1299 cell lines with IC50 values 2.7-8.0 µM.

Steroidal alkaloids efficient aromatase inhibitors with potential for the treatment of postmenopausal breast cancer.

Plant-derived natural products are of great interest due to their diversity in modern drug discovery. Sarcococca saligna has been used for the treatment of different diseases. The present study was aimed at isolating phytochemical constituents including Alkaloid-C (a), Dictyophlebine (b), Sarcovagine-D (c) and Saracodine (d) Holaphylline (e) from Sarcococca saligna to investigate the anticancer effect of these compounds. These compounds were evaluated for inhibition of aromatase enzyme of breast cancer in assistance by molecular docking simulations to understand molecular interaction between the enzyme and ligands. The IC50 values of compound 1 and 5 were found 138.27 ± 0.01 µl and 12.91 ± 0.01 µl, respectively, and both were found active due to their bulky structures in comparison to the active site of aromatase enzyme. The standard drug exemestane showed potent activity in comparison with the test compounds, having IC50 values of 0.052 ± 0.01 µl. Both compounds showed favorable electrostatic interactions with the active site of aromatase enzyme but the shape and steric bulk of the compounds was the limiting factor in their inhibitory effects. New lead compounds could be generated after extensive modifications guided by computational and experimental tools as a possible anticancer agents by targeting aromatase enzyme.

4-dehydroxyepisarcovagine A, a new steroidal alkaloid from Sarcococca pruniformis Lindl.

A new steroidal alkaloid, 4-dehydroxyepisarcovagine A (1), along with seven known alkaloids, sarcovagine D (2), sarcovagenine C (3), epoxysarcovagenine D (4), Pachysamine L (5), Pachysamine E (6), sarcovagine A (7) and sarcovagine B (8), was isolated from the roots and stems of Sarcococca pruniformis Lindl. The structure of compound 1 was elucidated by means of spectroscopic analysis.

Two New Cytotoxic Steroidal Alkaloids from Sarcococca Hookeriana.

Two new steroidal alkaloids, named hookerianine A (1) and hookerianine B (2) were isolated from the stems and roots of Sarcococca hookeriana Baill., along with two known compounds, sarcorucinine G (3) and epipachysamine D (4). On the basis of spectroscopic methods and by comparison with literature data, their structures were determined. As well as X-ray crystallography was performed to confirm compound 4. To identify novel antitumor inhibitors, all compounds were performed a CCK-8 assay against five human cancer cell lines SW480, SMMC-7721, PC3, MCF-7 and K562 in vitro. Compound 2 exhibited moderate cytotoxic activities to all cell lines with IC50 values in the range of 5.97⁻19.44 µM. Compound 3 was the most effective one against SW480 and K562 cell lines with IC50 values of 5.77 and 6.29 µM, respectively.

Three New Cytotoxic Steroidal Alkaloids from Sarcococca hookeriana.

Three new steroidal alkaloids with an unusual 3α tigloylamide group, named sarchookloides A⁻C (1⁻3), were isolated along with four known compounds (4⁻7) from the roots of Sarcococca hookeriana. Their structures and relative configuration were elucidated on the basis of spectroscopic methods including MS, UV, IR, 1D, and 2D NMR data. The isolated compounds were evaluated for their cytotoxicity against five human cancer cell lines: Hela, A549, MCF-7, SW480, and CEM in vitro. All three amide substituted steroidal alkaloids exhibited significant cytotoxic activities with IC50 values of 1.05⁻31.83 µM.

Antiinflammatory Steroidal Alkaloids from Sarcococca wallichii of Nepalese Origin.

Bio-assay guided isolation from the plant Sarcococca wallichii Staph. yielded two new steroidal alkaloids: wallichimine A (1) and wallichimine B (2), and five known ones: sarcodinine (3), N-methylpachysamine A (4), alkaloid C (5), dictyophlebine (6), and sarcorine (7). The structures of the compounds were determined using mass spectrometry and NMR spectroscopy techniques. The immunomodulatory potential of compounds was evaluated on different parameters including production of intracellular reactive oxygen species (ROS), nitric oxide (NO) and on proinflammatory cytokine TNF-α. All compounds were found to be potent inhibitors of intracellular ROS produced from isolated neutrophils, except compound 5, which showed a moderate level of inhibition. Compounds 2 and 4 potently inhibited the production of NO (67.9% and 62.5% respectively). Compound 2 showed potent suppression on production of proinflammatory cytokine TNF-α (76.7%). Among all the tested compounds the new compound 2 was found to be the most potent immunosuppressive agent. This study shows that steroidal alkaloids could be lead compounds for anti-inflammatory drug discovery.

