Herbal Drug Technology Practical 6th Sem PDF

# Particulars of the Experiments Performed ## Page 1 | Date | Serial No. | Experiment No. | Subject/Experiment | Page No. | Signature | Remarks | |---|---|---|---|---|---|---| | 13/09/2023 | 1 | 1 | To perform the preliminary phytochemical screening of aqueous extract of the given crude drug | 1-1...

# Particulars of the Experiments Performed ## Page 1 | Date | Serial No. | Experiment No. | Subject/Experiment | Page No. | Signature | Remarks | |---|---|---|---|---|---|---| | 13/09/2023 | 1 | 1 | To perform the preliminary phytochemical screening of aqueous extract of the given crude drug | 1-19 | | | | 20/09/2023 | | | | | | | | 27/09/2023 | 2 | 2 | To identify the chemical characters of given excipient (tragacanth) | 21-25 | | | | 11/09/2023 | 3 | 3 | To identify the chemical characters of a given excipient (Starich) | | | | | 18/09/2023 | 4 | 4 | To identify the chemical characters of a given excipient (Homey) | 33-37 | | | | 08/10/2023 | 5 | 5 | To determine the phenol content of the given sample | 39-41 | | | ## Page 2 | Date | Serial No. | Experiment No. | Subject/Experiment | Page No. | Signature | Remarks | |---|---|---|---|---|---|---| | 2/10/2023 | 6 | 6 | To determine the total alkaloid content of given sample | 43-47 | | | ## Preliminary Phytochemical Screening Test - **Aim of the experiment:** To perform the preliminary phytochemical screening of aqueous extract of the given crude drug **References:** - Textbook of Pharmacognosy by Kokate, Purohit A.P and Gokhale S.B - 58th edition page no 3 A.27 - A.27 **Requirements:** **Apparatus required:** - Beaker - Test tubes - Weighing balance - Spatula - Petri dish glass - Porcelain dish - Water bath - conical flask - Measuring cylinder - Glass rod - Measuring cylinder **Chemicals required:** - Dragon drop reagent - Mayer's reagent - Wagner's reagent - Tannic acid - Picrolonic acid - Sodium hydroxide - Fehling's solution A and B - Hagen's reagent - Sulphuric acid - Chloroform - Ammonia - Potassium chloride - Acetic acid - 3,5-Dinitro benzolic acid - Alcohol - Methanolic anhydrous - Magnesium acetate - Acetic anhydride - Hot Methanolic alkali - Pyridine - Sodium carbonate - Copper sulphate & HCl - Zinc dust - Leucon cyanides - Million's reagent - Ninhydrine solution - Ferrous sulphate - Sodium acid phosphate - Phenazone - Sodium Chloride - D-naphthol - Barfoed's reagent - Gelatin - Sodium acetate - Sudan II solution - Tincture Aloina - Phenylhydrazine HCl - Heconceinol - **Theory:** Preliminary phytochemical screening test are done to confirm presence of phytoconstituents also known as secondary metabolites that are present in the crude drug like Alkaloids, Glycoside, Triterpenoids, Steroids, Carbohydrates, Triterpenoid, volatile oil - **This process is mainly done to detect the bioactive principle present in medicinal plant and subsequently may lead to drug discovery and development. - In this experiment we mainly deal with the aqueous extract of the crude drug because of polarity in the crude drug and thus maximum no. of phytochemical constituent can be found out from extract. - The aqueous extract of given supplied drug sample were subjected to qualitative chemical analysin. The various chemical tests were performed on this extract for the identification secondary metabolites present in it as per Text book of pharmacognosy (Willison by Kokate, C.K et. al. **Procedure:** - Take 50g of powder crude drug and macerate it with 500ml of water for 48 hrs. with occasional stirring. - Third occasionally shake for 6 hrs. and allowed it to stand further. - After that filtration is done and the filtrate are evaporate to dryness in a flat bottom - Prep. of test soln.:- Take 500mg of extract and Dissolve it in 500ml of distilled water. Stir the solution in the beaker. is completely dissolve in water. The sample soln. is subjected to various qualitative tests to reveal the presence in absence of contaminating phytochemical constituents. **Phytochemical screening test:** 1. **Alkaloids:** - **Dragendroff’s test:** Alkaloids give reddish brown precipitate with Dragendroff ’s reagent. - **Mayer’s test:** Alkaloids gave cream colour precipitate with Mayer’s reagent - **Wagner’s test:** Alkaloids give reddish brown precipitate with Wagner’s reagent. - **Hager’s test:** Alkaloids gave yellow precipitate with Hager’s reagent. 