Pregnane steroids and steroid glycosides with multidrug resistance reversal activity from Marsdenia tenacissima.

Twenty compounds comprising four pregnane steroids (2-4 & 20) and 16 pregnane glycosides (1 & 5-19) have been obtained from the ethanol extract of the roots of a Dai ethnological herb, Marsdenia tenacissima. Their structures were characterized on the basis of comprehensive spectroscopic analyses with 17 ones (1-17) being reported for the first time, including the rare cases (2 & 3) of free C21 steroids with 17α-acetyl substitution, compounds 4-7 bearing an unusual 14α-OH, and the first examples with simultaneous 14α-OH/17α-acetyl substitution (7) and glycosylation at C-12 position (10 & 11). An empirical rule for the identification of C-17 configuration, in C21 steroids incorporating the marsdenin constitution structure, was also proposed. All the isolates, along with an array of previously reported analogues in our compound library, were screened for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and six compounds exhibited moderate MDR reversal activity with reversal folds ranging from 1.92 to 4.44.

seco-Pregnane Glycosides from Australian Caustic Vine (Cynanchum viminale subsp. australe).

Cynanchum viminale subsp. australe, more commonly known as caustic vine, is a leafless succulent that grows in the northern arid zone of Australia. Toxicity toward livestock has been reported for this species, along with use in traditional medicine and its potential anticancer activity. Disclosed herein are novel seco-pregnane aglycones cynavimigenin A (5) and cynaviminoside A (6), together with new pregnane glycosides cynaviminoside B (7) and cynavimigenin B (8). Cynavimigenin B (8) contains an unprecedented 7-oxobicyclo[2.2.1]heptane moiety in the seco-pregnane series, likely arising from a pinacol-type rearrangement. Interestingly, these isolates displayed only limited cytotoxicity in cancer and normal human cell lines, in addition to low activity against acetylcholinesterase and Sarcoptes scabiei bioassays, suggesting that 5-8 are not associated with the reported toxicity of this plant species.

New Pregnane Glycosides from Mandevilla dardanoi and Their Anti-Inflammatory Activity.

Mandevilla Lindl. is an important genus of the Apocynaceae family, not only as ornamental plants but also for its medicinal uses. In Brazil, Mandevilla species are indicated to treat asthma and skin infections, their anti-inflammatory potential and wound healing properties are also reported in the literature. Concerning their chemical composition, this group of plants is a conspicuous producer of pregnane glycosides. Mandevilla dardanoi is an endemic species from the Brazilian semiarid region not studied by any phytochemical methods. In view of the medicinal potential of Mandevilla species, this study aimed to isolate new pregnane glycosides from M. dardanoi. To achieve this main goal, modern chromatography techniques were employed. Five new pregnane glycosides, dardanols A-E, were isolated from the roots of M. dardanoi by HPLC. Their structures were determined using extensive 1D and 2D-NMR and mass spectrometry (MSn and HRESIMS) data. The cytotoxicity and the anti-inflammatory potential of these compounds were evaluated. The first was evaluated by measuring proinflammatory cytokines and nitric oxide production by stimulated macrophages. Dardanols were able to inhibit the production of nitric oxide and reduce IL-1ß and TNF-α. The current work demonstrates the chemodiversity of Brazilian semiarid species and contributes to amplifying knowledge about the biological potential of the Mandevilla genus.

Trichilones A-E: New Limonoids from Trichilia adolfi.

In addition to the trichilianones A-D recently reported from Trichilia adolfi, a continuing investigation of the chemical constituents of the ethanol extract of the bark of this medicinal plant yielded the five new limonoids 1-5. They are characterized by having four fused rings and are new examples of prieurianin-type limonoids, having a ε-lactone which in 4 and 5 is α, ß- unsaturated. The structures of the isolated metabolites were determined by high field NMR spectroscopy and HR mass spectrometry. The new metabolites were shown to have the ε-lactone fused with a tetrahydrofuran ring which is connected to an oxidized hexane ring joined with a cyclo-pentanone having a 3-furanyl substituent. As the crude extract possesses antileishmanial activity, the compounds were assayed for cytotoxic and antiparasitic activities in vitro in murine macrophage cells (raw 264.7 cells) and in Leishmania amazoniensis as well as L. braziliensis promastigotes. Metabolites 1-3 and 5 showed moderate cytotoxicity (between 30-94 µg/mL) but are not responsible for the antileishmanial effect of the extract.

