Solasodine Production Research Articles

Elicitation based enhancement of solasodine production in in-vitro cultures of different Solanum species

Aim: To study the enhancement of solasodine content using elicitors such as NaCl, pectin, salicylic acid and yeast extract in cell suspension cultures of Solanum incanum, Solanum nigrum, Solanum surattense and Solanum villosum. Methodology: In-vitro callus induction from leaf explants was carried out on MS media supplemented with auxin, 2, 4- Dichlorophenoxyacetic acid (2.0 mg l-1). MS liquid medium supplemented with 2, 4-D (2.0 mg l-1) and varied concentrations of different elicitors were used for cell suspension culture. Results: The elicitor NaCl (150 mM) indicated maximum increment in solasodine production in three Solanum species studied. Salicylic acid with 75 μM resulted in considerable elevation in solasodine content in Solanum spp. Response to elicitation by pectin was high at different concentrations for different species of Solanum. Biotic elicitor yeast extract at 3.0 g l-1 concentration considerably increased solasodine production in S. nigrum and S. villosum. Solanum villosum exhibited best results in terms of solasodine concentration enhancement in response to pectin and yeast extract elicitors whereas Solanum incanum responded best to pectin followed by NaCl. Interpretation: The current results indicated that NaCl, pectin, salicylic acid and yeast extract can be effectively applied as influential elicitors for the enhancing the production of solasodine in cell suspension culture of different Solanum species. Key words: Callus, Cell suspension culture, Elicitors, Solanum, Solasodine, Steroidal alkaloids

AGROBACTERIUM MEDIATED HAIRY ROOT INDUCTION AND SOLASODINE PRODUCTION IN SOLANUM MELONGENA L. VAR INSANUM (L.) PRAIN.

Investigation was carried out in Solanum melongena L. var. insanum (L.) Prain. in terms of hairy root induction and solasodine production. Hairy roots were induced from regenerated shoots using Agrobacterium rhizogenes ATCC 15834. The hairy roots were characterized by their vigorous growth branched in nature and negative geotropism. The hairy roots are isolated and subjected into mass cultivation. Mass cultivated hairy roots were harvested and dried for solasodine production. The solasodine content of hairy root was higher than that of field grown sample and callus derived samples.

Genetic transfection, hairy root induction and solasodine accumulation in elicited hairy root clone of Solanum erianthum D. Don

An efficient genetic transfection technique has been established using A4 strain of Agrobacterium rhizogenes for the first time in a medicinally valuable plant Solanum erianthum D. Don. The explants were randomly pricked with sterile needle, inoculated with bacterial suspension. The infected leaves were then washed and transferred to MS basal medium fortified with cefotaxime for hairy root induction. A maximum transformation efficiency of 72 % has been recorded after two days of co-cultivation period. The transfer of rolA and rolB genes from the bacterium to the plant genome has been confirmed in five transformed hairy rootlines by standard Polymerase Chain Reaction technique. On the basis of growth analysis and secondary metabolite study two potential rhizoclones (A4-HR-A and A4-HR-B) were selected. Rhizoclone A4-HR-A can produce highest amount of alkaloid, phenolic and flavonoid, whereas A4-HR-B was observed to be highest tannin producer. Alkaloid like solasodine, commercially important for steroidal drug synthesis, was quantified from leaf and A4-HR-A clone by an improved High Performance Liquid Chromatography method. This showed a sustainable increase (1.33 fold) in production of solasodine in hairy rootline.

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.

Methyl jasmonate elicits the solasodine production and anti-oxidant activity in hairy root cultures of Solanum trilobatum L.

Hairy root cultures of Solanum trilobatum L. were established from leaf explants infected with Agrobacterium rhizogenes strains viz., MTCC 532, MTCC 2364 and ATCC A4. Initially 27 independent root clones were obtained and 5 clones (ST-02, ST-09, ST-11, ST-13 and ST-17) were scrutinized based on their rapid growth in MS solid medium and exemplified for biomass and solasodine accumulation. MS liquid medium containing 3% sucrose with an initial pH of 5.8 favored the maximum biomass accumulation (27.5 fold increments than initial inoculums) of hairy roots. Amid 5 clones, ST-09 was found to the superior one, producing 2 fold greater biomass (3.85±0.11g FW) and 3.3 fold elevated solasodine content (4.76±0.05mg/g DW) than non transformed roots (control). PCR and southern hybridization analysis revealed the integration of rolA gene in transformed roots. Hairy roots of ST-09 elicited with 4μM methyl jasmonate (MJ) for 2 weeks resulted in 1.9 fold and 6.5 fold enhanced production of solasodine (9.33±0.04mg/g DW) than unelicited and non transformed roots, respectively. This was also corroborated by RT-PCR analysis, showing a considerable upregulation of hmgr (HMGCoA reductase) gene transcripts during 1st 2 weeks of elicitation. A significant increase was noticed in the total phenolics (150.42mg/g DE), total flavonoids (521.09mg/g DE) and radical scavenging activity (83.3%) of 4μM MJ elicited roots in comparison with control. Thus, the study illustrates that hairy roots of S. trilobatum could be exploited for scaling up in bioreactors for the large-scale production of solasodine.