2. **Glycosides** - **General tests:** - **Test A:** Extracted 200mg of drug with 5ml of dil. H₂SO₄ by warming on a water bath. Filtered the 50 ml. Then the acid extract was neutralized with 5% NaOH, 0.1ml of Fehling’s soln. A and B were added until it became alkaline and heated on water bath for 2 minutes. The quantity of red precipitate formed was noted and compared with that of formed in test B - **Test B:** Extracted 200mg of drug using 5ml of water instead of H₂SO_4. Boiled and salted equal amount of water as was used in above test. Added 0.1ml of Fehling’s soln. A and B until alkaline and heated on water bath for 2 min. The quantity of red precipitate formed was noted. The quantity of precipitate formed in test B was compared with test A. - If the precipitate in test A is greater than test B, then glycoside may be present. - **Chemical tests for specific glycosides:** - **Anthraquinone glycosides:** - Boiled the test material with 1ml of sulphuric acid in a test tube for 5 minutes. 3. **Saponin Glycosides:** - **Froth formation test:** Placed 20ml soln of drug in a test tube then shake well. Stable froth (foam) was formed. - **Hemolysis test:** 0.2ml of soln of saponin added to 0.2ml of blood in normal saline and mixed well. Centrifuged and the red supernatant was noted. Compared with control tube containing 0.2 ml of 10% blood in normal saline diluted with 0.2 ml of normal saline. 4. **Amino Acid** - **Millon’s test:** To the test soln. added about 2ml of million’s reagent/ White precipitate indicates present of amino acid. - **Ninhydrine test:** To the test soln. added ninhydrine soln. and boiled. Violet colour indicated the presence of amino acid. 5. **Carbohydrates** - **Molisch’s test:** To the test soln., added few drops of alcholic a-naphthol, then add few drops of conc.H₂SO₄ through sides of test tube. Purple to violet colour appears at the junction. - **Barfoed’s test:** 1ml of test soln. was treated with 1ml of Barfoed’s reagent on water bath. Red Cupric oxide was formed, monosaccharide. - **Selivanoff’s test:** To the test soln. add equivalent volm. of crystals of resorcinol and conc. HCl and heat on a water bath. No colour was produced. 6. **Tannins** - **Goldbeater’s skin test:** Added 2% HCl to a small piece of gold beater’s skin. Rinsed it with distilled water and place in the soln. to be tested for 5 minutes. Then given wash of distilled water and transfer to a 1% Fe soln. Brown to black colour on the skin indicated presence of tannins. - **Ferric chloride test:** The extract was treated with FeCl₃. Blue colour appeared when hydrolysable tannins were present and greenish when condensed tannins were present. - **Phenazone test:** Added about 0.6gm of sodium acid phosphate to 5ml of aqueous extract. Warmed it and filtered. To the filtrate added 2% phenazone soln. Bulky precipitate was formed. 7. **Flavonoids** - **Shinoda test:** To the test soln. added few drops of magnesium tunnings and conc. HCl. Drops wise & pink-carlet appeared - **Alkaline reagent test:** To the test soln. added few drops of NaOH soln. Intense yellow colour in formed which turned to colourless on addition of few drops of dil. acid. - **Zinc HCl test:** To the test soln. added a mixture of zinc dust and conc. HCl. Red colour appeared after few minutes 8. **Observation Table** | Sample | Alkaloid | Glycosides | Amino acid | Carbohydrates | Tannin | Flavonoids | Volatile oil | Steroid and Terpenoid | |---|---|---|---|---|---|---|---|---|---| | Fennel | -ve | -ve | -ve | +ve | -ve | +ve | +ve | -ve | | Coriander | +ve | +ve | -ve | +ve | +ve | +ve | +ve | +ve| | Clove | -ve | -ve | -ve | +ve | +ve | +ve | +ve | +ve | | **N.B** | +ve - Present | -ve - Absent| | | | | | | ## Observations ### Page 3 **General Tests:** - **Test A:** Extracted 200mg of drug with 5ml of dil. Hysoy by warming on a water bath. Filtered the 50 ml. Then the acid extract was neutralized with 5% NaOH, 0.1ml of Fehling’s soln. A and B were added until it became alkaline and heated on water bath for 2 minutes. The quantity of red precipitate formed was noted and compared with that of formed in test B - **Test B:** Extracted 200mg of drug using 5ml of water instead of H₂SO_4. Boiled and salted equal amount of water as was used in above test. Added 0.1ml of Fehling’s soln. A and B until alkaline and heated on water bath for 2 min. The quantity of red precipitate formed was noted. The quantity of