C21, C22 pregnane glycosides and cytotoxic C27 spriostanol steroids from Asparagus cochinchinesis.

A preliminary chemical investigation on 70% MeOH extract of the roots of Asparagus cochinchinensis resulted in the isolation of nine steroids. These isolates comprised of four new C21 (1-4) and one new pregnane (5) glycosides, and four known C27 (6-9) spirostanol steroids. Their structures were identified via analysis of the spectroscopic data and the results of hydrolytic cleavage. The cytotoxic activities of the compounds were tested toward the human tumor cell line Hela (cervical cancer), and compounds 7 and 8 displayed moderate activity with IC50 values of 35.5 and 39.6 µM, respectively.

Pregnane Steroids from the Leaves of Melia Azedarach and Apoptotic Activity against T47D Cells.

OBJECTIVE: Nature has provided us with many pharmaceutical resources so far. Breast cancer shows an increasing trend in the world for the last decade and becomes one of five leading causes of death. Among the plants, Melia azedarach L. has been used widely in traditional medicine for many ailments including breast cancer. Following our previous findings that the ethyl acetate fraction was the most active cytotoxic fraction against T47D cells, we aimed to isolate the cytotoxic compounds and further elucidate their apoptotic mechanisms. METHODS: The compounds were isolated through a series of chromatography with cytotoxicity evaluations. Identification of the isolated compounds was achieved by intensive spectroscopic analysis such as NMR, MS, and IR spectra. Cytotoxicity was evaluated by MTT method using doxorubicin as a reference compound. The expression of apoptosis-related factors was quantified by flow cytometry and immunocytochemistry. RESULTS: Two isomers of pregnane steroids with molecular weight 330.2087 (C21H30O3) were isolated from the EtOAc extract. Spectroscopic analysis revealed the structures as 17-ethylene-3,4-dihydroxy-14-methyl-18-norandrostene-16-one (1) and 17-ethylene-3,4-dihydroxy-5-pregnene-16-one (2), respectively. These compounds showed moderate cytotoxicity (IC50 172.9 and 62.2 µg/mL, respectively) comparable to doxorubicin (IC50 3.08 µg/mL). The execution of apoptosis may be related to the increase of the ratio of BAX/bcl-2 of the cells.  Conclusion: The EtOAc fraction of Melia azedarach L. leaves and the isolated 5-pregnene-16-one steroids are promising reagents for breast cancer treatment by introducing apoptosis to tumor cells. However, further researches are required to highlight its safety and usage in vivo.
.

Synthesis and Characterization of Bone Binding Antibiotic-1 (BBA-1), a Novel Antimicrobial for Orthopedic Applications.

Osteomyelitis and orthopedic infections are major clinical problems, limited by a lack of antibiotics specialized for such applications. In this paper, we describe the design and synthesis of a novel bone-binding antibiotic (BBA-1) and its subsequent structural and functional characterization. The synthesis of BBA-1 was the result of a two-step chemical conjugation of cationic selective antimicrobial-90 (CSA-90) and the bisphosphonate alendronate (ALN) via a heterobifunctional linker. This was analytically confirmed by HPLC, FT-IR, MS and NMR spectroscopy. BBA-1 showed rapid binding and high affinity to bone mineral in an in vitro hydroxyapatite binding assay. Kirby-Baur assays confirmed that BBA-1 shows a potent antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus comparable to CSA-90. Differentiation of cultured osteoblasts in media supplemented with BBA-1 led to increased alkaline phosphatase expression, which is consistent with the pro-osteogenic activity of CSA-90. Bisphosphonates, such as ALN, are inhibitors of protein prenylation, however, the amine conjugation of ALN to CSA-90 disrupted this activity in an in vitro protein prenylation assay. Overall, these findings support the antimicrobial, bone-binding, and pro-osteogenic activities of BBA-1. The compound and related agents have the potential to ensure lasting activity against osteomyelitis after systemic delivery.

Pregnane glycosides from the leaves of Dregea volubilis and their α-glucosidase and α-amylase inhibitory activities.

