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EVALUATION OF CRUDE DRUGS

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Evaluation of drug means confirmation of its identity and determination of its quality and purity and detection of nature of adulteration.Evaluation of herbal drug is an important tool in the formulation of high quality herbal products. Quality of herb is
depends upon on many factors like cultivation, collection, drying, storage, processing for market etc. Now a day’s
substitution and adulteration of herb is very common due to scarcity of drug and its high price prevailing in the
market. Owing to medicinal properties attributed to an herb, it is necessary to maintain its quality and purity in the
commercial market. A present overview covering various tool like morphological, microscopical, physical, chemical
and biological employed for evaluation of herbal drugs.

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EVALUATION OF CRUDE DRUGS

  1. 1. K.Sudheer Kumar, Assistant professor. Dept.of Pharmacognosy Chilkur Balaji college of Pharmacy Hyderabad. E-mail:sudheer.y2k8@gmail.com EVALUATION OF CRUDE DRUGS K.Sudheer Kumar, Assistant professor. Dept.of Pharmacognosy Chilkur Balaji college of Pharmacy Hyderabad. E-mail:sudheer.y2k8@gmail.com
  2. 2. INTRODUCTION Crud drugs : Vegetable or animal drugs that consist of natural substances that have undergone only the processes of collection and drying. Natural substances: 1- Plant origin: leaves, flowers, seeds and barks. Or vegetable saps, extracts and secretions. 2- Animal origin: whole animals, glands or organs, extracts and secretions. INTRODUCTION Crud drugs : Vegetable or animal drugs that consist of natural substances that have undergone only the processes of collection and drying. Natural substances: 1- Plant origin: leaves, flowers, seeds and barks. Or vegetable saps, extracts and secretions. 2- Animal origin: whole animals, glands or organs, extracts and secretions.
  3. 3. Drug evaluation may be defined as the determination of identity, purity and quality of a drug.  Identity – identification of biological source of the drug.  Quality – the quantity of the active constituents present.  Purity – the extent of foreign organic material present in a crude drug. • Importance of evaluation of crude drugs: • Determination of Biochemical variation in the drugs • Identification of deterioration due treatment and storage • Repoting Substitution and adulteration, as result of carelessness, ignorance and fraud Drug evaluation may be defined as the determination of identity, purity and quality of a drug.  Identity – identification of biological source of the drug.  Quality – the quantity of the active constituents present.  Purity – the extent of foreign organic material present in a crude drug. • Importance of evaluation of crude drugs: • Determination of Biochemical variation in the drugs • Identification of deterioration due treatment and storage • Repoting Substitution and adulteration, as result of carelessness, ignorance and fraud
  4. 4. METHODS OF DRUG EVALUATION The evaluation of a drug is drug done by studying its various properties. The various properties are: (1) Organoleptic evaluation (2) Microscopic evaluation (3) Physical evaluation (4) Chemical evaluation (5) Analytical evaluation (6) Biological evaluation METHODS OF DRUG EVALUATION The evaluation of a drug is drug done by studying its various properties. The various properties are: (1) Organoleptic evaluation (2) Microscopic evaluation (3) Physical evaluation (4) Chemical evaluation (5) Analytical evaluation (6) Biological evaluation
  5. 5. 1.Organoleptic (Morphological) Evaluation • This refers to drug evaluation by means of organs of sense and includes other sensory organs like color, odour, taste ,size ,shape and texture. • It includes the study of morphology and other sensory characters. S.NO: CHARACHTER DRUG EXAMPLES.NO: CHARACHTER DRUG EXAMPLE 1 Brown colour Cinnamon 2 Aromatic odour Umbelliferous fruits 3 Sweet taste Liquorice 4 Fractured surface Cinchona 5 Wavy shape Rauwolifia 6 7 to 8mm width 25 to 60 mm length (size) Senna leaf
  6. 6. (a) Study of Morphology • It includes the visual examination of drug. S.NO PART OF DRUG EXAPLE 1 BARK KURCHI 2 UNDERGROUND TURMERIC,ZINGER 3 LEAVES DIGITALIS 4 FLOWERS SAFFRON 5 FRUITS FENNEL5 FRUITS FENNEL 6 SEEDS NUX-VOMICA 7 RESIN ASAFOETIDA 8 WOOD SANDAL WOOD 9 GUMS ACACIA 10 ENTIRE DRUG ERGOT
  7. 7. 1- Shape and size. Flowers: Floral parts: stigmas, corollas, anther, ovary, receptacle. Leaves and leaflets: Length, width, apex, margin, base, venation, the texture of the leaf and the hairs in upper and lower surface. The feel of the surface described as soft, hairy smooth. Bark: The barks occur in three shapes: •Flat or curved pieces. • Single quill. •Double quills. ii- Barks have two surfaces, an outer and inner. iii- The inner surface is usually lighter in color than the outer surface 2- Odor and taste. Odor: 1- distinct 2- indistinct aromatic-balsamic,- spicy 1- Shape and size. Flowers: Floral parts: stigmas, corollas, anther, ovary, receptacle. Leaves and leaflets: Length, width, apex, margin, base, venation, the texture of the leaf and the hairs in upper and lower surface. The feel of the surface described as soft, hairy smooth. Bark: The barks occur in three shapes: •Flat or curved pieces. • Single quill. •Double quills. ii- Barks have two surfaces, an outer and inner. iii- The inner surface is usually lighter in color than the outer surface 2- Odor and taste. Odor: 1- distinct 2- indistinct aromatic-balsamic,- spicy
  8. 8. Taste: 1) Acidic (sour) 2) Saccharine (sweet): indicates sugar or sugar like substances 3) e.g., liquorice. 4) Saline (salty) 5) Alkaline 6) Bitter: indicates presence of substances such as bitter principle 7) e.g., glycoside, alkaloids. 8) Tasteless 9) Distinctive sensations to the tongue I. Mucilaginous and oily (soft feeling) e.g., linseed. II.Astringent indicates presence of tannin. III.Pungent (warm biting sensation) e.g., ginger. IV.Acrid (irritant sensation) e.g., Aconite, coca. V.Nauseous (those tending to excite vomiting), Ipecac. Taste: 1) Acidic (sour) 2) Saccharine (sweet): indicates sugar or sugar like substances 3) e.g., liquorice. 4) Saline (salty) 5) Alkaline 6) Bitter: indicates presence of substances such as bitter principle 7) e.g., glycoside, alkaloids. 8) Tasteless 9) Distinctive sensations to the tongue I. Mucilaginous and oily (soft feeling) e.g., linseed. II.Astringent indicates presence of tannin. III.Pungent (warm biting sensation) e.g., ginger. IV.Acrid (irritant sensation) e.g., Aconite, coca. V.Nauseous (those tending to excite vomiting), Ipecac.
  9. 9. 3- Color and external markings. I. 1- White: e.g., starch, II. 2- Pale yellow:e.g., ginger,squill,white pepper. III. 3- Deep yellow: e.g., peeled liquorice. IV. 4- Light pale brown e.g., nux-vomica, fennel. V. 5- Dark brown: e.g., cloves buds. VI. 6- Dark reddish brown: cinchona. VII.7- Red: (brick red). e.g., cinnamon bark inner portion VIII.8- Pale green e.g., lobelia. IX. 9- Greenish brown: most of the leaf herbs. 3- Color and external markings. I. 1- White: e.g., starch, II. 2- Pale yellow:e.g., ginger,squill,white pepper. III. 3- Deep yellow: e.g., peeled liquorice. IV. 4- Light pale brown e.g., nux-vomica, fennel. V. 5- Dark brown: e.g., cloves buds. VI. 6- Dark reddish brown: cinchona. VII.7- Red: (brick red). e.g., cinnamon bark inner portion VIII.8- Pale green e.g., lobelia. IX. 9- Greenish brown: most of the leaf herbs.
  10. 10. 2. Microscopic or Anatomical Evaluation • This method allows a more detailed examination of a drug and it can be used to identify organized drugs by their known histological characters. • Before examination through a microscope the material must be suitably prepared. • This can be done by powdering, cutting thin sections of the drug or preparing a macerate. 2. Microscopic or Anatomical Evaluation • This method allows a more detailed examination of a drug and it can be used to identify organized drugs by their known histological characters. • Before examination through a microscope the material must be suitably prepared. • This can be done by powdering, cutting thin sections of the drug or preparing a macerate.