Immunosuppressive and hepatoprotective potential of Sarcococca saligna and its biomarker components.

Sarcococca saligna methanolic extract, fractions and isolated pure compounds saracocine (1), saracodine (2), pachyximine-A (3) and terminaline (4) were found to possess potent immunosuppressive activities. The fractions and compounds were tested in-vitro for their effects on human T-cell proliferation, and cytokine (IL-2) production. All the fractions, sub-fractions and purified compounds showed significant suppressive effect on IL-2 production in a dose-dependent manner. They also exhibited a suppressive effect on the phytohemagglutinin-stimulated T-cell proliferation. None of the extracts and purified compounds showed any cytotoxicity effects on the 3T3 mice fibroblast cell line. The crude extract, DCM fraction (pH9), DCM fractions (pH7) and one of the steroidal alkaloids (terminaline) were checked in-vivo for their hepato-protective potential against CCl4-induced liver injury. In in-vivo experiments, the basic and neutral DCM fractions and terminaline (4) significantly reduced inflammation in the liver. DCM fraction (pH9), DCM fractions (pH7) and compound 4 reduced the serum enzyme levels (ALT, AST, and ALP) down to control levels despite CCl4 treatment. They also reduced the CCl4-induced damaged area to almost zero as assessed by histopathology. The pale necrotic areas and mixed inflammatory infiltrate which are seen after CCl4 treatment were absent in the cases of basic, neutral fractions and terminaline treatment. These hepato-protective effects were better than the positive control silymarin. Our results suggest the therapeutic effect of S. saligna extract, fractions and bioactive steroidal alkaloids against CCl4-induced liver injury in vivo and their immunosuppressive function in vitro.

Pregnane alkaloids from Sarcococca hookeriana var. digyna.

Fourteen pregnane-type steroidal alkaloids were isolated from the ethanolic extracts of whole Sarcococca hookeriana var. digyna plants. Their structures were elucidated on the basis of spectral data. Three of them were identified as new steroidal alkaloids: (S)-20-(N,N-dimethylamino)-16α,17α-epoxy-3ß-methoxy-pregn-5-ene (1), (20S)-20-(N,N-dimethylamino)-3ß-tigloylamino-5α-pregn-11ß-ol (2), and (20S)-2α,4ß-bis(acetoxy)-20-(N,N-dimethylamino)-3ß-tigloylamino-5α-pregnane (3). Some of the isolated compounds showed estrogen biosynthesis-promoting effects in human ovarian granulosa-like KGN cells. The EC50 values for the most effective compounds, vagnine B (6) and funtumafrine C (12), were 71 µM and 67 µM, respectively.

Syringe test (modified larval immersion test): a new bioassay for testing acaricidal activity of plant extracts against Rhipicephalus microplus.

We report a new bioassay "syringe test" (modified larval immersion test) for in vitro evaluation of acaricidal activity of crude plant extracts. Prepared syringes, containing eggs of tick, were incubated until 14 d after hatching of eggs, when the bioassay was performed on the larvae. Lethal concentrations for 50% of larvae (LC(50)), LC(90) and LC(99) values were calculated for each tested product. 95% confidence intervals for LC(50) were very narrow, indicating a high degree of repeatability for the new bioassay on larvae of R. microplus. Bioassays were applied to six crude aqueous-methanol extracts from five plants (Acacia nilotica, Buxus papillosa, Fumaria parviflora, Juniperus excelsa, and Operculina turpethum), of which three showed discernible effects. Twenty-four hours post exposure, LC(99) values were 11.9% (w/v) for F. parviflora, 20.8% (w/v) and 29.2% (w/v) for B. papillosa and A. nilotica, respectively. After six days of exposure these values were; 9.1% (w/v), 9.2% (w/v) and 15.5 (w/v) for F. parviflora, A. nilotica and B. papillosa, respectively.

Rapid characterization and identification of steroidal alkaloids in Sarcococca coriacea using liquid chromatography coupled with electrospray ionization quadropole time-of-flight mass spectrometry.

