Silymarin Production Research Articles

Silymarin Production in Inoculated Silybum marianum L. Hairy Roots Culture with Piriformospora indica

Silymarin Production in Inoculated Silybum marianum L. Hairy Roots Culture with Piriformospora indica

Callus induction and silymarin production in of Silybum marianum L. under tissue culture medium

Callus induction and silymarin production in of Silybum marianum L. under tissue culture medium

Salicylic Acid Foliar Spray Enhanced Silybum marianum Growth and Yield, as Well as Its Chemical Constituents and Chalcone Synthase Gene Activity

Silymarin, a secondary metabolite found mainly in the Silybum marianum L. fruits, has been associated with the hepatoprotective activity of the plant. Among various elicitors, salicylic acid, a “Generally Regarded As Safe” compound recognized by the Food and Drug Administration, is one of those being used in the induction and enhancement of valuable plant secondary metabolite production in various plant species. In this study, two concentrations (10−4 and 10−3 molar) of salicylic acid have been applied to the S. marianum plants as foliar spray to investigate their effects on plant growth and yield, as well as the production of its bioactive compound, silymarin. Our results indicated that both concentrations of salicylic acid increased the plant height, the number of branches, leaves, and capitula, as well as the dried weight of roots, aerial parts, and fruits. The enhancement effects in plant growth and yield were accompanied by an increase in photosynthetic pigments such as chlorophyll-a, b, and carotenoids as well as element contents such as nitrogen, phosphorus, and potassium. The potential of salicylic acid as an elicitor for the enhancement of secondary metabolites in S. marianum was supported by the increase in silymarin’s major components, silybin (A + B), in the salicylic acid-treated plants. Concomitant expressions of CHS1, 2, and 3 genes that have been associated with the production of silymarin in S. marianum were also observed in the salicylic acid-treated plants. A lower concentration (10−4 M) of salicylic acid was found to be a better elicitor as compared with the 10−3 M salicylic acid. An increase of 3.4 times in capitula number and fruit dried weight as well as 2.6 times in silybin (A + B) contents were observed in plants sprayed with 10−4 M of salicylic acid as compared with the control.

Characterization and potential utilization of recently available milk thistle, Silybum marianum (L.) Gaertn., wild type and mutant accessions

AbstractMilk thistle, Silybum marianum (L.) Gaertn. (Asteraceae), is an economically important medicinal plant utilized for silymarin production. Moreover, the species has been positively evaluated for vegetable oil and biomass production. Despite these positive characteristics, milk thistle is still marked by traits that are typical of undomesticated species (most importantly natural fruit dispersal at maturity) and requires further genetic improvement for its complete exploitation. This manuscript summarizes all the information collected through time about a collection of nine milk thistle wild and mutant lines and it discusses the possible further utilization of these genotypes. The accessions are characterized by interesting traits related to: fruit silymarin composition (S. marianum chemotype A and B), fruit fatty acid composition (high oleic and high stearic acid lines), fruit condensed tannins content, vegetative biomass composition (modification of xylans or lignin content), vegetative biomass structure (dwarf and tall lines), modifications of leaf variegation (hypervareigated line) and different types of fruit shatter resistance at maturity. All the lines underwent subsequent generations of selfing and are stable for all the described traits. The accessions will be made available at the Genebank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK, Gatersleben) and may prove to be a useful genetic material for the improvement of qualitative fruit traits (silymarin quality, fatty acid composition) and for the further development of shatter-resistant S. marianum genotypes towards the complete domestication of this promising species.

