Introduction of secondary metabolities.pptx

PHARMACOGNOSY & Phytochemistry-I (BP405T)Unit-IVPart-2INTRODUCTION OF SECONDARY METABOLITE
PRIMARY AND SECONDARY METABOLITE
Alkaloids: GENERAL PROPERTIES OF THE ALKALOIDS CHEMICAL PROPERTIES
CLASSIFICATION OF ALKALOIDS
Biosynthetic Classification:
Pharmacological Classification
Taxonomic Classification
Chemical Classification
Identification test of alkaloids
Mayer’s reagents
Wagner’s reagents
Dragendorff’s reagent
Hager’s reagent
GENERAL METHODS OF EXTRACTION AND ISOLATION OF ALKALOIDS FUNCTIONS OF ALKALOIDS IN PLANT Pharmacological activity and uses
GLYCOSIDES:Physical properties Chemical properties
Classification
On the basis of the type of the sugar or the glycone part
Glycosides are classified on the basis of the pharmacological action
Glycosides are also classified on the basis of linkage between glycone and aglycone part
Identification test of glycosides
ISOLATION-(STAS-OTTO METHOD
TANNINS:Physical Properties
Chemical Properties
Classification of Tannin
Identification test of Tannin

1. PHARMACOGNOSY & Phytochemistry-I (BP405T) Unit-IV Part-2 INTRODUCTION OF SECONDARY METABOLITE Name: Mrs. Pooja Deepak Bhandare Assistant Professor Dadasaheb Balpande College of Pharmacy Besa Nagpur

2. PRIMARY AND SECONDARY METABOLITE PRIMARY METABOLITE Widely distriuted in nature practically in all oraganisms Directly involved in growth, development and reproduction. They are not poisonous. Generally non-expensive to produce ( to isolate from plant and plant product) Example starch froms potato Present in plant large quantity Simple in nature Generally do not have any pharmacological action. Example Carbohydrate, lipids, proteins and amino acid. SECONDARY METABOLITE Biosynthesized derived from primary metabolites more limited distributed in taxanomic plant. eg. Reserpine from Rauwolfia serpentine They do not directly involved in the normal growth, development and reproduction Some of these compounds are poisonous Generally expensive to produce example quinine from cinchona bark Present in plant small quantity complex in nature Have marked pharmacological action on human body responsible for responsible for medicinal value of plant Example Alkaloids, Glycosides, tannins, Volatile oil , Resins and Resins combination

3. Alkaloids Coined by W. Meissner German Pharmacist in 1819. The term derived from the word “ alkali like ” so they have some character similar to naturally occurring complex amines. Defination Alkaloids are the naturally occurring organic products of plant origin, basic in nature and contain one or more n itrogenous atoms normally of heterocyclic in nature, and have marked physiological action when administered internally

4. GENERAL PROPERTIES OF THE ALKALOIDS PHYSICAL PROPERTIES Crystalline solid (some are amorphous soilid example emetin Some alkaloids like nicotine and coniine are liquid and volatile in nature. Pilocarpine ( nonvolatile Colourless , (some are coloured) Some alkaloids are coloured like berberine is yellow and bentanidin is red. Solubility : Free alkaloids bases are fairly soluble in Organic solvent , Non polar solvent and practically insoluble OR sparingly soluble in water. Alkaloids salt are freely soluble in water, less soluble in alcohol and very springily soluble in organic solvent . ( Strychine Hcl .) Isomerization: Optically active isomers isomers may show different physiological activities l- ephedrine > d- ephidrine Most alkaloids are bitter in taste and many are posionus .

5. CHEMICAL PROPERTIES Basic in nature because on lone pair of electron in nitrogen atom. Nitrogen exist as Primary, secondary, terytary and Qutenrnary ammonium compound Basicity : Secondary alkaloids > Primary alkaloids > Tertary alkaloids The normal elements present in the alkaloids are carbon, hydrogen and oxygen but every alkaloid should essential contain at least one nitrogen imparts for basic properties. Chemical Test Mayer’s reagents : Potassium – Mercuric iodide solution gives cream coloured precipitate Wagner’s reagents : Potassium Tri iodide solution gives reddish brown precipitate. Dragendorff’s reagent : Potassium bismuth iodide solution gives reddish brown precipitate or Orange red precipitate. 4. Hager’s reagent : Saturated solution of picric acid gives Yellow coloured precipitate.

