Higher antioxidant activity has been measured in ginseng leaves and fruits than in roots, possibly in association with the 4e9-fold higher phenolic contents of fruits and leaves than roots. However, the beneficial effects of these phenolic compound-rich fruits and leaves remain unclear. Short-term hydroponic-cultured ginseng is a widely available, eco-friendly plant that cultivated in pesticide-free indoor farms. Unlike typically grown ginseng, which is usually sold in the form of dried roots, sHCG is harvested as a whole plant, consisting of roots, stems, and leaves. Ginseng roots contain high levels of ginsenosides, whereas the stems and leaves contain high levels of phenolic compounds; therefore, sHCG has a nutritional advantage over ginseng because all parts of the plant are used. Our previous study reported that sHCG, in the form of 1-year-old ginseng seedlings grown hydroponically for 21 days, exhibited higher antioxidant activity than 5-year-old ginseng plants. Contents of total ginsenosides and four ginsenosides were significantly higher in sHCG than in 5-year-old ginseng. Additionally, sHCG has an almost fourfold higher total phenolic content compared to 5-year-old ginseng.
However, the beneficial physiological effects of sHCG are not clear. Several variable factors determine the extractability of saponin, such as temperature, solvents, extraction time, and the liquid to solid ratio. However, it is difficult to determine which factor is most important due to their complex interactions. Therefore, led grow lights response surface methodology has been used to determine optimal extraction conditions in studies attempting to maximize the yield of saponins from medicinal plants and Rg1 and phenolics from RG. However, to date, no study has evaluated the extraction conditions for sHCG and the bio-activity of the extract. In this study, the optimal extraction conditions for sHCG were evaluated using RSM modeling, with the aim of maximizing the yields of bioactive compounds abundant in sHCG but present at lower levels in white ginseng and RG. Additionally, pilot-scale sHCG extracts were produced using the optimal extraction conditions for future commercial production and the effects of the extracts on TNF-a/IFN-ginduced TARC levels in human keratinocytes were evaluated as indicators of AD disease severity.Fifty-one phenolic compounds were extracted from sHCG, WG, and RG, and analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry by reference to a database of mass-fragmentation patterns. The CGA content was markedly higher in sHCG than in WG and RG.
HPLC-UV quantification revealed a CGA content of 12.7 mg/100 g dry sHCG, which was 2.5- and 13.3-fold higher than the contents in WG and RG, respectively . Similarly, Chung et al. reported that of 23 phenolic compounds, CGA and p- and m-coumaric acids were the major phenolic compounds detected, particularly in leaves of ginseng aged 3e6 years. Because only roots of WG and RG were analyzed, the higher content of CGA in sHCG may be attributed to the leaves. CGA is biosynthesized and stored during the early stages of leaf development in other plants, such as coffee trees and perforate St John’s wort. Therefore, it is reasonable to assume that the ginsenoside and CGA contents are higher in sHCG containing leaves and stems cultured in the early growth period than in conventionally grown ginseng and RG. In addition to CGA, 22 ginsenosides of sHCG were profiled in our previous study, and 4 ginsenosides were selected as representative bioactive compounds. These four ginsenosides and CGA are known to be anti-inflammatory, antioxidant and immune enhancing agents. AD is a chronic inflammatory skin disease caused by impaired immune regulation, genetic abnormalities, and hypersensitivity to environmental factors such as allergens. Keratinocytes are epidermal cells that play a critical role in maintaining epidermal barrier function, thus protecting the body from allergens and pathogens. When stimulated by immune triggers , keratinocytes release inflammatory mediators and macrophage-derived chemokines.
TARC/CCL17 and thechemokines promote penetration of inflamed tissue by T cells. In particular, TARC/CCL17, which has high affinity for CCR4þ T cells, is found only in AD lesions; higher levels are associated with more severe AD. To examine the AD-preventing effects of CGA and the four ginsenosides, it was evaluated whether secretion of TNF-a/IFN-ginduced TARC/CCL17 was decreased by the treatment of HaCaT cells with CGA and the four ginsenosides. Treatment of HaCaTs with 2 and 4 mM CGA signifificantly reduced TNF-a/IFN-g-induced production of TARC/CCL17 by 32% and 45%, respectively . Also, Re, Rg1, Rb1, and Rd at 10 mM significantly reduced TNF-a/IFN-ginduced TARC/CCL17 production in HaCaTs by 28%, 38%, 31%, and 22%, respectively .The milk thistle Silybum marianum Gaertn., synonymous Carduus marianus L., is an annual or biennial plant. The plant is native to the Mediterranean region; however, it was naturalized elsewhere in the world . The fruits of this plant accumulate isomeric mixture of flavonolignans in their outer shells. Silibinins, isosilibinins, silydianin and silychristin are the major flavonolignans produced by S. marianum fruits. The standardized extract of the fruits is known as silymarin, which is an important pharmaceutical raw material used for oral treatment of liver disorders. This plant was placed in 2016 at the sixth position among the top-selling herbal dietary supplements in the natural and health food market and the sixteenth position in the mainstream multi-outlet channel market in U.S.A., at about $9.968 million and $17.077 million, respectively .