Yield and silymarin content in milk thistle (Silybum marianum (L.) Gaertn.) fruits affected by the nitrogen fertilizers

Milk thistle (Silybum marianum (L.) Gaertn.) is a common weed that has been introduced as a medicinal plant for silymarin production. This renders it important to optimize cultivation techniques, including fertilization. In the present study, a two-year field experiment was conducted to evaluate the influence of conventional and stabilized nitrogen fertilizers on plant growth, fruit yield, and fruit quality. The experiment was conducted in a randomized complete block design with five fertilization treatments (control, conventional nitrogen fertilizer applied at 75 kg/ha and 125 kg/ha, and stabilized nitrogen fertilizer applied at 75 kg/ha and 125 kg/ha. Nitrogen fertilization had a significant effect on vegetative and yield parameters. Specifically, the application of nitrogen fertilizers positively influenced rosette diameter, height, aboveground dry biomass, total chlorophyll content, number of capitula per plant, number of fruits per central capitulum, and fruit yield. In particular, in most traits, the usage of high nitrogen levels of the two types of fertilizer had the most pronounced impact. The application of the stabilized fertilizer at a rate of 125 kg N/ha induced an increase in fruit yield of up to 29.6 %. Nitrogen fertilization had no impact on the accumulation of oil and silymarin in the fruits, although some effects on oil and silymarin yield were noted. In general, the application of nitrogen fertilizers had no effect on the antioxidant activity or total phenolic content of the fruit extracts. Finally, climatic conditions affected the growth and productivity of milk thistle, as fruit yield was favoured by wet conditions.

Enrichment of Therapeutically Significant Flavonolignans of Silybum Marianum in Vegetative Parts by Applying Fungal Elicitors, Methyl Jasmonate and Silver Nanoparticles as Elicitor in Hydroponic Culture

Introduction: Medicinal plants are being used to treat several diseases for many decades and this is an ancient method to treat the patients. Herbal plant Silybum marianum found most effective one to cure liver disorders. This plant produces silymarin which is a secondary metabolite and have hepatoprotective properties. Silymarin is a mixture of flavonolignans (silybin A, silybin B, isosilybin A isosilybin B, silychristin, silydianin apigenin 7-D glucose and taxifolin) that has antiviral, antibacterial, antifungal and antiallergenic properties. Therefore, the excessive production of silymarin is necessary to cure different types liver disorders, so the present study executed to boost up production of Silymarin flavonolignans in vegetative parts of the Silybum marianum plant by using different elicitors.
 Materials and Methods: In the present study, elicitation technique in hydroponics system was used to enhance the production of pharmacologically active flavonolignans in Silybum marianum. Fungal elicitors prepared from lyophilized Aspergillus niger biomass, methyl jasmonate, silver nanoparticles and combination of silver nanoparticles and methyl jasmonate were added (0.2g/l), (100µM/l), (1 ppm) and (100µM/lppm) in hydroponics with Hoagland’s solution in hydroponics to enhance the production of flavonolignans of Silybum marianum. Controls were also set for each treatment. Plants were harvested after 72 hours of introduction of elicitors. High performance liquid chromatography technique was used for analytical purpose. Four solvents (methanol, acetonitrile, chloroform and 2% Trifloro acetic acid) were used in HPLC. Column was C18 and run time of sample was 1 hour. Silybum marianum’s seed extract was used as a standard. Extract of control and treated plants were run on the same polarity in HPLC.
 Results: Results showed that after elicitation significant increase was observed in production of silymarin’s flavonolignans (silybin A, silybin B, isosilybin A, isosilybin B and apigenin 7-D glucose) in vegetative part of the plant but rate of production was different for each elicitor, fungal elicitors prepared from lyophilized Aspergillus niger biomass proved best among all treatments.

​Optimizing Cultivation Technique of Silybum marianum (L.) Gaertn. under Tropical Climate

