chapter -1 pharmacognosy bpharm 4th sem cognosy

Pharmacognosy

1. Pharmacognosy & Phytochemistry I BP405T-1 Ms. Sapna kumari Assistant Professor School of Pharmacy

2. Introduction to Pharmacognosy Classification of drugs Quality Control of Drugs of Natural origin CHAPTER-1

3. Definition, History, scope and development of Pharmacognosy A

4. Definition Study of crude drugs obtained from plant, animals and minerals and study of their active constituents. Derived from Latin term Pharmakon - A drug Gignosco - To Acquire knowledge of Pharmacognosy word first used as Pharmacognosis . Term pharmacognosy first used by  John .Adam .Schmidt , A Austrian Physicist and he used the term Pharmacognosist in 1811 in his work Lehrbuch der Materia Medica“. After J. A. Schmidt, C.A Seydler used the term Pharmacognosis in his work Anlactica Pharmacognostica in 1815.

5. WHAT IS PHARMACOGNOSY? Pharmacognosy is the study of crude drugs from natural sources including  information  regarding  History,  Distribution,  Cultivation & collection  Sensory, physical and physicochemical characterization  Structural features (Phytochemical Analysis)  Medicinal uses,  Processing of crude drug for commercial market  Storage  Preservation of drug.

6. AD (ANNO DOMINI) In the year of lord BC (BEFORE CHRIST) BIRTH OF JESUS CHRIST Concept of AD and BC was given by POP DIONYSIUS , he was a Christian. Before the year of Christ

7. History  In India medicinal properties of plants described in  Rigveda and Ayurveda  (3500-1500 B.C.)  Charaka Samhita  – plants  Sushruta   Samhita -surgery  Papyrus Eber  (1500 B.C.)- it is an oldest written document of egypt it contains 876 formulations and 700 crude drugs.  In china medicinal plants had been used since 5000 B.C. Egyptians  ( Ebers papyrus, 1550 BC)

8. History Hippocrates – Father of medicine (460-360 BC) He was a Greek scientist and has described human anatomy and physiology. He has also describe collection, identification, uses, property, of around 300 drugs.

9. Aristotle-  Father of biology(384-322 BC) Student of Pluto & known for his writing on  animal kingdom. Theophrastus  - Father of botany (384-322 BC) Student of Aristotle wrote about  plant kingdom . Galen - Described method of preparations of plant and animal drugs known as “ Galenicals ”

10. History Dioscorides “De Materia Medica ” (600 medicinal plants) Described some medicinal plants like Belladona , Ergot, Opium, Colchicum etc.

11. History Swede linnaeus   –Classified plants and introduced  binomial system  for naming plants. It is a system used to name species Eg - genus-homo, species- sepiens To ensure that scientific name of an organism is same all over the world. Classification further developed by  Bentham & Hooker- Monocotyledon, dicotyledon & gymnosperm. Eicher & Engler. Mendel  – Study on hybrids. Paracelus –  Developed mineral salts. Le’mary - Importance of extraction method and alcohol as extractant . Willium withering  – Published some of medicinal properties of fox glove leaves. Stass & otto - Developed extraction process for alkaloids.

12. Development 1934-1960 Development of modern pharmacognosy with integration of chemistry, biotechnology, analysis, cology etc. Around 75 substance from plant were isolated and structures were determined along with pharmacological studies. 1928 Penicillin Antibiotic 1952 Reserpine Hypotensive and tranquilizer ( drug reduce reduce tension) 1955 Vincrisitin and vinblastin Anticancer Blood and lymph cancer 1960 Diosgenin Preparation of sterodal hormones

13. Development 1960-1970 Antibiotics like Streptomycin, Chloramphenicol , Tetracycline , Gresiofulvin etc isolated so called  antibiotic age . Phytoconstituents from Digitalis Senna Aloe Belladona Ergot were isolated and their clinical used were established

14. Development SAR  Semi synthetic preparations E.g. Ergometrin    Methyl Ergometrin Better oxitocic activity Study of biosynthetic pathways Shikimmic acid & other biosynthetic path way Constituents Acivity Responsible group Reserpine Hypotensive Trimethoxy benzoic acid Tubocurarine Muscle relaxant Quaternary ammonium group

15. Development Natural molecule as model for synthesis of new drug E.g. During world war –II  Quinine  as an antimalarial drug was short in supply so similar type of compound chloroquine was isolated Cocaine  to lignocaine , xylocaine prepared as local analgesic Silicin   to salicylic acid (Aspirin) prepared which is used as Analgesic, Anti thrombolytic and Anti inflammatory agent.

16. Development 1970-1990 Phytoconstituents Plant Name Activity Podophllotoxin (Etoposide, Teniposide) Podophyllum Lung and testicular cancer Silymarin Milk thistle Liver disease Taxol Taxus Ovarian, Breast and lung cancer Guggulosterol Guggul Arthritis, Hypercholesteremia Ginkolide Ginko Increase blood circulation in brain Ginsenoside Ginseng Tonic

17. Development 1990 onwards Herbal drug demand increase due to less toxicity, side effect and cost. To control quality WHO published  guideline  and prepared  monograph. Gov. Of India published  Ayurvedic pharmacopoeia  part I &II. Indian drug manufacturers published 3 volume of  Indian herbal pharmacopoeia. USP 2010- 18 plants IP 2009 - 27 plants TKDL – Traditional knowledge digital library Prepared by india from 2 feb 2009 to protect traditional knowledge. 7 crore TKDL project took 9 year to enlist 2 lakh formulations from Ayurveda Siddha and unani . Available in english , German, Japaniese , french and spanish .

18. Scope Identification  of proper drug by morphology and microscopy. Quality  of herbs known by  evaluation . Good quality of drug obtained by  proper cultivation and harvesting, drying & storing  at proper time. Cultivation  of drug which are in demand and earn by selling it. Active constituents are isolated from plants and can be marked to earn.

