Investigations on the content of steroidal alkaloids and sapogenins withinSolanum sect.Solanum ( = sect.Morella) (Solanaceae)

Plant material of 55 strains from 32 species ofSolanum sect.Solanum ( = sect.Morella) has been investigated for the content of steroidal alkaloids and sapogenins. The leaf-extracts of all species contain diosgenin resp. tigogenin or both together in considerable amount. On the contrary the characteristic steroidal alkaloid solasodine was absent in these samples. For 29 species the presence of steroidal sapogenins has been proved for the first time. In unripe fruits, however, solasodine could be detected in most of the species examined. New occurences of solasodine are reported for seven further species. These chemical results may contribute to a better comprehension of the difficult genusSolanum.—Ontogenetic studies on different organs ofS. atriplicifolium show the relationship between accumulation of steroidal compounds and plant maturity.     

Rapid separation of solasodine glycosides by an immunoaffinity column using anti-solamargine monoclonal antibody

Immunoaffinity column using anti-solamargine monoclonal antibody for separation of solasodine glycosides was established. This method was specific for solasodine glycosides which was detected by thin layer chromatography and the western blotting. Total solasodine glycosides have been separated directly from the crude extract of Solanum khasianum fruit by the newly established immunoaffinity column. This revised version was published online in August 2006 with corrections to the Cover Date.     

Studies on Indian Solanum I. Alkaloid content and detection of solasodine

Fruits and/or leaves of 31 species ofSolanum that grow in India have been assayed for total alkaloid content and the presnce of solasodine in them has been determined by TLC. This survey revealed that 12 species contained solasodine and thatS. khasianum, S. elaeagnifolium, S. auriculatum andS. giganteum contained sufficient quantity of total alkaloid to warrant further studies on these plants in order to determine whether they could be developed as sources of raw materials for the steroid industry.     

Plantlet regeneration enhances solasodine productivity in hairy root cultures of <Emphasis Type="Italic">Solanum khasianum</Emphasis> Clarke

Summary Shoot regeneration in hairy root cultures of Solanum khasianum Clarke influences root growth, solasodine production. and permeabilization of solasodine into the medium. These parameters are dependent on exogenously supplied auxin and cytokinin: the effect being both concentration-and clone-dependent. Hairy root cultures with no shoot regeneration showed high permeabilization of solasodine into the medium by the sixth week of incubation, suggesting the medium acts as a sink for the solasodine synthesized by the roots. Solasodine in the culture medium was toxic to the transformed roots and caused browning of root tips. In a separate set of experiments, the hairy root cultures showed regeneration of approximately 50–70 mm long shoots after treatment with indole-3-acetic acid and kinetin. These hairy root cultures had inereased levels of solasodine production, compared to cultures without shoot regeneration. The plantlets formed in the hairy root cultures accumulated some of the solasodine, thereby reducing its permcabilization into the medium. Transport of solasodine from root to shoot reduced the toxic effect of solasodine in the root zone and extended the exponential growth phase by 8-10d.     

Effects of short-term treatment of solasodine on cauda epididymis in dogs

Oral administration (80 mg/kg body wt/day for 30 days) of solasodine (extracted and isolated from the berries of the Solanum xanthocarpum) to intact dogs significantly decreased the epithelial cell height of cauda epididymides. The cells became atrophic and the lumen was devoid of spermatozoa. Castration followed by the adminstration of solasodine further reduced the epithelial cell height in comparison to castrated controls. Concurrent treatment of solasodine along with testosterone propionate was unable to restore the normal epithelial lumen parameters. Total protein, sialic acid, glycogen and acid phosphatase activities were significantly reduced in solasodine treated cauda epididymides. These result suggest antiandrogenic potency of solasodine.     

