Silymarin Content Research Articles

Analysis of metabolic variations throughout growth and development of adventitious roots in Silybum marianum L. (Milk thistle), a medicinal plant

Silybum marianum L. is a medicinal plant used in the treatment for jaundice and liver diseases. In this study, an adventitious root culture was developed for the production of health promoting phytochemicals. Adventitious roots were induced from nodal explants on solid Murashige and Skoog medium supplemented with 1.0 mg l−1 of α-Naphthalene acetic acid. Growth kinetics of the roots was investigated every week, for 8 weeks of culture period. Highest fresh biomass formation (153 mg l−1) was observed in 6-week old cultures. Adventitious roots were harvested from different growth stages as control (CTR), lag phase (LAG), logarithmic phase (LOG) or stationary phase (STN). Metabolite profiling of the samples was investigated using electro spray ionization time of flight mass spectrometry. Significant phenylpropanoids such as cinnamic acid and di-hydro kaempferol were predominantly found in LOG phase, whereas the highest amount of malonic acid was detected in STN as compared to other growth phases. More sucrose content was detected in CTR, while the tryptophan content was higher in LOG phase. Among the vital fatty acids, prostaglandin A1 and phenyl acetic acid were at highest levels in STN phase. However, more brassicasterols were observed in LAG phase than other growth phases. Punicic acid and lignan pinoresinol were detected abundantly in the LOG phase. Biochemical characterization revealed significant correlations between silymarin content and DPPH as well as TPC and TFC in the growth curve. Interestingly, among all growth stages there was no correlation of PAL activity with TFC and silymarin content.

Accumulation of silymarin in milk thistle seeds under drought stress.

According to the results obtained in this study, drought stress can enhance the accumulation of silymarin in milk thistle seeds. Moreover, under drought stress, the share of silybin increased which possess the greatest degree of biological activity among the silymarin components. Silymarin, an isomeric mixture of flavonolignans found in milk thistle (Silybum marianum (L.) Gaertn) seeds, has been used for its hepatoprotective effects for more than 2,000years. Biosynthesis and accumulation of active substances like silymarin in plant tissues highly interacts with the environmental conditions. Effects of moderate and severe drought stress (based on soil moisture depletion) on silymarin content and composition in milk thistle seeds were evaluated in a field study. Averaged across treatments, milk thistle seeds contained 19.3gkg(-1) silymarin. Drought stress enhanced silymarin accumulation in milk thistle seeds. Plants grown under moderate and severe drought stress treatments contained 4 and 17% greater silymarin than those grown in well-watered condition, respectively. Greater content of sylimarin in stressed plants was attributed to more contents of silybin, isosilybin and silychristin, while silydianin content was lower under drought condition. According to the results obtained in this study, drought stress enhanced accumulation of silymarin in milk thistle seeds and improved its quality by increasing the share of silybin, which possess the greatest degree of biological activity among the silymarin components.

Interactive effect of deficit irrigation and soil organic amendments on seed yield and flavonolignan production of milk thistle (Silybum marianum L. Gaertn.)

Milk thistle is a recognized medicinal plant cultivating for production of silymarin and oil. Responses of milk thistle to deficit irrigation and organic soil amendments were studied in a 2-year field experiment. Three levels of irrigation regimes including normal irrigation (I100), moderate deficit (I75) and severe deficit irrigation (I50) and two sources of soil amendments consisted of vermicompost (VM) and poultry manure (PM) plus no amendments comprised nine treatments laid out in a factorial pattern in a randomized complete block design. Moderate and severe deficit irrigation resulted in 7 and 27% seed yield reduction, respectively. Among yield components, number of seeds head−1 showed the highest sensitivity to deficit irrigation followed by number of heads plant−1 while seed weight remained stable. As irrigation deficiency intensified, Irrigation water use efficiency followed an increasing trend. Among the two soil organic amendments, only PM significantly improved the seed yield where plots amended with PM produced 8, 12 and 17% higher seed yield under I100, I75 and I50, respectively. Application of PM also improved irrigation water use efficiency. Implementation of deficit irrigation improved silymarin content of the seeds; however, silymarin yield declined due to the lower productivity in stressed conditions. Oil yield was also adversely affected by both moderate and severe deficit irrigation and reduced by 9 and 32%, respectively. Application of soil amendments had no significant effect on silymarin and oil content of the seeds, but PM improved silymarin and oil yield due to its beneficial effect on seed yield. The results of this study indicated that milk thistle can be grown under moderate deficit irrigation (saving 25% of irrigation water) and amending soil with PM can improve the yield and minimize the negative impact of drought stress.

