New physalins from Physalis angulata and Physalis lancifolia. Structure and reactions of physalins D,I, G and K

The structures of physalins I, G and K are established respectively as 5,6-dihydro-5α-methoxy-6β-hydroxy, 4,5-dehydro-5,6-dihydro-4β-hydroxy and 5,6-dihydro-4α,5α-epoxy-6α-hydroxy derivatives of physalin B. The chemistry of physalin D, and the synthesis of physalins D and I from physalin F and physalin K from physalin G are described.     

Physalins E and H,new physalins from Physalis angulata and P. lancifolia

From the stems and leaves of Physalis angulata and P. lancifolia, the isolation of five new physalins E, F, G, H and I is reported. The structures of physalins E and H are established respectively as 5,6-dihydro-5α,7α-dihydroxy and 7β-hydroxy derivatives of physalin B.     

A practical radiochromatographic assay for cholesterol epoxide hydrase.

A method for the assay of cholesterol epoxide hydrase activity is described. The assay involves the thin-layer chromatographic separation and quantitation of radiolabeled cholestan-3β,5α,6α-epoxide and its major hydration product, cholestan-3β,5α,6β-triol. Radiochromatographic scanning is employed to quantitate the reaction. The procedure is sensitive, rapid, and nondestructive.     

Evidence for the formation of a steroid S-glutathione conjugate from an epoxysteroid precursor.

Biotransformation of [4-¹⁴C]cholesterol α-epoxide (5α,6α-epoxycholestan-3β-ol) to the S-glutathione conjugate, 3β,5α-dihydroxycholestan-6β-yl-S-glutathione, by S-glutathione transferase of the rat liver soluble supernatant fraction, has been described. After the isolation from the biological incubation mixture by n-butanol extraction, followed by the Amberlite XAD-2 column treatment, the conjugate was chromatographically identified with a synthetic specimen which was prepared from cholesterol α-epoxide and glutathione in an ethanolic solution of sodium hydroxide. Further identification of the biologically formed conjugate with the synthetic one, including structural assignment, was established from the result that they yielded the same desulfrization product, 3β,5α-dihydroxycholestane, by the treatment with Raney nickel in an atmosphere of hydrogen.     

The recently proposed 20,22-epoxycholesterol as the intermediate in the conversion of cholesterol to pregnenolone by adrenal cortex mitochondria must be 5α,6α-epoxycholestan-3β-ol

Recently proposed 20,22-epoxycholesterol as the obligatory intermediate in the NADPH-dependent conversion of cholesterol to pregnenolone by bovine adrenal cortex mitochondria was isolated and found to be a misassigned metabolite. It was identified as 5α,6α-epoxycholestan-3β-ol by gas-liquid chromatography-mass spectrometry as well as by the reverse isotope dilution method. The thin-layer chromatographic behavior of the proposed 20,22-epoxide which had been reported to have a higher polarity than pregnenolone was in good accordance with that of the identified 5α,6α-epoxycholestan-3β-ol. All four diastereoisomers of 20,22-epoxycholesterol had much lower polarity than pregnenolone in thin-layer chromatograms obtained in various solvent systems. Endogeneous cholesterol, contained originally in the mitochondria, was also converted in the presence of NADPH to pregnenolone and 5α,6α-epoxycholestan-3β-ol as observed with radioactive cholesterol added as the exogeneous substrate.     

Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography—mass spectrometry with selective ion monitoring

SUMMARY

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis. We have developed an analytical method using capillary gas chromatography-mass spectrometry (GC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL). The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra. The major oxidation products of cholesterol in oxidized LDL were identified as 7β-hydroxy-cholesterol and 7-keto-cholesterol. Minor products included 4β-hydroxy-cholesterol, 6β-hydroxy-cholesterol and cholesterol-5α,6α-epoxide. Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7β-hydroxy-cholesterol and 7-keto-cholesterol. 7-Lathosterol was not detected in oxidized LDL. A time course oxidation of native LDL with 8 μM CuCl₂ demonstrated a rapid increase in 7β-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h. Cholesterol—5α,6α-epoxide, and β4-hydroxy- and 6β-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period. This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range. This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.     