Rapid characterization of 23 pregnane-type steroidal alkaloids was studied using a positive ion electrospray ionization quadropole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. ESI-QqTOF-MS (positive ion mode) showed the presence of the protonated molecules [M+H](+) which through low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) analysis showed the characteristic loss of dimethylamine moiety [M+H-45](+) followed by the sequential lossess of attached substituents. Steroidal alkaloids having tigloyl or senecioyl group at C-3 produced diagnostic fragment ions at m/z 100 and 83. Our study also demonstrates the influence of unsaturation, and number and nature of substitutents on product ion abundance and fragment ions. Moreover, the generalization of the fragmentation pattern was linked with the structural features in steroidal skeleton. This strategy was successfully applied in LC-ESI-QqTOF-MS/MS analysis of Sarcococca coriacea extract to investigate and characterize pregnane-type steroidal alkaloids in complex mixture.

Cytotoxic steroids from Sarcococca saligna.

Two new C21-steroidal esters, sarsaligates A(1) and B(2), and two new steroidal alkaloids, sarsaligenines A(3) and B(4), together with four known compounds (sarcovagine, sarcorucinine, dimethylamino-3 ß-pregnane-20-one, and ß-sitosterol 5- 8, respectively), were isolated from the leaves and stems of Sarcococca saligna. The structures of compounds 1-4 were elucidated by NMR and MS spectroscopic analysis. Of the compounds tested, 5 and 6 were the most cytotoxic against the cell lines K562, SK-BR-3, and PANC-1, with IC50 values in the range of 2.25-5.00 µM, while 3 and 4 selectively inhibited HL-60 cells with IC50 values of 2.87 and 3.61 µM, respectively. Compounds 3-6 therefore deserve further evaluation of their cytotoxic potentials.

Two new steroidal alkaloids from the roots of Sarcococca ruscifolia.

Two new steroidal alkaloids, 20alpha-dimethylamino-2alpha-hydroxyl-3beta-tigloylamino-5alpha-pregnane (1) and Delta(16)-20alpha-dimethylamino-3beta,4alpha-diol-5alpha-pregnane (2), along with five known alkaloids, sarcovagine D (3), pachysamine G (4), pachysamine H (5), pachysamine A (6), and terminaline (20S)-20-(N,N-dimethylamino)-5alpha-pregn-3beta,4alpha-diol (7), were isolated from the roots of Sarcococca ruscifolia. Their structures were elucidated on the basis of NMR and MS data, and the relative stereochemistry of 1 was finally determined by X-ray crystallographic analysis.

Bioactive steroidal alkaloids from Sarcococca hookeriana.

Four new 5 alpha-pregnane-type steroidal alkaloids, hookerianamides L(1), M(2), N(3), and O(4), and a known N-formylchonemorphine (5) have been isolated by acid-base extraction of the dichloromethane extract of Sarcococca hookeriana. The structures of all compounds were determined with spectroscopic techniques and by comparison with literature data. All compounds displayed antileishmanial and antibacterial properties. Compounds 1, 4, and 5 were found to be more potent than standard pentamidine (IC (50) = 9.59 microg/mL) with respect to leishmanicidal activity. The minimum inhibitory concentration of most of the compounds against Bacillus subtilis, Streptococcus minor, and Streptococcus ferus was lower than that of the standard ampicillin.

The antifungal activity of Sarcococca saligna ethanol extract and its combination effect with fluconazole against different resistant Aspergillus species.

Microbial resistance is a major drawback in chemotherapy of microbial or fungal infection disease. In this study, the antifungal activity of ethanol extract of a selected plant (Sarcococca saligna) has been investigated against clinical isolates of Aspergillus niger, Aspergillus treus, Aspergillus flavus, and Aspergillus fumigatus. Also, the enhancement of the antifungal activity of fluconazole by this extract was further evaluated against mentioned test strains. Conventional disk diffusion method was used to assay the antifungal activity of S. saligna ethanol extract in the absence and presence of fluconazole. The highest antifungal activity was observed against A. treus. The ethanol extract of S. saligna enhanced the antifungal activity of fluconazole against A. niger and A. treus and A. flavus. At the highest tested contents (4 mg/disk), 1.15-, 0.64-, and 2.47-fold increases in inhibition zone surface area were observed for A. niger, A. treus, and A. flavus, respectively. However, no enhancing effect was observed for this plant extract against Aspergillus fumigates at tested contents (0.5, 1, 2, 3, and 4 mg/disk). In a separate experiment, the general cytotoxicity of the ethanol extract of S. saligna was examined with brine shrimp assay. This plant extract showed low cytotoxicity against Artemia salina (LC(50) = 186 microg/ml).

Bioactive 5alpha-pregnane-type steroidal alkaloids from Sarcococca hookeriana.