In consideration of the market requirements for a standardized product with a high content of bioactive principles, several efforts are directed to the setup of suitable growing conditions for stimulation of plant secondary metabolite production.Therefore, the development of an alternative growing system could be an effective tool to overcome the drawbacks linked to open-field cultivation . Hydroponics is a growing system, in which the nutrient elements that are normally found in the soil are dissolved in a proper quantity of the irrigation water supplied to the plants. Hydroponic is also known as ’soilless culture’, because the plants are cultivated in pure nutrient solutions or in artificial growing media instead of the common agricultural soil . With more isolated condition and well-defined composition in this technique, the management of important growing parameters such as surrounding climate or nutrition represents a significant tool for the regulation of secondary metabolism. In particular, a proper change in the composition of the nutrient solution could stimulate the secondary metabolism and favor the accumulation of bioactive compounds inside the tissues.A further major advantage of hydroponics is the possibility to expose the plants to stress factors that can elicit an increase in the concentrations of secondary metabolites .
Among the most remarkable representatives of elicitors is salicylic acid which is a natural plant stress mediator that can highly improve the production of pharmaceutically active compounds in plants . It successfully enhanced silymarin production in S. marianum in vitro cultured cells in MS liquid medium but not previously used in hydroponic culture. Nutrient film technique is one of the hydroponic types in which the plants are housed in net pots that are placed on channels having the nutrient solution. The nutrient solution is pumped though the channels and constantly running along the bottom of the channel and the roots are not completely submerged . Hydroponic culture of medicinal plants aiming at production of important secondary metabolites is rarely reported.Nuclear magnetic resonance spectroscopy can provide useful qualitative and quantitative information in analysis of complex mixtures such as plant extracts. Quantitative proton nuclear magnetic resonance spectroscopy can offer an overview of the sample composition through quantification of multiple metabolites without the need for chromatographic separation. This method, orthogonal to the high-performance liquid chromatography analysis, allows quantification of targeted compounds without the need of reference materials . So, the objectives of the present study were to establish a hydroponic culture protocol for S. marianum as one of the medicinally valuable plant species using nutrient film technique; enhance plant capacity to accumulate flavonolignans using salicylic acid as elicitor and determine the influence of the hydroponic system, vertical grow system as a different cultivation technique and elicitation on seed productivity and silymarin yield which was achieved by comparing the flavonolignan content in different cultures using qHNMR and HPLC techniques.
In addition to, provide a demonstration of the effect of salicylic acid on biosynthetic pathway leading to flavonolignan production.Different salicylic acid treatments were supplied to each single plant and the plants were randomized on one nutrient film apparatus so that each single plant was a biological repetition. Salicylic acid was added at concentrations of 100, 200 and 400 lM, into the nutrient medium when the plant is at BBCH growth stage 75. Nonelicitor treatment was considered as the control culture. The mature fruits of the cultured S. marianum plants were collected at BBCH growth stage 88, 5 d after the elicitor addition. Sampling was performed individually from each single plant and further fruit analysis was performed at single plant level.In the past 40 years, numerous strategies have been developed to improve plant productivity using in vitro culture techniques such as elicitation . However, there are no reports for the use of these strategies in hydroponic system. In the present study, hydroponic culture was successfully established from the seedlings at BBCH growth stage 14 using nutrient film technique system . The results of measuring different factors for flower-head and seed proved that milk thistle productivity significantly affected by hydroponic system and elicitation treatments . Considerable increase in these factors was achieved by elicitation of the hydroponically cultured plant using 200 mM salicylic acid than field-grown plant . The most interesting observations for the hydroponic culture was its ability to produce fast plant growth with high biomass. In addition to, the feasibility to control the growing environment and to change the composition of the nutrient solution, when it was necessary to treat the nutrient solution with elicitors, because the nutrient elements are readily available at the root zone and can be easily taken up by the plants. The flavonolignans contents in the fruits of different cultures of S. marianum were analyzed using both qHNMR and HPLC. Both techniques were reported to be used interchangeably to quantitate flavonolignans in the silymarin complex .Quantitative analysis in NMR depends on the fact that the integrated intensity of a signal due to the analyte nuclei is directly proportional to its molar concentration and to the number of nuclei that give rise to this signal. The direct proportionality of the analytical response and molar concentration is one of the main advantages of qNMR as a method for quantification over other spectroscopic methods. In UV spectroscopy, for example, the concentration is related to the molar absorptivity which is different for each molecule. Therefore, we have to obtain pure reference standard for each compound of interest. In qNMR, a single standard can be used to quantify many components in a mixture, which can be even structurally unrelated to the analytes under investigation, contains the nucleus of interest and has a resonance that does not overlap those of our target analytes. Silibinin was used as a reference standard.
The 1 H NMR data of the compounds under investigation in this study has been previously published . The chemical structures of flavonolignans under investigation in the present study are shown in Fig. 3. The flavonolignan contents in the fruits of hydroponically cultured plant are shown in Table 1. Silychristin, silibinin and isosilibinin were the major flavonolignans detected in the fruits of the cultured plants while silydianin was not detected. estimated the silymarin content in the field-grown fruits collected from Cairo – Alexandria Road between 11.02 and 15.54 mg/g DW in which silychristin and silibinin/ isosilibinin were 2.77–3.17 and 3.03–6.38 mg/g DW respectively. Taking in consideration the variation due to re-cultivation in different habitat in Beni-Suef governorate. Elicitation of the hydroponic culture with salicylic acid at different concentrations enchanced the production of these flavonolignans in the fruits. A dose of 100 mM salicylic acid added to the hydroponic cultures of S. marianum for 5 d increased total silymarin content 1.4-fold higher than the control. The highest content of total silymarin was observed after addition of 200 mM salicylic acid for 5 d. This is considered as 1.7-fold higher than in the control.