Immune depression in Rhodnius prolixus by seco-steroids, physalins

A comparative study of the effects of physalins, seco-steroidal substances of Physalis angulata (Solanaceae), on the immune reactions of R. prolixus was carried out. Ecdysis and mortality were not affected by treatment with physalins B, D, F or G (1-10 microg/ml of blood meal). R. prolixus larvae fed with blood containing physalins and inoculated with 1 microl of Enterobacter cloacae beta12 (5 x 10(3)/insect) exhibited mortality rates three times higher than controls. The insects treated with physalin B, and F (1 microg/ml) and inoculated with E. cloacae beta12 showed significant differences on lysozyme activity in the hemolymph compared to untreated insects. Furthermore, physalin D (1 microg/ml) significantly reduced the antibacterial activity. Concerning cellular immune reactions, all insects treated with physalins (1 microg/ml), exhibited drastic reductions in the quantity of yeast cell-hemocyte binding and subsequent internalization. Insects inoculated with bacteria and treated with physalins B, F and G showed reductions of microaggregate formation but physalin D did not. Physalins B and F also reduced total hemocyte count in the hemolymph. These results suggest that, in different ways, probably due to their different chemical structures, physalin B, D, F and G are immunomodulatory substances for the bloodsucking insect, R. prolixus.     

Physalins with anti-inflammatory activity are present in Physalis alkekengi var. franchetii and can function as Michael reaction acceptors

Michael reaction acceptors (MRAs) are a class of active molecules that are directly or indirectly involved in various cellular processes, including the regulation of many signaling pathways. In this study, the inducible nitric oxide synthase (iNOS) assay was used to demonstrate that the dichloromethane extract of Physalis alkekengi var. franchetii (DCEP) possesses anti-inflammatory activity that might be attributed to the modification of key cysteine residues in IKKβ by the MRAs in DCEP. To isolate these MRAs, glutathione (GSH) was employed, and a simple ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) screening method was developed to investigate the GSH conjugates with potential MRAs. Five physalins, including one new compound isophysalin A (2), together with four known steroidal compounds, physalin A (1), physalin O (3), physalin L (4) and physalin G (5), were isolated to evaluate the GSH conjugating abilities, and it was indicated that compounds 1, 2 and 3, which had a common α,β-unsaturated ketone moiety, exhibited conjugating abilities with GSH and also showed significant nitric oxide (NO) production inhibiting activities. The anti-inflammatory activities of compounds 1, 2 and 3 might be attributed to their targeting multiple cysteine residues on IKKβ; therefore, the alkylation of IKKβ by compound 1 was further studied by micrOTOF-MS. The result showed that six cysteine residues (C(59), C(179), C(299), C(370), C(412), and C(618)) were alkylated, which indicated that IKKβ is a potential target for the anti-inflammatory activity of physalin A.     

Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles

We recently established that drugs used for the treatment and the prophylaxis of breast cancers, such as tamoxifen, were potent inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), which led to the accumulation of 5,6α-epoxy-cholesterol (5,6α-EC) and 5,6β-epoxy-cholesterol (5,6β-EC). This could be considered a paradox because epoxides are known as alkylating agents with putative carcinogenic properties. We report here that, as opposed to the carcinogen styrene-oxide, neither of the ECs reacted spontaneously with nucleophiles. Under catalytic conditions, 5,6β-EC remains unreactive whereas 5,6α-EC gives cholestan-3β,5α-diol-6β-substituted compounds. These data showed that 5,6-ECs are stable epoxides and unreactive toward nucleophiles in the absence of a catalyst, which contrasts with the well-known reactivity of aromatic and aliphatic epoxides. These data rule out 5,6-EC acting as spontaneous alkylating agents. In addition, these data support the existence of a stereoselective metabolism of 5,6α-EC.     

Cytotoxic terpenoids from Juglans sinensis leaves and twigs

Bioassay-guided fractionation of an 80% MeOH extract of Juglan sinensis leaves and twigs has resulted in the isolation of three new triterpenes (1-3) and two new sesquiterpenes (4-5) along with two known sesquiterpenes (6-7). The new compounds were determined to be 3β, 11α, 19α, 24, 30-pentahydroxy-20β, 28-epoxy-28β-methoxy-ursane (1), 1α, 3β-dihydroxy-olean-18-ene (2), 2α, 3α, 23-trihydroxy-urs-12-en-28-oic acid 28-O-β-d-glucopyranoside (3), (4S, 5S, 7R, 8R, 14R)-8, 11-dihydroxy-2, 4-cyclo-eudesmane (4), 15-hydroxy-α-eudesmol-11-O-β-d-glucopyranoside (5), by spectroscopic analysis. The cytotoxicity of compounds (1-7) against four cancer cell lines such as B16F10, Hep-2, MCF-7 and U87-MG was evaluated. Compounds 1, 2, 6 and 7 showed potent cytotoxicity against all of four cancer cell lines, respectively.     