  11. 11. 1. Palisade Ratio 2. Stomatal Number 3. Stomatal Index 4. Stomata 5. Vein-islet Number 6. Vein-termination Number 7. Trichomes or plant hairs 8. Calcium oxalate crystals Quantitative Microscopy 1.Lycopodium spore method 1. Palisade Ratio 2. Stomatal Number 3. Stomatal Index 4. Stomata 5. Vein-islet Number 6. Vein-termination Number 7. Trichomes or plant hairs 8. Calcium oxalate crystals Quantitative Microscopy 1.Lycopodium spore method
  12. 12. 1.Palisade ratio: • It represents the average number of palisade cells beneath one epidermal cell, using four continuous epidermal cells for the count. • It is determined from powdered drugs with the help of camera-Lucida. • Examples: Atropa belladona – 05-70 Adhatoda vasica –5.5-6.5 Cassia angustifolia –5.5-10.0 upper,4.0-7.4 lower(senna) Digitalis lanata –2.5-6.5 1.Palisade ratio: • It represents the average number of palisade cells beneath one epidermal cell, using four continuous epidermal cells for the count. • It is determined from powdered drugs with the help of camera-Lucida. • Examples: Atropa belladona – 05-70 Adhatoda vasica –5.5-6.5 Cassia angustifolia –5.5-10.0 upper,4.0-7.4 lower(senna) Digitalis lanata –2.5-6.5
  13. 13. 2.Stomatal Number: • The average number of stomata present per square millimeter of the epidermis is known as stomatal number Examples: a)Atropa belladonna upper epidermis---07-10 lower epidermis---77-115 b)Datura metel upper epidermis---147-160 lower epidermis---200-209 c)Ocimum sanctum upper epidermis---64-72 lower epidermis---175-250 2.Stomatal Number: • The average number of stomata present per square millimeter of the epidermis is known as stomatal number Examples: a)Atropa belladonna upper epidermis---07-10 lower epidermis---77-115 b)Datura metel upper epidermis---147-160 lower epidermis---200-209 c)Ocimum sanctum upper epidermis---64-72 lower epidermis---175-250
  14. 14. 3.Stomatal Index: • It is the percentage proportion of the number of stomata to the total number of epidermal cells. • Stomatal number varies considerably with the age of the leaf but stomatal index is relatively constant for a given species. • Stomatal index calculated by • S.I = Example: a)Atropa belladonna upper epidermis--- nil lower epidermis---20.2-23-0 3.Stomatal Index: • It is the percentage proportion of the number of stomata to the total number of epidermal cells. • Stomatal number varies considerably with the age of the leaf but stomatal index is relatively constant for a given species. • Stomatal index calculated by • S.I = Example: a)Atropa belladonna upper epidermis--- nil lower epidermis---20.2-23-0 S E+S S.I = Stomatal index S= Number of stomata per unit area E=Number of epidermal cells in the same unit area
  15. 15. 4.Stomata: (primary and important function is gaseous exchange) a minute epidermal opening present on arial parts of plants, Stomata consists of central pore ,two kidney shaped similar cells(guard cells) & varying number of subsidiary cells. Epidermis of leaf shows different characteristics e.g.cuticle,stomata,trichomes, Types of stoma: 1.Moss type 2.Gymnospermous type 3.Gramineous type 4.Dicoteyledonous---it is having diagnostic significance and classified based on form of arrangement of subsidiary cells. 4.Stomata: (primary and important function is gaseous exchange) a minute epidermal opening present on arial parts of plants, Stomata consists of central pore ,two kidney shaped similar cells(guard cells) & varying number of subsidiary cells. Epidermis of leaf shows different characteristics e.g.cuticle,stomata,trichomes, Types of stoma: 1.Moss type 2.Gymnospermous type 3.Gramineous type 4.Dicoteyledonous---it is having diagnostic significance and classified based on form of arrangement of subsidiary cells.
  16. 16. a) Paracytic or rubiaceous or parallel-cell stomata: in this stomata two guard cells covered by two subsidiary cells, e.g. senna b) Diacytic or caryophyllaceous or cross-celled stomata: in this stomata the guard cells are covered by two subsidiary cells on right angle to that of stomata e.g. peppermint c)Anisocytic or cruciferous or unequal-celled stomata: in this stomata number of guard cells is two but covered by three subsidiary cells and in that one is small in size with other two e.g.Datura d) Anomocytic or ranunculaceous or irregular- celled:in this type stoma is surrounded by varying number of subsidiary cells. e.g. digitalis e)Actinocytic or radiate-celled stomata: two guard cells are surrounded by radiating subsidiary cells. a) Paracytic or rubiaceous or parallel-cell stomata: in this stomata two guard cells covered by two subsidiary cells, e.g. senna b) Diacytic or caryophyllaceous or cross-celled stomata: in this stomata the guard cells are covered by two subsidiary cells on right angle to that of stomata e.g. peppermint c)Anisocytic or cruciferous or unequal-celled stomata: in this stomata number of guard cells is two but covered by three subsidiary cells and in that one is small in size with other two e.g.Datura d) Anomocytic or ranunculaceous or irregular- celled:in this type stoma is surrounded by varying number of subsidiary cells. e.g. digitalis e)Actinocytic or radiate-celled stomata: two guard cells are surrounded by radiating subsidiary cells.
  17. 17. 5.Vein-islet Number: • Vein-islet number is defined as the number of vein-islets per sq.mm. of leaf surface. S.NO NAME OF DRUG Vein-islet Range 1 Andrograohis paniculata 9-12 2 Bacopa monniera 6-13 3 Cannabis sativa 18-243 Cannabis sativa 18-24 4 Digitalis purpurea 2.5-3 5 Eucalpytus globules 8-13.5 6.Vien-termination Number: It is defined as the number of veinlet terminations per.sq. mm of the leaf surface between midrib and margin.
  18. 18. 8.Calcium oxalate crystals: Several cell contents present in vegetable drugs. the inorganic crystalline compounds by virtue of their specific shapes can be utilized for the identification of herbal drugs. due to this reason they are called as diagnostic characters of the plant. 1.Cubical (cube shape) e.g,senna,Glycyrrhiza. 2.Rhombic (diamond shape) e.g., 3.Tetragonal e.g,onion. 4.Mono clinic(all three axes are un equal) e.g,Gall. 5.Acicular (long slender, pointed , budles) e.g, Squill, Cinnamon. 6.Rosettes –clusters (aggregation of crystals) e.g, Clove,Arjuna. 7.Microsphenoidal (minute in structures) e.g, Henbane. 8.Calcium oxalate crystals: Several cell contents present in vegetable drugs. the inorganic crystalline compounds by virtue of their specific shapes can be utilized for the identification of herbal drugs. due to this reason they are called as diagnostic characters of the plant. 1.Cubical (cube shape) e.g,senna,Glycyrrhiza. 2.Rhombic (diamond shape) e.g., 3.Tetragonal e.g,onion. 4.Mono clinic(all three axes are un equal) e.g,Gall. 5.Acicular (long slender, pointed , budles) e.g, Squill, Cinnamon. 6.Rosettes –clusters (aggregation of crystals) e.g, Clove,Arjuna. 7.Microsphenoidal (minute in structures) e.g, Henbane.