19. Pharmacognosy is an important link between Pharmacology and Medicinal chemistry. Crude drugs also provides the intermediate precursor for synthesis of new drug. Better active constituent’s  semisynthetic derivatives  can be prepared. Eg . Ergotamine to Methyl Ergotamine Synthetic molecule  based on plant active constituent provide better activity. Eg . Quinine – Chloroquine  ( Antimalarial activity) Vasicine  – Bromohexine  (Bronchodilator) Scope

20. we can obtained  new medicine  for new diseases like AIDS, Cancer etc. Plant tissue culture and genetic engineering gives more advance natural products. Natural products as  Pharmaceutical aid.  Accacia & tragacanth – Binding, emulsifying & suspending agent.  Carbohydrates- Sweetening agent  Volatile oil – Flavoring agent  Fixed oil- Ointment base, emolient  Starch- Disintigrating agent  Piperine - Bioavaibility enhancer. Scope

21. Sources of drugs B

22. Exploration of medicinal plant in ancient time Trial and error- try and discard until its serves the purpose While searching for food- herbs and species are identified. Signature of nature- similarity between human organ and plant part Zoo pharmacognosy-animal self medication power Accidental discovery- cinchona,penicillin

23. Drugs are substances that are used or intended to be used in the diagnosis, prevention, treatment or cure of diseases. In early times, these substances were derived from natural sources, of which plants took up the major share. With the introduction of technology, most drugs today are manufactured synthetically in the laboratory. The major sources of drugs can be grouped into the following   1. Plant source 2. Animal source 3. Microbial source 4. Mineral Source 5. Marine source 6. Tissue culture Sources of drug

24. Plant Source Plant source is the oldest source of drugs. Most of the drugs in ancient times were derived from plants. Almost all parts of the plants are used i.e. leaves, stem, bark, fruits and roots. Leaves: a. The leaves of Digitalis Purpurea are the source of Digitoxin and Digoxin , which are cardiac glycosides. b. Leaves of Eucalyptus give oil of Eucalyptus, which is important component Sources of drug

25. Plant Source c. Tobacco leaves give nicotine. d. Atropa belladonna gives atropine. Flowers: Poppy papaver somniferum gives morphine ( opoid ) Vinca rosea gives vincristine and vinblastine Rose gives rose water used as tonic. Photo of Papaver somniferum by Evelyn Simak Fruits: Senna pod gives anthracine , which is a purgative (used in constipation) Calabar beans give physostigmine , which is cholinomimetic agent. of cough syrup. Sources of drug

26. Seeds: Seeds of Nux Vomica give strychnine , which is a CNS stimulant. Castor oil seeds give castor oil. Calabar beans give Physostigmine , which is a cholinomimetic drug. Roots: Ipecacuanha root gives Emetine, used to induce vomiting as in accidental poisoning. It also has amoebicidal properties. Rauwolfia serpentina gives reserpine , a hypotensive agent. Reserpine was used for hypertension treatment. syrup. Bark: Cinchona bark gives quinine and quinidine , which are antimalarial drugs. Quinidine also has antiarrythmic properties. Atropa belladonna gives atropine , which is anticholinergic . Hyoscyamus Niger gives Hyosine , which is also anticholinergic .

27. Sources of drug Animal source Drugs obtained from animals sources are whole animals, glandular products (thyroid organ), liver extract, polypeptide venoms, non-peptide toxins, etc. Fish liver oil, musk, beeswax, hormones, enzymes, and antitoxins sera are the products obtained from animal sources ( kokate ). A large number of other natural products from animal sources are used as pharmaceutic excipient and others are used as important drugs or as nutritional supplements. The examples are as given below ( Annamalaiuniversity ): Pancreas is a source of Insulin, used in treatment of Diabetes. Urine of pregnant women gives human chorionic gonadotropin ( hCG ) used for the treatment of infertility. Sheep thyroid is a source of thyroxin, used in hypertension .

28. Sources of drug Cod liver is used as a source of vitamin A and D. Anterior pituitary is a source of pituitary gonadotropins , used in treatment of infertility. Blood of animals is used in preparation of vaccines. Stomach tissue contains pepsin and trypsin , which are digestive juices used in treatment of peptic diseases in the past. Nowadays better drugs have replaced them. cough syrup.

29. Sources of drug Microbial Source Many life-saving drugs are obtained from microbes, Penicillium notatum is a fungus which gives penicillin. Actinobacteria give Streptomycin. Aminoglycosides such as gentamicin and tobramycin are obtained from streptomycis and micromonosporas .

30. Sources of drug Mineral Source Metallic and Non metallic sources. Iron is used in treatment of iron deficiency anemia. Mercurial salts are used in Syphilis. Zinc is used as zinc supplement. Zinc oxide paste is used in wounds and in eczema . Iodine is antiseptic. Iodine supplements are also used. Gold salts are used in the treatment of rheumatoid arthritis. Miscellaneous Sources Fluorine has antiseptic properties. Borax has antiseptic properties as well. Selenium as selenium sulphide is used in anti dandruff shampoos. Petroleum is used in preparation of liquid paraffin

31. Marine Source   . Sources of drug Marine Pharmacognosy is a sub-branch of Pharmacognosy , which is mainly concerned with the naturally occurring substances of medicinal value from marine.   Generally the drugs are obtained from the marine species of bacteria, virus, algae fungi, sponges etc. In the western medicine Agar, Alginic acid, Carrageenan , Protaminesulphate , Spermaceti and Cod & halibut liver oils are the marinemedicinal established products. Macroalgae or seaweeds have been used as crude drugs in the treatment of Iodine deficiency states such as Goiter etc

32. Some seaweeds (algae growing in sea or rock below high water mark) have also been utilized as sources of additional vitamins and in the treatment of Anemia during pregnancy. Marine flora and fauna play significant role as a source of new molecular entity   During the past 30-40 years, Numerous novel compounds have been isolated from marine organisms having biological activities such as antibacterial ,antiviral, antitumour , antiparasitic , anticoagulants, antimicrobial, antiinflammatory and cardiovascular compounds. of cough syrup. Sources of drug

33. Tissue Culture Tissue culture is invitro cultivation of plant cell or tissue under aseptic and controlled environmental condition, in liquid and semi solid well defined nutrient media for production of primary and secondary metabolites or to regenerate whole plant.   Eg . Ten times more production of anthraquinone derivatives from cassia tora (6%) as compared to crude drug. The suspension culture of Dioscorea deltoidea produce up to 1.5 % dry content of diosgenin . The catharanthus roseus cell culture yielded 4 times more ajamalicene and serpentine than the whole plant. (Shah B, 2010) Sources of drug

34. Types of crude drugs 2 types exist depending on this type of classification; Organized drugs Unorganized drugs

35. Organized drugs These are drugs obtained from the direct parts of the plants and containing cellular tissues E.g. Rhizomes, barks, leaves, fruits, entire plants, hairs and fibers

36. Examples of organized drugs Leaves– Digitalis, Eucalyptus, Mint, Senna , Spearmint, Squill , Tulsi , Vasaka , Coca, Buchu , Hyoscyamus , Belladonna, Tea. Barks– Cascara, Cassia, Cinchona, Cinnamon, Quillia . Flowering parts– Clove, Pyrethrum, Chamomile .