Three new pregnane glycosides, drevoluosides O-Q (1-3) along with five known volubiloside C (4), dreageoside A11 (5), 17ß-marsdenin (6), stavaroside H (7), and hoyacarnoside G (8) were isolated from the methanol extract of the Dregea volubilis leaves. Their structures were elucidated by chemical and spectroscopic methods. Compounds 6-8 showed significant anti α-glucosidase activity with the inhibitory percentages ranging from 32.6 to 47.1% at the concentration of 200 µM. Compound 3 showed significant inhibitory α-amylase activity with IC50 value of 51.3 ± 2.1 µM.

Anti-nociceptive, anti-inflammatory and anti-arthritic activities of pregnane glycosides from the root bark of Periploca sepium Bunge.

ETHNOPHARMACOLOGICAL RELEVANCE: Periploca sepium Bunge (P. sepium) is used in traditional Chinese medicine (TCM) for the treatment of autoimmune diseases, particularly rheumatoid arthritis. Periploca sepium periplosides (PePs), isolated from the root bark of P. sepium, characterized as the cardiac glycosides-free pregnane glycosides fraction, is expected to possess therapeutic potential on inflammatory arthritis. AIM OF THE STUDY: The current study is designed to evaluate the anti-nociceptive, anti-inflammatory and anti-arthritic activities effects of the PePs. MATERIALS AND METHODS: The anti-nociceptive activity of PePs was examined in the writhing test and hot-plate test in mice. The anti-inflammatory activity of PePs was determined by the 2, 4-dinitro-1-fluorobenzene (DNFB)-induced ear edema model and the carrageenan induced paw edema model in mice. The anti-arthritic activity of PePs was investigated by evaluating the joint inflammation and arthritis pathology in rat adjuvant induced arthritis (AIA) and murine collagen induced arthritis (CIA). Phytohaemagglutinin M (PHA-M) -elicited human peripheral blood mononuclear cells (PBMCs) were further applied to assess the suppressive activity of PePs on IFN-γ and IL-17 production. RESULTS: PePs treatment markedly decreased the acetic acid-induced visceral nociceptive response and increased the hot-plate pain threshold. Further, oral administration of PePs exhibited anti-inflammatory activity by decreasing DNFB-induced ear edema in mice and carrageenan-induced paw edema in rats. Moreover, oral treatment of PePs ameliorated joint swelling and attenuated bone erosion in rodent arthritis, and the therapeutic benefits were partially attributed to the suppression of proinflammatory cytokines such IFN-γ and IL-17. Moreover, PePs suppressed the proliferation as well as IFN-γ and IL-17 secretion in PHA-M-elicited human PBMCs in a concentration dependent manner. CONCLUSIONS: Taken together, our results justified the traditional use of Periploca sepium Bunge for the treatment of diseases associated with inflammation and pain.

Two new pregnane alkaloids from Pachysandra terminalis Sieb. et Zucc.

Two new pregnane alkaloids, (20S)-20α-cinnamoylamino-3ß-dimethylamino-5-en-pregnane (1) and (20S)-20α-cinnamoylamino-3ß-dimethylamino-pregnane (2), and four known alkaloids (+)-(20S)-20-(dimethylamino)-3-(3'R-isopropyl)-lactam-5α-pregn-2-en-4-one (3), axillaridine A (4), pachysamine M (5) and 20α-dimethylamino-16ß-hydroxy-3ß-senecioylamino-pregn-5-ene (6) were obtained from the whole herb of Pachysandra terminalis Sieb. et Zucc. Their structures were determined by various spectral techniques and computed electronic circular dichroism (ECD) data. Compounds 1-4 were tested for cytotoxicity against three human tumor cell lines and a human umbilical vein endothelial cell (HUVEC) line. Compound 4 exhibited moderate cytotoxicity against MCF-7, U251 and A549 cells with IC50 values of 15.01 ± 0.47 µM, 20.13 ± 1.34 µM and 20.04 ± 1.16 µM, respectively; compounds 1-3 showed weak cytotoxic activity against three tumor cells.

Five New Pregnane Glycosides from Gymnema sylvestre and Their α-Glucosidase and α-Amylase Inhibitory Activities.