precipitate formed in test B was compared with test A. - If the precipitate in test A is greater than test B, then glycoside may be present. ### Page 5 - **Alkaloids:** - **Dragendroff's test:** Alkaloids gave reddish brown precipitate with Dragendroff’s reagent. - **Mayer’s test:** Alkaloids gave cream colour precipitate with Mayer’s reagent - **Wagner’s test:** Alkaloids give reddish brown precipitate with Wagner’s reagent. - **Hager’s test:** Alkaloids gave yellow precipitate with Hager’s reagent. ### Page 6 **Gluconides:** - **General tests:** - **Test A:** Extracted 200mg of drug with 5ml of dil. H₂SO₄ by warming on a water bath. Filtered the 50 ml. Then the acid extract was neutralized with 5% NaOH, 0.1ml of Fehling’s soln. A and B were added until it became alkaline and heated on water bath for 2 minutes. The quantity of red precipitate formed was noted and compared with that of formed in test B - **Test B:** Extracted 200mg of drug using 5ml of water instead of H₂SO_4. Boiled and salted equal amount of water as was used in above test. Added 0.1ml of Fehling’s soln. A and B until alkaline and heated on water bath for 2 min. The quantity of red precipitate formed was noted. The quantity of precipitate formed in test B was compared with test A. - If the precipitate in test A is greater than test B, then glycoside may be present. - **Chemical tentus for specific glycosider:** - **Anthraquinone Glycosides: ** - Boiled the test material with 1ml of sulphuric acid in a test tube for 5 minutes. ### Page 7 - **Saponin Glycosides:** - **Froth formation test:** Placed 20ml soln of drug in a test tube then shake well. Stable froth (foam) was formed. - **Hemolysis test:** 0.2ml of soln of saponin added to 0.2ml of blood in normal saline and mixed well. Centrifuged and the red supernatant was noted. Compared with control tube containing 0.2 ml of 10% blood in normal saline diluted with 0.2 ml of normal saline. - **Cardiac glycosides (Cardinalides):** - **Kellen – Killiani test (test for deoxy sugar):** - Extract the drug with chloroform and evaporated it to dryness. Add .4ml of glacial acetic acid containing trace amount of .5ml of conc. H₂ SO₄ to a small test tube, add carefully 0.5ml of conc. H₂ SO₄ by the side of the test tube. Acetic a layers shows blue colour. - **Raymond’s test:** Treated the soln. with hot metallic alkali Violet Colour appeared. - **Libermann Burchhard’s test:** Extract of powdered drug when treated with acidic anhydride and conc. H₂SO₄, bluish green colour was produced. ### Page 8 **Amino acids:** - **Million’s test:** To the test soln. added about 2ml of million’s reagent/ White precipitate indicates present of amino acid. - **Ninhydrine test:** To the test soln. added ninhydrine soln. and boiled. Violet colour indicated the presence of amino acid. ### Page 9 **Carbohydrates:** - **Holisch’s test:** To the test soln., added few drops of alcholic a-naphthol, then add few drops of conc.H₂SO₄ through sides of test tube. Purple to violet colour appears at the junction. - **Barfoed’s test:** 1ml ของ test soln. was treated with 1ml of Barfoed’s reagent on water bath. Red Cupric oxide was formed, monosaccharide. - **Selivanoff’s test:** To the test soln. add equivalent volm. of crystals of resorcinol and conc. HCl and heat on a water bath. No colour was produced. **Tannins** - **Goldbeater’s skin test:** Added 2% HCl to a small piece of gold beater’s skin. Rinsed it with distilled water and place in the soln. to be tested for 5 minutes. Then given wash of distilled water and transfer to a 1% Fe soln. Brown to black colour on the skin indicated presence of tannins. - **Ferric chloride test:** The extract was treated with FeCl₃. Blue colour appeared when hydrolysable tannins were present and greenish when condensed tannins were present. - **Phenazone test:** Added about 0.6gm of sodium acid phosphate to 5ml of aqueous extract. Warmed it and filtered. To the filtrate added 2% phenazone soln. Bulky precipitate was formed. ### Page 10 **Flavonoids** - **Shinoda test:** To the test soln. added few drops of magnesium tunnings and conc. HCl. Drops wise & pink-carlet appeared - **Alkaline reagent test:** To the test soln. added few drops of NaOH soln. Intense yellow colour in formed which turned to colourless on addition of few drops of dil. acid. - **Zinc HCl test:** To the test soln. added a mixture of zinc dust and conc. HCl. Red