37. Fruits– Amla , Anise, Bitter Orange peel, Capsicum, Caraway, Cardamom, Coriander, Dill, Fennel, Lemon peel, Star anise. Seeds– Bitter almond, Black Mustard, Cardamom, Colchicum, Ispaghula ,, Linseed, Nutmeg, Nux vomica . Roots and Rhizomes – Ashwagandha , Colchicum corm, Dioscorea , Garlic, Ginger, Ginseng, Glycyrrhiza . Plants- Ephedra , Ergot, Bacopa . Hairs & fibres : cotton, jute, silk.

38. Unorganized drugs These are drugs which are prepared from plants by some intermediate physical process such as incision, drying or extraction and not containing any cellular plant tissue E.g. Aloe, honey, beeswax, essential oil

39. Differences between organized and unorganized drugs Organized Drugs Unorganized Drugs These may be of plant or animal origin. These may be of plant, animal or mineral origin. These are direct part of plants or animals. These are the product of plant or animals. These have well defined cellular structure. These do not have well defined cellular structure. Generally identified by morphological character. Generally identified by organoleptic properties.

40. Dried latex The latex is a product contained in special secretory tissues of certain plants. It is usually a white aqueous suspension, wherein microscopically small particles of oil globules are suspended. These natural suspensions of milky consistency may contain proteins, sugars, minerals and alkaloid salt in the true solution where gums, starch and resins are in the suspended form. Examples of commercially important lattices are opium, papain , ficin and gutta percha .   Dried Juice   The juices are obtained by fleshy leaves(aloe) or from stems of the tree( kino ). In all cases incisions are made to respective part of the plant and juice coming out is collected and dried. Dried Extract   The extract covered under crude drugs differ from galenical extracts. The extract of pharmacognostic origin consisting of extracting the parts of the plants with water followed by concentration , while pharmaceutical preparations known as extracts are prepared by using alcoholic or hydroalcoholic solutions and adjusting the product to a standard strength. Agar, sodium alginate and catechu are few extracts of plant origin while gelatin is the extract from animal source.

41. Gums Gums are translucent and amorphous substances produced by plants. Gums are usually pathological products and are produced when the plant is growing under unfavourable conditions or is injured. Thus they are the abnormal products of plant metabolism. The gums are produced by the process known as “Gummosis”.   Gums are soluble or partly soluble in water. They are insoluble in alcohol and in most of the organic solvents. They form viscous adhesive solution with water either by swelling or due to absorption. Aqueous solutions of gums are usually leavorotatory . Gums are plant hydrocolloids and may be anionic or non ionic polysaccharides. On hydrolysis(acidic hydrolysis or prolonged boiling with water), gum yield sugar and uronic acids which form salts with calcium and magnesium. The uronic acids are glucuronic acids are glucoronic acid, galactouronic acid or aldobionic acid. Pharmaceutically important gums are gum acacia, tragacanth , gum karaya , gum ghatti and guar gum

42. Mucilages Mucilages are also plant products similar to gums and regarded to be the normal products of plant metabolism. They are produced inside the cells of the plant. Mucilages form slimy masses with water but do not dissolve. Mucilages are esters of sulphuric acid wherein ester group is a polysaccharide complex. Various examples along with methods of formation of mucilages are given below:   From the cell wall of seed epidermis : Isabgol and linceed From endodermis : Fenugreek From leaf epidermis : Senna From bark : Cinnamon, slipper elm From special secretion cells : Squill From algae : Agar and chondrus  

43. Oleo resin When the natural plant resins are accompanied with the volatile oil in homogeneous form are known as oleo resin. Canada balsams and copaiba are suitable example of ole resin – do not compare these with prepared oleo resins which are prepared by percolating drugs containing volatile oil and resin together. The examples of this type are capsicum and podophyllum and ginger oleo resin. Oleo-gum-resin These are the combinations of volatile oil, gum and resins. Sometimes they also contain other substances like enzymes e.g. myrrh and asafoetida . The resins are found in different parts of plant or animal. External glands : Indian hemp In the heart wood of certain woods : guaiacum and red sanders Internal glands of plants : Male ferns Glands on surface of insects : shellac

44. B. Classification of crude drugs CHAPTER-2

45. classification Classification of crude drugs may include; Alphabetical classification Morphological classification Pharmacological classification Chemical classification Taxonomic classification Chemo taxonomic classification

46. Characteristics of an ideal system of classification An ideal method of classification of crude drugs should be; Simple Easy to use Free from confusion

47. 1. Alphabetical classification Alphabetical classification is the simplest way of classification Crude drugs are arranged in alphabetical order of their: Latin names English names Local/ vernacular names

48. The following pharmacopoeia classify crude drugs according to this system: British Pharmacopoeia British Herbal Pharmacopoeia Herbal Pharmacopoeia United States Pharmacopoeia and National Formulary British Pharmaceutical codex European Pharmacopoeia

49. Advantages of alphabetical system of classification It is easy and quick to use There is no repetition of entries and is devoid of confusion. In this system location, tracing and addition of drug entries is easy

50. Disadvantage There is no relationship between previous and successive drug entries E.g. Acacia, Benzoin, Cinchona, Dill, Ergot, Fennel, Gentian, Hyoscyamus, Ipecacuanha, Jalap, Kurchi , Liquorice , Mints, Nuxvomica , Opium, Podophyllum, Quassia, Rauwolfia, Senna, Vasaka , Wool fat, Yellow bees wax. Can not identify source of drug –plant, animal, mineral.

51. 2. Morphological classification In this system, the drugs are arranged according to the plant parts or animal parts. Part of the plant is used as a drug e. g. leaves, roots, stem, fruit, flower, rhizome.

52. ADVANTAGE Convenient for practical study. Helpful in identification & detection of adulteration. Disadvantage: No corelation between chemical constituents and therapeutic action. Repetition of drugs.

53. 3. Pharmacological classification This involves grouping of drug according to their pharmacological action. This is also referred to as therapeutic classification of drugs. Drugs like digitalis, squill and strophanthus having cardiotonic action are grouped together irrespective of their parts used or their phytoconstituents .

