五种茄科糖苷生物碱及其混合物的抗真菌活性研究

本文研究了五种茄科糖苷生物碱(茄碱、查茄碱、边缘茄碱、澳洲茄碱和番茄碱)对两种植物病原真菌白菜白斑病菌和葱紫斑病菌的抑制活性.结果表明番茄碱的抗真菌活性最强,其后依次是查茄碱、边缘茄碱和澳洲茄碱,茄碱的活性最弱;不同浓度茄碱和查茄碱(马铃薯中的两种糖苷生物碱)的混合物均具有协同抗真菌作用,且低浓度的混合物产生的协同作用效果较大;边缘茄碱和澳洲茄碱(龙葵中的两种糖苷生物碱)的混合物基本没有协同抗真菌作用;边缘茄碱和查茄碱的混合物以及澳洲茄碱和茄碱的混合物(均为来自不同植物的糖苷生物碱的混合物)在抗真菌活性上都呈现了相加关系.     

Amelioration by glucose-6-phosphate and NADP of potato glycoalkaloid inhibition in cell, enzyme and liposome assays.

Lysis of human erythrocytes by 20 microM chaconine was reduced by 0.5 mM glucose-6-phosphate (G6P) and NADP. Both compounds caused approximately 50% inhibition of haemolysis at 1 mM. Glucose, glucose-1-phosphate, rhamnose, galactose and galactose-6-phosphate were ineffective; NAD was effective, although not to the extent of NADP. Of the tested sugars, only G6P reduced solanine-induced haemolysis. G6P also reduced the synergistic haemolytic action of solanine and chaconine in combination. G6P and NADP at or above 5 mM antagonised chaconine-induced betanin loss from excised red beet root discs; NADP was more effective than G6P. Disruption of PC/cholesterol liposomes by chaconine and inhibition of acetylcholinesterase by chaconine or solanine, were unaffected by up to 10 mM NADP or 50 mM G6P.     

Synergistic interaction between the potato glycoalkaloids α-solanine and α-chaconine in relation to lysis of phospholipid/sterol liposomes

At pH 7.2, the steroidal glycoalkaloid α-chaconine disrupted phosphatidylcholine/cholesterol liposomes whereas α-solanine was virtually without effect. A glycoalkaloid mixture extracted from potato sprouts and comprising approximately equal amounts of solanine and chaconine had, at 150 μM, a lytic effect the same as a 150 μM solution of chaconine only. The apparent synergistic interaction between the two compounds was confirmed using 1:1 mixtures of authentic solanine and chaconine from different sources and of different batches. Combinations (1:1) of solanine or chaconine and tomatine or digitonin (both of which lysed liposomes) or β₂-chaconine (which is non-lytic) did not produce synergistic effects. The synergism between solanine and chaconine was observed only when the two compounds were present together, although the order of addition into the test system did not appear crucial. Pretreatment of liposomes with one glycoalkaloid and its subsequent removal did not permanently sensitize the membranes to the second glycoalkaloid. The magnitude of the synergism was dependent on the relative amounts of solanine and chaconine with maximal effects where chaconine comprised 40% or more of the mixture.     

Effects of glycoalkaloids from Solanum plants on cucumber root growth

The phytotoxic effect of four glycoalkaloids and two 6-O-sulfated glycoalkaloid derivatives were evaluated by testing their inhibition of cucumber root growth. The bioassays were performed using both compounds singly and in equimolar mixtures, respectively. Cucumber root growth was reduced by chaconine (C), solanine (S), solamargine (SM) and solasonine (SS) with IC₅₀ values of 260 (C), 380 (S), 530 (SM), and 610 μM (SS). The inhibitory effect was concentration-dependent. 6-O-sulfated chaconine and 6-O-sulfated solamargine had no inhibitory effects, which indicated that the carbohydrate moieties play an important role in inhibiting cucumber root growth. The equimolar mixtures of paired glycoalkaloids, both chaconine/solanine and solamargine/solasonine, produced synergistic effects on inhibition of cucumber root growth. By contrast, mixtures of unpaired glycoalkaloids from different plants had no obviously synergistic effects. The growth inhibited plant roots lacked hairs, which implied that inhibition was perhaps at the level of root hair growth.     

Effect of steroidal glycoalkaloids of the potato on the permeability of liposome membranes

At pH 7.2, the potato glycoalkaloid α-chaconine caused release of entrapped peroxidase from phosphatidylcholine liposomes containing different free sterols but was ineffective against sterol-free liposomes. The alkaloid was able to complex with all the tested sterols in vitro although there was no close correlation between the extent of sterol binding and liposome disruption. α-Solanine also complexed with sterols in vitro but had no effects on sterol-containing liposomes under these conditions. Both sterol concentration and alkaloid concentration were limiting factors in the action of chaconine but did not markedly affect that of solanine. Solanine destabilized liposome membranes only at pH values of 8 and above but was less effective than chaconine. The importance of the carbohydrate moiety of glycoalkaloids was further demonstrated by the inability of β₂-chaconine to complex with sterols or disrupt liposomes.     