Gymnema sylvestre, a medicinal plant, has been used in Indian ayurvedic traditional medicine for the treatment of diabetes. Phytochemical investigation of Gymnema sylvestre led to the isolation of five new pregnane glycosides, gymsylosides A-E (1-5) and four known oleanane saponins, 3ß-O-ß-D-glucopyranosyl (1→6)-ß-D-glucopyranosyl oleanolic acid 28-O-ß-D-glucopyranosyl ester (6), gymnemoside-W1 (7), 3ß-O-ß-D-xylopyranosyl-(1→6)-ß-D- glucopyranosyl-(1→6)-ß-D-glucopyranosyl oleanolic acid 28-O-ß-D-glucopyranosyl ester (8), and alternoside XIX (9). Their structures were identified based on spectroscopic evidence and comparison with those reported in the literature. All compounds were evaluated for their α-glucosidase and α-amylase inhibitory activities. Compounds 2-4 showed significant α-amylase inhibitory activity, with IC50 values ranging from 113.0 to 176.2 µM.

New calogenin pregnane glycoside derivative from Huernia saudi- arabica and its Lipase and α-Glucosidase Inhibitory Activities.

As ongoing investigation of Huernia saudi-arabica D.V.Field (Asclepiadaceae), a new steroidal pregnane glycoside (Huernioside A) was isolated from dichloromethane fraction (DCM); it was identified as 3ß, 11, 14ß, 20(R)-tetrahydroxy-pregna-5,9(11)-diene-3-O-ß-D-thevetopyranosyl-(1-4)-ß-D-cymaropyranoside(HCP) through analysis of 1D, 2D NMR besides ESI-MS data. The alcoholic extract of the aerial part (ALE), DCM and HCP showed inhibitory potential against pancreatic lipase compared to orilstat. Among the tested samples, the ALE and HCP exhibited a promising pancreatic lipase inhibitory commotion through IC50 values of 0.61 ±â€¯0.15, 1.23 ±â€¯0.07 mg/ml (equivalent to 88.8 µM), respectively. HCP was prevailed to have a mixed mode of inhibition as exposed by enzyme kinetic studies. Hydrophobic interactions were the major forces involved in ligand enzyme interactions. In contrast, moderate α-glucosidase inhibitory activities were evidenced for ALE and HCP (% inhibition: 24.8 ±â€¯1.8 and 26.6 ±â€¯2.5, respectively) compared to acarbose. This investigation is the first to report on the possible in vitro anti-obesity and anti-diabetic impact of H. saudi-arabica.

Pregnane glycoside from Huernia saudi-arabica as latent schistosomicidal mediator.

Development of a novel agent for control of schistosomiasis is a mandate. In-vitro anti-schistosomal activity of the aerial parts of Huernia saudi-arabica were examined. Chromatographic investigations of the ethanol extract (EE) were afforded three compounds. Pregnane glycoside (CI) 12-ß-p-hydroxy-benzoyl-20-O-acetyl-boucerin-3-O-ß-D-glucopyranosyl-(1→4)-ß-D-cymaropyranosyl-(1→4)-ß-D-cymaropyranoside, in addition to two flavonoids (CII) luteolin-4'-O-ß-D-neohesperidoside and (CIII)quercetin-3-rutinoside were recognized via spectral analysis. The schistosomicidal effects were evaluated using scanning electron microscope (SEM). In-vitro bioassays on the viability (mobility, morphological changes and mortality) of Schistosoma mansoni adults, cercariae, miracidia and eggs at different concentrations 2.5, 5, 12.5, 25 and 50 µg/ml of EE and 2.6, 5.2, 13, 26 and 52 µM of CI in incubation times 1,2,4,6,12hrs were carried out. EE and CI evidenced in-vitro anti-schistosomal activity with a dose and incubation time-dependent fashion. The effect of EE and CI was evident by the topography damage showed by SEM. EE proved moderate in-vitro cytotoxicity with IC50 of 8.48 µg/ml.

Steroids from herbs of Reineckia carnea and their anticomplement activities.