54. Pharmacological Action Drugs Anticancer Vinca , Podophyllum , Taxus Anti-inflammatory Colchicum, Turmeric Antiamoebic Ipecac root, Kurchi bark Antiasthmatic Ephedra , Lobelia Anthelminthic Male fern, Quassia wood Antispasmodic Datura , Hyoscyamus Astringent Catechu Analgesic Opium, poppy Bitter tonic Quassia wood, Nux-vomica , Gentian Carminatives Coriander, fennel, clove, peppermint Purgatives Senna , Rhubarb Expectorant Tulsi , Balsam of Tolu Cardiotonic Digitalis, Squill , Strophanthus Tranquilizers Rauwolfia Roots

55. ADVANTAGE This system of classification can be used for suggesting substitutes of drugs if they are not available at a particular place or point of time. DIS ADVANTAGE Drugs having different action on the body gets classified separately in more than one group that causes confusion. E.g. Cinchona is an antimalarial drug because of presence of quinine but can be put under the group of drug affecting heart because of antiarrythymic action of quinidine .

56. Chemical Classification The crude drugs are divided into different groups according to the chemical nature of their relevant constituent. The chemical classification of drugs is dependent upon the grouping of drugs with identical constituents .

57. Chemical classification Carbohydrates Gums - Acacia, Tragacanth , Guargum Mucilages - Plantago seed Others include Starch, Honey, Agar, Pectin, Cotton .

58. 2. Glycosides Anthraquinone Glycosides - Aloe, Cascara, Rhubarb, Senna Saponins Glycosides - Quillaia , Arjuna , Glycyrrhiza Cyanogenetic Glycosides – Bitter almond, wild cherry bark. Isothiocyanate Glycosides - Mustard Cardiac Glycosides - Digitalis, Strophantus , squill Bitter Glycoside- Gentian, quassia , Chirata , Kalmegh .

59. 3. Tannins Tannins are astringent, bitter plant based poly hydroxy phenols that either bind or precipitate proteins. E.g.-pale catechue Black catechue Amla , Behra,Harde , Myrobalan etc.

60. 4. Volatile oils Monoterpenes and sesquiterpenes obtained from plants. Examples- Cinnamon, Fennel, Dill, Caraway, Coriander, Cardamom, Orange peel, Mint, Clove

61. 5. Lipids Fixed oils – Castor, Olive, Almond, Shark liver oil Fats – Theobroma , Lanolin, Lard Waxes – Beeswax, Spermaceti , Wool fat.

62. 6. alkaloids Nitrogenous substances of plant origin Pyridine and Piperidine – Lobelia, Nicotiana Tropane - Coca, Belladonna, Datura , Stramonium, Hyoscyamus, Henbane Quinoline – Cinchona

63. Isoquinoline – Opium, Ipecac, Calumba Indole – Ergot, Rauwolfia Amines – Ephedra Purine bases – Tea, coffee

64. 7. Resin Complex mixture of resinol , resin acids, resitannols , resene etc. Eg . Colophony, podophyllum , jalap, capsicum, turmeric, asafoitida etc.

65. Taxonomic classification d on an accepted system of botanical classification Natural relationship Grouped into: Kingdom- Plant Division-Angiosperm Class- Dicotylendons Order- Tubiflorae Family- Solanaceae Genus- Datura Species- metal Variety- fastuosa

66. Plant kingdom Thallophyta Algae, Fungi Eg . Agar Red algae Pteridophyta Derived name from fern Silent feature of plants Rise in pot as ornamentals eg ,. Male fern Gymnosperm Gymno - naked Sperm-seed Plant body is sporophytes and differentiate into root, stem and leaves. Eg . Ephedra Angiosperm Enclosed seed Dicot Two cotylendons Coriander capsicum Monocot One cotylendons Colchicum Maize

67. Advantages Allows for precise and ordered arrangement of drugs. Accommodates any drug without ambiguity DISADVANTAGES Can not recognize drug is organized or unorganize

68. Chemotaxonomical classification Chemo- nature of active constituents of drug Taxonomy- science deal with identification, nomenclature and classification of plants. It involves study of chemical variation in different type of plant and used of this information in classification of crude drugs. Generally secondary metabolites are studied. e.g.. Tropane alkaloids- solanaceae family Isothiocynates - crucifareae Anthocynidines - red, blue and other colors of flower.

69. Major plant drugs currently used in medicine DRUG PLANT SOURCE USE(S) Vinblastine Catharanthus roseus Anticancer Vinblastine Catharanthus roseus Anticancer Reserpine Rauvolfia serpentina Antipsychoyic , antihypertensive Quinine Cinchona sp. Antimalarial , amoebic dysentery Pilocarpine Pilocarpus jaborandi Antiglucoma Cocaine Lidocaine , procain Erythroxylum coca Topical anesthetic

70. DRUG PLANT SOURCE USE(S) Codeine Morphine Papaver somniferum Antitussive , Narcotic analgesic Atropine Atropa belladonna Spasmolytic Artemisinin Artemesia annua Antimalarial Taxol Taxus baccata , T. brevifolia Breast and ovarian cancer Allicin Allium sativum Antifungal, amoebiasis Podophyllotoxin Etoposide , Teniposide Podophyllum Anticancer

71. Adulteration and Quality control of crude drug c

72. Inferiority is a natural substandard condition (e.g. where a crop is taken whose natural constituent is below the minimum standard for that particular drug) which can be avoided by more careful selection of the plant material. Eg - minimum value of strychnine is 1.15% Less than 1.15% strychnine content - inferior Introduction The term 'adulteration' or debasement of an article covers a number of conditions, which may be deliberate or accidental. Usually in crude drugs, this practice includes substitution of the original crude drugs partially or fully with other substances which is either free from or inferior in therapeutic and chemical properties. DEFINATION- comes from Latin word which means mixing the original drug material with other false material.

73. Spoilage is a substandard condition produced by microbial or other pest infestation, which makes a product unfit for consumption, which can be avoided by careful attention to the drying, and storage conditions. Deterioration D ue to age Less chemical component of Drug,also can be due to high temp., light, improper storage condition, bacterial growth or intentional extraction of the constituents and the sale of the residue as the original drugs. Admixture is the addition of one article to another through accident, ignorance or carelessness e.g. During collection of root and rhizome, stones are collected. inclusion of soil on an underground organ or the co- collection of two similar species.