Precursor-feeding strategy for the production of solanine,solanidine and solasodine by a cell culture of Solanum lyratum

Solanum lyratum, a medicinal plant, has been used to treat cancers, tumors, and warts for many years. Undifferentiated cell cultures were mainly used to study the precursor-feeding strategy for the production of secondary metabolites of α-solanine, solanidine, and solasodine for pharmaceutical usage. In this study, S. lyratum cells were fed with exogenous plant sterols including cholesterol, stigmasterol, and mixed sterols (β-sitosterol, campesterol, and dihydrobrassicasterol). The results showed that none of the plant sterols exhibited an effect on cell growth as compared to that of the control. Cellular concentrations of solanidine and solasodine were relatively higher than α-solanine levels in all the treatments. The maximal solasodine level in cells was 11.19 mg/g dry weight (DW) after 0.05–1 mg/l stigmasterol feeding, which was about 10-fold higher than the control. With regard to solanidine levels, the maximal level in cells was 5.84 mg/g DW after feeding with 20 mg/l cholesterol. This is the first report on the in vitro enhancement of solanidine and solasodine, steroidal alkaloids with medicinal value, from S. lyratum.     

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     

Production of solasodine by hairy root,callus, and cell suspension cultures of Solanum aviculare Forst.

The production of the steroidal alkaloid solasodine, an alternative to diosgenin as a precursor for the commercial production of steroid drugs, was studied in hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. through manipulation of culture medium. The individual and combined effects of medium components on the growth index and the production of solasodine were analyzed using factorial analysis of variance. Solasodine content was optimized to 6.2 mg g⁻¹in the hairy root, 1.4 mg g⁻¹callus, and 0.7 mg g⁻¹in cell suspension cultures (dry weight). An improved isocratic reversed phase high performance liquid chromatographic method provided selective determination of the solasodine content of these samples. Analysis of growth and solasodine content of hairy root cultures and callus cultures demonstrated that the production of solasodine was shown to be growth-dependent in hairy root cultures but not in callus cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of l-arginine,casein hydrolysate,banana powder and sucrose on growth and solasodine production in shoot cultures of Solanum laciniatum

The addition of l-arginine, casein hydrolysate, banana powder and the reduction of the concentration of sucrose in the media could increase the solasodine content in the shoot cultures of Solanum laciniatum. No linear correlations between growth index, chlorophyll content and solasodine content were observed, however a positive linear correlation between solasodine productivity and chlorophyll content occurred in these shoot cultures.Abbreviations Ch chlorophyll content - DW dry weight - fl flask - FW fresh weight - GI growth index - LOD limit of detection - LOQ limit of quantitation - SDc solasodine content - SDp solasodine productivity - w week     

Solasodine production from self-immobilised Solanum aviculare cells.

Procedures were developed for 'self-immobilisation' of Solanum aviculare cells to eliminate the need for artificial immobilisation supports. Depending on the cytokinin used in liquid medium, compact aggregates 0.4-2.0 cm in diameter were formed without dispersed cells also being present. Histochemical analysis showed that the aggregates were structurally organised to facilitate nutrient transport. Growth, sugar uptake and solasodine production were measured in shake-flask cultures. Most of the product was stored in the aggregates to reach a maximum concentration of 0.3% dry weight; this is between 1.5 and 10 times the levels reported for suspended cells under similar conditions. A substantial amount of solasodine was produced after growth ceased. The maximum rate of solasodine production was about 0.22 mg g-1 d-1. A simple air-driven bioreactor was tested for culture of the aggregates; solasodine yields were comparable to those measured in shake flasks.     

Production of solasodine by Solanum laciniatum using plant tissue culture technique

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.     

In vitro production of solasodine from Solanum trilobatum

Sucrose concentration in the culture medium affected chlorophyll content, trichome development and amount of solasodine in regenerated plantlets of Solanum trilobatum. High chlorophyll content and glandular trichomes were observed in the plants grown on Murashige and Skoog basal medium supplemented with 131.85 mM sucrose. The solasodine was quantified using reverse phase high performance liquid chromatography. The plantlets cultivated on this medium yielded 35.97 mg g⁻¹ (d.m.) solasodine whereas the field plants used as control yielded only 2.32 mg g⁻¹ (d.m.) of solasodine.     