Background: A high prevalence of global liver diseases compels the availability of affordable drugs with fewer side effects. Silybum marianum (L.) Gaertn. is being used as a traditional medicine for centuries to treat a range of liver and gallbladder disorders. In tropical regions especially in Indonesia, S. marianum is scarcely cultivated. Therefore, cultivation on a large scale to initiate the independency of providing domestic medicinal raw materials demand is needed. This research was conducted to find out a suitable method in producing silymarin from S. marianum by optimizing cultivation technique by using foliar fertilizer (FF) applied to different planting periods and various spacing protocols. Methods: The experiment of nine treatment packages consisted of a combination plant spacing (S) and foliar fertilizer/FF dosage (D) factors, also applied time of planting (P), were examined to obtain the highest silymarin content. This field study was designed using a randomized complete block design (RCBD). Result: The results showed that a combination of the plant spacing of 140 x 140 cm, FF of 2 mg per plant and planting period in May, obtained the best results in the silymarin content of 1.52 ± 0.07%. It also gave the highest estimated silymarin production per unit area that was 13.79 ± 1.42 kg.ha-1.

Relationship between different agro-climatic conditions and silymarin production in wild milk thistle (Silybum marianum L. Gaert.) in Pakistan

Relationship between different agro-climatic conditions and silymarin production in wild milk thistle (Silybum marianum L. Gaert.) in Pakistan

Effect of gamma rays and colchicine on silymarin production in cell suspension cultures of Silybum marianum: A transcriptomic study of key genes involved in the biosynthetic pathway.

The aim of this study was to investigate secondary metabolite production in Silybum marianum L. cell suspension cultures obtained from seeds treated with gamma rays (200 and 600Gy) and 0.05% colchicine. The effects of these treatments on callus induction, growth, viability, and silymarin production were studied, along with the changes in the transcriptome and DNA sequence of chalcone synthase (CHS) genes. The effect of gamma radiation (200 and 600Gy) on silymarin production in S. marianum dry seeds was also studied using HPLC-UV. All three treatments induced high callus biomass production from leaf segments. The viability of the cell suspension cultures was over 90%. The flavonolignan content measured in the extracellular culture medium of the S. marianum cell suspension was highest after treatment with 600Gy, followed by 0.05% colchicine, and finally, 200Gy, after a growth period of 12days. In general, an increased expression of CHS1, CHS2, and CHS3 genes, accompanied by an increase of silymarin content, was observed in response to all the studied treatments, although the effect was greatest on CHS2 expression. Bioinformatics analysis confirmed that the three CHS2 clones exhibited the highest genetic variation, both in relation to each other and to the CHS1 and CHS3 clones. Based on the results, S. marianum plants obtained from seeds previously exposed to 600 and 200Gy as well as colchicine constitute a renewable resource with the potential to obtain large amounts of silymarin.

Interactive effects of zinc oxide nano particles and different light regimes on growth and silymarin biosynthesis in callus cultures of Silybum marianum L.

Silybum marianum L. commonly known as milk thistle is a medicinally potent plant with a multitude of pharmacological applications. The present investigations demonstrated the effects of Zinc Oxide nanoparticles (ZnO NPs) on callus growth and biosynthesis of silymarin in milk thistle under various light conditions. The callus cultures developed on Murashige and Skoog (MS) basal media containing ZnO NPs (0.15 mg/L), under the dark condition maintained for two weeks, followed by transference into normal light produced the maximum callus fresh weight (2294 mg/L FW). Further, the metabolite profiling revealed that ZnO NPs significantly augmented the production of silymarin and upregulated the antioxidant system in the callus cultures. Maximum TPC (total phenolic content: 37 ± 0.20 mg/g DW), TFC (total flavonoid content: 8.9 ± 0.023), DPPH antioxidant activity (91.5 ± 1.75%), Superoxide dismutase activity (SOD: 4.1 ± 0.045 nM/min/mg FW) and the highest silymarin content (14.6 ± 0.023 mg/g DW) were recorded in the callus cultures developed on MS media supplemented with solitary ZnO NPs (0.15 mg/L). While the callus culture evolved in presence of only PGRs (2,4 D and BA: 2 mg/L, each) accumulated the lesser fresh weight (562 mg/L FW). A higher concentration of ZnO NPs (0.15 mg/L) enhanced the secondary metabolite accumulation and silymarin content in the callus of Silybum marianum. This is the first standardized protocol to be applied on the industrial level for the production of silymarin.