74. Sophistication intentional addition of other substance in original drug with intent to defraud; such materials are carefully produced and may appear at first sight to be genuine e.g. powder ginger may be diluted with starch with addition of little coloring material ( turmeric ) to give the correct shade of yellow colour. Substitution is the addition of an entirely different subastance in replacement of that which is required e.g. supply of cheap cottonseed oil in place of olive oil. Addition of water in cow milk- adultration Replacement of goat milk in place of cow milk- substitution

75. REASONS OF ADULTERATION N on availability Ignorance and inadequate knowledge of collectors Regional/ linguistic nomenclature Ecotype, genotype, chemo-type variation Making profit

76. Different methods used for adulteration may be grouped as follows: 1.Substitution with Inferior Commercial Varieties Due to morphological resemblance to the authentic drugs, different inferior commercial varieties are used as adulterant which may or may not have any chemical or therapeutic potential as that original natural drug. E.g. Arabian Senna ( Cassia angustifolia ),dog Senna ( Cassia obovata ) and avaram ( Cassia auriculata ) have been used to adulterate Senna ( Cassia senna ) ; TYPES OF ADULTERATION OR SUBSTITUTION OF HERBAL DRUGS: TYPES OF ADULTERATION OR SUBSTITUTION OF HERBAL DRUGS: TYPES OF ADULTERATION OR SUBSTITUTION OF HERBAL DRUGS:

77. Japanese ginger ( Zingiber mioga ), African and cochin ginger to adulterate medicinal ginger ( Zingiber officinale ). Strychnous nux-blanda or S. potatorum in place of S. nux-vomica Capsicum minimum replaced by C. annuum . Gentian substituted by kutki 2 . Adulteration by Artificially Manufactured Substitutes To provide the general form and appearance of various drugs, some materials are artificially manufactured and are used as substitute of the original one Generally this practice is for much costlier drugs.

78. E.g. artificial invert sugar for honey; paraffin wax after yellow coloration substituted for bees wax. Compressed chicory in place in place of coffee Properly cut and shaped baswood for Nutmeg 3. Substitution by Exhausted Drugs Here the same plant material is mixed which is having no active medicinal components as they have already been extracted out. This practice is most common in case of volatile oil containing materials like clove, fennel, coriander, caraway etc., where the dried exhausted material resembles the same like original drug (similarly with drugs like Cascara sagrada and ginger).

79. Sometimes when coloring matters have been extracted or removed during exhaustion, the residue is re-colored with artificial dyes as is done with saffron and red rose petals. Exhausted ginger made bitter with aloes 4.Substitution by Superficially Similar but Cheaper Natural Substances (Inferior drugs) Usually here the adulterated product has no relation with the genuine article, may or may not have any therapeutic or chemical component desired, Due to their morphological resemblance, they are marketed as adultrants .

80. - leaves of species - Ailanthus are substituted for belladonna, senna , mint etc.; -Leaves of Phytolacca and Scopolia for belladona ; -Leaves of Xanthium for stramonium and dandelion for henbane; -Indian dill with European dill or caraway etc. Mother cloves and clovestalks are mixed with clove -Beeswax is substituted by Japan wax. 5. Adulteration by Addition of Worthless Heavy Materials i.e. Harmful adulterants   The wastes from market are collected and admixed with authentic drug. This is particularly noticed for liquids or unorganised drugs. E.g. -A large mass of stone mixed with Liquorice root -pieces of limestone are found in asafoetida -

81. - lead shot has occurred in pieces of opium -Pieces of ambered coloured glass in colophony -White oil in coconut oil -Cocoa butter mixed with stearin or paraffin The addition of rodent faecal matter to cardamom seed is a very harmful adulterant. 6.Addition of Synthetic Principles Sometimes to fortify inferior natural products, synthetic principles are added E.g. adding citral to oil of lemon and orange; benzyl benzoate to balsam of Peru etc.  

82. 7. Usage of Vegetative Matter from the Same Plant This is done by mixing adventitious matters, miniature plants growing alongwith with medicinal plant in excessive amount or parts of plant other than that which constitutes the drugs due to their resembling colour , odour and in some cases constituents. E.g. The lower plants like moss,liver warts and epiphytes growing in bark portion are mixed with Cascara or Cinchona; - stems of buchu are sometimes cut into short lengths and added to the drug. - Stem portions are mixed alongwith leaf drugs like stramonium , lobelia and senna .

83. 8. Adulteration of Powders Powdered forms are frequently found to be adulterated. E.g. -Dextrin in Ipecacuanha -Powdered liquorice or gentian admixed with powdered olive stones -Exhausted ginger powder in powdered colocynth or ginger -Red sanders wood in capsicum The powdered bark is frequently found to be adulterated with brick powder.

84. EFFECT OF USING ADULTERANTS Effect of using adulteration EFFECT OF USING ADULTERANTS EFFECT OF USING ADULTERANTS Adulterated or spurious raw materials may not possess the therapeutically active constituents, or even may be having some toxic compounds that may not only affect the efficacy of the finished product but also cause deleterious effect on human health.   •Reliability of the finished product is at national and international level is affected and betraying the faith of people on the Indian System of Medicine

85. DRUG- EVALUATION Drug Evaluation means conformation of its identity and determination of its quality and purity and detection of nature of adulteration.   It is necessary because 1) Biochemical variation in the drug 2) Deterioration due to treatment and storage 3) Substitution and adulteration, as a result of carelessness, ignorance or fraud. Initially, crude drugs were identified by comparison only with the standard description available. Due to advancement, at present evaluation also includes method of estimating active constituent present in the crude drug, in addition to its morphological and microscopic analysis. Its possible to perform physical evaluation which could be both qualitative and quantitative nature

86. Different techniques involved are 1) Morphological and Organoleptic evaluation 2) Microscopic evaluation 3) Chemical evaluation 4) Physical Evaluation 5) Biological Evaluation Standardization (Drug Evaluation) of Crude drugs

87. MORPHOLOGICAL EVALUATION • Organoleptic evaluation means conclusions drawn from studies resulted due to impressions on organs of senses. Done for unorganized drug. Morphological evaluation means identification of drug by size, shape, specific textute ( done for organized drug) The study of form of crude drug is MORPHOLOGY, while description of the form is MORPHOGRAPHY. E.g. -Fractured surface in cinchona, quillaia , cascara barks and quassia wood

88. Morphological evaluation (SHAPE) Nux vomica- disc shape Sarpgandha –wavy shape (TASTE)Liquorice & stevia – sweet taste ODOUR- MENTHA & CLOVE-AROMATIC (COLOUR) Cardmom & coriander if shade dried- green colour Sun dried- pale green