Production of monoclonal antibodies and development of an ELISA for solamargine

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml^–1 to 11.5 nmol ml^–1 of solamargine.     

Steroidal glycoalkaloids in cell and shoot teratoma cultures ofSolanum dulcamara

Bittersweet (Solanum dulcamara L., Solanaceae) is of interest as a source of steroidal alkaloids for the commercial production of hormones. Since glycoalkaloid production is positively correlated to differentiation, tumor and teratoma cultures of the soladulcidine chemotype were established by transformation withAgrobacterium tumefaciens. A newly developed HPLC-system, which allowed separation and sensitive quantitation of the glycoalkaloids soladulcidine-tetraoside, solamargine and solasonine, was used to analyse glycoalkaloid profiles in plants and cultures. Tumors and teratoma were charcterized by a shift in their alkaloid pattern from soladulcidine tetraoside to the solasodine glycosides solamargine and solasonine. Shoot teratoma showed a total glycoalkaloid content of 1% dw, which is about fivefold higher than in the source plant. A regenerated plant retained the altered alkaloid spectrum; the levels, however, equalled those of the source plant. From the alteration of alkaloid pattern in the transformed cultures suggestions can be made concerning the biosynthetic pathway. Completion of the biosynthesis of the aglycone is likely to be complete before glycosylation occurs.     

QTL mapping of foliar glycoalkaloid aglycones in Solanum tuberosum×S. berthaultii potato progenies: quantitative variation and plant secondary metabolism

 Glycoalkaloids are quantitatively inherited in Solanum, and in high concentrations they can be toxic to humans. The increased use of wild potato germplasm to improve the pest resistance, yield, and quality characteristics of cultivated potato may elevate or introduce new, more toxic glycoalkaloids into the cultivated gene pool. Therefore, it is important to increase our understanding of their inheritance, accumulation, and biosynthesis. Glycoalkaloids have two basic constituents – a glycosidic grouping and a steroid alkaloid skeleton. Steroid alkaloids are classified as solanidanes and spirosolanes, of which solanidine and solasodine are, respectively, representatives. RFLP-mapped, diploid, reciprocal backcross potato progenies involving the parents S. tuberosum and S. berthaultii, which produce solanidine and solasodine, respectively, were analyzed for segregation of the glycoalkaloids solanine, chaconine, solasodine and solamargine to identify quantitative trait loci (QTLs) for the production of the aglycones solanidine and solasodine. The F₁ clone M200-30 exhibited low to nondetectable levels of solasodine and solanidine, suggesting that expression was controlled by recessive genes. In a backcross to berthaultii (BCB) and backcross to tuberosum (BCT), several QTLs for the accumulation of solasodine and solanidine were identified. Three QTLs explaining approximately 20% of the variation in solasodine were identified in BCB on chromosomes 4, 6, and 12. Similarly, three QTLs were identified in BCT on chromosomes 4, 8 and 11, but these accounted for only 10% of the variation observed in solasodine accumulation. Two QTLs for solanidine were identified in BCT on chromosomes 1 and 4. The QTL located on chromosome 1 was highly significant, accounting for 17% and 22% of the variation in solanidine accumulation in 1994 and 1995, respectively. This same QTL was also detected in BCB. The QTLs detected in this study probably represent structural and/or regulatory genes controlling the accumulation of solasodine and solanidine. Results are discussed in the context of steroid alkaloid accumulation and biosynthesis. Received: 27 August 1997 / Accepted: 16 March 1998     

In vitro Leishmanicidal and Cytotoxic Activities of the Glycoalkaloids from Solanum lycocarpum (Solanaceae) Fruits

Leishmaniasis is an infection caused by a protozoan parasite of the genus Leishmania and is the second most prevalent parasitic protozoal disease after malaria in the world. We report the in vitro leishmanicidal activity on promastigote forms of Leishmania amazonensis and cytotoxicity, using LLCMK₂ cells, of the glycoalkaloids from the fruits of Solanum lycocarpum, determined by colorimetric methods. The alkaloidic extract was obtained by acid‐base extraction; solamargine and solasonine were isolated by silica‐gel chromatography, followed by reversed‐phase HPLC final purification. The alkaloidic extract, solamargine, solasonine, as well as the equimolar mixture of the glycoalkaloids solamargine and solasonine displayed leishmanicidal activity against promastigote forms of L. amazonensis, whereas the aglycone solasodine was inactive. After 24 and 72 h of incubation, most of the samples showed lower cytotoxicities (IC₅₀ 6.5 to 124 μM ) as compared to leishmanicidal activity (IC₅₀ 1.1 to 23.6 μM ). The equimolar mixture solamargine/solasonine was the most active with an IC₅₀ value of 1.1 μM , after 72 h. Likewise, solamargine was the most active after 24 h with an IC₅₀ value of 14.4 μM , both in comparison with the positive control amphotericin B.     