Biotechnological production of silymarin in Silybum marianum L.: A review

Abstract Biotechnology approaches applied through plant tissue culture of milk thistle (Silybum marianum L. Gaertn.) can contribute to the production of biologically active substances, micropropagation and the study of genes involved in the biosynthetic pathway of silymarin. The fruits (achenes) of S. marianum contain a mixture of flavonolignans, collectively known as silymarin, a high value hepatoprotectant. Because of its high biological activities, the increasing pharmaceutical demand for silymarin has made it an important target to study its production through biotechnological systems. In vitro cell, tissue and organ cultures of medicinal plants provide an appropriate solution for the problems correlated to conventional production in the open field. However, the productivity of silymarin through different biotechnological systems is low in comparison to field production, which requires more efforts and attempts in this regard. Elicitation strategy has substantially enhanced the accumulation of silymarin substances in callus, cell and hairy root cultures, providing a continuous and promising production of such high value natural derived compounds in large-scale through bioreactor system. Few investigations have focused on studying the bioengineering of key genes involved in the biosynthetic pathway of silymarin in S. marianum plant. These studies have provided a rational approach and new insights into the regulation of silymarin biosynthetic pathway to improve the biotechnological production of silymarin and flavonoids through plant tissue culture system. This review comprehensively summarizes the recent advances on various approaches of biotechnology of S. marianum plant and discusses the advantages and possible challenges, which will provide useful clues and strategies to overcome the problems correlated to each. A brief description of the phytochemistry of milk thistle, biosynthesis of silymarin components and biological activity of in vitro cultures was also reviewed.

A New Strategy to Increase Production of Genoprotective Bioactive Molecules from Cotyledon-Derived Silybum marianum L. Callus.

There is a need to enhance the production of bioactive secondary metabolites and to establish new production systems, e.g., for liver-protective compounds of Silybum marianum seeds. Quantifying and identifying the produced phytochemicals, and examining their protective effects against genotoxic agents, is of great interest. This study established a protocol for the qualitative and quantitative production of hepatoprotective compounds in cotyledon-derived Silybum marianum callus through optimized supplementation of the MS medium with the growth regulators 2,4-D, benzylaminopurine, myoinositol, and asparagine. High-performance liquid chromatography (HPLC) coupled with electrospray ionisation mass spectrometry (ESI-MS) allowed for identification and quantification of the produced compounds. None of the growth medium combinations supported a detectable production of silymarin. Instead, the generated calli accumulated phenolic acids, in particular chlorogenic acid and dicaffeoylquinic acid, as revealed by HPLC and mass spectrometric analysis. 4-Nitro-o-phenylenediamine (NPD) was employed in the AMES-test with Salmonella typhimurium strain TA98 because it is a potent mutagen for this strain. Results revealed that callus extract had a high anti-genotoxic activity with respect to standard silymarin but more evident with respect seed extract. The callus produced chlorogenic acid and dicaffeoylquinic acid, which revealed higher bioactivity than silymarin. Both compounds were not formed or could not be detected in the seeds of Silybum marianum Egyptian ecotype.

Identification of Milk Thistle Shatter‐Resistant Mutant Lines with Altered Lignocellulosic Profile for the Complete Domestication of the Species

Milk thistle [Silybum marianum (L.) Gaertn.] is used as a medicinal plant for the production of silymarin. Other than silymarin, the fruit also contains valuable compounds such as oil and protein, and the lignocellulosic biomass of this species has been positively evaluated for biogas, syngas, and energy production. Despite these positive characteristics, the plant is still marked by traits that are typical of undomesticated species. Specifically, fruit shattering at maturity severely hampers its possible utilization as a crop. Therefore, for the first time, an ethyl methanesulfonate mutagenesis program was implemented to identify milk thistle lines with reduced fruit shattering, and its effectiveness was evaluated through genome‐wide analysis. The candidate nonshattering lines were multiplied up to M6 for the validation of the favorable traits. The lignocellulosic biomass of the selected shatter resistant lines was analyzed according to compositional analysis. The natural milk thistle fruit dispersal mechanism was shown to be modulated by changing air relative humidity. Three vigorous, stable, shatter‐resistant lines were identified. This is the first report that describes milk thistle nonshattering lines. At this stage in the three lines, the morphological description and the compositional analysis of the lignocellulosic biomass indicated that shatter resistance was concomitant to changes in vegetative biomass structure and composition. This observation is compatible with the described natural fruit dispersal mechanism, and it is of interest for the design of new milk thistle crop ideotypes for the complete domestication of the species.