Effects of Auxins on the Production of Steroidal Alkaloids in Rapidly Proliferating Tissue and Cell Cultures of Solanum lyratum

Solanum lyratum, a rare species, is used to treat cancer, tumours and warts. Plant cell and tissue culture of S. lyratum, producing steroidal alkaloids, could be useful supplements to natural sources. To study the production of solanine, solanidine and solasodine by adding auxin-type phytohormones including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) to cell and callus cultures of S. lyratum. Methanolic extracts were made from callus and cell cultures of S. lyratumand and analysed using RP C₁₈ HPLC with UV detection. 2,4-D-induced calli from roots led to a significant enhancement in solanine production with a value of 4.13 mg/g dry weight (DW). The maximal solanidine and solasodine levels of 6.26 and 7.69 mg/g DW were respectively obtained with IBA- and IAA-treated S. lyratum cells at concentrations of 1 and 5 mg/L. Auxins were found to be useful phytohormones for the production of steroidal alkaloids. The callus and cell culture system developed is simple and can hence be a method of production of steroidal alkaloids in S. lyratum and other Solanaceae species.

Comparison and evaluation of steroid alkaloid solasodine on in vivo and in vitro cultures of Solanum surattense Burm L.

Steroid alkaloid solasodine is a nitrogen analogue of diosgenin that is used as a raw material for synthesis of steroid drugs. Plant medicinal Solanum surattense (Solanaceae) is a rich source of solasodine. The purpose of this study was production and maintenance of callus from leaves and comparison of solasodine steroid alkaloid production in outdoor plants and in vitro calli. The leaves were collected, sterilized and placed on MS medium supplemented with BAP (1 to 2 mg/L) and NAA (1 to 3 mg/L). The samples were kept in a growth chamber at 25°C with 16/8 (day/night) photoperiod and irradiance of 3000 to 2000 lux. The methanol extracts was performed to obtain solasodine from 2 g dry weight of each leaves and calluses and then injected into the high-performance liquid chromatography (HPLC) system. The results showed that solasodine produced by leaves was 0.3316 mg/g whereas, the solasodine content obtained from calluses were 0.3951 mg/g (in 1 mg/L BAP + 2 mg/L NAA) and 0.3908 mg/g (in 1 mg/L BAP + 3 mg/L NAA). Plant tissue cultures have been suggested as a potential tool for solasodine production. Key words: Callus induction, Solanum surattense Burm, in vitro, in vivo, solasodine,high-performance liquid chromatography (HPLC).

Solasodine accumulation in regenerated plants of Solanum torvum Sw

A nodal segment culture was developed in order to assess Solanum torvum Sw. regeneration and solasodine levels. The influence of auxins (indoleacetic acid, 1-Naphthaleneacetic acid) and benzyl adenine on S. torvum growth in micropropagation was investigated. A nodal segment culture was initiated with seeds germinated in MS basal medium added of GA3 and grown in different concentrations of IAA, IAA + BAP and NAA + BAP. Sixty-day-old plants from the in vitro culture were collected, frozen and lyophilized; then, the methyl orange method was used to quantify solasodine for the spectrophotometric assay. The best results regarding plant regeneration and solasodine accumulation were obtained by using the MS basal medium without addition of plant growth regulators; however, there was great production of calluses presenting friable bases. Based on these results, cell cultures can be initiated from such calluses with application of other auxins and cytokinins to enhance solasodine production, besides different elicitors, light intensities and sucrose concentrations.

Salinity Stress Enhances Production of Solasodine in Solanum nigrum L.

Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot derived leaves) of Solanum nigrum L. were cultured under salinity stress (0-150 mM NaCl) for enhanced production of solasodine, a steroidal alkaloid and an alternative to diosgenin, which is used as a precursor for the commercial production of steroidal drugs. The role of plant growth regulators and various concentrations of NaCl during in vitro production of solasodine was studied. The in vitro yield was compared with the yield from leaves of field grown plant. Solasodine content was maximum (2.39 mg/g dry wt.) in regenerative callus when grown on medium added with 150 mM NaCl; followed by in vitro raised leaf of microshoot. Quantitative estimation of solasodine was carried out using a new HPTLC method, which is validated for its recovery and precession. The proposed HPTLC method showed a good linear relationship (r(2)=0.994) in 50-2000 ng/spot concentration ranges. The data demonstrate that the solasodine production in cultures was growth dependent.

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.

Plantlet regeneration enhances solasodine productivity in hairy root cultures of Solanum khasianum Clarke

Abstract 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 increased levels of solasodine production, compared to cultures without shoot regeneration. The plantlets formed in the hairy root cultures accumulated some of the solasodine, thereby ...

Manipulation of MS and B5 components for enhancement of growth and solasodine production in hairy root cultures of Solanum khasianum 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 Gamborg’s 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 Gamborg’s 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.