Effect of Manure and Bio-fertilizers on Growth, Yield, Silymarin content, and protein expression profile of Silybummarianum.

The impact of the bio-fertilizer (humic acid, EM and yeast), organic fertilizer (cattle and chicken residues) and a combination of both on the growth and yield of Silybum marianum were studied. Plant height, number of branches, number of roots, root length and dry matter accumulation in plant organs significantly increased with the application of bio-fertilizer in combination with organic one. Both bio -fertilizers, organic fertilizer significantly affected the dry weight of herbs and root of Silybum marianumas compared with the control treatments. The plants treated with 10m 3 cattle ma- nure /fed plus EM or 10 m 3 Chicken manure showed significantly higher silymarin content in the second season as com- pared with the other bio , manure fertilization and control treatments . SDS-PAGE analysis of total cellular proteins was used for detecting changes in gene expression on dry seeds. The data showed no variation in protein pattern in terms of number of bands and their relative intensities among all the studied treatments .

In vitro Production of Silymarin from Silybum marianum L.

Silybum marianum L. is a wild medicinal herbal plant in Jordan. It is widely used in folk medicine due to its high content of silymarin. In vitro production of silymarin was experimented at different concentrations (0.0, 0.4, 1.0, 1.6 or 2.0 mg/l) of growth regulators (6-furfurylaminopurine (kinetin), 6-benzylaminopurine (BA), or 6-(gamma,gamma- Dimethylallylamino) purine (2ip)) and different concentrations (15, 30,45,60 mg/l) of carbon sources (glucose, fructose, and sucrose). HPLC (High Performance Liquid Chromatography) analysis was used to identify silymarin components. In vitro grown S. marianum on MS (Murashige and Skoo 1962) medium supplemented with (1.6 mg/l) kinetin and (0.1 mg/l) 1-Naphthaleneacetc acid (NAA) gave the highest silymarin content of (0.84%) silybin and (0.49%) silydanin as compared with cultures grown on hormone-free MS media which contained (0.36%) silybin and (0.30%) silydanin. In vitro grown S. marianum on MS medium supplemented with (2.0 mg/l) BA and 0.1 mg/l NAA yielded (0.67%) silybin and (0.37%) silydanin, while MS media supplemented with (1.0 mg/l) of 2iP gave (0.72%) silybin and (0.24%) silydanin. Otherwise, the in vivo (wild) grown shoots of S. marianum gave (1.07%) for silybin and (0.46%) sildyanin. Among carbon source, glucose at (45 g/l) gave (1.63%) of silymarin content. Results indicated a significant use of in vitro grown cultures for silymarin production.

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis. Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L −1 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L −1 Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L −1 each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.

Breeding Objectives and Selection Criteria for Milk Thistle [<i>Silybum marianum</i> (L.) Gaertn.] Improvement

Milk thistle [Silybum marianum (L.) Gaertn.] is an important medicinal-industrial plant. The medicinal compounds of milk thistle are derived from its seeds. The plant is commercially cultivated for the production of silymarin. Cultivation offers the opportunity to optimise achene yield and silymarin content. Furthermore, efforts should be made to develop new cultivars. The main objective of milk thistle breeding is to develop high yielding cultivars with elevated silymarin content. There are few developed cultivars of milk thistle (e.g. Argintiu, Budakalaszi, Szibilla, Khoreslo, Babak Castle, Mirel, Silma and Silyb). Different genotypes of milk thistle have variable amount of silymarin e.g. a ‘Royston’ genotype is rich in silymarin (6-10%). Further progress can be made by using the genetic diversity available in abundance in different genotypes. The main concern of the breeder should be to increase the yield and silymarin contents of the seeds. Asynchronous flowering and seed shedding are also major problems in milk thistle cultivation. At the time of harvest, the plants have flower heads at all stages of development resulting non-uniform maturation of seeds. Therefore, a breeding effort in milk thistle should be planned to obtain plants with simultaneous flowering and reduced crop losses. The developed cultivar Argintiu is characterised by simultaneous seed maturation in flower heads. The wild populations, as valuable gene pools, could be exploited for the improvement of milk thistle crop.