The influence of methyl jasmonate,cholesterol and l‐arginine on solasodine production in hairy root culture of Solanum mammosum

The increasing demand of diosgenin for high‐revenue synthesis of steroid hormones by the pharmaceutical industries has driven researchers to look for other alternatives. Solasodine which was reported to be present in Solanum mammosum is known to be a potential source. The present study highlighted that added methyl jasmonate, cholesterol and l ‐arginine into the modified liquid full‐strength Murashige and Skoog (MS) medium (with ammonium to nitrate ratio 10.3 mM: 39.4 mM, and 4% (w/v) sucrose) could influence the solasodine production in the hairy roots of S. mammosum. The findings showed that both hairy root line‐ATCC31798 and line‐A4 (which were separately induced by Agrobacterium rhizogenes strain ATCC31798 and A4) acquired solasodine productivity of 4.5 mg/g dry weight roots with average dry biomass of 190 mg after 32 days culture, when using 50 mg fresh weight roots as initial inoculum size, with 100 mM cholesterol, 1000 μM l ‐arginine and 300 μM methyl jasmonate added simultaneously into the culture medium on day 20 of culture. The amount of solasodine obtained was five times higher than those without both the elicitor and precursor treatment. The improved solasodine production with a high‐biomass growth could reduce the production cost of steroid synthesis in the long run.     

Solaverbascine—a new 22,26-epiminocholestane alkaloid from Solanum verbascifolium

Besides solasodine and tomatidine the new alkaloid solaverbascine has been obtained from the leaves of Solanum verbascifolium and identified as (22S:25R)-22,26-epiminocholest-5-ene-3β, 16β-diol by physical data and direct comparison with synthetic material.     

Effect of elicitation on the accumulation of solasodine by immobilized cells of Solanum eleagnifolium Cav.

The effect of a fungal elicitor obtained from Alternaria sp. on growth and solasodine production by free and alginate-entrapped cells of Solanum eleagnifolium Cav. was studied. Fourteen-day-old cultures were elicited with 1% FW/V autoclaved homogenates. The solasodine production increased from 0.9 to 1.5 mg g^-1 DW (65%) in suspension cultures and from 0.75 to 1.4 mg g^-1 DW (about 95%) in entrapped cells. The maximum accumulation was obtained after 72 h of elicitation. In order to induce alkaloid release from cells (suspension and entrapped cells), permeabilization with 10% dimethylsulfoxide (DMSO) for 30 min was used. In both cases (free and entrapped cells), about 50–60% of intracellular solasodine was released into the medium. The reuse of elicited and permeabilized entrapped cells was also carried out for three production cycles.     

Phospholipids modulate the substrate specificity of soluble UDP-glucose:steroid glucosyltransferase from eggplant leaves.

UDP-glucose-dependent glucosylation of solasodine and diosgenin by a soluble, partially purified enzyme fraction from eggplant leaves is affected in a markedly different way by some phospholipids. While glucosylation of diosgenin and some closely related spirostanols, e.g. tigogenin or yamogenin, is strongly inhibited by relatively low concentrations of several phospholipids, the glucosylation of solasodine is unaffected or even slightly stimulated. These effects depend both on the structure of the polar head group and the nature of the acyl chains present in the phospholipid. The most potent inhibitors of diosgenin glucosylation are choline-containing lipids: phosphatidylcholine (PC) and sphingomyelin (SM) but the removal of phosphocholine moiety from these phospholipids by treatment with phospholipase C results in an almost complete recovery of the diosgenin glucoside formation by the enzyme. Significant inhibition of diosgenin glucoside synthesis and stimulation of solasodine glucosylation was found only with PC molecular species containing fatty acids with chain length of 12-18 carbon atoms. PC with shorter or longer acyl chains had little effect on glucosylation of either diosgenin or solasodine. Our results indicate that interaction between the investigated glucosyltransferase and lipids are quite specific and suggest that modulation of the enzyme activity by the nature of the lipid environment may be of importance for regulation of in vivo synthesis of steroidal saponins and glycoalkaloids in eggplant.     