Isolation of solasodine and other steroidal alkaloids and sapogenins by direct hydrolysis-extraction of Solanum plants or glycosides therefrom

Concomitant extraction and hydrolysis of Solanum steroidal glycoalkaloids in a two-phase system containing an aqueous mineral acid and an organic water immiscible solvent, and having a boiling point under 100 oC, is described. It is essentially a “one-pot” process, combining direct acid hydrolysis of the glycosides in the plant material and in situ extraction of the released aglycones after alkali treatment, in a single step, the various ingredients being added simultaneously or sequentially, as required. Application of the process to fruits, leaves, and tissue cultures of Solanum khasianum Clarke plants, either fresh or in dried, finely ground form, using a two-phase aqueous hydrochloric acid-toluene system, proved to be very suitable for continuous production of pure solasodine which is a valuable raw steroid for the preparation of steroidal drugs. Pure Solanum glycoalkaloids were also prepared and hydrolysed accordingly. The process has analytical applications too, and could be extended in a general way to glycosides in other series for preparation of their related aglycones.

The regulation of solasodine production by Agrobacterium rhizogenes-transformed roots of Solanum aviculare.

Transgenic roots of Solanum spp. containing extra copies of an hmgr gene derived from Artemisia annua have been obtained via transformation with Agrobacterium rhizogenes. Hairy root clones of Solanum aviculare which were transgenic for hmgr typically grew faster than those which did not contain extra copies of this gene and also accumulated up to 4.2 times more solasodine when grown under dark, but not light, conditions. The implications of these findings with respect to the regulation of solasodine production in Solanum spp. are considered.

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.

Effect of sucrose, exogenous product concentration, and other culture conditions on growth and steroidal alkaloid production by Solanum aviculare hairy roots

Growth and steroidal alkaloid production by Solanum aviculare hairy roots were investigated as a function of temperature, initial sucrose and exogenous solasodine concentrations, and medium osmolality. Root growth measured as dry weight was significantly affected by initial sucrose concentrations between 2 and 12%; however, sucrose levels below 6–9% did not significantly alter the specific alkaloid content. Biomass production was maximum and about 60% higher than in 3% sucrose medium when the initial sucrose concentration was 4–6%. The effect of sucrose concentration on growth could not be attributed to osmotic effects, starch accumulation in the roots, or sugar depletion at the lower sugar levels. No significant difference at the 0.01 level in growth rate or alkaloid yield was found between seven root clones cultured in either 3 or 6% sucrose medium. The minimum root doubling time occurred at 25°C; doubling times at 20°C, 30°C, and 37°C were between 1.1 and 3.4 times higher. Experiments with exogenous solasodine added to the medium revealed that growth is strongly inhibited by extracellular product concentrations as low as 2 mg l −1. Nett steroidal alkaloid production declined to negative values when more than 10 mg l −1 solasodine was added, indicating that degradation or conversion of solasodine occurred. As extracellular concentrations can reach at least this level in batch culture, in situ removal of product is recommended for solasodine production systems.

Initiation of and solasodine production in hairy root cultures of Solanum mauritianum Scop.

Initiation and establishment of hairy root cultures from leaf or seedling hypocotyl explants of Solanum mauritianum Scop., using six strains of Agrobacterium rhizogenes was attempted. Success was only achieved following hypocotyl inoculation with strain LBA 9402. Transformation frequency was very low, with only one instance out of a possible 90 being recorded. Resultant hairy root cultures grew rapidly and could be maintained using a Murashige and Skoog (1962) medium supplemented with 0.1 g L−1myo-inositol and 3% sucrose, either as a solid or liquid culture. Under these conditions, the roots had a solasodine content of 126 μg g−1 DW. Lower levels of solasodine and decreased root growth rates were recorded when the medium strength was reduced by half or 3% glucose substituted for the 3% sucrose.

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.

Solasodine Production bySolanum eleagnifoliumCav.in vitroCultures: Influence of Plant Growth Regulators, Age and Inoculum Size. Large-Scale Production

Abstract The effect of different auxins (2,4-D, 2,4,5-T, NAA and IAA) on calli maintenance and plant cell suspension initiation as well as the influence of auxins in combination with citoquinines on growth and solasodine productivity by cell suspensions of Solanum eleagnifolium Cav. was studied. 2,4-D and 2,4,5-T produced a friable and productive calli line and a fine and homogeneous cell suspension cultures. 2,4-D (0.5 μM or 5 μM) or NAA (50 μM) produced the highest growth index and solasodine productivity with values of about 0.5 mg 1−1 day−1 to 0.8 mg 1−1 day−1. Simultaneous administration of auxin and cytokinin increased the solasodine productivity with a diminution of cell growth. The combination of NAA (50 μM) with kinetin (0.25 μM) produced an increase of solasodine productivity from 0.8 mg 1−1 day−1 to 2.54 mg 1−1 day−1. An inoculum size of 5% V/V and 19-day-old was selected to be used in fermentation processes in order to obtain solasodine production by S. eleagnifolium Cav. suspension cultures. ...

Stimulation of solasodine production by combining fungal elicitors and immobilized cell suspension cultures ofSolanumsurattense Burm

Combining the use of fungal elicitors likeAspergillus andFusarium with immobilized cell suspension cultures ofSolanum surattense Burm. showed a stimulation in solasodine production, of ablout 17 times as compared to cell suspensions.