89. ORGANOLEPTIC EVALUATION Aromatic odor of Umbelliferous fruits - Sweet taste of Liquorice -Brown color of cinnamon -Odor and taste of spice-drugs like, asafoetida , black pepper, nutmeg, caraway, cummin etc

90. MICROSCOPIC EVALUATION Allows more detailed examination of a drug BY USE OF MICROSCOPE • Can be used to identify the organised drugs by their known histological characters (cellular) • Mostly used for qualitative evaluation of organised crude drugs in entire and powdered forms . CHEMOMICROSCOPY: • For the effective results, various reagents or stains can be used to distinguish cellular structure (chemo-microscopy) A drop of phloroglucinol and conc. HCL give red stain with lignin. Mucilage is stained pink with Ruthenium red

91. QUANTITATIVE MICROSCOPIC EVALUATIONS- it involves a specific histological characters. LEAF CONSTANT CONTAINS 5 PARAMETERS STOMATAL NUMBER STOMATAL INDEX VEIN ISLET NUMBER VEIN TERMINATION NUMBER PALISADE RATIO •1. LEAF CONSTANT : Stomatal number and index Why - to identify crude leafy drugs & to distinguish between some closely related species. Evaluation and purity determination of crude leafy drug Eg - Indian senna & Alexandrian senna. i ) Stomatal number – is average number of stomata per sq. mm of the leaf. Example- Indian senna stmatal no. in upper epidermis 220-260, lower epidermis-240-265 Stomatal no. can increase or decrease as age of leaf.

92. ii) Vein islet number – is defined as the number of vein-islets per sq. mm of the leaf surface midway between midrib and the margin. Levin in 1929 determined vein islet no. Name of Plant Range Adhatoda vasica 6-8 Datura stramonium 12-16 Digitalis purpurea 2.5-3.0 Eucalyptus globulus 8-13.5 Ocimum sanctum 19-23 2 ) Stomatal Index – it is the percentage proportion of number of stomata form from the total no. of epidermis cell. Stomatal index are constant. Calculated by using the following equation: S.I. = [S / (E + S) ] X 100 Where, S.I.= Stomatal Index S = No. of stomata per unit area E = No. of epidermal cells in the same unit area

93. Vein islet and vein termination

94. iii) Vein-termination number - is defined as the number of veinlet terminations per sq. mm of the leaf surface midway between midrib and margin.

95. Leaf constants or Diagnostic characters of leaves i )Palisade ratio – is defined as average number of palisade cells beneath each epidermal cell. It can be determined with powdered drugs also. Table: Palisade ratios of various leaf drugs Name of Plant Range Adhatoda vasica 5.5-6.5 Datura stramonium 4.2-6.5 Digitalis purpurea 3.7-4.2 Eucalyptus globules 5.5-6.5 (upper) Ocimum sanctum 02-3.5

96. Palisade ratio

97. STOMATA It is a minute epidermal opening present on aerial parts of the plants , with following characteristics: i ) A central pore ii) Two kidney shaped similar cells containing chloroplasts known as guard cells and varying number of subsidiary (epidermal) cells covering the guard cells. Function of stomata is gaseous exchange and the secondary function is transpiration. It is not essential that each plant must have stomata.   EG-They are also present in stems (Ephedra), flowers (clove), and fruits (fennel). It is generally observed that stomata are abundantly present in dicot leaves.

98.   In some cases, they are present on the upper surface of leaves, while in others on lower surface only (coca and cherry). In some, the stomata are present on both surfaces of the leaves (senna, belladonna, datura etc.). The distribution of stomata between upper and lower epidermis in dicot leaves shows great variation. Types of Stomata •Depending upon the guard cells and arrangement of subsidiary cells, stomata are divided into four types: 1. Moss type 2. Gymnospermous type 3. Gramineous type 4. Dicotyledonous type  

99.  ACCORDING TO ARRANGEMENT OF SUBSIDARY CELLS: i ) Paracytic or Rubiaceous stomata This type of stomata comprises of two guard cells covered by two subsidiary cells, the long axis of which are parallel to that of stoma e.g. coca and senna leaves ii) Diacytic or caryophyllaceous or cross-celled stomata The guard cells are covered by two subsidiary cells, as in case of paracytic stoma, but the arrangement of subsidiary cells on the guard cell is at right angle to that of stoma, e.g. Peppermint, spearmint and Vasaka

100. iii) Anisocytic or cruciferous or unequal celled stomata : The guard cells are covered by three subsidiary cells, of which one is markedly smaller than the other two , e.g. Belladonna, datura and stramonium   iv) Anomocytic or ranunculaceous or irregular celled stomata : Stoma is surrounded by varying number of subsidiary cells resembling other epidermal cells e.g. Digitalis, lobelia, buchu IP recognise one more type of stomata,   v) Actinocytic or radiate celled stomata Two guard cells are surrounded by a circle of radiating subsidiary cells.

101. TRICHOMES Trichomes are the tubular elongated or glandular outgrowth of the epidermal cells . Trichomes are also called as plant hairs. trichomes consists of two parts root and body. Ttrichomes present in most of plant parts and are function less but some times perform secretory function. Depending up on the structure and the number of cells present in trichomes,they are classified in to following. 1.Covering Trichomes or non glandular trichomes: 2.Glandular Trichomes: eg dhatura 3.Hydathode or special Trichomes: Trichomes absent in coca, less in senna leaves.

102. TRICHOMES

103. QUANTITATIVE MICROSCOPY Lycopodium spore method : It is use to determination of percentage of foreign organic matter, mainly use for powdered drugs. Developed by T.E WALLIS. It is used when especially chemical and other methods of evaluation of drugs fails to determine quality. Lycopodium spores are very characterized in shape and appearance and uniform in size (25μm) on avg., 94000 spores present/mg of lycopodium powder. It consists of 1.well defined particles which may be counted. 2.Single layered cells or tissues the area of which may be traced under suitable magnification and actual area calculated 3.The objects of uniform thickness, the length of which can be measured, and actual area calculated.

104. Lycopodium spore method

105. PHYSICAL EVALUATION These are rarely constant for crude drug , but may help in evaluation with reference to crude drug. 1. Moisture content 2. Specific gravity 3. Density 4. Optic rotation 5. Refractive index 6. Melting point 7. Viscosity Moisture content is responsible for the decomposition of crude drug due to chemical change or microbial attack. It is necessary to determine and control the moisture content of crude drug . It is determined by heating the drug at 105°C in an oven to a constant weight. Moisture content of is 5% w/w.