  19. 19. 7.Trichomes or plant hairs 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.trichomes 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 2.Glandular Trichomes 3.Hydathode or special Trichomes 7.Trichomes or plant hairs 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.trichomes 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 2.Glandular Trichomes 3.Hydathode or special Trichomes
  20. 20. The most basic terms used are glabrous—lacking hairs— and pubescent— having hairs. Details are provided by: a. glabrous, glabrate – lacking hairs or trichomes; surface smooth b. hirsute – coarsely hairy c. hispid – having bristly hairs d. articulate – simple pluricellular-uniseriate hairs e. downy – having an almost wool-like covering of long hairs f. pilose – pubescent with long, straight, soft, spreading or erect hairs g. puberulent – minutely pubescent; having fine, short, usually curly, hairs h. pubescent – bearing hairs or trichomes of any type i. strigillose – minutely strigose j. strigose – having straight hairs all pointing in more or less the same direction as along a margin or midrib k. tomentellous – minutely tomentose l. tomentose – covered with dense, matted, woolly hairs m. villosulous – minutely villous n. villous – having long, soft hairs, often curved, but not matted The most basic terms used are glabrous—lacking hairs— and pubescent— having hairs. Details are provided by: a. glabrous, glabrate – lacking hairs or trichomes; surface smooth b. hirsute – coarsely hairy c. hispid – having bristly hairs d. articulate – simple pluricellular-uniseriate hairs e. downy – having an almost wool-like covering of long hairs f. pilose – pubescent with long, straight, soft, spreading or erect hairs g. puberulent – minutely pubescent; having fine, short, usually curly, hairs h. pubescent – bearing hairs or trichomes of any type i. strigillose – minutely strigose j. strigose – having straight hairs all pointing in more or less the same direction as along a margin or midrib k. tomentellous – minutely tomentose l. tomentose – covered with dense, matted, woolly hairs m. villosulous – minutely villous n. villous – having long, soft hairs, often curved, but not matted
  21. 21. Quantitative microscopy Lycopodium spore method: 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. Quantitative microscopy Lycopodium spore method: 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.
  22. 22. The percentage purity of an authentic ginger powder calculated as follows N X W X 94,000 X 100 N= NUMBER OF CHARACTERISTIC STRUCTURES(STRACH GRAINS) IN 26 FIELDS W=WEIGHT IN mg OF LYCOPOSIUM TAKEN S=NUMBER OF LYCOPODIUM SPORES IN THE SAME 25 FIELDS M=WEIGHT IN mg OF SAMPLE CALCULATED ON BASIS OF DRIED SAMPLE AT 105 C P=2,86,000 IN CASSE OF GINGER STARCH GRAIN POWDER S x M x P = % Purity of drug The percentage purity of an authentic ginger powder calculated as follows N X W X 94,000 X 100 N= NUMBER OF CHARACTERISTIC STRUCTURES(STRACH GRAINS) IN 26 FIELDS W=WEIGHT IN mg OF LYCOPOSIUM TAKEN S=NUMBER OF LYCOPODIUM SPORES IN THE SAME 25 FIELDS M=WEIGHT IN mg OF SAMPLE CALCULATED ON BASIS OF DRIED SAMPLE AT 105 C P=2,86,000 IN CASSE OF GINGER STARCH GRAIN POWDER
  23. 23. 3. Physical Evaluation • Physical contents such as elasticity in fibres, viscosity of drugs containing gums, swelling factor for mucilage containing materials, froth number of saponin drugs, congealing point of volatile and fixed oils, melting and boiling points and water contents are some important parameters used in the evaluation of drugs. • Ultraviolet light is also used for determing the fluorescence of extracts of some drugs. 3. Physical Evaluation • Physical contents such as elasticity in fibres, viscosity of drugs containing gums, swelling factor for mucilage containing materials, froth number of saponin drugs, congealing point of volatile and fixed oils, melting and boiling points and water contents are some important parameters used in the evaluation of drugs. • Ultraviolet light is also used for determing the fluorescence of extracts of some drugs.
  24. 24. • Physical constants are extensively applied to the active principles of drugs, such as alkaloids, volatile oils, fixed oils etc. A few of them are I. Moisture Content II. Viscosity III. Melting point IV. Solubility V. Optical Rotation VI.Refractive Index VII.Ash values VIII.Extractive values IX.Volatile oil Content X.Foreign organic matter XI.swelling factor • Physical constants are extensively applied to the active principles of drugs, such as alkaloids, volatile oils, fixed oils etc. A few of them are I. Moisture Content II. Viscosity III. Melting point IV. Solubility V. Optical Rotation VI.Refractive Index VII.Ash values VIII.Extractive values IX.Volatile oil Content X.Foreign organic matter XI.swelling factor
  25. 25. I. Moisture Content: • Presence of moisture in a crude drug can lead to its deterioration due to either activation of certain enzymes or growth of microbes. • Moisture content can be determined by heating the drug at 150⁰C in an oven to a constant weight and calculating the loss of weight. I. Moisture Content: • Presence of moisture in a crude drug can lead to its deterioration due to either activation of certain enzymes or growth of microbes. • Moisture content can be determined by heating the drug at 150⁰C in an oven to a constant weight and calculating the loss of weight. S.NO DRUGS MOISTURE CONTENT W/W 1. Aloes Not more than 10 2. Digitalis Not more than 5 3. Starch Not more than 15
  26. 26. II.Viscosity: • Viscosity of a liquid is constant at a given temperature and is an index of its composition. • Hence, it is used as a means of standardising liquid drugs. i)Liquid paraffin-kinematic viscosity not less than 64-centistokes at 37.8° ii)Pyroxylin-kinematic viscosity,1100-2450 centistokes II.Viscosity: • Viscosity of a liquid is constant at a given temperature and is an index of its composition. • Hence, it is used as a means of standardising liquid drugs. i)Liquid paraffin-kinematic viscosity not less than 64-centistokes at 37.8° ii)Pyroxylin-kinematic viscosity,1100-2450 centistokes
  27. 27. III.Melting Point: • It is one of the parameters to judge the purity of crude drugs containing lipids as constituents. They may of animal or plant origin and contain fixed oils, fats and waxes. The purity of the following crude drugs can be ascertained by determining their melting points in the range shown against each of them. III.Melting Point: • It is one of the parameters to judge the purity of crude drugs containing lipids as constituents. They may of animal or plant origin and contain fixed oils, fats and waxes. The purity of the following crude drugs can be ascertained by determining their melting points in the range shown against each of them. S,NO DRUGS MELTING POINT (°C) 1 COLOPHONY 75-85 2 BEES WAX 62-65 3 WOOL FAT 34-44
  28. 28. IV.Solubility : The presence of adulterant in a drug could be indicated by solubility studies S.NO DRUG SOLUBILITY 1 Castor oil Soluble in 3 volumes of alcohol 2 Balsam of Peru Soluble in chloral hydrate solution 2 Balsam of Peru Soluble in chloral hydrate solution 3 Asafoetida Soluble in carbon disulphide 4 Alkaloid bases Soluble in chloroform 5 colophony Soluble in light petroleum
  29. 29. V.Optical Rotation: • Many substances of biological origin, having a chiral centre, can rotate the plane of polarised light either to right(dextro rotatory)or to the left(laevo). The extent of rotation is expressed in degrees, plus(+) indicating rotation to the right and minus(-) indication rotation in the left. Such compound are optically active and hence called optical rotation. V.Optical Rotation: • Many substances of biological origin, having a chiral centre, can rotate the plane of polarised light either to right(dextro rotatory)or to the left(laevo). The extent of rotation is expressed in degrees, plus(+) indicating rotation to the right and minus(-) indication rotation in the left. Such compound are optically active and hence called optical rotation. S.NO Drugs Angles of Optical Rotation 1. Caraway oil + 75° to +80° 2. Clove oil 0° to +6.0° 3. Honey +3° to -15°
  30. 30. VI.Refractive Index: When a ray of light passes from one medium to another medium of different density, it is bent from its original path. Thus, the ration of velocity of light in vaccum to its velocity in the substance is said to the Refractive index of the second medium. It is measured by means of refractometer. RI of a compound varies with the wavelength of the incident light, temperature and pressure. VI.Refractive Index: When a ray of light passes from one medium to another medium of different density, it is bent from its original path. Thus, the ration of velocity of light in vaccum to its velocity in the substance is said to the Refractive index of the second medium. It is measured by means of refractometer. RI of a compound varies with the wavelength of the incident light, temperature and pressure. S.NO DRUGS REFRACTIVE INDEX 1 Arachis oil 1.4678 to 10470 2 Castor oil 104758 to 10527 3 Clove oil 1.527 to 10535
  31. 31. VII.Ash values 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 a criterion to judge the identity OR purity of the crude drug 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 a criterion to judge the identity OR purity of the crude drug S.NO Drugs total ash(% w/w) acid insoluble ash % (w/w) 1 Agar - 1.00 2 Bael 03.5 - 3 Cannabis 15.0 5.00 4 Gelatin 03.6 - 5 Valerin 12.0 -
  32. 32. TYPES OFASH VALUES 1.Total ash value Useful for detecting low grade products, exhausted products, excess of sandy and earthy matter with drug. 2.Acid insoluble ash value Used for the determination of earthy matter present on roots, rhizomes, and also on the leaves, Crude drugs contain calcium oxalate crystals the amount may varies depending on the environmental conditions. 3.Sulphated ash value Used for the detection of low grade products. 4. Water soluble ash value Water soluble ash value Used to detect either material exhausted by water or not ( Tea leaves, Ginger rhizomes). 1.Total ash value Useful for detecting low grade products, exhausted products, excess of sandy and earthy matter with drug. 2.Acid insoluble ash value Used for the determination of earthy matter present on roots, rhizomes, and also on the leaves, Crude drugs contain calcium oxalate crystals the amount may varies depending on the environmental conditions. 3.Sulphated ash value Used for the detection of low grade products. 4. Water soluble ash value Water soluble ash value Used to detect either material exhausted by water or not ( Tea leaves, Ginger rhizomes).