Silymarin content in Silybum marianum fruits at different maturity stages

Silymarin is one of the most investigated plant extracts with known mechanism of action. It is used for oral treatment of toxic liver damage. Silybin, isosilybin, silychristin and silydianin are the main flavonolignan components of silymarin isolated from the fruits of Silybum marianum. A reversed-phase high-performance liquid chromatography (HPLC) method for determination of flavonolignans was developed. The method depended on an isocratic solvent system comprising acetonitrile and water containing 0.5% (v/v) phosphoric acid. The method was used for analysis of the flavonolignans content in the fruits of S. marianum at different maturity stages. A correlation between fruits color, maturity stage and silymarin content is demonstrated. Key words: Silybum marianum, silymarin; , flavonolignans; , fruits; , maturity; , high performance liquid chromatography (HPLC).

An integrated proteomic approach to decipher the effect of methyl jasmonate elicitation on the proteome of Silybum marianum L. hairy roots

Jasmonate and its methyl derivative, methyl jasmonate (MeJA), are naturally occurring compounds that mediate several plant physiological processes in response to pathogen attack, wounding, and ozone. Exogenous application of jasmonates triggers defense responses that resemble those initiated by pathogen infection and also modulates the production of certain secondary metabolites in a variety of plant species. In this study, we treated the hairy root cultures of Silybum marianum L. with 100μM MeJA and then measured the content of Silymarin (SLM). We observed that the SLM content increased significantly after 48h of MeJA treatment and remained constant for 120h. However, MeJA treatment caused a significant growth reduction after 96h incubation. The activity of lipoxygenase as a key enzyme in the jasmonate biosynthesis pathway and anti-oxidative enzymes; peroxidase and ascorbate peroxidase was also significantly increased after MeJA treatment. To elucidate the global effect of jasmonate on gene expression of S.marianum, we employed high resolution two-dimensional gel electrophoresis coupled with tandem mass spectrometry. Out of 670 reproducibly detected protein spots which were analyzed on each given gel, 32 spots were up- or down regulated upon MeJA treatment. Of them, ten proteins such as ER binding protein, glutamine synthetase, pathogenesis-related protein, caffeoyl CoA O-methyltransferase, and profilin-1 could be identified by mass spectrometry analysis. The possible implications of the identified proteins on physiological outcome of MeJA application in S.marianum hairy root culture will be discussed.

Effects of light regimes on in vitro seed germination and silymarin content in Silybum marianum

Silybum marianum is an economically important crop worldwide. It is renowned for production of biologically important silymarin. Average sale of silymarin is about US$ 8 billion/annum and its demand varies from 18 to 20tons/year. Despite of its demand, there is lack of research efforts on cultivation and improvement of this plant. We hereby established feasible seed germination protocol for production of healthier and chemically consistent plantlets. Combination of benzyladenine (BA, 0.5mgl−1)+gibberellic acid (GA3, 1.5mgl−1)+thidiazuron (TDZ, 1.0mgl−1) produced optimum germination frequency in seeds kept in 2 weeks dark and subsequently transferred to 2 weeks light (16h photoperiod) conditions. Correlation among mean shoot length, mean root length, set of antioxidative enzyme activities was also observed in current report. Silymarin was determined by high performance liquid chromatography (HPLC). Considerable amount of silymarin (5.48mgg−1DW) was detected in our study, which was comparative to other reports available. Antioxidant activity (1,1-diphenyl-2-picrylhydrazyl; FRSA) and phenylalanine ammonia lyase (PAL) activity was also determined. Silymarin content had shown direct relationship with these activities. It showed that silymarin was a major antioxidant in current report. This study provides basis for expedited production of S. marianum plantlets with feasible content of silymarin.

Antioxidant and Anti-Hepatitis C Viral Activities of Commercial Milk Thistle Food Supplements.

Milk thistle dietary supplements that contain silymarin are widely marketed and used in the USA and other countries for liver enhancement and recovery. More recently, silymarin has also been identified as a possible antiviral for the treatment of hepatitis C virus (HCV) infection. To assess different brands of commercially sold silymarin, 45 products were collected from local stores and analyzed for their silymarin content, antioxidant activities, and antiviral activity against HCV. Antioxidant activity was measured as radical scavenging activity using DPPH and by estimating their antioxidant capacity as trolox equivalent. Anti-HCV activity was measured in an HCV genotype 1b replication inhibition assay. Samples were found to vary widely in their silymarin content, with some samples having none or very low concentrations while silymarin represented higher than 80% of other samples. Both antioxidant and anti-HCV activity correlated with the overall level of silymarin.

Improved silymarin content in elicited multiple shoot cultures of Silybum marianum L.

