How we can extract and isolate steroids form dried plant tissue? Please provide all necessary details i.e. solvent, procedure, instruments etc.
Use petroleum ether / hexane for extracting steroids. Use Soxhlet extraction procedure for extraction. Easy and can be done without any specific instrumentation. Once you get the extracts, check for the presence of steroids via chemical tests. Once confirmed, perform TLC and then can do the analysis via spectrometric and chromatographic techniques as per your convenience.
You can isolate steroids relatively easy, using a common column cromatography, Silica gel and starting with n-hexane and gradually increase the polarity with CHCl3 . Steroids have low-mid polarity so you may see them in Hexane-CHCl3 2:8. Also, they can be easily spotted using sublimated Iodine over the TLC.
Dear Vinesh
Most of the steroids have great solubility in non polar solvent. Try to begin your extraction with non-polar eg. Pet ether. As non -polar solvent also solubilise most of the fatty material from the sourced plant, which interfere the isolation process. For this saponfication of fats is required. Then you work ahead with non-saponified extract for column chromatography with either polarity based eluent or isocratic, as method optimized.
By the following procedure, I have isolated 2 steroidal compounds from C.coromandelicum leaves
Extraction and isolation: The powdered leaves (1kg) were soaked in 3 litres of ethanol for 3 days. The whole mixture was then filtered through filter paper and the filtrate was then evaporated under reduced pressure at (40-50)oC using Rotary Evaporator to provide 130 gm of a gummy concentrate of the crude extract. A portion of the ethanol extract (10 gm) was dissolved in 90% ethanol. It was partitioned with n-hexane, then with chloroform (CHCl3) and finally with ethylacetate . All the extracts were filtered through a cotton plug followed by Whatman filter paper number 1 and then concentrated by using a rotary vacuum evaporator to provide n-hexane (2.5 gm), then with chloroform (CHCl3) (2.0 gm) and finally with ethylacetate (1.0 gm) extractives.
Chromatographic separation: The column was packed with fine TLC grade silica gel (Kiesel gel 60H) was used as the packing material. A column having 40 cm length and 3 cm in diameter was packed with the silica gel (70 gm) up to a height of 23 cm under reduced pressure. The column was washed with n-hexane to facilitate compact packing. The sample was prepared by adsorbing 3.5 gm of n-hexane soluble extract onto silica gel (Kiesel gel 60H, mesh 70-230), allowed to dry and subsequently applied on top of the adsorbent layer. The column was then eluted with n-hexane followed by mixtures of n-hexane and dichloromethane and then dichloromethane and methanol . The polarity was gradually increased by adding increasing proportions of dichloromethane and methanol. A total of 25 fractions were collected each in 100 ml beakers. The Fractions 11 to 13 of the crude n-hexane extract was subjected to Sub-Column Chromatography (Kieselgel 60, mesh 70-230) for further fractionation. The column was eluted with n-hexane, ethylacetate and methanol mixtures of increasing polarities to provide 17 fractions. Fraction-7 was found to yield crystals on the wall of the beaker. The crystals were washed with n-hexane carefully. As a result mother solution was obtained leaving back the needle shape crystals which were isolated as compound 1. The column fractions 12-13 of crude n-hexane extract were bulked together as they showed similar TLC feature. The mixed fraction was found to yield crystals on the wall of the beaker. The crystals were washed with n-hexane carefully. As a result mother solution was obtained leaving back the colorless sharp crystals. These sharp crystals provide the compound 2.
Test for steroid:Salkowski reaction: A few crystals of compounds 1 and 2 were dissolved in chloroform and a few drops of concentrated sulphuric acid were added to the solution, both compounds 1 and 2 formed a reddish color in the upper chloroform layer , indicating presence of steroids.
Liebermann-Burchard reaction: A few crystals of compounds 1 and 2 were dissolved in chloroform and few drops of concentrated sulfuric acid were added to it followed by the addition of 2-3 drops of acetic anhydride. In this case both compounds 1 and 2 turned to violet blue and finally formed green color which indicates the presence of steroids .
Spectroscopic characterization : Different spectroscopic methods were used to elucidate the structure of isolated compounds 1 and 2. Among the spectroscopic techniques IR, 1H and 13C-NMR were carried out.
Use petroleum ether / hexane for extracting steroids. Use Soxhlet extraction procedure for extraction. Easy and can be done without any specific instrumentation. Once you get the extracts, check for the presence of steroids via chemical tests. Once confirmed, perform TLC and then can do the analysis via spectrometric and chromatographic techniques as per your convenience.
Most of steroids exhibit fluorescence character in UV light after treatment with AS, VS or Sulfuric acids.
Dear Vinesh
I hope the attached information is useful to you in future as well for pursuing your research on natural products.
Best wishes.
Dear Vinesh,
In our lab, we have extracted steroids from plant powder using various solvents like petroleum ether, hexane and sometimes solvent like methanol and ethanol too (for extracting steroids like stigmasterol, betasitosterol, etc.). As suggested by Alok Sir, further qualitative determination, isolation and characterization can be carried out using these extracts.
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