  33. 33. Determination Total ash value 1.Weigh accurately about 3gms of the powdered drug in a tared silica crucible 2.Incinerate the powdered drug by gradually increasing the heat until free from carbon and cool. Keep it in desiccators 3. Weigh the ash and calculate the % of the total ash with reference to the air dried sample 1.Weigh accurately about 3gms of the powdered drug in a tared silica crucible 2.Incinerate the powdered drug by gradually increasing the heat until free from carbon and cool. Keep it in desiccators 3. Weigh the ash and calculate the % of the total ash with reference to the air dried sample
  34. 34. 1. Boil the total ash obtained as above for 5 minutes with 25ml of dilute HCL 2.Filter and collect the insoluble matter on the ashless filter paper , wash the filter paper with hot water, ignite in tared crucible, cool and kept in desiccators 3.Weigh the residue and calculate the acid insoluble ash of the drug Determination of Acid insoluble ash value 1. Boil the total ash obtained as above for 5 minutes with 25ml of dilute HCL 2.Filter and collect the insoluble matter on the ashless filter paper , wash the filter paper with hot water, ignite in tared crucible, cool and kept in desiccators 3.Weigh the residue and calculate the acid insoluble ash of the drug
  35. 35. VIII.Extractive values In crude drugs, sometimes the active chemical constitutes cannot be determined by normal procedures. In such cases, water, alcohol or ether soluble extractive values are determined for evaluation of such drugs. Significances : 1.Useful for the evaluation especially when the constituents of the drugs can not be readily estimated by any other means 2.It also helps to indicate the nature of chemical constituents present in the drug 3. Also helps in the identification of adulterants VIII.Extractive values In crude drugs, sometimes the active chemical constitutes cannot be determined by normal procedures. In such cases, water, alcohol or ether soluble extractive values are determined for evaluation of such drugs. Significances : 1.Useful for the evaluation especially when the constituents of the drugs can not be readily estimated by any other means 2.It also helps to indicate the nature of chemical constituents present in the drug 3. Also helps in the identification of adulterants
  36. 36. Types of extractive values A. water soluble extractive values B. Alcohol soluble extractive values C. Ether soluble extractive values Types of extractive values A. water soluble extractive values B. Alcohol soluble extractive values C. Ether soluble extractive values
  37. 37. Determination of water soluble extractive value 1. Macerate about 5gm of the accurately weighed coarse powder with 100ml of chloroform water in a 100ml volumetric flask for 24 hours . 2. Shake frequently for first 6 hours 3. Filter rapidly through filter paper and evaporate 25ml of water extract to dryness in a tared flat-bottomed shallow dish. 4. Dry the residue at 105 and weigh. Keep it in a desiccators 5. Dry the extract to constant weight ,finally , calculate the % W/W of Water soluble extractive value with reference to the air dried drug. Determination of water soluble extractive value 1. Macerate about 5gm of the accurately weighed coarse powder with 100ml of chloroform water in a 100ml volumetric flask for 24 hours . 2. Shake frequently for first 6 hours 3. Filter rapidly through filter paper and evaporate 25ml of water extract to dryness in a tared flat-bottomed shallow dish. 4. Dry the residue at 105 and weigh. Keep it in a desiccators 5. Dry the extract to constant weight ,finally , calculate the % W/W of Water soluble extractive value with reference to the air dried drug.
  38. 38. A. Water soluble extractive value Water soluble extractive value is applied for the drugs which contain water soluble constituents such as tannins, sugars, plant acids and mucilage. S.NO DRUG WATER SOLUBLE EXTRACTIVE (% W/W) S.NO DRUG WATER SOLUBLE EXTRACTIVE (% W/W) 1 Aloe Vera NLT 25.0 2 Linseed NLT 20.0 3 Senna leaves NLT 30.0 4 Ginger NLT 10.0 5 Glycyrrhiza NLT 20.0 NLT= Not less than
  39. 39. Determination of Alcohol soluble extractive values 1. Macerate about 5gm of the accurately weighed coarse powder with 100ml of 90% alcohol in a 100ml stoppered flask for 24 hours . 2. Shake frequently for first 6 hours 3. Filter rapidly through filter paper and collect the filtrate evaporate 25ml of alcohol extract to dryness in a tared flat-bottomed shallow dish. 4. Dry the residue at 105 and weigh. Keep it in a desiccators 5. Dry the extract to constant weight ,finally , calculate the % w/w of alcohol soluble extractive value with reference to the air dried drug. Determination of Alcohol soluble extractive values 1. Macerate about 5gm of the accurately weighed coarse powder with 100ml of 90% alcohol in a 100ml stoppered flask for 24 hours . 2. Shake frequently for first 6 hours 3. Filter rapidly through filter paper and collect the filtrate evaporate 25ml of alcohol extract to dryness in a tared flat-bottomed shallow dish. 4. Dry the residue at 105 and weigh. Keep it in a desiccators 5. Dry the extract to constant weight ,finally , calculate the % w/w of alcohol soluble extractive value with reference to the air dried drug.
  40. 40. B.Alcohol soluble extractive value Alcohol soluble extractive value is applied for the drugs which contain alcohol soluble constituents such as tannins, resins and alkaloids .Official method for the assay of myrrh & asafoetida. Generally,95% ethyl alcohol is used for determination of Alcohol soluble extractive. B.Alcohol soluble extractive value Alcohol soluble extractive value is applied for the drugs which contain alcohol soluble constituents such as tannins, resins and alkaloids .Official method for the assay of myrrh & asafoetida. Generally,95% ethyl alcohol is used for determination of Alcohol soluble extractive. S.NO DRUG Alcohol soluble extractive. (% W/W) 1 Aloe vera NLT 10.0 2 Benzoin NLT 90.0 3 Asafoetida NLT 50.0 4 Ginger NLT 04.0 5 Myrrh NLT 24.0 NLT= Not less than
  41. 41. C.Ether soluble extractive value Ether soluble extractive value is applied for the extraction of volatile oils, fixed oils and resins. 1.Volatile ether soluble extractive value –(volatile oil) 2.Non volatile ether soluble extractive value –(resin, fixed oils, coloring matter) C.Ether soluble extractive value Ether soluble extractive value is applied for the extraction of volatile oils, fixed oils and resins. 1.Volatile ether soluble extractive value –(volatile oil) 2.Non volatile ether soluble extractive value –(resin, fixed oils, coloring matter) S.NO DRUGS LIMIT FOR NON-VOLATILE ETHER SOLUBLE EXTRACTIVES(% W/W) 1 CAPSICUM NLT 12.0 2 MALE FERN NLT 01.0 3 LINSEED NLT 25.0 NLT= Not less than
  42. 42. IX.Volatile oil content: Efficiency of several drugs is due to their odorous principle (volatile oils).Such crude drugs are standardized on the basis of their volatile oil contents. Weighed quantity of the drug is boiled with water in a round bottomed flask fitted with clevenger apparatus. The distillate collected is graduated into volatile oil. The amount thus obtained is recorded from the tube. IX.Volatile oil content: Efficiency of several drugs is due to their odorous principle (volatile oils).Such crude drugs are standardized on the basis of their volatile oil contents. Weighed quantity of the drug is boiled with water in a round bottomed flask fitted with clevenger apparatus. The distillate collected is graduated into volatile oil. The amount thus obtained is recorded from the tube. S.NO DRUGS VOLATILE OIL CONTENT (% W/W 1 CARAWAY NLT 2.5 2 CLOVE NLT 15.0 3 FRESH LEMON PEEL NLT 205 4 FENNEL NLT 1.4 5 DILL NLT 205 NLT= Not less than
  43. 43. x.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 exceeds the limits, deterioration in quality of the drug takes place.  The physical or Physico chemical parameters useful in quality profile of a crude drug evaluation x.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 exceeds the limits, deterioration in quality of the drug takes place.  The physical or Physico chemical parameters useful in quality profile of a crude drug evaluation
  44. 44. XI.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 XI.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
  45. 45. 4. 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 4. 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
  46. 46. 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.Microchemical tests: These are the tests which are carried on slides. Example: Euginol in clove oil is precipitated as potassium euginate crystals. 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.Microchemical tests: These are the tests which are carried on slides. Example: Euginol in clove oil is precipitated as potassium euginate crystals.
