Pharmacognosy Lab Lab1,2,3 Comparative determination of glycosides in Senna using different methods of extraction ( Soxhlet, Maceration, & Ultrasonic bath ) Senna s Advantages : I. Used as Stimulant Laxative ( Stimulate or facilitate bowel evacuation ) Used for patients who aren t responsive for milder laxatives Senna s Disadvantages : I. May cause laxative dependence when its used for more than one week I Electrolytes imbalance Abdominal cramps Sennoside A is the dimer of Rhein, Rhein is more toxic and have less activity than sennoside A Senna s Mechanism of Action : Contains sennosides which acts as a stimulant laxative and increase the tone of the smooth muscle contractions within the wall of the colon which enhance the water secretion into the large intestine. In Soxhlet extraction ( Boiling chips ) are used to reduce the pressure inside the apparatus, without the boiling chips the apparatus may explode Ultrasonic Extraction s Advantages : I. High yield I Short time Suitable for heat liable substances Ultrasonic Extraction s Disadvantages : I. Consumes a large amount of solvent Unfriendly environmentally Maceration s Advantages : I. Suitable for heat liable substances Simple and cheap method Maceration s Disadvantages : I. Time consuming method I Consumes a large amount solvent Poor extraction for the substances which have limited solubility in room temperature because in this method heat is avoided. 3 rd Pharmacy Rafalmalik 1
Quizzes/Report Questions Why in Soxhlet extraction there s minimum loss of solvent? Because : I. Only one patch of solvent is recycled I Repetitive extraction cycles which enables large amount of the substance to be extracted + minimum loss of solvent The system is closed system What s the best method to extract heat sensitive crude drugs? Soxhlet extraction because there s no direct contact between heat and the crude drug Mention the hazards/properties of Ethanol? Hazards : I. Irritation and redness of the eye Skin dryness I Respiratory infections IV. Toxic V. Highly flammable Properties : I. Polar, miscible in water Highly flammable I Toxic Mention the scientific name and the family of Senna leaves? Scientific name : Cassia acutifolia, Family : Leguminosae Which method should give the highest yield/lowest yield and why? Soxhlet extraction gives the highest yield while maceration gives the lowest yield, Soxhlet gives the highest yield due to the repetitive extraction cycles which enables larger amount of the substance to be extracted, while maceration gives the lowest yield because heat and agitation are both avoided in this method. Why Ethanol was used as a solvent in this procedure? Because it has a high ability to dissolve the active ingredient and less ability to dissolve the impurities, and it can be easily removed from the active ingredient by using rotatory evaporator, and its available and cheap and less toxic than other organic solvents. 3 rd Pharmacy Rafalmalik 2
Lab 4 Liquid-Liquid Extraction Partition Co-efficient : Is the ratio of the concentration of the solute within the organic layer to the ratio of the concentration of the solute within the aqueous layer. P.C = [ Conc ]org / [ Conc ]aq Factors affecting the partition co-efficient : I. Temperature Particle size of solute & solvent I Concentration of solute Partition Co-efficient Applications : I. Pharmaceutical drugs, knowing the drug hydrophilicity & hydrophobicity which will give us an idea about the drug distribution Purification of essential oils I Inorganic and organic chemistry IV. Agriculture, insecticides, partition co-efficient gives us an idea about the half life of the insecticide, if the conc of insecticide in the organic layer is larger than that in the aqueous layer it means that the half-life of the insecticide is long which is not good because it may remain on the vegetables or fruits when drawing them into the market which may poison the individuals who consume them. that s why its preferred when the conc of the insecticide in the aqueous layer is higher than its in the organic layer. 3 rd Pharmacy Rafalmalik 3
Liquid-Liquid Extraction Procedure Senna Contents Anthraquinone + Glycoside ( Polar Contents ) Lipids + Chlorophyll + Other contents ( Non-Polar Contents, the other contents MOST of them are non-polar ) Organic Layer Add N-Hexane ( will separate the lipids, chlorophyll and the other non-polar contents from Anthraquinone and the glycoside ) Aqueous Layer Aqueous Layer Add HCl ( will break the glycosidic linkage between the glycon part ( sugar ) and the aglycone part ( Anthraquinone ), in another meaning it will separate the polar part from the non-polar part of Sennoside A ) Organic Layer Organic Layer Add Chloroform or petroleum ether ( will break down the bond between the Anthraquinone and convert it to mono-anthraquinone ) Separate the organic layer to obtain the active metabolite ( Anthraquinone ) but still its not very pure because it may contain other polar contents 3 rd Pharmacy Rafalmalik 4