6. CLASSIFICATION OF ALKALOIDS Biosynthetic Classification: Pharmacological Classification Taxonomic Classification Chemical Classification

7. Biosynthetic classification Given to the Precursor from which the alkaloids are produced in plant biosynthetically. So all the alkaloids derived from the same precursor can be brought under same group even they have different taxonomic distribution and pharmacological activity. e. g. piperidine alkaloids derived from lysin, pyrrolidin alkaloids derived from ornithin and indole derived from tryptophan. Pharmacological Classification Classification depends upon Pharmacological action e.g. CNS stimulant or depression and anti malarias etc Individual alkaloids may exhibit different action within the same drug for e.g. in cinchona, quinine is an anti malarial where as quinidine is a cardiac depressant. In opium morphine is narcotic analgesic where as codine is antitussive.

8. Taxonomic classification : Classification deals with the ‘ Taxon ’ Like genus, subgenus, species and subspecies etc Large number of alkaloids are classified on their like various family like rubiaceous alkaloids and solanceous alkaloids. Some phytochemist have stepped further and classified alkaloids based on chemotaxonomic classification

9. Chemical Classification Widely accepted classification of alkaloids. The basic of classification is the ring structure (normally heterocyclic ring) present in the alkaloids. Heterocyclic or typical alkaloids : Contain nitrogen in heterocyclic ring Non-heterocyclic or atypical alkaloids: Contain nitrogen in aliphatic chain Heterocyclic or typical alkaloids : a) Mononuclear Type Example Pyrrole &Pyrrolidine Hygrine Stachydrine Pyridine & Piperdine Arecoline Lobeline, Connine Pyrolizidine Sympthiline Aporphine Boldine

10. b) Polynuclear Class Examples 6. Isoquinoline Emetine Papaverine 7. Qunaline Quinine 8. Norlupinane lupanine 9. Quinazoline Vasicine 10. Tropane Atropine, Hyoscine 11. Indole Reserpine vincristine strychine 12. Purine Caffine 13. Steroidal Solnidine Conessine 14. Terpenoids Aconitine Aconine

11. 2.Non-heterocyclic or atypical alkaloids: Contain nitrogen in the aliphatic chain 1. Phenyl ethyl amine (Amino alkaloid) Example : Ephedrine, Mescaline Capsaicin 2. Tropolone Example Colchicine 3. Modified diterpene Example Taxol Classification based on origine True Alkaloids Proto Alkaloids Pseudo Alkaloids

12. True alkaloid Contain Heterocyclic N atom (N in Heterocyclic Ring) Derived directly from amino acid ( tryptophan,tyrosine , phenylalanine, lysine, ornithine, histidine etc) Basic in nature from water soluble salt Example quinine, atropine, morphine Morphine Tryptophan Tyrosine

13. Proto alkaloid Contain N atom but not in the ring system (heterocyclic ring) Derived from Amino acid. Sometimes considered as biological amine. Basic in nature Colchinie , Ephidrine Phenylalanine (amino acid precursor)

14. Pseudo Alkaloid Contain Heterocyclic N atom Donot derived from amino acid ( derived from non-amino acid precursor) Weakly basic nitrogen compound Example: Purine alkaloids: Caffine Steroidal alkaloids : Solanidine , conessine Purine precurssor

15. Identification test of alkaloids 1. Precipitation reaction Mayer’s reagents : Potassium – Mercuric iodide solution gives cream coloured precipitate Wagner’s reagents : Potassium Tri iodide solution gives reddish brown precipitate. Dragendorff’s reagent : Potassium bismuth iodide solution gives reddish brown precipitate or Orange red precipitate. 4. Hager’s reagent : Saturated solution of picric acid gives Yellow coloured precipitate.