Steroidal alkaloids from Solanum khasian um: Application of 13C NMR spectroscopy to their structural elucidation

The solasodine glycosides, solasonine, solamargine and khasianine have been isolated from berries of Solanum khasianum and characterized by ¹³C NMR spectroscopy. By application of this method, the structure of khasianine has been elucidated as O-α-l-rhamnosyl (1→4_glu)-O (3)-β-d-glucopyranosyl-solasodine (β₂-solamargine).     

Synergism between the potato glycoalkaloids alpha-chaconine and alpha-solanine in inhibition of snail feeding

Snails (Helix aspersa L.) were fed filter paper treated with the potato glycoalkaloids, alpha-solanine and alpha-chaconine, singly or together. In pure form, both glycoalkaloids deterred feeding, with chaconine being the more active compound. In combination, authentic solanine and chaconine interacted synergistically in their inhibition of feeding. The antifeedant activities of methanolic extracts of tuber peel of the potato varieties Majestic and Sharpe's Express presented via filter paper discs did not differ significantly from those of authentic glycoalkaloid solutions of comparable concentration and ratio. In contrast, feeding inhibition by diluted tuber peel extracts of the variety Homeguard was greater than that elicited by comparable authentic glycoalkaloid solutions suggesting additional inhibitory compound(s) in the peel of this variety. Comparison of data from peel extracts of all three potato varieties and authentic glycoalkaloids indicated that the level of feeding inhibition by the extracts was, at least in part, a consequence of a synergism between solanine and chaconine.     

Silverleaf nightshade,Solarium elaeagnifolium,origin, distribution,and relation to man

Silverleaf nightshade (Solanum elaeagnifolium) is a perennial weed that has become increasingly troublesome over the past several decades. Extensive use of soil-applied herbicides, accompanied by a reduction in annual weed competition and reduced tillage, have contributed to its spread and establishment as a serious pest. Crop plants are affected directly via competition and allelopathy or indirectly as the nightshade plants serve as hosts for destructive phytophagous insects or fungal pathogens. Probably native to the southwestern United States and northern Mexico,S. elaeagnifolium is now found in many semiarid regions of the world. Plants were used by the Pima, Kiowa and Navajo Indians in the preparation of food and in the tanning of leather. Containing the toxic glycoalkaloids solanine and solasonine, plants can cause livestock poisonings. The fruits, however, are a source of solasodine, which is used in the commercial manufacture of steroidal hormones.     

Identification of molecular markers associated with leptine production in a population of Solanum chacoense Bitter

  Solanum chacoense Bitter, a wild relative of the cultivated potato, produces several glycoalkaloids, including solanine, chaconine, and the leptines. The foliar-specific leptine glycoalkaloids are believed to confer resistance to the Colorado Potato Beetle (CPB). Using two bulked DNA samples composed of high- and low-percent leptine individuals from a segregating F₁ population of S. chacoense, we have identified two molecular markers that are closely linked to high percent solanine+chaconine and, conversely, to nil/low percent leptine. One of these, a 1,500-bp RAPD product (UBC370-1500), had a recombination value of 3% in the F₁ progeny, indicating tight linkage. UBC370-1500 mapped to the end of the short arm of potato chromosome 1, in the region of a previously mapped major QTL for solanidine, from a S. tuberosum (solanidine)×S. berthaultii (solasodine) cross. Taken together, these results suggest that either (1) a major locus determining solanidine accumulation in Solanum spp. is on chromosome 1 in the region defined by the RFLP markers TG24, CT197, and CT233, or (2) this region of chromosome 1 may harbor two or more important genes which determine accumulation of steroidal aglycones. These findings are important for the genetics of leptine (as well as other glycoalkaloid) accumulation and for the development of CPB-resistant potato varieties. Received: 5 March 1998 / Accepted: 28 July 1998     

Bioactive steroidal alkaloid glycosides from Solanum aculeastrum.

Solanum aculeastrum Dunal was investigated for the presence of molluscicidal compounds. This led to the isolation of solaculine A, from the root bark in addition to known steroidal alkaloids; solamargine and beta-solamarine from the berries. The structures were elucidated by spectroscopic techniques. Molluscicidal activity of the aqueous extracts of the berries and root bark, and the isolated compounds were investigated.     

