Steroidal Saponins Lab Report

2.1-Experimental notes:
2.1.1-Instruments and materials:
1. Sensitive electrical balance: Sartorious /Germany.
2. Oven: Memmert 854 / Germany.
3. Hot plate : NORST ACHTUNG
4. Water bath: MEMMERT
5. Centrifuge:
6. Chiller: Utratemperature 2000,Julabbo F30 , Vision Scientific co., Ltd
7. Rotatory evaporator: Buchi Rotatory evaporator attached to vacuum pump.
8. Ultraviolet light (DESAGA HEIDELBERG) , CAMAG of 254 and 366 nm wave lengths.
9. PH meter.
10. Electro-thermal melting point (Stuart / UK).
11. Thin layer chromatography (TLC).
A-Analytical TLC:
Silica gel GF 60 and silica gel GF 254; layer thickness 0.25 mm ;20 x20 cm aluminium cards ;made by MERCK, Germany.
B-Preparative TLC:
1. Silica gel GF 254 adsorbent, Himedia, India.
2. Preparative

1- Extraction method No. 1:
Fifty grams of powdered aerial parts of portulaca oleracea were hydrolyzed by using reflux for 9 hr. with 300 ml of 2N hydrochloric acid then the extract cool at room temperature ,filter and wash the residue with 2N of ammonia solution. The residue dried overnight at 60ºC ,the final step involve the extraction of the residue with 250 ml of chloroform by using soxhlet ,the final extract cool at room temperature ,then filter and evaporate to dryness by using rotatory evaporator at 40ºC to yield (2.264 gm),as show in scheme (2-1).
2.3.1.2-Extraction method No.2:
Fifty grams of powdered aerial parts of portulaca oleracea extracted by soxhlet with 500ml 0f 70% ethanol for 8 hr. then the extract cool at room temperature ,filter and evaporate to dryness by rotatory evaporator at 60ºC .The dried extract hydrolyzed by reflux with 250 ml of 2N of hydrochloric acid for 3 hr. The final extract cool at room temperature ,filter then partitioning with ethyl acetate three times each one with 50 ml of ethyl acetate ,combine the three lower layer and evaporate to dryness by rotatory evaporator to give the crude extract(12.765 gm) as shown in scheme

• Injection volume: 20μl
• Injection concentration: 1mg /ml
• Detection wavelength: 280nm
2.3.6-Isolation and purification of Flavonoids (Genistien, Rutin, Quercetin) by:
2.3.6.1- Preparative TLC plate:
Isolation and purification of genistein,rutin, quercetin were carried out by preparative TLC.
On a glass plates (20 cm x20 cm) a slurry of 60 gm of silica gel GF 254 suspended in 120 ml of distilled water was applied in 0.75 mm thickness manually by using Jobling laboratory division plate coater. The freshly coated plates were left until the transparency of the layer disappears. After 10 minutes, the plates heated for 1 hour at 110ºC. The completely dried and activated plates are kept in a dry place for use.
The crude extract applied in a row or bands of spots as a concentrated solution by using the capillary tube at least five times on each plate with concerning the drying of each spot before the another application.The solvent system (S5) ( rutin and quercetin) , and S7 (genistein), put in a glass tank (22.5 x22 x7)cm, covered with glass lid and allowed to stand for 45 min.in order to saturate the tank before use. By the aid of Liebermann burchard reagent and UV at 254 nm the bands can detected. The target bands after detection were scratched off the developing plate , collected in dry, clean beaker and mixed with mobile solvent of chloroform- methanol (60:40), then heated with continuous stirring and filtered. After the evaporation of the mobile solvent