colour appeared after few minutes ### Page 11 **Observation Table** | Sample | Alkaloid | Glycosides | Amino acid | Carbohydrates | Tannin | Flavonoids | Volatile oil | Steroid and Terpenoid | |---|---|---|---|---|---|---|---|---|---| | Fennel | -ve | -ve | -ve | +ve | -ve | +ve | +ve | -ve | | Coriander | +ve | +ve | -ve | +ve | +ve | +ve | +ve | +ve| | Clove | -ve | -ve | -ve | +ve | +ve | +ve | +ve | +ve | ### Page 12 **Volatile oil:** - **Test a:** To a thin section of drug added Sudan III. Red coloured obtained - **Test b:** To a thin section of drug added few drops of tincture alkana, red colour indicated the presence of volatile oil **Stenoids and triterpenoids:** - **Libermanan Burchard test:** Treat the extract with few drops of acetic anhydride, boil and cool. Then add conc. H₂SO₄ from the side of the test tube. Brown ring was formed at the junction of two layers. Showa upper layers turn green which indicates presence of Steroid and formation of deep red colour indicates presence of triterpenoids. - **Salkowahi test:** Treat the extract with few drops of conc. H₂SO_4. Red colour at lower layer indicates presence of steroid and formation of yellow coloured lower layer indicates presence of triterpenoids. ### Page 13 - **Report:** After the chemical test for fennel, it was found that alkaloid, glycoside, amino a, Tannin and steroids were absent and carbohydrate (Volatile oil and flavonoid were present. - **reference:** Badgujar, Sham Kant. B. Vaimou, Patel and Atmaram H., "Feniculum Vulgare." *A review of its Botany, Phytochemistry, Pharmacology, and toxicology*, *Biomed. Res. Int*. 2014, *1*, *1-32*. ### Page 14 - **Aim of the experiment:** To identify the chemical characters of a given excipient (Tragacanth) - **Reference:** *Practical Pharmacognosy* by Kokate C.K. 5th edition, Vallabh Prakashan. Reprint 2018, page no. = 98-99. - **Requirements:** - **Apparatus and glass ware needed:** Test tube, measuring cylinder, water bath, weighing balance, beaker, etc. - **Chemicals required:** HCl , Fehling’s solm., Barium chloride, sodium hydroxide , lead acetate, Ruthenium red, iodine , etc. - **Theory:** Tragacanth is a natural gum. It is primarily used in the food industry as a thickening and gelling agent. - **Biological source:** A dried exudation obtained from the stems and branches of *Astragalus gummifer* belong to family Leguminosae. - **Description:** - **Color**: White to slightly yellow colour. - **Odour**: Odourless - **Taste**: Mucilaginous taste - **Shape:** The gum keeps from the plant in twisted ribbons on flakes that can be powdered. - **Solubility:** 1 gm of the sample in 50 ml of water swells to form a smooth, stiff, opalescent mucilage. It is soluble in ethanol but does not swell in 60.1 (w/v) aq. ethanol. - **Chemical constituents:** Tragacanthin (water soluble part), Bassonin (water insoluble part) - **Uses:** Demulcent, emollient, thickening agent, emulsifying agent, binding agent. - **Chemical Tests:** | Tests | Observation | Inference | |---|---|---| | 1. To 4ml of 0. 5-1 w/v soln. added 0.5ml of HCl and heated 30 minutes on a water bath. Divided the liquid into two parts. | | | | a) To the first part added 1.5ml of NaOH soln and Fehling’s solm. and Warimed in water bath. | Red precipitate was produced | Tragacanth was present | | b) To the second part, added Backy (10%) soln. | No precipitate was obtained (distinction from agar.) | Tragacanth was present. | | 2. To 0.51.w/vsoln. of the gum,added 20% w/v som. of lead acetate | A voluminous flocculent precipitate was obtained (distinction from acacia) | Tragacanth was present. | | 3. Mounted a small quantity of powder in nuthenium net and examined microscopically. | Particles did not acquired pink colour (distinction from Indian tragacanth) | Tragacanth was present. | | 4. To 0.1g of powder added ON/50 Iodine | The mixture acquired an olive green colour | Tragacanth was present. | - **Report:** From the above morphological characters and chemical tests the given excipient was identified as Tragacanth. ### Page 15 - **Aim of the experiment:** To identify the chemical characters of a given excipient (starch). - **Reference:** *Herbal Drug Technology* by Agarwal S.S. 2^nd edition, Universities Press (India) Pvt. Ltd, Page no. = 102. - **Requirements:** -**Apparatus required:** Test tube, measuring cylinder, weighing balance, beaker, conical