106. 2. Viscosity : Resistance of the fluid to flow . Viscosity of a liquid is constant at given temperature . Hence it is used as a means of standardizing liquid drugs. PYROXYLIN kinematic viscosity is 1100-2450 centistokes . LIQUID PARAFFIN kinematic viscosity is 64 centistokes at 37.8°C.   3. Melting Point : It is one of the parameter to judge the purity of crude drug. In case of pure chemical or phytochemicals , melting point are very sharp and constant . Since the crude drug from animals and plants origin contain the mixed chemicals . Purity of crude drug can be determined by their melting points. Eg . COLOPHONY 75-85°C, BEES WAX 62-65°C, WOOL FAT 34-44°C COCOA BUTTER 30-33°C. Certain drugs fluoresce when the cut surface or the powder is exposed to

107. Certain drugs fluoresce when the cut surface or the powder is exposed to ultraviolet radiation , And it is useful in the identification of those drugs . Indian and Chinese Rhubarb are very difficult to distinguish and is very difficult form , but examination in ultraviolet light gives such marked differences in florescence that the varieties can be easily distinguish from each other .   4. Solubility: The number of ml of solvent required to dissolve 1g of drug . The presence of adulteration in a drug could be indicated by solubility studies. Eg . Balsam of peru is soluble in chloral hydrate solution, Colophony is freely soluble in light petroleum, Asafoetida is soluble in carbon disulphide., Alkaloidal bases are soluble in chloroform.

108. The following are the most important parameter with respect to crude drug.   Ash Value: The residue remaining after incineration is the ash content of the drug (inorganic salts of carbonates , phosphates , silicates of sodium , potassium, calcium and magnesium ) is known as ash content. Ash value is the criterion to judge the identity and purity of crude drug. It is also useful for detecting low grade products, exhausted drug and drugs from earthy matter .   There are three types of Ash value: Total Ash Acid Insoluble Ash Water soluble Ash  

109. The total ash includes both “physiological ash”, which is derived from the plant tissue itself, and “non-physiological” ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the plant surface. Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth. Water-soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water.  

110. Extractive Value It is used to detect either material exhausted by water or not (tea leaves , ginger rhizomes) The extract obtained by exhausting crude drugs with different solvents are approximate measures of their chemical constituents . Various solvents are used according to the type of the constituents to be analyzed . Useful for the evaluation especially when the constituents of the drugs can not be readily estimated by any other means . It also helps to indicate the nature of chemical constituents present in the drug. Also helps in the identification of adulteration of drug . Solubility of drug is also detected . There are three types of extractive value . Water soluble extractive value Alcohol soluble extractive value Ether soluble extractive value.

111. 1. Water soluble Extractive It is applied for the drugs which contain water soluble constituents such as tannins , sugar , plant acids and mucilage. 2. Alcohol soluble Extarctive It is applied for drugs which contain alcohol soluble constituents such as tannins, resins and alkaloids. 3. Ether soluble Extractives : It is applied for the extraction of volatile oils , fixed oils and resins . Volatile ether soluble extractive value Non volatile ether soluble extractive value.   .

112. Foreign Organic matter : The parts of the organ or organs other than those named in the definition and description of the drug are defined as foreign organic matter. The maximum limit for the foreign organic matter is defined in the monograph of crude drug. If it exceedsthe limits, deterioration in quality of the drug takes place.   Swelling factor : Significances : Useful in the evaluation of crude drugs containing mucilage Useful for the detection of purity of the crude drug Determination : 1. Transfer 1 gm of the seeds to a 25ml stoppered cylinder 2. Fill up to the 20ml mark on the cylinder with water. Agitate gently and occasionally during 24 hours and allowed to stand 3.Measure the volume occupied by the swollen seeds. and occasionally during 24 hours and allowed to stand 3.Measure the volume occupied by the swollen seeds.

113. Spectrophotometric methods: i ) UV- Ultra violet /visible spectroscopy ii)IR-Infra Red spectroscopy iii) Fluorescence analysis iv) NMR-nuclear magnetic resonance spectroscopy v) MS-Mass spectroscopy vi) X-ray diffraction vii) RIA-radio immuno assay. Above mentioned all spectrphotometric evaluation techniques are useful for structure elucidation of Phytoconstituents.  

114. Chromatographic techniques: TLC-Thin layer chromatography Principle :Adsorption Adsorbent silica gel G/C coated to a thickness of minutes and used. After development of chromatography spots are revealed by spraying with suitable detecting agent TLC is useful to analyse Alkaloids, Glycosides like all bio- constituents.   The Rf value vary depend on the nature of substance, composition of solvent and impurities. Rf value =    TLC/HPTLC are micro analytical techniques used for determination of natural products Advantages : simple in operation and rapid Chromatographic techniques.

115. CHEMICAL EVALUATION Determination of the active constituent in a drug by chemical tests is referred to as chemical evaluation. The following are various methods of chemical evaluation 1. Instrumental methods 2. Chemical tests 3. Individual constituent chemical tests 4. Micro chemical tests 1. Instrumental methods: They make use of various instruments for evaluation like colorimetry , flourimetry , spectrophotometry etc. 2. Chemical constants tests: These are like acid value, iodine value and ester value etc are used for the identification of fixed oils and fats. 3. Individual chemical tests: These are the tests which are used for identifying particular drugs. 4. Micro-chemical tests: These are the tests which are carried on slides. Example: Euginol in clove oil is precipitated as potassium euginate crystals.

116. Method for chemical evaluation Extract obtained using petroleum ether, chloroform, ethanol and water was prepared using the respective solvent. These extracts along with positive and negative controls were tested for the presence of active phytochemicals viz : tannins, alkaloids, phytosterols , triterpenoids , falvonoids , cardiac glycosides, anthroquinone glycosides, saponins , carbohydrates, proteins, amino acids and fixed oils & fats following standard methods   Tannins 1. Ferric chloride Test: Added a few drops of 5% ferric chloride solution to 2 ml of the test solution. Formation of blue color indicated the presence of hydrolysable tannins.   2. Gelatin Test: Added five drops of 1% gelatin containing 10% sodium chloride to 1 ml of the test solution. Formation of white precipitates confirmed the test.