Quizzes/Report Questions What s the difference between chloroform and petroleum ether? which one of them will give the highest yield in sennoside A extraction and why? Chloroform s polarity index is 4.1 while petroleum ether s polarity index is 0.1, so according to polarity index values chloroform can mainly dissolve non-polar substances and small extent of polar substances but petroleum ether can only dissolve non-polar substances. chloroform will give a higher yield than petroleum ether when the most other contents in senna leaves are polar, but petroleum ether will give a higher yield when the most other contents in senna leaves are non-polar or when there s no any other contents beside anthraquinone and glycoside. Why Chloroform always occupy the lower layer in the separatory funnel? Because of its high density, due to the molecular weight of the chlorine atoms and the large intermolecular forces. Why HCl was used after N-Hexane? If HCl was used firstly, it would break the glyosidic linkage between the sugar ( Glucose ) and the anthraquinone, upon adding N-Hexane, the anthraquinone will dissolve with the lipid and chlorophyll and it would be impossible to isolate the anthraquinone with the aqueous layer. Which extraction method gave the highest yield and why? Soxhlet gave the highest yield because of the greater yield ( in the first lab ) of the thin extract which is formed as a result of the repetitive cycles and heat was used. then sonication gave lesser yield than Soxhlet but more than that of maceration because of the high yield ( in the first lab ) which was formed due to agitation by sound frequency. maceration method gave the least yield because in this method heat and agitation weren t used. 3 rd Pharmacy Rafalmalik 5
Lab 5- Stem Distillation Stem Distillation s Principles : I. The liquids should be miscible, if they re immiscible we use liquid-liquid extraction The liquids should have different boiling points Stem-Distillation is used for extraction, purification and identification of unknown compounds depending on their standard boiling point. Stem-Distillation is used to extract volatile oils because it s totally closed system. Hear liable oils are extracted by Expression method, such as ( Orange oil, olive oil, lemon oil ). Expression method procedure : cut the peel ( incision ) then apply a pressure on the peel, wash the peel with water, because the water is immiscible with the volatile oil it would be easy to observe the volatile oil which is separated from the water. Cardamom ( 7el ) Advantages : I. Flavoring agent Carminative Eugenol ( Common name : Clove Oil ) Eugenol belongs to phenylpropanoids ( Family of organic compounds ) Eugenol Uses : I. Used for Toothache, Local Anesthetic I IV. Has Carminative effect With zinc oxide, it can be used as temporary dental filling Counter irritant Eugenol side effects : ( Long term use + Overdose ) I. Hepatotoxicity I Nausea & Vomiting Unconsciousness In this lab, water was used as a solvent because its immiscible in volatile oils, have a high boiling point and no hazards. Eugenol Structure 3 rd Pharmacy Rafalmalik 6
Lab 6- Chromatography Thin layer chromatography s ( TLC ) mechanism is Adsorption Circular Filter paper chromatography s mechanism is Partition In TLC the compounds that have more affinity for the mobile phase will have small Rf value, unlike the compunds that have more affinity for the stationary phase those will have high Rf value. In Circular filter paper, the compounds that can make bonds with the mobile phase will move with the mobile phase and have small Rf value, while the compounds that can make bonds with the stationary phase will remain on the stationary phase for a period of time and give high Rf value. Rf value should not be more than 1 The stationary phase in this lab is silica gel, which contain S-OH in its structure. Oven is used for more efficient separation, because the -OH in the silica gel will make hydrogen bonding with the H20 which exists in the air, when this happens there will be no spots, because the silica won t be able to react with compound to produce spot. Visualization of the spots can be achieve by : I. Uv light which is non destructive Specific coloring agent which is destructive The main difference between preparative TLC and analytical TLC is that in preparative TLC you can detect and collect you re the product that made a spot, but in analytical you can only detect with no any collection. Solvent system composed of 40% chloroform and 30% ethanol and 30% water, if compound C is more polar than compound B but less polar than compound A, the spot of which compound will have the highest Rf value? and why? Compound A, because the solvent system is non-polar. 3 rd Pharmacy Rafalmalik 7
Lab 7- Column Chromatography The mechanism of separation is Adsorption Columns varies in their size according to the amount of the sample, they could be either made from glass or steel, steel column are used when the sample require a pressure to move with the mobile phase. some columns contain glass filter while others aren t, you should prepare a filter by yourself and adding it before the stationary phase. Isocratic solvent system means that the amount of the solvent doesn t change until the end of the process, while Gradient solvent system means that the amount of the solvent changes during the process. The first color indicate that this compound in the mixture/sample has more affinity for the mobile phase while the last color indicate that this compound in the mixture/sample has more affinity for the stationary phase. 3 rd Pharmacy Rafalmalik 8