flask, etc. - **Chemicals required:** HCl, iodine, NaOH, Fehling’s solm. etc. - **Theory:** Starch consists of polysaccharide granules obtained from the grains of maize *Zea mays* or wheat *Triticum sativum*, belonging to family Gramincae - on the tubers of the potato *Solanum tuberosum* belonging to family Solanaceae. - **Description:** - **Color:** White (Rice and maize starch), creamy (wheat), slight yellowish (potato) - **Odour:** Odourless - **Taste:** Mucilaginous taste - **Shape:** Fine powder or innegular, angular masses. - **Solubility:** It is sparingly soluble in cold water and mostly soluble in hot water. After cooling it forms gel. - **Chemical constituents:** Amylose, amylopectin. -**Uses:** Dusting powder, pharmaceutical aid, protective and demulcent, tablet disintegrating agent and diluents. - **Chemical Tests:** | Tests | Observation | Inference | |---|---|---| | 1. Iodine test:- Mount a few starch granules in water, added a drop of iodine. | Starch granules show bluish colouration when examined microscopically. | Starch was present. | | 2. Starch mucilage:- Make a suspension of 0.5g of starch with about 5 ml of water. | | | | 3. Fehling’s soln. + Starch mucilage + heat | | | | 4. Starch mucilage + 0.5ml HCl + Heat (30mins). After 30 mins. added NaOH until the mixture is alkaline to litmus and warmed for few minutes. | | | | 5. 5ml of starch mucilage + 4 drops of iodine water. | | | - **Report:** From the above morphological characters and chemical test the given excipient was identified as Starch. ### Page 16 - **Aim of the experiment:** To identify the chemical characters of a given excipient (Honey) - **Reference:** *Herbal Drug Technology* by Agrawal S.S. 2nd edition, Univerisitics Press (India) Pvt. Ltd, Page no. = 228 - **Requirements:** - **Apparatus and glass wares needed:** Test tube, china dish, measuring cylinder, etc. - **Chemicals required:** Ether, alptra Napthol, conc. H₂SO₄, Fehling’s solm. A and B, HCl, Resorcinol. - **Theory:** Honey is the saccharine liquid prepares from the nectar of the flowers by the honey bee *Apis mellifera*, *Apis densata* and other species of Apis belonging to the family Apidas. - Honey is a golden elixir (melted by bees and in more than just a delectable sweetmen; it’s natural maruel with a rich tapestry of flavours and health benefits. Honey’s composition is a complex is a complex blend of sugars enzymes in form acids, vitamin and minerals. - Its unique chemical composition gives honey antimicrobial properties, making it a natural preservative and a remedy for various aliments. From soothing sore to aiding wound healing, honey has been a go-to traditional medicine for centuries. - **Description:** - **Color:** Light yellow to brown yellow - **Odour:** Pleasant - **Taste:** Sweet - **Solubility:** Soluble in H₂O and insoluble in alcohol. - **Chemical constituents:** Glucose 35% (±3%), Fructose 45% (±5%), Sucrose (2-3%), and water (14-20%), Dextrin, maltose, gums, traces of succinic acid, acetic acid, volatile oil, amino acid, protein, colouring matter, etc. - **Uses:** - It is used as an emollient, nutritive, mild laxative, sweetening agent. - It is used as an important component of linctures and cough mixture. - It is used as antiseptic and bactericidal. - It is used as a vehicle in ayurvedic and unani prep. - **Chemical test:** | Tests | Observation | Inference | |---|---|---| | 1. Ficke’s test: Take about 3 gm of honey +2ml of ether and shaked throwghly and allowed the two layers to separate and evaporate to drymen | Transient pink colour was observed | Pure honey was present. | | 2. **Molinchin test:** Honey is treated with alpha Napthal and conc. H₂SO₄ | Purple colour was observed | Carbohydrate was present. | | 3. **Reducing sugar test:** Heated honey boiled 1 drop of mixture of Fehling’s soln. A and B | Brick red colour of Cupric oxide | Monosaccharide was present. | - **Report:** From the above morphological characters and chemical tests the given excipient was found to be Honey. ### Page 17 - **Aim of the experiment:** To determine the phenol content of the given sample - **Reference:** William C. Evans, *Trease and Evans’ Pharmacognosy*, 16th edition / Elsevier, 2009, page no. = 29. - **Requirements:** - **Apparatus and glassware needed:** Volumetric flask, measuring cylinder, beaker, water bath, heating mantle etc. - **Chemicals required:** Cinnamon oil, KOH