  47. 47. 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 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
  48. 48. I. 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. II. 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 I. 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. II. 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
  49. 49. 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. 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.
  50. 50. III. 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. IV. Triterpenoids 1. 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. III. 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. IV. Triterpenoids 1. 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.
  51. 51. V. 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. V. 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.
  52. 52. VI. 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. VII. 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. VI. 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. VII. 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.
  53. 53. VIII. Test for 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. 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 VIII. Test for 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. 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
  54. 54. 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 IX. 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. X. Test for proteins 1. 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. 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 IX. 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. X. Test for proteins 1. 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.
  55. 55. XI. 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. XII. 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. XI. 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. XII. 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.
  56. 56. 5.Analytical evaluation Chromatographic techniques: a)TLC-Thin layer chromatography b)HPTLC-High performance thin layer chromatography c)HPLC-High performance/pressure liquid chromatography d)GLC-Gas chromatography e)CC-column chromatography f)Gel permeation chromatography g)Affinity chromatography 5.Analytical evaluation Chromatographic techniques: a)TLC-Thin layer chromatography b)HPTLC-High performance thin layer chromatography c)HPLC-High performance/pressure liquid chromatography d)GLC-Gas chromatography e)CC-column chromatography f)Gel permeation chromatography g)Affinity chromatography
  57. 57. Spectrophptometric 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 Spectrophptometric 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
  58. 58. Chromatographic techniques: a)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 pirity,nature,of substance, composition of solvent and impurities  TLC/HPTLC are micro analytical techniques used for determination of natural products Advantages :simple in operation and rapid Chromatographic techniques: a)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 pirity,nature,of substance, composition of solvent and impurities  TLC/HPTLC are micro analytical techniques used for determination of natural products Advantages :simple in operation and rapid
  59. 59. • Thin layer chromatography(TLC), has become increasingly popular for both qualitative and quantitative evaluation of drugs. • Rf values refers to the ration of distance travelled by the solute to the distance moved by the solvent on a thin layer adsorbent. Distance travelled by the compound(solute) Distance travelled by the solvent • Thin layer chromatography(TLC), has become increasingly popular for both qualitative and quantitative evaluation of drugs. • Rf values refers to the ration of distance travelled by the solute to the distance moved by the solvent on a thin layer adsorbent. Distance travelled by the compound(solute) Distance travelled by the solventRf =
  60. 60. b)HPTLC-High performance thin layer chromatography:  It is very useful qualitative/quantitative method for pharmaceutical analysis  HPTLC is a major advancement of TLC principle requiring shorter time better resolution  HPTLC plates available in the form of pre coats  Silica gel-Gel with very small particle size used as a stationary phase gives rapid separation with sensitivity  About 36 cm solvent front migration is sufficient to effect proper seperation  Whatmann-HPTLC plates are produced from 4-5μm layer  About 7cm distance achieved in about 4 minutes b)HPTLC-High performance thin layer chromatography:  It is very useful qualitative/quantitative method for pharmaceutical analysis  HPTLC is a major advancement of TLC principle requiring shorter time better resolution  HPTLC plates available in the form of pre coats  Silica gel-Gel with very small particle size used as a stationary phase gives rapid separation with sensitivity  About 36 cm solvent front migration is sufficient to effect proper seperation  Whatmann-HPTLC plates are produced from 4-5μm layer  About 7cm distance achieved in about 4 minutes
  61. 61. Sample preparation :HPTLC needs high concentration sample. small amounts of sample need to apply, sample spot size 1 mm in diameter and sample applied by capillaries. solvent systems (component and mobile phases) S.NO COMPONENT MOBILE PHASES.NO COMPONENT MOBILE PHASE 1 Amino acids, alkaloids Butanol-acetic acid-water(4:5:1) 2 Cardiac glycoside Dichloromethane-methnol- formamide(8:2:1) 3 Flavonoids,coumarin glycosides Ethyl acetate-methyl ketone-acetic acid- water(5:3:1:1) 4 Saponins Chloroform-methanol-water(7:4:1:) 5 Terpenes,essential oils ,sterols Hexane-acetone-(9:1)
  62. 62. c) HPLC-High performance/pressure liquid chromatography  The term liquid chromatography used to refer to those methods in which the separation takes place with packed column.(stationary) A liquid mobile phase used eluent.  In HPLC mobile phase forced to column under high pressure  Derivatisation in HPLC undertaken to increase sensitivity of detection for a given compound  Colum used in HPLC narrow (1 mm or less) flow rate of mobile phase is (100μl /min) Advantages : most versatile ,safest. Uses :quality control of drugs like morhine,emetine,steroids c) HPLC-High performance/pressure liquid chromatography  The term liquid chromatography used to refer to those methods in which the separation takes place with packed column.(stationary) A liquid mobile phase used eluent.  In HPLC mobile phase forced to column under high pressure  Derivatisation in HPLC undertaken to increase sensitivity of detection for a given compound  Colum used in HPLC narrow (1 mm or less) flow rate of mobile phase is (100μl /min) Advantages : most versatile ,safest. Uses :quality control of drugs like morhine,emetine,steroids
  63. 63. d) GLC-Gas liquid chromatography  GLC separates volatile substances by percolating a gas stream over a stationary phase. Principle :GLC works on partitioning Carrier gas used as mobile phase (Nitrogen, Helium) A film of a liquid spread over an inert solid. Acts as stationary phase. GLC applied for i.Assay of impurities ii.Examination of volatile oils plant alkaloids. d) GLC-Gas liquid chromatography  GLC separates volatile substances by percolating a gas stream over a stationary phase. Principle :GLC works on partitioning Carrier gas used as mobile phase (Nitrogen, Helium) A film of a liquid spread over an inert solid. Acts as stationary phase. GLC applied for i.Assay of impurities ii.Examination of volatile oils plant alkaloids.
  64. 64. e)CC-column chromatography  Liquid chromatography in which mobile phase in form of liquid passes over the stationary phase packed in a column.  Colum is either a glass, metallic column. the column adsorption chromatography is oldest and still practiced to day for extraction process. f)Gel permeation chromatography  Size-exclusion chromatography.seperation occurs not on the basis of adsorption /partition ,but on the effective size of solutes present in solution for the separation purpose e)CC-column chromatography  Liquid chromatography in which mobile phase in form of liquid passes over the stationary phase packed in a column.  Colum is either a glass, metallic column. the column adsorption chromatography is oldest and still practiced to day for extraction process. f)Gel permeation chromatography  Size-exclusion chromatography.seperation occurs not on the basis of adsorption /partition ,but on the effective size of solutes present in solution for the separation purpose
  65. 65.  Stationary phase used are cross linked polymers which give an open network with large number of pores during flow large size particles can’t enters in to pores hence excluded.  Various types of gels are used sofgel,semi-rigid gels, rigid gels. Use :separates biomolecules,protiens,poly-peptides.  Stationary phase used are cross linked polymers which give an open network with large number of pores during flow large size particles can’t enters in to pores hence excluded.  Various types of gels are used sofgel,semi-rigid gels, rigid gels. Use :separates biomolecules,protiens,poly-peptides.