Physalin F induces cell apoptosis in human renal carcinoma cells by targeting NF-kappaB and generating reactive oxygen species

Background

The aim of this study was to determine the molecular mechanisms of physalin F, an effective purified extract of Physalis angulata L. (Solanacae), in renal carcinoma A498 cells.

Methodology/Principal Findings

Physalin F was observed to significantly induce cytotoxicity of three human renal carcinoma A498, ACHN, and UO-31 cells in a concentration-dependent manner; this was especially potent in A498 cells. The physalin F-induced cell apoptosis of A498 cells was characterized by MTT assay, nuclear DNA fragmentation and chromatin condensation. Using flow cytometry analysis, physalin F induced A498 cell apoptosis as demonstrated by the accumulation of the sub-G1 phase in a concentration- and time-dependent manner. Moreover, physalin F-mediated accumulation of reactive oxygen species (ROS) caused Bcl-2 family proteins, Bcl-2, and Bcl-xL degradation, which led to disruption of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-3 and caspase-9 activity, which led to poly(ADP-ribose) polymerase cleavage. However, the antioxidant N-acetyl-_L-cysteine (NAC) and glutathione (GSH) resulted in the inhibition of these events and reversed physalin F-induced cell apoptosis. In addition, physalin F suppressed NF-κB activity and nuclear translocation of p65 and p50, which was reversed by NAC and GSH.

Conclusion

Physalin F induced cell apoptosis through the ROS-mediated mitochondrial pathway and suppressed NF-κB activation in human renal cancer A498 cells. Thus, physalin F appears to be a promising anti-cancer agent worthy of further clinical development.     

Trypanosoma rangeli: effects of physalin B on the immune reactions of the infected larvae of Rhodnius prolixus

Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED(50)) volume, was 15.2+/-1.6 microg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 microg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 microg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 x 10(3) parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 microg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte micro-aggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.     

Oligofuro- and spiro-stanosides of Asparagus adscendens

Two oligofurostanosides and two spirostanosides, isolated from a methanol extract of Asparagus adscendens (leaves), were characterized as 3-O-[{α-l-rhamnopyranosyl (1 → 4)} {α-l-rhamnopyranosyl (1 → 6)}-β-d-glucopyranosyl]-26-O-[β-d-glucopyranosyl]-22α-methoxy-(25S)-furost-5-en-3β,26-diol (Adscendoside A), 3-O-[{α-l-rhamnopyranosyl (1 → 4)} {α-l-rhamnopyranosyl (1 → 6)}-β-d-glucopyranosyl]-26-O-[β-d-glucopyranosyl]-(25S)-furost-5-en-3β,22α,26-triol-(Adscendoside B), 3-O-[{α-l-rhamnopyranosyl (1 → 6)}-β-d-glucopyranosyl]-(25S)-spirostan-5-en-3β-ol (Adscendin A) and 3-O-[{α-l-rhamnopyranosyl (1 → 4)} {α-l-rhamnopyranosyl (1 → 6)}-β-d-glucopyr anosyl]-(25S)-spirostan-5-en-3β-ol (Adscendin B), respectively. Adscendin B and Adscendoside A are the artefacts of Adscendoside B formed through hydrolysis and methanol extraction respectively.bl]     

Pubescenol,a withanolide from Physalis pubescence

A new withanolide, named pubescenol, and also physalin E and physalin E acetate have been isolated from Physalis pubescence. The structure of pubescenol is assigned as 4α,7α-dihydroxy-l-oxo-24α,25α-epoxy-2,3-dihydrowithanolide.     

Germacranolides from Viguiera microphylla

Three new germacranolides, including two heliangolides (niveusin C-2′,3′-epoxide and 1,2-dehydroniveusin C-2′,3′-epoxide) and a germacrolide (3β-hydroxy-8β-epoxyangeloyloxycostunolide-1β,10α-epoxide) were isolated from Viguiera microphylla. Their structures were established by spectroscopic analyses, including extensive ¹H NMR and ¹³C NMR decoupling experiments and chemical transformations. X-ray diffraction analysis confirmed the structure of niveusin C-2′,3′-epoxide.     

Investigations of carotenoids in some animals of the Adriatic Sea V. Echinodermata

The author investigated the carotenoids in the Echinodermata from Adriatic sea by means of columnar and thin-layer chromatography. The following carotenoids were identified:

- in Coscinasterias tenuispina: β-carotene, isocryptoxanthin lutein, lutein-5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin and asterinacid.