Silybum marianum L. extracts are being used as antihepatotoxic therapy for liver diseases. Silymarin is a polyphenolic flavonoid mixture isolated from milk thistle which is believed to be responsible for the plant's hepatoprotective action. Regeneration of Silybum marianum plants from shoot tip explants and assessment of their morphogenic potential, silymarin total concentration and its major constituents upon exposure to medium composition alteration and different elicitors' application was targeted. Different concentrations of NaCl, quercetin, gamma irradiation and dried fungal extracts were used to elicit silymarin production in the cultures. The chemical composition of silymarin and its total concentration was investigated through HPLC at all the experiment stages. Multiple shoots were recorded after 3weeks of culture on MS medium containing various concentrations of BA and/or NAA. IAA was more effective than NAA and IBA in inducing robust roots in shoot cultures. The flowering plants recorded 20% and 40% of the total plants number in the multiplication and rooting stages respectively. The highest total silymarin concentration reached its peak with (10Gy) gamma-irradiation to be 6.598% dry weight in the in vitro regenerated shoot tip explants. The in vitro grown flowers showed 1.7 times more sylimarin productivity as compared to that of the wild grown congruent.

국내에 자생하는 큰엉겅퀴와 고려엉겅퀴의 분자유전학적 및 화학적 분석

국내에 자생 중인 큰엉겅퀴(Cirsium pendulum)를 강원도의 홍천, 원주, 평창, 양양 및 경기도 가평과 충청북도의 충주에서 채취하고, 고려엉겅퀴(Cirsium setidens)를 강원도의 태백 및 경상북도의 봉화에서 채취하였다. 채취된 식물들의 genomic DNA를 분리하여서 18S rDNA, ITS1, 5.8S rDNA, ITS2 및 28S rDNA의 일부를 증폭하고, 그 염기서열을 분석하였다. 이렇게 얻어진 염기서열을 GenBank에 등록하고, 서로 비교해 보았다. 그 결과 큰엉겅퀴는 6개체 모두에서 18S rDNA 및 5.8S rDNA의 염기서열은 서로 완전히 일치하고 있으나, ITS1과 ITS2의 염기서열에서는 약간의 차이를 보였다. 고려엉겅퀴의 경우에는 18S rDNA, ITS1 및 ITS2에서 2개체가 서로 비교적 큰 차이를 보였다. 일본의 홋카이도에서 채취된 큰엉겅퀴와 중국의 안휘성에서 채취된 엉겅퀴(Cirsium japonicum)를 포함하여서 ITS1과 ITS2 염기서열을 비교한 결과, 엉겅퀴, 큰엉겅퀴, 고려엉겅퀴는 확연하게 서로 다른 그룹을 형성하는 것으로 나타났다. 또한 채취된 큰엉겅퀴 및 고려엉겅퀴들의 silymarin 함량을 분석해 본 결과, 모두에서 그 함량이 2 mg/ml가 넘는 것으로 나타났다. 따라서 엉겅퀴뿐만 아니라 큰엉겅퀴나 고려엉겅퀴도 여러 가지 약리작용을 가진 것으로 보고된 silymarin을 상당히 높은 농도로 함유하고 있는 것으로 나타났다. Cirsium pendulum plants were collected from Hongcheon, Pyeongchang, Wonju, Yangyang in Kangwondo, Gapyeong in Gyeongkido, and Choongju in Choongcheongbukdo. Cirsium setidens plants were collected from Taebaek in Kangwondo and Bonghwa in Kyeongsangbukdo. Genomic DNA was prepared from those plants and used for the amplification of 18S rDNA, ITS1, 5.8S rDNA, ITS2, and part of 28S rDNA. The PCR products were sequenced, and the sequence was deposited in the GenBank. The comparison of those sequences has revealed that the rDNA sequences are identical for all six C. pendulum plants, but that the ITS1 and ITS2 sequences contain variable nucleotides. The two C. setidens plants had different nucleotides in 18S rDNA, ITS1, and ITS2. The comparison of the DNA sequences of C. pendulum and C. setidens collected in this study with C. pendulum of Hokkaido in Japan and C. japonicum of Anhui in China indicated that the plants of those three species are clearly divided into three distinct groups. The silymarin content of the collected plants was analyzed and turned out to be quite high. Therefore, it has been found that both C. pendulum and C. setidens plants are producing large amounts of silymarin, which has been reported to have various medicinal effects.