  66. 66. g)Affinity chromatography  This technique is mainly used for the separation of protiens,enzymes,antigens,antibodies.  The adsorbent used is one of biological substance having a specific affinity for other substance .  These two substances are biologically interacting pairs such adsorbent is attached to a porous stationary phase and placed in a column, when mixture containing the other complement of adsorbent passed through stationary phase g)Affinity chromatography  This technique is mainly used for the separation of protiens,enzymes,antigens,antibodies.  The adsorbent used is one of biological substance having a specific affinity for other substance .  These two substances are biologically interacting pairs such adsorbent is attached to a porous stationary phase and placed in a column, when mixture containing the other complement of adsorbent passed through stationary phase
  67. 67. Spectrophptometric methods: i) UV- Ultra violet /visible spectroscopy Ultra violet –visible absorption techniques encompass analytical methods based up on measurement of light absorption by substances in wave length region from 190 to 900 nm 190-380 nm UV region. 380-900 nm visible region. Applications :we can analyze variety of pharmaceutical phytoconstituents like Lobeline-244 nm, Morphine-286 nm Antharaquinone 505 nm Spectrophptometric methods: i) UV- Ultra violet /visible spectroscopy Ultra violet –visible absorption techniques encompass analytical methods based up on measurement of light absorption by substances in wave length region from 190 to 900 nm 190-380 nm UV region. 380-900 nm visible region. Applications :we can analyze variety of pharmaceutical phytoconstituents like Lobeline-244 nm, Morphine-286 nm Antharaquinone 505 nm
  68. 68. ii)IR-Infra Red spectroscopy  IR is the study of reflected,absorbed,transmitted radiant energy in region of electromagnetic spectrum 0.8 to 500 nm  It is divided in to three regions Near IR-12,500-400 cm-1, Mid IR-4000-400 cm-1 Far IR-400-20cm-1  IR spectrophotometer can be divided in to single and double beam and Fourier transform spectrophotometer(FTIR) Applications :Identification of drugs, polymorphs Raw materials,excipients. ii)IR-Infra Red spectroscopy  IR is the study of reflected,absorbed,transmitted radiant energy in region of electromagnetic spectrum 0.8 to 500 nm  It is divided in to three regions Near IR-12,500-400 cm-1, Mid IR-4000-400 cm-1 Far IR-400-20cm-1  IR spectrophotometer can be divided in to single and double beam and Fourier transform spectrophotometer(FTIR) Applications :Identification of drugs, polymorphs Raw materials,excipients.
  69. 69. iii) Fluorescence analysis  The organic molecules absorbs light usually over a specific range of wave length, and many of them re-emits such radiation known as luminescence.  The phenomina when the re-emission of absorbed light losts only when substance receiving exiting rays, and called as fluorescence. iii) Fluorescence analysis  The organic molecules absorbs light usually over a specific range of wave length, and many of them re-emits such radiation known as luminescence.  The phenomina when the re-emission of absorbed light losts only when substance receiving exiting rays, and called as fluorescence. S.NO HERBAL DRUG NATURE OF FLUORESCENCE. 1 CINCHONA PURPLE BLUE 2 RHUBARB VIOLET 3 QUASSIA WHITISH BLUE Fluorescence characters under UV light
  70. 70. iv) NMR-nuclear magnetic resonance spectroscopy  NMR absorbs radio frequency radiation by substance held in a magnetic field.  Absorption results from interaction of radiation with magnetic moment of nuclei in sample and it occurs at different frequencies for nuclei with chemically different environment. Applications NMR is imp tool in elucidation of molecular structure It is applicable in identification of impurities. It reveals position of protons in a complex molecule iv) NMR-nuclear magnetic resonance spectroscopy  NMR absorbs radio frequency radiation by substance held in a magnetic field.  Absorption results from interaction of radiation with magnetic moment of nuclei in sample and it occurs at different frequencies for nuclei with chemically different environment. Applications NMR is imp tool in elucidation of molecular structure It is applicable in identification of impurities. It reveals position of protons in a complex molecule
  71. 71. v) MS-Mass spectroscopy  Mass spectrometry concerned with the electron ionisation,subsequent fragmentation of molecules, determination of the mass to charge ratio (m/e).and relative abundances of ions which are produced. Applications  It determines molecular weight of compound  It helps in identification of drug constituents v) MS-Mass spectroscopy  Mass spectrometry concerned with the electron ionisation,subsequent fragmentation of molecules, determination of the mass to charge ratio (m/e).and relative abundances of ions which are produced. Applications  It determines molecular weight of compound  It helps in identification of drug constituents
  72. 72. vi) X-ray diffraction: Many compounds are capable of crystallising in more than one type of crystal lattice at a particular temperature and pressure, since the rate of phase transformation of a metastable polymorph to the stable one can be quite slow.Polymorphs plays a very imp role in pharmaceutical science vii) RIA-radio immuno assay  This technique uses on antibody specific for the drug being assayed and a labeled form of the same drug. The label may be particular radio-isotope, active-enzyme or a C14 and iodine I125 commonly used isotopes in RIA  RIA is method of choice for identification of cardiac glycoside, insulin, vi) X-ray diffraction: Many compounds are capable of crystallising in more than one type of crystal lattice at a particular temperature and pressure, since the rate of phase transformation of a metastable polymorph to the stable one can be quite slow.Polymorphs plays a very imp role in pharmaceutical science vii) RIA-radio immuno assay  This technique uses on antibody specific for the drug being assayed and a labeled form of the same drug. The label may be particular radio-isotope, active-enzyme or a C14 and iodine I125 commonly used isotopes in RIA  RIA is method of choice for identification of cardiac glycoside, insulin,
  73. 73. 6.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 • Digitalis 1IU=76mg of standard • Vit-A 1IU=0.344 of standard • Vit-D 1IU=0.025of standard 6.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 • Digitalis 1IU=76mg of standard • Vit-A 1IU=0.344 of standard • Vit-D 1IU=0.025of standard
  74. 74. 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 arte evaluated by this method on cats, frogs or pigeons. 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 arte evaluated by this method on cats, frogs or pigeons.
  75. 75. 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) 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)
  76. 76. 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 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
  77. 77. EVALUATION OF HEAPATOPROTECTIVITY ANIMALS USED: Male and Female Albino rats Heapatotoxicity indused by: Chemicals Industrial pollutants ccl4 Drugs (paracetamol,rifampicin) PARAMETERS FOR ESTIMATION 1.PHYSIOLOGICAL—HEXOBARBITAL HYPNOSIS 2.BIO-CHEMICAL—SERUM ESTIMATION ENZYMES LIKE SGOT(serum glutamic oxaloacetic transaminase) SGPT (serum glutamic pyruvic oxaloacetic transaminase) 3.BLOOD CHELESTEROL,TRIGLYCERIDES LEVELS 4.HISTOPATHOLOGICAL METHODS (liver tissue necrosis) for testing cultured heaptocytes are used for In-vitro studies EVALUATION OF HEAPATOPROTECTIVITY ANIMALS USED: Male and Female Albino rats Heapatotoxicity indused by: Chemicals Industrial pollutants ccl4 Drugs (paracetamol,rifampicin) PARAMETERS FOR ESTIMATION 1.PHYSIOLOGICAL—HEXOBARBITAL HYPNOSIS 2.BIO-CHEMICAL—SERUM ESTIMATION ENZYMES LIKE SGOT(serum glutamic oxaloacetic transaminase) SGPT (serum glutamic pyruvic oxaloacetic transaminase) 3.BLOOD CHELESTEROL,TRIGLYCERIDES LEVELS 4.HISTOPATHOLOGICAL METHODS (liver tissue necrosis) for testing cultured heaptocytes are used for In-vitro studies
  78. 78. 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. 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.