117. Alkaloids Approximately 50 mg of extract was dissolved in 5 ml of distilled water. Further 2M hydrochloric acid was added until an acid reaction occurred and filtered. The filtrate was tested for the presence of alkaloids as detailed below 1.Dragendorff’s Test : To 2 ml of the filtrate was added 1 ml of Dragendorff’s reagent. Formation of orange or reddish brown precipitate indicated the test as positive. 2.Mayer’s Test : To 1 ml of test solution or filtrate was added a drop or two of the Mayer’s reagent. white or a creamy precipitate confirmed the test as positive. 3.Hager’s Test : To 1 ml of test solution or filtrate, a drop or two of Hager’s reagent formation of yellow precipitate indicated the test as positive. 4.Wagner Test: Two drops of Wagner’s reagent was added to 1ml of the test solution. The formation of yellow or brown precipitate confirmed the test as positive for alkaloids.

118. Phytosterols 1. Liebermann- Burchard’s Test: The extract (2 mg) was dissolved in 2 ml of acetic anhydride, heated to boiling, cooled and then 1 ml of concentrated sulfuric acid was added. A brown ring formation at the junction and the turning of the upper layer to dark green color confirmed the test for the presence of phytosterols .   Triterpenoids Salkowski Test: Approximately 2 mg of dry extract was shaken with 1 ml of chloroform and a few drops of concentrated sulfuric acid were added.A red brown color formed at the interface indicated the test as positive for triterpenoids . Saponins 1.Foam Test: 5 ml of the test solution taken in a test tube was shaken well for five minutes. Formation of stable foam confirmed the test. 2. Olive oil test: - Added a few drops of olive oil to 2ml of the test solution and shaken well. The formation of a soluble emulsion confirmed the test.

119. Flavonoids 1.Shinoda test : A few magnesium turnings and 5 drops of concentrated hydrochloric acid was added drop wise to 1 ml of test solution. A pink, scarlet, crimson red or occasionally green to blue color appeared after few minutes confirmed the test. 2. Alkaline reagent test: Addition of 5 drops of 5% sodium hydroxide to 1 ml of the test solution resulted an increase in the intensity of the yellow color which became colorless on addition of a few drops of 2 M hydrochloric acid which indicated the presence of falvonoids . 3.Lead acetate test : A few drops of 10% lead acetate added to 1ml of the test solution resulted in the formation of yellow precipitate confirmed the presence of falvonoids .

120. Cardiac glycosides 1.Keller -Killiani test : Added 0.4 ml of glacial acetic acid and a few drops of 5% ferric chloride solution to a little of dry extract. Further 0.5 ml of concentrated sulfuric acid was added .The formation of blue color in acetic acid layer confirmed the test.. Carbohydrates 1.Molisch’s test : To 1 ml of test solution added a few drops of 1 % alpha- napthol and 2-3 ml concentrated sulfuric acid. The reddish violet or purple ring formed at the junction of two liquids confirmed the test. 2. Barfoed’s test: 2ml of reagent was added to 2 ml of the test solution, mixed & kept a in boiling water bath for 1 min. Red precipitate formed indicates the presence of monosaccharide's.

121. 3. Seliwanoffs test : To 3 ml of Seliwanoffs reagent was added to 1 ml of the test sample and heated on a water bath for one minute. The formation of rose red color confirmed carbohydrates 4 . Fehlings test : Dissolved 2 mg dry extract in 1 ml of distilled water and added 1ml of Fehling’s(A+B) solution, shooked and heated on a water bath for 10 minutes. The brick red precipitate formed confirmed the test.   Anthraquinone glycosides Hydroxyanthraquinone Test: To 1 ml of the extract, added a few drops of 10% potassium hydroxide solution. The Formation of red color confirmed the test. .

122. Test for proteins: Biuret test : To 2 ml of the test solution added 5 drops of 1% copper sulphate solution and 2 ml of 10% NaOH .Mix thoroughly. Formation of purple or violet color confirmed proteins.   Test for amino acids 1. Millon’s test : Added 5 drops of millons reagent to 1 ml of test solution and heated on a water bath for 10 min, cooled and added 1% sodium nitrite solution. Appearance of red color confirmed the test.   Fats and fixed oils : To 5 drops of the sample was added 1 ml of 1% copper sulphate solution and a few drops of 10% sodium hydroxide. The formation of a clear blue solution confirmed the test.

123. BIOLOGICAL EVALUATION It is employed when the drug cannot be evaluated satisfactorily by chemical and physical methods. In this method, the response produced by the test drug on a living system is compared with that of the stranded preparation. Such an activity is represented in units as International Units (I.U). Dose is termed as International units IU eg . Digitalis 1IU=76mg of standard , Vit -A 1IU=0.344 of standard , Vit -D 1IU=0.025of standard.

124. Indication of Biological Evaluation When the chemical nature of the drug is not known but is has an biological action. When chemical methods are not available. When the quantity of the drug is small and so it cannot be evaluated chemically. Drugs which have different chemical composition but same biological activity. Example: Cardiac glycosides are evaluated by this method on cats, frogs or pigeons.

125. SIGNIFICANCE 1.The method is generally used when standardization is not done satisfactory by chemical or physical methods 2.When the quantity of the drug /sample are very less then the drugs are evaluated by biological methods. 3.These methods are performed on living animals, isolating living organ and tissue, animal preparation, and micro-organism ( Bioassay). METHODS OF STUDIES 1)Toxic----animals are used 2)Symptomatic-----animals are used 3)Tissue-------isolated tissue is used  To estimate potency of drug ,With entire animal or with tissue, To conform therapeutic activity.

126. Evaluation of Hypoglycemic activity deficiency of glucose in the bloodstream . Traditional Diabetic drugs - Momordica charantaka,Fenu greek,Gudmar . Diabetis is induced in animals by Alloxan & Streptazocin . Alloxan cause necrosis of pancreatic islet-B cells which shows 180-250mg/ml fasting blood glucose levels Streptazocin cause formation of streptomycin they produce cytotoxic nitrourcido glucopyranose which cause diabetes ANIMALS USED: Rabbits,Rats,Mice —(4 to 7 days) Dose :rats--80mg/kg ,mice—150mg/kg of streptazocin single oral injection 140-180 mg/kg of alloxan for rabbits at marginal ear vein for 7 days for rats and mice intraperitoneally 2 days Insulin levels are noted by tests like RIA.ELISA. RIA-detects antibody antigen reaction by using radioisotopes. ELISA-detects antibody antigen reaction by using enzyme.

127. Reference WHO guidelines for assessing quality of herbal medicines By World Health Organization THANK YOU

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