A new polyhydroxylated pregnane, named lß,2ß,3ß,4ß,5ß,6ß-hexolhydroxy-pregn-16-en-20-one (1), along with nine known (2-10) steroidal saponins were isolated from the whole plant of Reineckia carnea. Structure elucidations of all compounds were established by interpretation of their NMR spectral data, HR-ESI-MS and comparing with literatures. In addition, these compounds were evaluated with anticomplement activity. The result showed that compound 1 exhibited anticomplement effects with the CH50 values of 0.043 mg/mL, but saponins (2-10) showed no inhibition. Interestingly, hydrolysis of steroidal saponins (2-10) resulted in its aglycones (2a-10a) correspondingly which showed anticomplement activity with the CH50 values of 0.049-0.156 mg/mL.

Calotroposid A: a Glycosides Terpenoids from Calotropis gigantea Induces Apoptosis of Colon Cancer WiDr Cells through Cell Cycle Arrest G2/M and Caspase 8 Expression

Objective: This study aims to isolate the active anticancer compound from ethyl acetate fraction extracted from the roots of Calotropis gigantea and to determine the operating mechanism of the isolates towards WiDr colon cancer cells. Methods: the isolation was conducted by using bioassay guided isolation approach method. The cytotoxic potential was determined by using MTT method. The chemical structure was identified by using UPLCMS/MS and NMR-1H spectroscopy. The cell cycle arrest and apoptosis induction were determined by flow cytometry method. The expression of caspase-8 was determined by immunocytochemistry method. Results: The results showed that the active compounds are obtained calotroposid A compound which is glycosides terpenoids. Calotroposide A is capable of inhibiting the growth of WiDr colon cancer cells at IC50 17.23µg/ml. Cell apoptosis induction took place and was indicated by cell apoptosis increase, S and G2/M accumulation and by caspase-8 expression. Conclusion: Calotroposide A induces anticancer activity against WiDr colon cancer cells by means of apoptosis induction mechanism through extrinsic pathway with increased expression of caspase-8.

Melanogenesis inhibitory pregnane glycosides from Cynanchum atratum.

Bioassay-guided fractionation of the methanolic extract from the roots of Cynanchum atratum has resulted in the isolation of three new pregnane glycosides (1-3) along with four known compounds (4-7). Their structures were identified by analysis of the spectroscopic data including extensive 2D NMR. All of the isolates were evaluated for their potential to inhibit the melanin production in α-melanocyte stimulating hormone (α-MSH)-activated B16 melanoma cells. Of these, compounds 4-7 dose-dependently inhibited the melanin production with the IC50 values ranging from 4 µM to 33 µM.

Rapid structure prediction by HPLC-ESI-MSn of twenty-five polyoxypregnane tetraglycosides from Dregea sinensis with NMR confirmation of eight structures.

Dregea sinensis Hemsl is an important herbal medicine in the Dai nationality of China. Its prominent clinical application has generated interest in the polyoxypregnane glycosides of the plant. This paper describes an extension of previous research on the polyoxypregnane di- and triglycosides of D. sinensis, aiming at identifying related tetraglycosides. On the basis of HPLC-ESI-MSn analysis in positive mode, twenty-five previously undescribed polyoxypregnane tetraglycosides were characterized (regarding molecular masses and fragmentation in MSn) from an ethyl acetate fraction that was not previously investigated. Guided by MSn fragmentation and known structures of related di- and triglycosides from D. sinensis, tentative structures were predicted from the MS data. In order to test the predictions, eight of the glycosides were isolated and their structures were elucidated by 1D and 2D NMR methods, confirming the tentative predictions. Finally, the cytotoxicity of the isolates was evaluated on several human cell lines with little effect in general, even though slight inhibitory effects of four polyoxypregnane glycosides were detected at 10 µM against the human leukemia cell line HL-60.

A new antiviral pregnane from a gorgonian-derived Cladosporium sp. fungus.

A new pregnane, 3α-hydroxy-7-ene-6,20-dione (1), and five known steroids (2-6), along with one known steroidal glycoside (7) were obtained from the fungus Cladosporium sp. WZ-2008-0042 cultured from a gorgonian Dichotella gemmacea collected from the South China Sea. The structure and absolute configuration of the new compound (1) were elucidated by comprehensive spectroscopic data and X-ray diffraction data. The compound has a rare configuration of 3α-OH that is different from most of pregnanes. All of the isolated compounds were evaluated for their antiviral activities against respiratory syncytial virus (RSV). Among them, 1 exhibited potential antiviral activity with the IC50 value of 0.12 mM.