Interactive Effects of Light and Melatonin on Biosynthesis of Silymarin and Anti-Inflammatory Potential in Callus Cultures of Silybum marianum (L.) Gaertn.

Silybum marianum (L.) Gaertn. is a well-known medicinal herb, primarily used in liver protection. Light strongly affects several physiological processes along with secondary metabolites biosynthesis in plants. Herein, S. marianum was exploited for in vitro potential under different light regimes in the presence of melatonin. The optimal callogenic response occurred in the combination of 1.0 mg/L α-naphthalene acetic acid and 0.5 mg/L 6-benzylaminopurine under photoperiod. Continuous light associated with melatonin treatment increased total flavonoid content (TFC), total phenolic content (TPC) and antioxidant potential, followed by photoperiod and dark treatments. The increased level of melatonin has a synergistic effect on biomass accumulation under continuous light and photoperiod, while an adverse effect was observed under dark conditions. More detailed phytochemical analysis showed maximum total silymarin content (11.92 mg/g dry weight (DW)) when placed under continuous light + 1.0 mg/L melatonin. Individually, the level of silybins (A and B), silydianin, isolsilychristin and silychristin was found highest under continuous light. Anti-inflammatory activities were also studied and highest percent inhibition was recorded against 15-lipoxygenase (15-LOX) for cultures cultivated under continuous light (42.33%). The current study helps us to better understand the influence of melatonin and different light regimes on silymarin production as well as antioxidant and anti-inflammatory activities in S. marianum callus extracts.

Determination amount of silymarin and pharmaceutical products from milk thistle waste obtained from cold press

Determination amount of silymarin and pharmaceutical products from milk thistle waste obtained from cold press

Localization of phenolic compounds in the fruits of Silybum marianum characterized by different silymarin chemotype and altered colour

Silybum marianum (L.) Gaertn (Asteraceae) is a valuable medicinal plant utilized for silymarin production. However, only fragmentary and contradictory information about silymarin localization within S. marianum fruit are available. In this work, a twofold research approach was adopted in order to investigate the distribution and quantification of silymarin and of other phenolic compounds within the different fruit regions (pericarp, seed integument, cotyledon). Two S. marianum wild accessions with contrasting silymarin chemotype (A and B) and a mutant line (C) with an altered fruit colour were analysed. Fruits of Cynara cardunculus were studied as a reference.Firstly, the fruit morpho-anatomy was reviewed by means of light microscopy digital imaging and, secondly, a comprehensive histolocalization of the different classes of polyphenols within the fruit was carried out. The experimental evidences confirmed that silymarin, and its precursor taxifolin, are only accumulated in the seed integuments. The dark colour of fully-ripened fruits is due to the accumulation of condensed tannins in the pericarp subepidermal cell layer. On the contrary, the studied mutant line shows reduced condensed tannin content that probably result from impairment at the level of flavonoid biosynthetic pathway.Condensed tannins content is comparatively low in S. marianum fruits and very low in the identified mutant line. This could represent an advantage for the possible employment of S. marianum fruits and of silymarin extraction by-products in the feed and food sector.