  79. 79. Evaluation of Anti-inflammatory activity Inflammation is caused by mechanical,infections,auto-immune Types of inflammations:Rhemated arthritis,gout,dysmenorrhoea PRINCIPLE:Anti-inflammatory activity is reduction of local edema induced in rat paw by injecting irritant or inflammatory substance Inflammation is induced by carrageenan and croton oil Methods 1: Carrageenan is a muco-polysaccharide isolated from sea moss which induces inflammation by giving through intraperitoneally saline in a dose of 0.1 ml. animal is treated with herbal extract given orally (antagonist) Volume of paw is measured five times with plethysmo meter Methods 2:Here albino rats or mice are used, edema is produced pinna of ear with croton oil(1 ml/ear) After induce herbal extract is added to the same area Edema is measured by using verniercallipers and record the changes 0---no effect +ve---slight ++ve---pronounced Evaluation of Anti-inflammatory activity Inflammation is caused by mechanical,infections,auto-immune Types of inflammations:Rhemated arthritis,gout,dysmenorrhoea PRINCIPLE:Anti-inflammatory activity is reduction of local edema induced in rat paw by injecting irritant or inflammatory substance Inflammation is induced by carrageenan and croton oil Methods 1: Carrageenan is a muco-polysaccharide isolated from sea moss which induces inflammation by giving through intraperitoneally saline in a dose of 0.1 ml. animal is treated with herbal extract given orally (antagonist) Volume of paw is measured five times with plethysmo meter Methods 2:Here albino rats or mice are used, edema is produced pinna of ear with croton oil(1 ml/ear) After induce herbal extract is added to the same area Edema is measured by using verniercallipers and record the changes 0---no effect +ve---slight ++ve---pronounced
  80. 80. Evaluation of Anti fertility activity Abortificient activity, contraceptives Traditional drugs like embelin from embilica and gossypol from gossypium produce abortificient activity Types of Anti fertility evaluations In-females : Destruction of zygotes &prevention of ovulation In-males : estimation of Spermicidal activity & Anti-androgenic activity Protocols for anti- spermatogenic activity After acclimatization male rats are feed by herbal extract for 60 days Between 12 day and 15 day & 60 th day male rats are mated with female (morphological weight of rat noted) Histopathlogical study of sperm RESULT If No-fertilization b/w 12-15 days means functional sterility, after 56 days means anti-spermatogenic Evaluation of Anti fertility activity Abortificient activity, contraceptives Traditional drugs like embelin from embilica and gossypol from gossypium produce abortificient activity Types of Anti fertility evaluations In-females : Destruction of zygotes &prevention of ovulation In-males : estimation of Spermicidal activity & Anti-androgenic activity Protocols for anti- spermatogenic activity After acclimatization male rats are feed by herbal extract for 60 days Between 12 day and 15 day & 60 th day male rats are mated with female (morphological weight of rat noted) Histopathlogical study of sperm RESULT If No-fertilization b/w 12-15 days means functional sterility, after 56 days means anti-spermatogenic
  81. 81. Spermicidal activity (in-vitro) Take human semen on a slide and add herbal extract and sorensens phosphate buffer Microscopic examination Identification of Immobility means spermicidal Spermicidal activity (in-vitro) Take human semen on a slide and add herbal extract and sorensens phosphate buffer Microscopic examination Identification of Immobility means spermicidal
  82. 82. Protocols for anti-fertility in female rats Acclimatized 8-10 days ( I-group) Vaginal smears are taken to measure cycle Animals are feed by herbal extract (oestrogenisity leads increase weight of uterus / cornifiction of vagina) Further the rats are mate with males (mating detected by smears) Ovulation is absent due to contraceptive activity) In case of fertilization ova are examined to see the transportation and implantation Acclimatized 19 days ( II-group) If weight gain contraception If bleeding abortion Protocols for anti-fertility in female rats Acclimatized 8-10 days ( I-group) Vaginal smears are taken to measure cycle Animals are feed by herbal extract (oestrogenisity leads increase weight of uterus / cornifiction of vagina) Further the rats are mate with males (mating detected by smears) Ovulation is absent due to contraceptive activity) In case of fertilization ova are examined to see the transportation and implantation Acclimatized 19 days ( II-group) If weight gain contraception If bleeding abortion
  83. 83. Testing of anti-ulcer activity Causes of ulcer -- improper diet, alcohol consumption,stress,drugs(NSAIDS) Traditional drugs like liquorice,atropine,hyoscine and in less extent Gafaranate extracted from cabbage juice shows anti ulcer effect Chemical used to induce ulcer: Alcohol 1ml/kg orally Aspirin -200mg/kg orally Stress induced: animals are immobilized in cylindrical cage Animals are devided in to 3 groups 1.Those treated with normal saline 2.Those treated with ulcerogenic solution 3.Strss produced Animals are sacrificed after inducing All the groups are treated with herbal extracts and standard is treated with ranitidine Stomach or duodenum is isolated Organs are opened to know ulcer effect and gastric juice is measured and ulcer effect express in table 0-no damage 0-absence 1.redness of mucosa 1.slight 2.Erosin of mucosa 2.One ulcer 5mm length 3.ulceration 3.More than one Testing of anti-ulcer activity Causes of ulcer -- improper diet, alcohol consumption,stress,drugs(NSAIDS) Traditional drugs like liquorice,atropine,hyoscine and in less extent Gafaranate extracted from cabbage juice shows anti ulcer effect Chemical used to induce ulcer: Alcohol 1ml/kg orally Aspirin -200mg/kg orally Stress induced: animals are immobilized in cylindrical cage Animals are devided in to 3 groups 1.Those treated with normal saline 2.Those treated with ulcerogenic solution 3.Strss produced Animals are sacrificed after inducing All the groups are treated with herbal extracts and standard is treated with ranitidine Stomach or duodenum is isolated Organs are opened to know ulcer effect and gastric juice is measured and ulcer effect express in table 3.ulceration 3.More than one 4.One above 5 mm 10.Total ulcerations
  84. 84. Evaluation of Neuropharmacologocal activity Testing of herbal drugs on CNS&ANS Drugs and actions actions on CNS:cocaine,cannabis,morphine(stimulants,tranquilisers) METHODS FOR TESTING: 1.LOCOMOTAR ACTIVITY-----ACTIVITY CAGE(locomotion count is noted) 2.LOCOMOTAR CO-ORDINATION-----ROTATING ROD 3.PENTETRAZOLE CONVULSION IN MICE 80-120 mg/kg- intraperitonelly a)time of onset of convulsions b)number of convulsions c)mortality rate 4.BARBITURATE SLEEPING TIME stimulant or depressant effects on CNS Phenobarbitone ----55mg/kg Hexobarbitone -----33mg/kg Evaluation of Neuropharmacologocal activity Testing of herbal drugs on CNS&ANS Drugs and actions actions on CNS:cocaine,cannabis,morphine(stimulants,tranquilisers) METHODS FOR TESTING: 1.LOCOMOTAR ACTIVITY-----ACTIVITY CAGE(locomotion count is noted) 2.LOCOMOTAR CO-ORDINATION-----ROTATING ROD 3.PENTETRAZOLE CONVULSION IN MICE 80-120 mg/kg- intraperitonelly a)time of onset of convulsions b)number of convulsions c)mortality rate 4.BARBITURATE SLEEPING TIME stimulant or depressant effects on CNS Phenobarbitone ----55mg/kg Hexobarbitone -----33mg/kg
  85. 85. AUTONOMIC NERVOUS SYSTEM in-vitro studies 1.Guinea pig ileum prepatiopn: (non-specific anti-spasmodic activity) Guinea pig ileum is useful for the evaluations of nerve mediated and directly stimulated contractions because it contains non-adrenergic (cholinergic)nerves called as intrinsic parasympathetic nerves, present between circular and longitudinal muscle layers. Protocol : Food is withdrawn over night Animal is killed (suitable length of ileum is separated –1.5 to 2 cm long) Kept in salt solution(physiological salt) Jacketed organ bath at 32-37 c Guinea pig ileum shows a response to para-sympathomimetics&para-sympatholytics(ACH given to ileum) Herbal drug is given AUTONOMIC NERVOUS SYSTEM in-vitro studies 1.Guinea pig ileum prepatiopn: (non-specific anti-spasmodic activity) Guinea pig ileum is useful for the evaluations of nerve mediated and directly stimulated contractions because it contains non-adrenergic (cholinergic)nerves called as intrinsic parasympathetic nerves, present between circular and longitudinal muscle layers. Protocol : Food is withdrawn over night Animal is killed (suitable length of ileum is separated –1.5 to 2 cm long) Kept in salt solution(physiological salt) Jacketed organ bath at 32-37 c Guinea pig ileum shows a response to para-sympathomimetics&para-sympatholytics(ACH given to ileum) Herbal drug is given
  86. 86. Due to drug treatment if ACH type contractions are produced, persist even after giving a dose of histamine H1-receptor antagonist, then it may be indicated that it is a cholinergic drug. it can be verified by blocking effect with atropine.(it does not distinguish between muscarinic and nicotinic receptor activity. If contractions caused by herbal drug are blocked by ganglion blocking agent then it may be indicated that herbal drug is nicotinic receptor mechanism . Besides cholinergic activity the contractions occurred due to drug may be because of its activity at other receptors like histamine. Due to drug treatment if ACH type contractions are produced, persist even after giving a dose of histamine H1-receptor antagonist, then it may be indicated that it is a cholinergic drug. it can be verified by blocking effect with atropine.(it does not distinguish between muscarinic and nicotinic receptor activity. If contractions caused by herbal drug are blocked by ganglion blocking agent then it may be indicated that herbal drug is nicotinic receptor mechanism . Besides cholinergic activity the contractions occurred due to drug may be because of its activity at other receptors like histamine.