The bioassay-guided phytochemical investigation of Sarcococca hookeriana with respect to cholinesterase inhibitory properties has yielded two new 5alpha-pregnane-type steroidal alkaloids, hookerianamides J (1) and K (2), along with eight known compounds (3-10). The structures of 1 and 2 were elucidated by spectroscopic methods. These compounds displayed good to moderate activities in vitro against the enzymes acetylcholinesterase (IC 50 8.1-48.5 microM) and butyrylcholinesterase (IC 50 0.4-4.0 microM). Compounds 1-10 were also tested in vitro for their leishmanicidal activity against Leishmania major and for their antibacterial activities against Bacillus subtilis, Micrococcus luteus, Streptococcus faecalis, and Pseudomonas pallida.

Terpenoid alkaloids of the Buxaceae family with potential biological importance.

The plants of the family Buxaceae are widely used in traditional medicine and constitute rich sources of terpenoidal alkaloids. Compounds of this family have been the subject of numerous chemical and pharmacological studies over past decades because of their interesting biological activities such as cholinesterase inhibition, as well as antibacterial and antileishmanial activities. The chemical and biological properties of these alkaloids, including data relevant to straightforward structure determination and information on biosynthesis, are highlighted in this review, with 144 references being cited.

Cholinesterase inhibiting and antiplasmodial steroidal alkaloids from Sarcococca hookeriana.

Bioguided phytochemical investigation of Sarcococca hookeriana with respect to the cholinesterase enzyme inhibitory assay yielded two new pregnane-type steriodal alkaloids hookerianamide H (1) and hookerianamide I (2), along with three known alkaloids N(a)-methylepipachysamine D (3), sarcovagine C (4) and dictyophlebine (5). Their structures were determined with the aid of extensive spectroscopic analysis. All compounds showed good inhibitory activities against the enzymes acetylcholinesterase (IC(50) 2.9-34.1 microM) and butyrylcholinesterase (IC(50) 0.3-3.6 microM). These compounds also showed moderate antiplasmodial activity (IC(50) 2.4-10.3 microM) against the Plasmodium falciparum chloroquine resistant W2 strain.

Structure activity relationship studies on antileishmanial steroidal alkaloids from Sarcococca hookeriana.

The search for antileishmanial constituents from the medicinal plant Sarcococca hookeriana (Buxaceae) of Nepalese origin has resulted in the isolation of 17 (1-17) active steroidal alkaloids. Compounds 1, 2, and 10 were subjected to derivatization and five chemically derived derivatives (1a, 2a, 10a, 10b, 10c) were also obtained. All these natural compounds and derivatives were found to have potent to mild antileishmanial properties. The IC(50) values were found to be in the range of 0.20-61.44 microg mL(-1) (IC(50) value of standard drug amphotericin B = 0.12 microg mL(-1)). The structure activity relationship indicated that the varieties of functionalities present in ring A of the steroidal alkaloids were found to play a characteristic role to increase the antileishmanial activity.

Studies on cardio-suppressant, vasodilator and tracheal relaxant effects of Sarcococca saligna.

Sarcococca saligna is a shrub that is traditionally used for its medicinal properties in Pakistan. In this study we report the cardio-suppressant, vasodilator and tracheal relaxant activities of the aqueous-methanolic extract (Ss.Cr) of the plant. Ss.Cr, that tested positive for the presence of saponins, flavonoids, tannins, phenols, and alkaloids, exhibited a dose-dependent (0.3-5 mg/mL) negative inotropic and chronotropic effect on the isolated guinea-pig atrium which was resistant to atropine (1 microM) and aminophylline (10 microM) pretreatment. In rabbit thoracic aorta, Ss.Cr dose-dependently (0.1-3 mg/mL) relaxed the high K+ (80 mM) and phenylephrine (PE, 1 microM)-induced contractions, indicating a possible Ca++ channel blocking (CCB) effect. When tested against PE (1 microM) control peaks in normal Ca++ and Ca++-free Kreb's solution, Ss.Cr exhibited dose-dependent (0.1-3 mg/mL) inhibition, being more potent in relaxing the PE responses in Ca++-free Kreb's solution, thus indicating specific blockade of Ca++ release from the intracellular stores. Ss.Cr also relaxed the agonist-induced contractions in: a) rat aorta irrespective of the presence of endothelium or nitric oxide synthase inhibitor L-NAME and b) rabbit and guinea-pig tracheal strips. The data shows that Ss.Cr possesses possible Ca++ channel blocking activity which might be responsible for its observed cardio-suppressant, vasodilator and tracheal relaxant effects though more tests are required to confirm this Ca++ channel blocking effect.