16. B . Specific reagent test 1. Vitali Morine test: Alkaloid + Vitali Morine reagent Violet colour (Tropane) (Fuming HNO3 + KoH solution) 2. Van Urk’s test Alkaloid + Parasimethyl amino benzaldehyde Blue colour (Ergot –Indole) (in dil H2SO4 + Traces of Fecl3) 3. Thalleoquin test Cinchona Powder + + Emerald green colour 4.Murexide test Alkaloid sample + KClO3 + HCl Dried resiude + Ammonia Purple colour Bromine water Dilute ammonia solution

17. GENERAL METHODS OF EXTRACTION AND ISOLATION OF ALKALOIDS Methods 1 :- Drug (Powdered) Moisten with H2O extracted with organic solvent Filtrate &treated with lime (Petroleum spirit or ether) . Reject the organic layer Add water Separate Organic layer Shaken with aqueous acid(allow to separate) Aqueous layer (Contains the alkaloids salts.)

18. Methods 2:- Drug (Powdered) Moisten with H2O extracted with alcohol Filtrate &treated with acid or water Add acetone Reject the organic layer Aqueous layer ammonia or sodium (Contains several impurities) (contains alkaloids) bicarbonate

19. FUNCTIONS OF ALKALOIDS IN PLANT They may have a vital role in growth regulatory factor. The alkaloids are poisonous in nature thus they protect the plant grazing animals or insects They act as a reserve substance in plant and supply nitrogen or other elements. They might be the by-product of various detoxification reaction in plants and by this way they cease the formation of harmful substance in plant. They are present in association with plant acid like quinic acid, cinchotannic acid etc. Hence they may provide the means of storing or transportation of such acids.

20. Pharmacological activity and uses Alkaloids are particularly interesting substances because of their multiple pharmacological activities: 1. on the CNS, whether they are depressants (morphine) or stimulants (caffeine); 2. on the autonomic nervous system: sympathomimetics (ephedrine) or sympatholytics (yohimbine, certain ergot alkaloids), parasympathomimetic (pilocarpine), anticholinergics (atropine, hyoscyamine), or ganglioplegics (nicotine). • In addition, alkaloids include local anesthetics (cocaine), agents to treat fibrillation (quinidine), antitumor agents (vinblastine), antimalarials (quinine), antibacterials (berberine), and amebicides (emetine).

21. GLYCOSIDES Definition The organic compounds mainly of plant origin and rarely of animal which on enzymatic or acid hydrolysis yields one or more sugar moieties( Glycon ) and a non sugar moieties (Aglycon or Genin). The sugar ( glycon ) present in glycosides are monosaccharides like glucose, rhamnose and rarely deoxysugars such cymarose found in cardiac glycosides.

22. Nonsugar + sugar = Nonsugar – sugar = glycoside Aglycon: Anthraquinone, steroid, flavonoids, alcohol, phenol, Isothiocynate etc Glycon : Glucose, galactose mannose digitoxoses rhamnose Distribution: Scropholraceae (Digitalis), Leguminosae (seen)a, Licorice ), Gentianaceae (Gentian), Dioscoraceae (Dioscorea) Rutaceae ( citras ) Glycosidic linkage Pharmacologically active

23. Physical properties Crystalline or amorphous, Solid Nonvoltile Solubility Soluble in Polar solvent – water and alcohol Exception : resin glycoside Insoluble in other organic solvent Chloroform ether etc Aglycon solubility soluble in nonpolar solvent (Benzene, ether) Insoluble in water Optically active usually laevorotatory Mostly bitter in taste Except glycyrrhizin stevioside More sugar unit in glycoside leads in more soluble in polar solvent

24. Chemical properties Hydrolysis - Easily hydrolyzed by water, mineral acid, and enzyme Glycoside Aglycon + Glycon Except- C glycoside are stable need oxidative hydrolysis (Aloin) Alkali hydrolysis- Strong and mild alkali hydrolysis ester group. They open and lactone ring cardiac glycoside Enzymtic hydrolysis Emulsin - Hydrolyse β -glycoside Nastose , Invertase- hydrolysis α glycoside Hydrolysis Acid/enzyme

25. Classification On the basis of the type of the sugar or the glycone part e.g. glucoside with glucose, fructoside with fructose and pentosides with pentose etc. Glycosides are classified on the basis of the pharmacological action exhibited by them For e,g Purgative glycosides – Aloe, Senna Cardiac glycosides – Digitalis, Thevetia Diuretics – Gokhru Glycosides are also classified on the basis of linkage between glycone and aglycone part O- glycosides - In these glycosides sugar is connected to OH or phenol group -OH + HO-C6H11O5 → -O- C6H11O5 + H2O Aglycon -O- Glycon Example- Rhubarb and Senna etc C- glycosides – In this type of glycosides sugar is connected to carbon atom CH + HO-C6H11O5 → -C-C6H11O5 + H2O Aglycon -C- Glycon Example- Aloe and Cascara