soln., sulphurica, distilled H₂O etc. - **Theory:** Phenolic compounds are important plant constituents with redox properties responsible for anti-oxidant activity. The phenol content of the given extract is determined by using alkaline medium. The determination of phenol content depends on the fact that phenolic compounds containing volatile oil are reduced in volm. when shaken with KOH due to solubility of phenols in alkali but non-phenolic portion of oil remains undisolved in KOH soln. The amount of phenol content can be determined by difference in volm. - **Procedure:** - In a volumetric flask added 75ml of KOH soln. - Now, added 10ml (cinnamon oil) in the same volumetric flask. - The solution was shaken properly so that both are completely mixed and the shaking was done for 5 minutes. - Kept the volumetric flask on boiling water bath for 3-5 minutes shaken the volumetric flask 3 times during the heating procedure. - Both the oil layer and KOH layer started to separate out. - Now, added sufficient quantity of KOH soln. to separate oil layer. - Kept the soln. overnight for 24 hours and then measure the volume of oil. - Aftern 24 hours, filter it and measured the obtained filtrate. Heated the filtrate in heating mantle. - Powder is obtained. Measure the total weight of the powder and calculate the total phenol content. - **Report:** Determination of total phenol content of given sample (Cinnamon oil) was done and the phenol content was found to be 45.8%. ### Page 18 - **Aim of the experiment:** To determine the total alkaloid content of given sample - **Reference:** *Practical Pharmacognory* by Kokate C.K., 5th edition, Vallabh Prakashan, Reprint 2019, page no. = 173. - **Requirements:** - **Chemicals required:** Sample extract, 1N H₂SO₄, chloroform, strong ammonia soln, Mayer’s reagent, NaCl, methyl red indicator, HCl etc. - **Apparatus required:** Separating funnel, beaker, measuring cylinder, flask, - **Theory:** Alkaloids are basic nitrogen containing compounds obtained from plants, animals and micro-organinm having manked physiological action. Alkaloids have diverse and important physiological effects on human and other animals. The term “alkaloid” is derived from the word “al -qali”. And they have some of the characteristic of natural amino a. The definition of alkaloid in the organic compound from natural or synthetic origin. Which are basic in nature and contains one or more nitrogen atom normally in heterocyclic ring and possess physiological action on human and animal body when used therapeutically. - **Alkaloid present in cinchona bark still play ** an important role in medicine for example anti-malaria and anti - arrhythmic drug. Six respective derivatives. ### Page 19 - **Calculation:** - Total volm. obtained after titration with 0.1 N NaOH = 4.5 ml - For blank: - - Total volm. obtained after titration against 0.1N NaOH = 6.5 ml - Therefore, blank sample = (6.5 - 4.5) ml = 2 ml - 1 ml of 0.1N HCl = 0.1629 g of alkaloid equivalent - 2 ml of 0.1N HCl = (0.01629 x 2) g of alkaloid = 0.03258 g of alkaloid = 32.58mg of alkaloid ### Page 20 - **Clidhydroquinine, dihydroquinine, quainidne, quunine, cindenine and cinchoardine has been qualified* in cruda plant extract. - Total alkaloids are determined volumetrically by acid bas titration and calculated as quinine. - **Procedura:** 1. If 10 ml of sample extract was taken into a separating funnel and 1N H₂SO₄ (10ml) and 10 ml water was added. 2. Then it was shaken the mixture. Then the chloroform and allowed to reprate the mixture. Then the chloroform layer was discarded. 3. The acid wash was transferred to the motion ligar and was basified with 5ml of strong ammonia soln. 4. Then it was shaken with successive portions of chloroform (30, 20, and 10 ml). The task for completion of extraction was done by Mayern neagent. 5. The combine chloroform extract was washed with 10ml of water. Then the extract was transfered to a distillation flask to remove the solvent on a boiling water bath. 6. 5ml of alchohol was added to the residue and evaporated the alcohol on a water bath. 7. The residue was dimolved in 2ml of chloroform and 10ml of standand N/10 HCl. Then it was heated on a water bath to remove the chlorofenm and was back titrated the excess acid against N/10 NaOH wing 3-5 drops of methyl red as indicator. - **Repont:** The total alkaloid content in the given sample was found to be 32. 58mg.