High performance liquid chromatographic determination of glycoalkaloids in callus and fruits ofSolanum eleagnifolium

Summary A method for the determination by HPLC of the triglycosides, solamargine and solasonine, in fruits and callus was developed. It was used to determine the content of triglycosides in samples fromSolanum eleagnifolium. The method involves ion-pair extraction of glycoalkaloids, clean-up of the samples and HPLC analysis. Recoveries ranged between 94–98%, and the coefficient of variation was approximately 5%.     

Release of vesicles containing acetylcholinesterase from erythrocyte membranes by treatment with dilauroylglycerophosphocholine

The shedding of acetylcholinesterase-enriched vesicles from erythrocytes of various species of animals occurred when cells were treated with C12:0PC. The response was observed shortly after a morphological change of erythrocytes without any accompanying detectable K+ leakage or hemolysis. The vesiculation was inhibited by the presence of serum albumin or by the incorporation of cholesterol into C12:0PC liposomes, indicating that the insertion of C12:0PC into the erythrocyte membrane causes the vesiculation. The ratio of C12:0PC to total phospholipid determined in vesicle fractions was almost the same as that observed in non-hemolyzed cell fractions. This finding suggests that the vesicles were not shed from portions of membranes rich in C12:0PC. The vesicles showed similar characteristics to those generated by ATP depletion; their diameter is 150-200 nm and they are enriched with acetylcholinesterase activity. Erythrocytes became denser when they lost acetylcholinesterase activity on treatment with C12:0PC.     

Freeze-etch electron microscopy of erythrocytes,Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B

Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150–250Åin diameter, in the membranes of rat erythrocytes, in cholesterol-containing membranes ofAcholeplasma laidlawii cells and in egg lecithin-cholesterol liposomes. No change in fracture faces was observed when cholesterol was absent in the membranes ofA. laidlawii, and lecithin liposomes.Amphotericin B does not visibly affect the freeze-etch morphology of erythrocytes, cholesterol-containingA. laidlawii cells and lecithin-cholesterol liposomes.     

Induction of acetylcholinesterase release from erythrocytes in the presence of liposomes

When human erythrocytes are incubated with liposomes, the release of acetylcholinesterase (AChE) occurs following an induction period [Cook et al. (1980) Biochemistry 19, 4601-4607]. However, the mechanism of the induction has not been elucidated. We examined the relationships among the lipid transfer from liposomes to erythrocytes, the morphological change of erythrocytes, the fluidity of the erythrocyte membrane and the start of AChE release. The AChE release into the liposomes and into shed-vesicle fractions started simultaneously after an induction period. The morphological index (MI) of erythrocytes was approximately 2.8 at the beginning of the release, regardless of the induction period. AChE was not released from the erythrocytes of index 2.8 even in the presence of liposomes if the MI remained at 2.8. Therefore, for the release, erythrocytes needed a further increase of the MI from 2.8. As the rate of lipid transfer increased, the induction period became shorter. No significant lipid release from erythrocytes was detected during the induction period. The initiation of the AChE release was not simply affected by the change in the membrane fluidity of erythrocytes upon interaction with liposomes. These results first demonstrate that AChE release into the shed-vesicle and liposome fractions is triggered by a further increase of the MI from 2.8, which is induced by lipid transfer from liposomes to erythrocytes.     

Anticholinesterase effect and toxicity of bis(trichloromethyl) sulfone (N-1386 Biocide) in rats

The oral LD50 for bis(trichloromethyl) sulfone (N-1386 Biocide) in male rats was 691 mg/kg. Deaths occurred 1-5 days after treatment and signs of toxicity suggestive of an anticholinesterase effect were noted. However, neither plasma cholinesterase nor brain acetylcholinesterase was inhibited 2, 4 or 24 hours after a single, oral dose of 500 mg/kg. Atropine (300 mg/kg, s.c.) or scopolamine (670 mg/kg, s.c.) pretreatments did not protect against the acute lethality of bis(trichloromethyl) sulfone although signs of toxicity were alleviated by both pretreatments. Bis(trichloromethyl) sulfone produced in vitro inhibition of rat plasma cholinesterase and brain acetylcholinesterase. The inhibition was competitive in brain. IC50's for these 2 enzymes were 8 microM in plasma and 25 microM in brain. In summary, bis(trichloromethyl) sulfone produced in vitro cholinesterase inhibition not demonstrated in vivo. Doses of anticholinergic compounds that ameliorated many toxic signs did not protect against lethality produced by bis(trichloromethyl) sulfone.