26. S- glycosides – In this type of glycosides sugar is connected to sulphur of SH group -SH + HO-C6H11O5 → S-C6H11O5 + H2O Example- Sinigrin from black mustard N- glycosides – In this type of glycosides sugar is connected to nitrogen of NH group N-H + HO-C6H11O5 → N-C6H11O5 + H2O Example- Nucleosides examples of N-glycoside

27. Glycosides are classified on the basis of the chemical nature of aglycon part

28. Identification test of glycosides A. Powdered drug sample + Dil H2SO4Cl → Acid extract+ Add NaOH chloride + Add Fehling A & Fehling B → Red ppt B. Powdered drug sample + Water → Aqueous extract+ Add water + Add Fehling A & Fehling B → Red ppt (glycoside present Test A Red ppt > Test B Red ppt 1.Anthraquinone glycoside ( i ) Modified brontrager’s test (C-glycoside) 1 gm of drug sample + 5ml of dilute HCl + 5 ml of ferric chloride →Boil for 10 min. on water bath → cool (on room temperature) →filter → extract of filterate with carbon tetrachloride or benzene + equal amount of ammonia solution →appearance of pink to red colour → presence of anthraquinone moiety. Extract Filter Filter Boil

29. (ii) Borntrager’s test : O-glycoside 1gm of drug sample + 5-10 ml of dilute HCl + 10 min. boil on waterbath and filter + extract of filterate with CCl4 or benzene + equal amount of ammonia solution to filterate + shake → appearance of pink to red colour → indicate presence of anthraquinone moiety. 2. Cardiac glycosides : (iii)Keller – kiliani test : ( digitoxose ). Alcoholic extract of drug + equal volume of water + 0.5 ml of strong lead acetate solution → shake → filter→ filtrate extracted with same amount of CHCl3 → CHCl3 extract evaporated to dry → remainder dissolved in 3 ml of glacial acetic acid + few drops of FeCl3 solution + 2ml of conc. H2SO4→ reddish brown layer → turns to bluish green → indicate the presence of aglycon ( digitoxose ). (ii) Baljet test : Section of drug containing cardiac glycoside ( thick section of leaf of digitalis) →dipped into sodium picrate solution → appearance of yellow to orange colour → indicate the presence of aglycon moiety.

30. iii) Legal Test : Crude drug + pyridine + Alkaline sodium nitroprusside →Blood red colour 3. Saponin glycoside Foam test- Forth formation test Haemolytic test: RBC rupture in the presence of saponin 4. Flavonoids Glycoside ( Hesperidine ) a) Shinoda test → Alcoholic extract + Conc.Hcl + Mg turning → Pink Red 5. Cyanogenetic Glycoside: Amygdalin (Bitter almond) Drug Powdered drug + suspended sodium picrate treated filter paper → Warm (37ºC) → Paper turns reddish 6. CoumarinGlycoside ( Khellin Visnaga ) 1. Fluorescence Test: Sample in test tube cover with alkali moisten filter paper → heat → filter paper observe under UV light → Green fluroscence

31. ISOLATION-(STAS-OTTO METHOD) Powder crude drug Extracted with alcohol and filter it Residue Filtrate ( Tannis+Impurities+Glycosides ) Add lead acetate solution (Excess of lead acetate is precipitated by passing hydrogen sulphide gas) Filter it Tannin and Impurity Filtrate Fractional crystallization Fraction Solubility Chromatography Pure glycoside

32. TANNINS Derived from the French word Tannin as the name indicate they posses the property of tan i.e to convert hide and skin into the leather Natural Polyphenols – Sufficient hydroxyl group and other suitable group (such as carboxyl) to form strong complex with protein and other macromolecule. Term first used by Seguin (1796) Complex non-nitrogenous plant product Astringent property - precipitate protein (Latin astringent means bind fast) Mostly high molecular weight compound water-soluble. DEFINATION:- Tannin is one of the type secondary metabolitie and it is a non-nitrogenous complex polyphenolic organic compound which is having high molecular weight and is water soluble and has astringent property