The effect of phosphate,nitrogen and sucrose on the production of phenolics and solasodine in callus cultures of solanum laciniatum

In leaf derived callus cultures of Solanum laciniatum Ait. both phenolics and solasodine concentrations increased when medium phosphate or nitrogen concs. were reduced to one-eighth or when sucrose concentration was increased from 3 to 4–8 %. Under these conditions growth was reduced and final FW:DW fell. Growth was inhibited by sucrose depletion and nitrogen supple -mentation. On additional nitrogen the concentrations of phenolics and protein significantly increased, FW:DW was reduced and solasodine concentration was unaffected. In seedling derived cultures phosphate depletion resulted in a significant increase in phenolics concentration, an inhibition of growth and a rise in solasodine concentration.     

Solasodine Yield in Callus Cultures of Solanum Iaciniatum Ait. Selected for Growth on Hormone-free Medium

Callus autotrophic for both auxin and cytokinin was selectedfrom a hormone-requiring culture of Solanum laciniatum Ait.The control of solasodine yield, callus growth and adventitiousshoot initiation by some exogenous growth regulators and/orlight in the habituated culture was determined. Solasodine concentrationvaried from 0.3 to 1.4 mg g^–1 d.wt. Solanum laciniatum, callus, habituation, solasodine     

Tomatidine inhibits iNOS and COX-2 through suppression of NF-kappaB and JNK pathways in LPS-stimulated mouse macrophages

We use the LPS-stimulated macrophage as a model of inflammation to investigate the anti-inflammatory effects of tomatidine and solasodine, whose structures resemble glucocorticoids. We found that tomatidine exhibited a more potent anti-inflammatory effect than solasodine. Tomatidine could decrease inducible nitric oxide synthase and cyclooxygenase-2 expression through suppression of I-kappaBalpha phosphorylation, NF-kappaB nuclear translocation and JNK activation, which in turn inhibits c-jun phosphorylation and Oct-2 expression. Here, we demonstrate that tomatidine acts as an anti-inflammatory agent by blocking NF-kappaB and JNK signaling, and may possibly be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.     

Manipulation of MS and B5 components for enhancement of growth and solasodine production in hairy root cultures of <Emphasis Type="Italic">Solanum khasianum </Emphasis>Clarke

Hairy root cultures of Solanum khasianumClarke were studied for growth and solasodine production by manipulating the major and minor salts, Fe-EDTA and vitamins of MS and B5 medium. Tissue growth and solasodine production are strongly affected by the culture medium. The experiments demonstrated that in part the effect of each componenet of B5 and MS is dependent on the growth phase of the roots. Solasodine production increased with a decrease in total nitrogen. The ratio between ammonium nitrate and potassium nitrate was crucial. Phosphate concentration greatly influences growth, although it showed no significant affect on solasodine production. Half strength of magnesium sulphate and calcium chloride as in MS medium increased growth and solasodine production as compared to the control. This combination of the major salts with minor salts from the MS medium and chelated iron and vitamins from Gamborgs B5 medium was found to be most suitable. Hence it was possible to enhance both the specific and volumetric yield of solasodine by formulating a medium that maximized secondary metabolite production and growth in hairy root cultures. The novelty of this article lies in manipulating the components in MS medium, which supported growth, and Gamborgs B5 medium, which supported secondary product formation in hairy root cultures of S. khasianum and combining the two for maximizing both the parameters through out the growth phase. Thus it shall find immense practical applicability in hairy root scale up, where the secondary metabolite production is directly related to growth.