English

Silybum marianum is a wild medicinal plant containing silymarin, which is a known drug, used as the hepatoprotective agent. S. marianum growing in different areas of NWFP were collected and analyzed for the silymarin and oil contents. In all the samples analyzed for the silymarin, high yield was found in the sample collected from Swat district (3.85%) while high yield of oil was recorded in the sample collected from Kohat district (27.87%). The rest of samples for silymarin contents were found between 2.98 to 3.85% while the oil contents were found to be between 11 to 27.87%. Both the yield of silymarin and oil contents found in all the samples depend on the environment in which the plants were grown and the environmental conditions affecting both the contents of silymarin and oil of this highly medicinal plant. The analysis is of particular importance for collecting this medicinal plant for silymarin content and utilization in the pharmaceuticals.

Effect of sowing date and rate on the yield and flavonolignan content of the fruits of milk thistle (Silybum marianum L. Gaertn.) grown on light soil in a moderate climate

In the moderate climate of Poland it is recommended that milk thistle (Silybum marianum L. Gaertn.) be grown on fertile soils. The plant, however, develops a strong root system, so a working hypothesis has developed that cultivation can be extended to light soils with periodic water deficits. The aim of the present research was to determine the effects of sowing milk thistle on light soil at different dates and rates on the achene yield and flavonolignan content. This experiment was carried out during 2004–2006 at the Mochelek Experiment Station of the University of Technology and Life Sciences in Bydgoszcz (53°13′ N; 17°51′ E). The average fruit yields were 1.23tha−1; those of silymarin were 26.5kgha−1. The moisture and thermal conditions during the research years caused the fruit yields to range from 0.55 to 1.68tha−1 and silymarin yields from 13.3 to 35.4kgha−1. Delaying sowing from early to mid-April increased the plant density and decreased numbers of inflorescences and fruits per inflorescence; as a result, no effect of sowing date on fruit yield was found. Delaying the sowing date increased silymarin content by about 0.4% and its yield by 5.3kgha−1. Increasing the sowing rate from 12 to 24kgha−1 resulted in a slight (40kgha−1) but significant increase in achene yield; however, it did not affect the silymarin content. The average silymarin content in fruits was 2.18%. The ratio of silydianin to silychristin was 1:2.2, and the ratio of silydianin to the sum of silybinin and isosilybinin was 1:3.3.

INFLUENCE OF FOLIAR FERTILIZATION AND GROWTH REGULATOR ON MILK THISTLE SEED YIELD AND QUALITY

The effects of foliar fertilization and a growth regulator 5-tert-butyl-N-m-tolylpyrazine-2-carboxamide (MD148/II) on the growth, seed yield, and silymarin content of milk thistle (Silybum marianum Gaertn.) plants were evaluated. The study was conducted over two years at an experimental field on a slightly acid-leached cinnamonic meadow soil. The MD148/II was applied in the beginning of milk thistle flowering stage. Foliar fertilizer was applied at different plant developmental stages with different proportions of nitrogen (N), phosphorus (P), and potassium (K). Treatments with foliar fertilizer and MD148/II resulted in improvement of plant biomass, number of plant lateral shoots, flowering rate, and seed yield and the content of some active substances in milk thistle seeds. A reduction of high molecular fatty acids was observed. The increase of seed yield was a result of the flower head setting enhancement. Therefore the combined treatment of foliar fertilizer and MD148/II was efficient in elicitation milk thistle production under field conditions.

Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum

Silymarin is one of the most potent antioxidant so far developed from plant sources used as hepatoprotectants. Influence of different concentrations (0, 1, 2, 4, 6 and 8mg/50ml culture) and exposure time (24, 48, 72, 96 and 120h) of salicylic acid on lipoxygenase activity, linoleic acid content, growth and production of silymarin in hairy root cultures of S. marianum were investigated. Detection and identification of flavonolignans was carried out by high performance liquid chromatograph method. Salicylic acid enhanced silymarin production (1.89mgg(-1) DW). The optimal feeding condition was the addition of salicylic acid (6 mg/50 ml culture) after 24h in which the silymarin content was 2.42 times higher than the control (0.78mgg(-1) DW). The content of silybin, isosilybin, silychristin, silydianin and taxifolin were 0.703, 0.017, 0.289, 0.02 and 0.863mgg(-1) DW respectively in these samples, while in non-treated hairy roots were 0.027, 0.046, 0.23, 0.022 and 0.453 respectively. Lipoxygenase activity also affected by elicitation. lipoxygenase activity increased 24h after treatment by approximately 1.57- fold (0.21 Delta OD(234)/mgproteinmin(-1)). Upon elicitation with salicylic acid, linoleic acid content of hairy roots (38.26mgg(-1) DW) were also elevated after 24h, in which the linoleic acid content was 2.37 times higher than the control (16.1mgg(-1) DW). It is feasible that elicitation with salicylic acid regulates the jasmonate pathway, which in turn mediates the elicitor-induced accumulation of silymarin.