33. Occurrence and Distribution: Widely distributed in plant kingdom higher plant – Combretaceae (Arjuna, Bahera, Myrobalam ) Leguminosae ( Black catechu, Pterocarpus, Ashoka) Found in seed leaf bark root Present in solution form Cell sap + Distinct vacuole Medicinal use : Due to astringent property. Externally: healing agent- Formation of new tissue on ( wound and inflamed mucosa ) Minor burn inflamed mucosa. Treatment of Varicose, ulcers, hemorrhoids , wounds, Antiseptic Haemoratatic Internally – Diarrhoea Antidote- Heavy metal Poisoning alkaloid

34. Physical Properties Amorphus , non crystalline Molecular weight 500> 20000 Soluble- Water, Alcohol, Dil alkali Sparingly soluble – Ethyl acetate Insoluble – Organic solvent except acetone Form colloidal solution with water Astringent taste Combine with skin/ Hide to form leather Chemical Properties Tannins form precipitate with protein, gelatine, alkaloids Tannin precipitated by salts of Copper, Tin, lead Antioxidant property – Polyhydroxy phenolic compound Astringent property Precipitated by strong potassium or chromic acid solution. Show acidic reaction due to phenol Colour reaction with Iron salt Hydrolysable tannin +Fecl3 → Bluish black colour Condensed tannin +Fecl3 → Brownish green colour

35. Classification of Tannin On the identify of phenolic nuclei linkage True Tannin (high molecular weight) Hydrolysable tannin Hydrolysed by mineral acid of enzyme ( Tannase ) Ester of phenolic acid linked to polyhydric alcohol (β-D-glucose) Two types on the basis phenolic acid 4. Hydrolysable Tannin → Dark Bluish black Pyrogallol 5. Example: Myrobalan, Bahera, Amla, Arjuna Gallotannin Hydrolysis Gallic acid (Rhubarb, clove etc) Ellagitannin Hydrolysis Ellagic acid ( Eucalyptus,Myrobalan ) Dry Distillation FeCl3 Condensed Tannin Resistance to Hydrolysis Derived from Flavonoids (Flavanol,Catechin,Flavan-3-4-diol)- polymer of flavanoids Condensed tannin Enzyme/Mineral acid Phlobaphenes (Red insoluble Compound ) 4. Condensed Tannin → Brownish Green colour Condensed Tannin → Catechol 5. Example : Cocoo Bean, Ptero crpous , Cinchona bark, Black catechu, Pale catechu FeCl3 Dry Distillation Tannin like substance Simple Phenolic compound Do not give positive Gold beater Skin test Respond to most test of tannin Example : Chlorogenic acid- Coffee, Nux vomica, Ipecacuonic acid- Ipecac root Pseudo tannin (Low molecular weight)

36. Identification test of Tannin Goldbeater’s skin test- The Goldbeater’s skin (membrane prepared from the intestine of ox) is soaked with HCl(5min) → Rinsed with distilled water → Place in solution of tannin (5 min) → Wash with distilled water → treat with 1% ferrous sulphate solution → A brown black colour on the skin confirms the presence of tannins. Phenazone test- 10ml of aqueous extract of tannins + sodium Hydrogen phosphate →Heat →Cool → filterate → 2% phenazone solution → Bulky colour precipitated. Gelatin test- Tannin solution + 1% gelatin solution +10% sodium chloride. →White Buff colour precipitate

37. Ferric chloride test- Tannin solution + ferric chloride solution → Blue colour → Hydrolysable tannin Tannin solution + ferric chloride solution → Brownish Green colour → Condensed tannin Match-Stick test- Dip a match stick in plant extract → dried near flame → Moisten it with conc. HCl → warm near the flame → Match stick wood turns → pink or red in colour → which confirms the presence of tannins Chlorogenic acid test → Drug extract + Aqueous ammonia → Green colour Vanillin-HCl acid test → Sample solution + Vanillin HCl reagent (Vanillin 1gm + alcohol 10ml + 10ml Con.c Hcl ) →Pink or red colour ( Pholoroglucinol )

38. Thank you!