Reversal of multidrug resistance by Marsdenia tenacissima and its main active ingredients polyoxypregnanes.

ETHNOPHARMACOLOGICAL RELEVANCE: Multidrug resistance (MDR) of cancer is often associated with the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP or ABCG2), in cancer cells, which facilitates the active efflux of a wide variety of chemotherapeutic drugs out of the cells. Marsdenia tenacissima is a traditional Chinese medicinal herb that has long been clinically used for treatment of cancers, particularly in combinational use with anticancer drugs. Polyoxypregnanes (POPs) are identified as main constituents of this herb, and three of them have been reported to exhibit P-gp modulatory effect and thus reverse MDR. Therefore, it is of great necessity to investigate more POPs that have potential to reverse transporters-mediated MDR. AIM OF THE STUDY: We aimed to identify POPs as the chemical basis responsible for circumventing ABC transporters-mediated MDR by M. tenacissima. MATERIALS AND METHODS: The MDR reversal effects of M. tenacissima crude extract together with a series of isolated POPs were evaluated on several MDR cancer cell lines that overexpress P-gp, MRP1 or ABCG2. The activities of P-gp, MRP1 and ABCG2 were determined by the flow cytometry-based substrate efflux assay. Molecular docking of POPs to a three-dimensional human P-gp homology structure was also performed. RESULTS: The crude extract of M. tenacissima was firstly found to circumvent P-gp-mediated MDR. Then, 11 polyoxypregnane compounds (POPs) isolated from this herb were found to overcome P-gp-, MRP1- and/or ABCG2-mediated MDR. Further mechanistic study delineated that the reversal of MDR by these POPs was due to significant increase in the intracellular concentrations of the substrate anticancer drugs via their inhibition of different ABC transporter-mediated efflux activities. Furthermore, molecular docking revealed that POPs with P-gp modulatory effect bound to P-gp and fitted well into the cavity between the alpha and beta subunit of P-gp via forming hydrogen bonds. In addition, several key structural determinants for inhibition of P-gp, MRP1 or ABCG2 by POPs were illustrated. CONCLUSIONS: Our findings advocated the rational use of M. tenacissima to enhance efficacies of conventional anticancer drugs in tumors with ABC drug transporters-mediated MDR. Furthermore, 11 POPs were found to contribute to MDR reversal effect of M. tenacissima via inhibition of different ABC efflux transporters.

Pregnane Glycosides from Cynanchum marnierianum Stimulate GLP-1 Secretion in STC-1 Cells.

In the framework of the search for natural glucagon-like peptide-1 secretagogues, the bioassay-guided fractionation of the ethanolic extract from Cynanchum marnierianum led to the isolation of two new pregnane glycosides named marnieranosides A (1) and B (2). The structures were determined based on spectroscopic data and were established as 12ß,20 S-O-dibenzoyl-pregn-6-en-5α,8ß,14ß,17ß-tetraol-3-O-ß-D-oleandropyranosyl-(1 → 4)-ß-D-cymaropyranoside (1) and 12ß,20R-O-dibenzoyl-pregn-6-en-5α,8ß,14ß-triol-3-O-ß-D-oleandropyranosyl-(1 → 4)-ß-D-canaropyranosyl-(1 → 4)-ß-D-cymaropyranoside (2). They present structural analogies to pregnanes previously described in species known for their appetite suppressant and antihyperglycemic effects, such as P57 from Hoodia gordonii. Lupeol (3), a known dipeptidyl peptidase-4 inhibitor, and the insulinomimetic kaempferol-3-O-neohesperidoside (4) were also identified in C. marnierianum. In an in vitro assay on secretin tumor cell line-1 cells, compounds 1, 2, and P57 were found to stimulate the secretion of GLP-1 by 130 % (all tested at 100 µM). These results suggest that C. marnierianum could be of great interest in the treatment of type 2 diabetes, and that pregnane derivatives should be partly responsible via the stimulation of glucagon-like peptide-1 secretion.