- in Marthasterias glacialis: β-carotene, echinenone, cryptoxanthin, lutein, lutein 5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin ester, astaxanthin and 3, 4-didehydro-α-carotene.

- in Paracentrotus lividus: β-carotene, echinenone, cryptothin, isocryptoxanthin, lutein, lutein-5, 6-epoxide, 4-hydroxy-4-keto-β-carotene, zeaxanthin, astaxanthin, astaxanthin ester and asterinacid.

- in Sphaerechinus granularis: ,β-carotene, echinenone, cryptoxanthin, lutein, lutein-5, 6-epoxide, astaxanthin and guaraxanthin.

     

A simple and convenient synthetic route to Ulipristal acetate

We set out to describe a new and efficient route for preparing Ulipristal acetate with a good yield. The selected epoxidization conditions gave out 80% of 5α,10α-epoxide 2a in the two diastereoisomers which greatly improved the yield of 11β-substituted isomer 4a. And phenyl–sulfinyl compound 6 was synthesized from ketone 5 directly treated with phenylsulfenyl chloride in the presence of triethylamine. These synthetic procedures is only 8 steps, less than currently reported in the literature, but more suitable for industrial process.     

Hepatic microsomal conversion of pregnenolone to 3β,5,6β-trihydroxy-5α-pregnan-20-one via pregnenolone α- and β-epoxides

In the presence of ferrous ion, ADP, and an NADPH-generating system, [4-¹⁴C]pregnenolone was oxidized by bovine liver microsomes to its α-epoxide (5,6α-epoxy-3β-hydroxy-5α-pregnan-20-one), β-epoxide (5,6β-epoxy-3β-hydroxy-5β-pregnan-20-one), trihydroxypregnanone (3β,5,6β-trihydroxy-5β-pregnan-20-one) which were separated, isolated on an octadecylsilicone column in 70% aq. methanol by high performance liquid chromatography, identified with respective synthetic specimens by gas-liquid chromatography-mass spectrometry. The microsomal Δ⁵-oxidation products of pregnenolone were detected in trace yield either when EDTA was added to the incubation mixture or when ferrous ion was omitted from the mixture. The microsomal oxidation system generated malondialdehyde significantly. It, however, was retarded to a negligible extent either by the addition of EDTA or by the omission of ferrous ion. Therefore, the microsomal formation of the significant yields of Δ⁵-oxygenated pregnenolones was reasonably attributed to a reaction linked to microsomal lipid peroxidation. The ratio of pregnenolone α- to β-epoxides formed was 1:3. A comparable study carried out under the same conditions by using [4-¹⁴C]cholesterol as the substrate resulted in the similar Δ⁵-epoxidation with concomitant formation of cholestane-3β,5α,6β-triol; cholesterol α- and β-epoxides formed were in the ratio 1:4.Both pregnenolone α- and β-epoxides were hydrolyzed by the microsomes to trihydroxypregnanone as the sole metabolite at a relative rate of 0.6:1. A similar relative value was also obtained in the microsomal hydrolysis of cholesterol α- and β-epoxides to the cholestanetriol.     

A taxoid from the needles of the Japanese yew,Taxus cuspidata

5α-Hydroxy-2α,9α,10β-triacetoxy-11,12-epoxy-taxa-4(20)-en-13-one (taxinine A 11,12-epoxide) was isolated from the needles of the Japanese yew, Taxus cuspidata Sieb et Zucc, together with 10 other taxoids. Its structure was established on the basis of spectroscopic analysis.     

苋科植物的丛枝菌根

有些种子植物如莎草科、十字花科、灯心草科、藜科、石竹科等20余科,以往曾被认为不能或不易形成丛枝菌根(郭秀珍等,1989;刘润进等,2000).随着对菌根的深入研究,曾被认为是不易与菌根菌组合的湿地生植物、寄生性植物、或一年生植物都被发现是可以形成内生菌根的(Trappe等,1992).此外,Allen等(1989)研究证实,Salsola kali,Atriplex roseum等生长于沙漠、海滨的藜科植物,进行接种处理后,也能形成丛枝菌根.我们在西双版纳调查热带雨林植物的丛枝菌根状况时,偶然发现刺苋(Amaranthus spinosus Linn.)的根系受到了丛枝菌根真菌的侵染,因此,对苋科植物作了扩大采样调查.本文主要报道从热带采集的5属6种苋科植物的根受丛枝菌根真菌感染形成丛枝菌根(arbuscular mycorrhiza,AM)和这些植物根际士壤中的丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)的状况.