  87. 87. 2.Isolated rabbit jejunum preparation With this study we can evaluate the nerve mediated and direct muscle stimulated adrenergic effects Rabbit jejunum is isolated along with mesenteric vessels as associated with sympathetic nerves Mesentery vessels are carefully cut off & then isolated rabbit jejunum with periarterial nerve is mounted in physiological solution in Jacketed organ bath 32-37c Nerves are connected to a electrode and stimulated If these are relaxed by herbal drug it may have noradrenalin effect or calcium channel blockade or potassium channel opening effect If the action of drug is blocked by adrenergic receptor antagonist it may have effect on adrenoreceptor. 2.Isolated rabbit jejunum preparation With this study we can evaluate the nerve mediated and direct muscle stimulated adrenergic effects Rabbit jejunum is isolated along with mesenteric vessels as associated with sympathetic nerves Mesentery vessels are carefully cut off & then isolated rabbit jejunum with periarterial nerve is mounted in physiological solution in Jacketed organ bath 32-37c Nerves are connected to a electrode and stimulated If these are relaxed by herbal drug it may have noradrenalin effect or calcium channel blockade or potassium channel opening effect If the action of drug is blocked by adrenergic receptor antagonist it may have effect on adrenoreceptor.
  88. 88. 3.Rat. Phrenic nerve- diaphragm preparation Phrenic diaphragm isolated from frontal part of right thoracic wall Mount to phrenic nerve electrode Muscle remains across platinum electrode Drug shows effected neuro muscular junction 3.Rat. Phrenic nerve- diaphragm preparation Phrenic diaphragm isolated from frontal part of right thoracic wall Mount to phrenic nerve electrode Muscle remains across platinum electrode Drug shows effected neuro muscular junction
  89. 89. Mosquito repellence test Protection period offered by herbal extract against mosquito bite in terms of first bite. 3-6 days old, blood starved sucrose fed yellow fever mosquito (Aedes aegypti) Human hand covered with snugly fitting polythene bag is introduced in a cage contains about a thousand hungry mosquitoes, and allowed to bite on muslin cloth which is treated wit herbal extract. Number of bites received in 5 minutes counted Mosquito repellence test Protection period offered by herbal extract against mosquito bite in terms of first bite. 3-6 days old, blood starved sucrose fed yellow fever mosquito (Aedes aegypti) Human hand covered with snugly fitting polythene bag is introduced in a cage contains about a thousand hungry mosquitoes, and allowed to bite on muslin cloth which is treated wit herbal extract. Number of bites received in 5 minutes counted
  90. 90. Anti insect activity WHO had given protocols for avoiding insect effects 1)Larvicidal activity: Aedes aegyptic ---yellow mosquito 100 ml of beaker Water Acetone extract of drug Larvae are feed with extract Estimation parameter: mortality rate in 24 hours 2) Adulticidal activity: Tribolem castaneum---Red flour beetle Take Red flour beetle in Petri dish In aria of 10 cm Dried at 27 c and 60% RH Add herbal extract Mortality count after 24 hrs Anti insect activity WHO had given protocols for avoiding insect effects 1)Larvicidal activity: Aedes aegyptic ---yellow mosquito 100 ml of beaker Water Acetone extract of drug Larvae are feed with extract Estimation parameter: mortality rate in 24 hours 2) Adulticidal activity: Tribolem castaneum---Red flour beetle Take Red flour beetle in Petri dish In aria of 10 cm Dried at 27 c and 60% RH Add herbal extract Mortality count after 24 hrs
  91. 91. Microbiological Assays Drug substances suppress or influence the growth of micro organism are generally analyzed by microbiological method.(Anti- biotics,Vitamins) 1.Cylindrical plate method Assay of antibiotics is based on measurement of the diameter of microbial growth inhibition surrounding the cylinders containing various dilutions of test compound which are placed on surface of solid nutrient medium . 2.Turbidimetric method Based on inhibition of microbial growth as indicated by measurement of turbidity(transmittance) of suspension of a suitable micro-organism in a fluid medium, to which have test compound. changes in transmittance is compared wit standard known compound. Microbiological Assays Drug substances suppress or influence the growth of micro organism are generally analyzed by microbiological method.(Anti- biotics,Vitamins) 1.Cylindrical plate method Assay of antibiotics is based on measurement of the diameter of microbial growth inhibition surrounding the cylinders containing various dilutions of test compound which are placed on surface of solid nutrient medium . 2.Turbidimetric method Based on inhibition of microbial growth as indicated by measurement of turbidity(transmittance) of suspension of a suitable micro-organism in a fluid medium, to which have test compound. changes in transmittance is compared wit standard known compound.
  92. 92. QUALITY CONTROL OF HERBAL DRUGS WHO guide lines to ensure quality of herbal drugs with modern techniques Monograph guide lines 1.Monograph title A) Botanical a)sensory evaluation b)foreign matter----(should free from it if possible) c)microscopy B )Physico-chemical a)TLC b)Ash----total, acid insoluble, water soluble c)Extractive matter---in hot water, cold water, ethanol d)water content and volatile matter----LOD e)volatile oil-------steam distillation C)Pharmacological a)Bitterness value—units equal to bitterness of std.solu of quinine hydrochloride b)Hemolytic activity----on Ox blood by comparison with std.ref.saponin c)Astringency---Fraction(tannin)that binds to std.hide powder d)swelling index---in water e)foaming index---foam height produced by 14 gm material under specified conditions QUALITY CONTROL OF HERBAL DRUGS WHO guide lines to ensure quality of herbal drugs with modern techniques Monograph guide lines 1.Monograph title A) Botanical a)sensory evaluation b)foreign matter----(should free from it if possible) c)microscopy B )Physico-chemical a)TLC b)Ash----total, acid insoluble, water soluble c)Extractive matter---in hot water, cold water, ethanol d)water content and volatile matter----LOD e)volatile oil-------steam distillation C)Pharmacological a)Bitterness value—units equal to bitterness of std.solu of quinine hydrochloride b)Hemolytic activity----on Ox blood by comparison with std.ref.saponin c)Astringency---Fraction(tannin)that binds to std.hide powder d)swelling index---in water e)foaming index---foam height produced by 14 gm material under specified conditions
  93. 93. D.Toxicological: a)Pesticide residue---chlorides, phosphorus estimation b)Arsenic—strain produced on HgBr2 paper in comparison to std.stain c)Heavy metals---cadmium, lead BW X ADI X Extraction factor safety factor 100 X MDI ADI = avg.daily intake, BW = body weight MDI = mean daily intake E.Microbial contamination: a)total viable aerobic count pathogen:E.coli, salmonella,P.aerogenosa,S.aures Aflatoxins:by TLC using std.Aflatoxins (B1,B2,G1,G2) mixtures----totally free F.Radioactive contamination: As per recommendations of international atomic energy agency IAEA Maximum residue limit = D.Toxicological: a)Pesticide residue---chlorides, phosphorus estimation b)Arsenic—strain produced on HgBr2 paper in comparison to std.stain c)Heavy metals---cadmium, lead BW X ADI X Extraction factor safety factor 100 X MDI ADI = avg.daily intake, BW = body weight MDI = mean daily intake E.Microbial contamination: a)total viable aerobic count pathogen:E.coli, salmonella,P.aerogenosa,S.aures Aflatoxins:by TLC using std.Aflatoxins (B1,B2,G1,G2) mixtures----totally free F.Radioactive contamination: As per recommendations of international atomic energy agency IAEA

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