Protective Effects of Nitric Oxide on Photosynthetic Stability and Performance of Silybum marianum under Water Deficit Conditions

Core Ideas Milk thistle is a medicinal plant cultivating for production of silymarin and oil.The results our work showed that with the application of SNP photosynthesis, pigments content, and water status were improved in the drought stress condition and as a result significant increases in secondary metabolites production and grain yield was observed.Overall, this study suggest that application of 100 µM SNP and using Sari ecotype can be recommended for maximizing crop yield under drought stress in semiarid environment. Milk thistle is a medicinal plant cultivated for production of silymarin and oil. Nitric oxide as an active molecule, plays an important role in plant’s reaction to drought stress. In this study, we investigated the role of sodium nitroprusside (SNP) as a nitric oxide donor for tolerance enhancement of drought in two genotypes of milk thistle in a 2‐yr field experiment. With increasing drought severity, photosynthetic rate and stomatal conductance of milk thistle decreased significantly. The exogenous application of SNP significantly compensated for the negative effects of drought on photosynthetic rate by 40%, but also reduced stomatal conductance during 6 d after withholding irrigation. Drought stress significantly decreased pigments content and this affect was compensated by applying SNP in both genotypes. Among yield components, the number of heads per plant showed the highest sensitivity to drought stress followed by seed weight, while all yield components responded significantly to the application of SNP. Withholding irrigation significantly decreased seed yield in both genotypes and years. Averaged over 2 yr, application of 100 µM SNP in Sari ecotype at the stage of stem elongation and anthesis significantly improved seed yield by 38 and 33%, respectively, compared with the non‐application of SNP. However, in the Hungarian cultivar, SNP only led to a significant increase of 29% in seed yield during anthesis stress. Overall, this study suggest that application of SNP at a rate of 100 μM had potential to ameliorate the adverse effect of drought stress on photosynthetic rate and seed yield of milk thistle.

A Mini Review on the Chemistry and Neuroprotective Effects of Silymarin

The plant milk thistle and silymarin has been traditionally used as a natural remedy for the treatment of various ailments including neurological disorders such as Alzheimer's and Parkinson's disease and cerebral ischemia for over 2000 years. In this article we review the neuroprotective effects of silymarin against various neurological dysfunctions. The neuroprotective effects conferred by silymarin include modulation of various antioxidant mechanisms, and several kinases involved in cell signaling pathways, inhibition of the inflammatory response generated during neurodegeneration, neurotropic effects, regulation of neurotransmitters and inhibition of apoptosis. The ease of availability, comparative low cost and safety profile provide additional advantages for the use of this compound as a potent drug with immense clinical benefit. However, there is a growing need for improvements in the bioavailability of silymarin and related products, and more consistent and reliable human trials are required to accurately validate the neuroprotective efficacy of this natural compound. The promising outcomes of the studies mentioned in this review provide renewed insight into the clinical relevance of silymarin in a variety of neurodegenerative disorders where neuroinflammation and oxidative stress are pathologically relevant to disease progression.

Induction of milk thistle (Silybum marianum L. Gaertn) growth and phytochemicals production by natural stimulants

Two field experiments were carried out through 2013/2014 and 2014/2015 seasons in Faculty of Agriculture experimental farm, Tanta University, to investigate the influence of chicken manure (CM) and yeast extract (YE) on Silybum marianum (milk thistle) growth, yield and phytochemical constituents. CM was applied to soil with rates of 0.0, 23.81, 35.71 and 47.62m3/hectare (ha) and YE was applied after 15 and 75days of seed sowing, with rates of 0, 25, 50 and 100g/L as foliar spray. The design of the experiment was as split plot with CM as the main plot. Results indicated that the applied bio-stimulants improved the vegetative traits and seed yield, but without obvious effect on chlorophyll content, though both treatments had resulted in a significant enhancement in PSII photochemical efficiency. Also, both bio-stimulants possessed positive impact on the antioxidant status of seed extracts and silymarin production, as well as the phytochemical constituents. However, these treatments caused a pronounced fluctuation in amino acids profile.

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t-resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated β-cyclodextrins.

The growing demand for t-resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large-scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated β-cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t-resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t-resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non-bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.