Screening some of plants from Silene Genus for 20-Hydroxyecdysone

Received: 04 November 2016 Revised: 24 December 2016 Accepted: 26 January 2017 Abstract: Eight Silene species include S. brachuica, S. guntensis, S. linicola, S. oreina, S. praemixta, S. pseudotites, S. viridiflora and S. wallichiana were screened for the main ecdysteroid 20-hydroxyecdysone by HPLC using the UV spectroscopy. HPLC analyses have shown all Silene plants except S. oreina contain 20hydroxyecdysone, but in different concentration. Studies have shown that S. praemixta and S. viridiflora are rich phytoecdysteroids containing plants and the yields of total ecdysteroids are 2.0% and 1.6%, respectively. The results of investigation species of Silene: S. brachuica, S. praemixta, S. viridiflora, S. guntensis, S. linicola, S. pseudotites and S. wallichiana showed that the yields of 20E of these plants are 0.03, 0.27, 0.35, 0.082, 0.367, 0.071 and 0.08% respectively. The plants of S. linicola, S. praemixta and S. viridiflora suggested to be used for production of ecdysteroids containing preparations.


Introduction
Phytoecdysteroids are contentedly widespread in the plant world.They are isolated from the main types of higher plants -ferns, gymnosperms and angiosperms, but their function in plants are yet studied insufficiently.The application of phytoecdysteroids is a promising alternative to the use of anabolic-androgenic steroids because of the apparent lack of adverse effects.Toxicity is very low, at an LD50 for 20-hydroxyecdysone (20E) of 6.4 g/kg (per os) and 9.0 g/kg (pre oral) [1].20E may be extended to treatments of pathological conditions where anabolic steroids are routinely applied.20E does not bind to the cytosolic steroid receptors, but rather is likely to influence signal transduction pathways, just as the anabolic steroids, possibly via membrane bound receptors.One of the most cited effects of phytoecdysteroid application is the increase of muscle size.
The tonic and anabolic preparations are produced from the plants Rhaponticum carthamoides (Asteraceae), Pfaffia irisinodes (Amaranthaceae), Ajuga turkestanica (Lamiaceae) and Serratula coronata (Asteraceae).The limited availability of the natural resources and relatively low content of 20E in these plants leads to a high cost of these preparations.Regional variations in the concentration levels of 20E range from 2.10-5-3% of dry weight with the average being between 0.001-0.01%.
More than 170 Silene species (family Caryophyllaceae) have been analyzed for their phytoecdysteroid content, and 140 of them were found to be positive and 93 different ecdysteroids have been detected from these plants.Some of them contain a high concentration of 20-hydroxyecdysone, such as Silene otites (almost 1%) and Silene multiflora (1.9%) [2].It established that the promising 20E containing species are plants of genus Silene L. and it is necessary to find novel plants among this genus.In this study we screened content of 20E from aerial parts 8 Silene species plants: S. brachuica Boiss., S. guntensis B. Fedtsch., S. linicola L., S. oreina Schischk, S. praemixta M. Pop., S. pseudotites, S. viridiflora L. and S. wallichiana Klotzsch.

Plant Material
Aerial parts of the Silene species such as S. brachuica Boiss., S. guntensis B. Fedtsch., S. linicola L., S. oreina Schischk, S. praemixta M. Pop., S. pseudotites, S. viridiflora L. and S. wallichiana Klotzsch.were collected in the Surkhan-Darya, Samarkand, Tashkent, Namangan regions and Botanical Garden of Uzbekistan in the summer time during 2011-2013.Aerial parts were collected when the plants were at the flowering stage.The plants were identified at the Department of Herbal Plants, Institute of the Chemistry of Plant Substances, Uzbekistan, by Dr. O.A. Nigmatullaev and voucher specimens were deposited at this Department.

Extraction
The collected plant material was air-dried away from direct sunlight at room temperature and then ground to a fine powder in a Waring blender.After grinding 100 g unit of the powdered plant material was extracted for 24 h in 500 ml of methanol.The extraction solvent were subsequently filtered to remove plant debris and then evaporated to dryness using a rotary vacuum at 40 o C. Solvents methanol, acetonitrile and water were purchased from Sigma (Italy).

HPLC analysis
The contents and quantity of the 20E in the extracts of Silene species were investigated by HPLC.Chromatographic profiles of the extracts were generated using a high performance liquid chromatograph LC-10ATvp connected to a UV-VIS detector SPD-10Avp (Shimadzu Co, Kyoto, Japan).Extracts were diluted to 1 mg/ml, filtered through 0.22 μm and 20 μl were injected.For separation of these extracts, a Nucleosil 100-5 C18 column with a size 250 mm × 4 mm (Macherey-Nagel GmbH & Co, KG) was used.Elution was carried out by a mobile phase consisted of A (water) and solvent B (acetonitrile) and the gradient profile was as follows: from 0% B to 5% B in 8 min, from 5% B to 85% B at 8-30 min, from 95% B to 100% B% at 30-35 min and at 100% B% until 40 min.Flow rate was 1 ml/min and detection was at 242 nm and 200 nm.The quantifications of 20-hydroxyecdysone in the extracts were carried out using a calibration curve of corresponding standards at different concentrations.Authentical phytoecdysteroid 20E was obtained from the Institute of the Chemistry of Plant Substance, Tashkent, Uzbekistan.The purity of the tested compound was >95 %, as determined by HPLC.Standard of 20E was dissolved in MeOH and stored in dark at 4 o C.