Production and quality of milk thistle (Silybum marianum[L.] Gaertn.) cultivated in cultural conditions of warm agri-climatic macroregion

A polyfactorial field experiment was established and investigated during vegetation periods from 2004 to 2007 at the Dolná Malanta locality (Nitra district, Slovakia). The following parameters were measured: (1) yields of milk thistle (Silybum marianum [L.] Gaertn.) achenes, i.e. fruits, in 2004–2007; (2) content of silymarin in dry fruits of milk thistle in 2006–2007; and (3) total yields of silymarin per ha in 2006–2007. Factors of the experiment were as follows: (1) crop residues of cultivated pre-crop (no crop residues – K, with crop residues – R); (2) cultivation of freezing-out intercrop (no intercrop – B, with intercrop – M); (3) fertilization using artificial fertilizers (no fertilization – O, with fertilization – F); (4) year of cultivation (2004–2007). The highest yields of milk thistle fruits were recorded in 2006: from 1,426.5 kg/ha (RBO variant – incorporated crop residues without intercrop, no artificial fertilizers) to 1,832.0 kg/ha (KBF variant – without crop residues, without intercrop and with application of artificial fertilizers). The highest content of silymarin complex in dry fruits of milk thistle was measured in 2007: from 15.14 mg/kg (RMF – with crop residues, intercrop and fertilization) to 20.01 mg/kg (KBO – without crop residues, intercrop and fertilization). The highest total yield of silymarin per ha was recorded in investigated variants in 2006; in variant without crop residues it ranged from 16.45 kg/ha (KMF – with intercrop, with fertilizers) to 24.62 kg/ha (KMO – with intercrop, no fertilization).

Elicitor-enhanced Silymarin production in hairy root cultures of Silybum marianum L. Gaertn

Silymarin is an antihepatotoxic polyphenolic substance isolated from the milke thistle (Silybum marianum) plant [1]. Hairy roots cultures of S. marianum produce silymarin. In an attempt to increase productivity, the effect of yeast extract was tested. The hairy roots induced by inoculation of leaf explants with A. rhizogenes (AR15834). One month old hairy roots were dissected from the explants and growth in the Erlenmeyer flask containing 50ml MS liquid medium [2]. PCR analysis using rolb gene and GUS assay were used for identification of transformed hairy roots [3]. The presences of flavonolignans in the hairy roots were determined by high-performance liquid chromatography (HPLC). There were five isolated components; taxifolin, silychristin, silydianin, silybin and isosilybin and the major flavonolignan was silybin that has not been reported in previous published work. For increasing the growth rate of hairy roots, effects of NAA, IBA and IAA (0.1, 0.5 and 1mg/L) were tested. The best media was achieved by 0.5mg/L NAA. Elicitation was carried out with yeast extract (0, 0.5, 1, 2.5 and 5mg/L) and different exposure times (24, 48 and 72h). The highest silymarin content (0.476mg/g DW) was obtained by 2.5mg/mL yeast extract, after 72h. This study indicates that elicitor treatment promotes silymarin production in hairy root culture of S. marianum.

Variation in Flavonolignan Concentration of Milk Thistle (Silybum marianum) Fruits Grown in Iran

ABSTRACT This study was conducted to analyze the genetic variation of silymarin and the corresponding flavonolignans in the fruit (achenes) of 32 ecotypes of milk thistle, Silybum marianum (L.) Gaertner, collected from the north, northwest, south, and southwest of Iran and two improved commercial varieties, one from England (CN Seeds, Ltd., Ely, United Kingdom) and one from Hungary (Budakalaszi, Budakalasz). A higher level of genotypic variation, heritability, and genetic gain was observed for silybin A and B in the studied ecotypes as compared with the two accessions, indicating that these traits could be further improved. In contrast, silymarin content and yield showed only moderate receptiveness for improvement. For silychristin and silydianin, high variation was obtained only in ecotypes originating from the south and southwest regions of Iran. Factor analysis resulted in two factors, accounting for about 91 percent of the total variation of the investigated characters. On this basis, three groups each with distinct chemotypic properties could be identified. Discriminant analysis verified the results of factor analysis and separated the groups.